Research ArticleConventional Rapid Latex Agglutination inEstimation of von Willebrand Factor Method Revisited andPotential Clinical Applications
Marianor Mahat1 Wan Zaidah Abdullah1 and Che Maraina Che Hussin2
1Departments of Haematology School of Medical Sciences Universiti Sains Malaysia Health Campus16150 Kubang Kerian Kelantan Malaysia2Department of Immunology School of Medical Sciences Universiti Sains Malaysia Health Campus16150 Kubang Kerian Kelantan Malaysia
Correspondence should be addressed to Wan Zaidah Abdullah wzaidahusmmy
Received 4 September 2014 Revised 30 November 2014 Accepted 30 November 2014 Published 25 December 2014
Academic Editor Peirong Jiao
Copyright copy 2014 Marianor Mahat et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Measurement of von Willebrand factor antigen (VWF Ag) levels is usually performed in a specialised laboratory which limitsits application in routine clinical practice So far no commercial rapid test kit is available for VWF Ag estimation This paperdiscusses the technical aspect of latex agglutinationmethod which was established to suit the purpose of estimating vonWillebrandfactor (VWF) levels in the plasma sample The latex agglutination test can be performed qualitatively and semiquantitativelyReproducibility stability linearity limit of detection interference and method comparison studies were conducted to evaluate theperformance of this test Semiquantitative latex agglutination test was strongly correlated with the reference immunoturbidimetricassay (Spearmanrsquos rho = 0946 119875 lt 0001 119899 = 132) A substantial agreement (120581 = 077) was found between qualitative latexagglutination test and the reference assay Using the scoring system for the rapid latex test no agglutination is with 0 VWF Ag(control negative) 1+ reaction is equivalent to lt20 VWF Ag and 4+ reaction indicates gt150 VWF Ag (when comparing withimmunoturbidimetric assay) The findings from evaluation studies suggest that latex agglutination method is suitable to be usedas a rapid test kit for the estimation of VWF Ag levels in various clinical conditions associated with high levels and low levels ofVWF Ag
1 Introduction
von Willebrand factor (VWF) a multimeric glycoproteinproduced by endothelial cells and megakaryocytes playsimportant roles in platelet adhesion at the sites of vascularinjury and in the coagulation process through stabilizationof factor VIII [1ndash3] VWF was first identified in hereditarybleeding disorder known as von Willebrand disease (VWD)and later it has been associated with other clinical conditionssuch as cancers clotting and vascular and liver disordersQuantitative and functional impairment of VWF may leadto bleeding disorder In contrast elevated levels of VWF pre-dispose to thrombotic complications [2 3] Epidemiologicalstudies have revealed that an increased level of VWF is sig-nificantly associated with cardiovascular diseases and acute
vascular events [4ndash8] making it a potential biomarker indisease progression and prognosis [9ndash11] Cardiovascular dis-ease is a global health problem and is a leading cause of deathin developed countries [12] In developing countries cardio-vascular disease has become increasingly prevalent [12 13]
Currently the available test for measurement of VWFlevel in plasma is VWF antigen (VWF Ag) assay that isusually performed by enzyme-linked immunosorbent assay(ELISA) and automated immunoturbidimetric proceduresuch as latex-immunoassay (LIA) which requires sophisti-cated instrumentation andwell-trained laboratory personnelFurthermore ELISA test is unsuitable for urgent testing [1415] Despite the advanced technology of testing VWF levelsin a fully equipped laboratory very little attention has beenfocused on testing the VWF levels in areas with limited
Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2014 Article ID 850810 10 pageshttpdxdoiorg1011552014850810
2 Journal of Immunology Research
laboratory facilities or at the bedside Thus the applicationof rapid simple and less expensive test method for thedetection of VWF might help in improving clinical manage-ment in resource-limited setting In the future estimationof VWF Ag could be possible as a point of care testing inclinical practice routinely
Latex agglutination test has been the method of choice inthe development of a rapid test kit in many fields includingclinical and veterinary medicines [16ndash20] In principle latexagglutination test is based on agglutination reactions betweenantigen and antibody Submicron microspheres or oftencalled ldquolatex beadsrdquo are used as a solid support for the anti-body (or antigen) to be adsorbed onto them The latex beadswith adsorbed antibody (or antigen) are used to detect anti-gen (or antibody) present in biological samples Polystyrenelatex beads are commonly used in the latex agglutinationtest because of their strong hydrophobic characteristic thatis ideal for the adsorption of materials such as proteins by asimple passive adsorption method [21]
The use of latex beads was first described for the detectionof rheumatoid factor by Plotz and Singer in 1956 [22] Sincethen latex tests have been developed to detect specific infec-tious diseases autoimmune diseases hormones drugs andserum proteins The original method for attachment of pro-teins (antibody) to latex beadswas passive adsorption Passiveadsorption using plain polystyrene latex beads has been usedin the development of many latex tests for the detection ofantibody or antigen such as Group D salmonellae [23] CRP[24] lactoferrin [25] and rotavirus [26] Covalent couplingmethod using functionalized latex beads such as carboxylatedpolystyrene latex beads can produce more stabilized protein-latex complex compared to passive adsorption method Ithas then become a method of choice in the development oflatex tests such as for the detection of anti-cysticercus anti-bodies [27] IgM quantification in cerebrospinal fluid [28]and detection of avian influenza virus subtype H5N1 [29]Although covalent coupling methods have many advantagesthe passive adsorption method is still widely used until todaybecause of its simplicity and flexibility
Conventional latex agglutination is not a new methodbut its application is limited in the field of hematology andhemostasisThe latexmethod hasmoved towards automationand currently the antigenic detection has been made easyand specific with this new technology especially using themonoclonal generated antibodies However the conventionalmethod may be useful to be applied as a rapid or screeningtest before running the usual laboratory investigations Thepresent study may provide evidence on the usage of a latexagglutination test for the detection of VWF Ag in the plasmasample that could be commercialized in the future In thisstudy surfactant-free latex beads were selected in order toavoid a variation in protein attachment condition that mightoccur due to the variation in the surfactant purityThe aggre-gation in the latex beads is prevented by the electrical chargethat is built onto the surface of the latex beads during theirsynthesis Chloromethyl latex beads have been selected to beused in the passive adsorptionmethodThe beads have a highdensity of chloromethyl groups attached to the styrenemono-meric unit and these functional groups can react directly
with amino groups in antibodiesThe beads are of hydropho-bic type and can be used at both high and low pH conditionsThe beads are stabilized by negatively charged sulfate groupsthat provide the colloid stability In terms of ease of washingand suitability in simple visible test latex beads with a size of1 120583m were used
This study was conducted with the aims to developand validate a latex agglutination test for the detection ofVWF Ag Method validation is a process to demonstratethe suitability of the test method for its intended purposeMethod validation is performed to determine the perform-ance characteristics of the test method for example repro-ducibility stability linearity limit of detection and compari-son ofmethod and also to estimate certain types of analyticalerrors by performing interference study Reproducibilitystudy estimates imprecision stability study determines sta-bility of the sample and reagent linearity study determinesreportable range detection limit study determines the lowestconcentration of analyte method comparison estimates inac-curacy and interference study estimates error caused by othermaterials that may be present in the specimen
2 Materials and Methods
21 Sample Collection and Plasma Preparation The studyprotocol was approved by the Human Research EthicsCommittee from Universiti Sains Malaysia Health Cam-pus Kubang Kerian Kelantan Malaysia [reference numberUSMKKPPPJEPeM2533(4)] Blood samples for validationstudies were collected into 32 buffered trisodium citrate(Becton Dickinson and Co Plymouth UK) and platelet-poor plasma was prepared by centrifuging the blood at2500 g for 15 minutes at room temperature Following cen-trifugation the top 23 of the plasma layer was transferredinto a plastic tube The collected plasma was recentrifuged at2500 g for 15 minutes to remove any remaining red cells orplatelets After centrifugation the top 23 portion of plasmavolume was transferred into cryogenic vials and the plasmasamples were frozen at minus70∘C until use On the day oftesting plasma samples were thawed at 37∘C for 10 minutesand mixed by gentle inversion prior to analysis which wasperformed within two hours after thawing
22 Preparation of VWF Antibody-Latex Reagent Chloro-methyl latex beads 10120583m in diameter were purchased fromLife Technologies USA Prior to use the latex beads werewashed according to the manufacturerrsquos instructions withsome modifications Briefly 25mL of 4 latex suspen-sion was washed twice in 10mL imidazole buffer pH 74(120mMNaCl 20mM imidazole and 5mM citric acid) bycentrifuging the latex suspension at 3000 g for 20 minutesat room temperature The final pellet was resuspended with5mL imidazole buffer to obtain a suspension of 2
VWF antibody-latex reagent was prepared by passiveadsorption method according to the manufacturerrsquos protocoland themethod described byMina et al 2012 [30] with somemodifications Briefly 10mL of 2 latex suspension was cen-trifuged at 3000 g for 20 minutes at room temperature and
Journal of Immunology Research 3
Table 1 Test score for qualitative latex agglutination test for VWF Ag
Test score Size of the agglutinated beads Appearance of the background Rapidity of the agglutination1+ Small clumps Cloudy gt2min2+ Small clumps Cloudy gt1min3+ Smalllarge clumps Clear gt30 sec4+ Large clumps Clear lt30 secVWF Ag von Willebrand factor antigen
the supernatant was discarded The pellet was resuspendedwith 1mL phosphate-buffered saline pH 74 (135mMNaCl26mMKCl 8mMNa
2HPO4 and 15mMKH
2PO4) that
was added with 200120583L of polyclonal rabbit anti-human vonWillebrand Factor from Dako Denmark (31 gL) The mix-ture was incubated with gentle mixing on a horizontal rotator(Barnstead Thermolyne Labquake Rotator USA) at roomtemperature for 24 hours After incubation the mixture wascentrifuged and the supernatant was kept for determinationof unadsorbed antibody by BCA method using Micro BCAProtein Determination Kit from Pierce Biotechnology USAThe concentration of unadsorbed antibody in the supernatantwas determined according to themanufacturerrsquos instructionsThe concentration of adsorbed antibody was estimated as thedifference between the initial concentration of antibody (ie620120583g) and the concentration of unadsorbed antibody in thesupernatant
Thepellet was thenwashed twice in 4mL imidazole bufferby centrifugation as stated earlier and resuspended with 3mLimidazole buffer containing 1 bovine serum albumin(Sigma-Aldrich USA) The mixture was incubated with gen-tle mixing at room temperature for one hour After incuba-tion the mixture was centrifuged and the pellet was washedtwice in 4mL imidazole buffer by centrifugation as statedearlier The final pellet was resuspended with 2mL imidazolebuffer containing 01 bovine serum albumin to obtain asuspension of 1 and the suspension was stored at 4∘C untiluse
The process described was carried out using differentconcentrations of the VWF antibody to determine the appro-priate concentration of antibody for the adsorption proce-dure As a control reagent the process was conductedwithoutthe antibody All reagents were tested on known negativeand positive samples Normal saline and buffers were usedas negative samples while commercial controls were used aspositive samples
23 Latex Agglutination Test Procedure and Result Interpre-tation VWF antibody-latex reagent was brought to roomtemperature and mixed gently before use The sedimentationof latex beads can occur during storage that can be reversedby pipetting the suspension of latex beads through a fine tippipette until they are returned to a uniform suspension Latexagglutination test for the detection of VWF in a plasma sam-ple was performed on a slide card (Thermo Scientific UK)Using a wooden applicator stick 20 120583L of VWF antibody-latex reagent was mixed with 20120583L of plasma sample on ablack reaction zone of the card The card was rotated on
Figure 1 Positive (number 5) and negative (number 6) reactions ofqualitative latex agglutination test for VWF Ag
a horizontal shaker (Boekel GrantMicroplate Shaker UK) forthreeminutes After threeminutes the agglutination reactionwas observed by visual inspection macroscopically with anaked eye
The detection and estimation of VWF by latex agglutina-tion test can be performed using a qualitative test and semi-quantitative test respectively Qualitative test was performedon undiluted plasma sample and the result was reportedas negative or positive Invisible or hardly visible agglutina-tion was reported as negative and visible agglutination wasreported as positive (Figure 1) For a positive result the degreeof agglutination was scored from 1+ to 4+ based on the sizeof the agglutinated beads the appearance of the backgroundand the rapidity of the agglutination development as shownin Table 1 Semiquantitative test was performed on dilutedplasma sample A positive plasma sample was serially dilutedtwofold with normal saline The highest dilution that stillshows a positive reaction is the end-titre of a sample
24 Validation Studies on VWF Antibody-Latex Reagent
241 Reproducibility Study Reproducibility studies wereperformedwithin a day and between daysWithin-day repro-ducibility study was carried out by testing VWF antibody-latex reagent on three plasma samples and the end-titreof each sample was determined three times Between-dayreproducibility study was conducted by testing the reagentin duplicate on two plasma samples and the end-titre of thesamples was determined on days 1 7 14 and 21 The reagentswere also tested on low and normal controls from Instrumen-tation Laboratories Italy (Hemosil Special Test Control Level2 and Hemosil Normal Control Assayed resp) on days 2 45 7 30 and 50
4 Journal of Immunology Research
242 Stability Study Stability of the reagent was checkedduring storage of VWF antibody-latex reagent at 4∘C for 21days Qualitative test was performed on two plasma sampleson days 1 2 3 4 5 6 7 14 and 21 using aliquots of samplesthat were stored at minus70∘C A new aliquot of sample was usedin each experiment Stability of plasma sample during storagewas also checked by testing VWF antibody-latex reagent(stored at 4∘C) on aliquots of plasma samples which wasstored at 4∘C After 0 (within two hours after blood collec-tion) 1 2 3 4 5 6 7 14 and 21 days the qualitative test wasperformed The reagents were also tested on Hemosil SpecialTest Control Level 2 and Hemosil Normal Control Assayed(Instrumentation Laboratories Italy) Semiquantitative latexagglutination test was performed on these controls for up to50 days Batch to batch variation was tested with the reagentsprepared on two different dates using two different lots of theVWF antibody
243 Linearity Study The linearity of latex agglutinationtest was determined by testing VWF antibody-latex reagenton two patient samples which were serially diluted twofoldThe VWF Ag level in diluted samples was measured byimmunoturbidimetric assay using Hemosil von WillebrandFactor Antigen kit (Instrumentation Laboratory Italy) Semi-quantitative latex agglutination test was performed ondiluted samples and the end-titres were compared with theVWF Ag levels Graphical presentations of the semiquan-titative VWF Ag by latex agglutination test (119884) versus theVWF Ag by immunoturbidimetric assay (119883) were made forindividual sample and also for a combination of the twosamples
244 Determination of Limit of Detection Limit of detectionof latex agglutination test was determined by testing VWFantibody-latex reagent on low-level samples (cryosuper-natant) Seven cryosupernatant samples were serially dilutedtwofold and the VWF Ag levels in the samples (neat) weremeasured by immunoturbidimetric assay using Hemosil vonWillebrand Factor Antigen kit (Instrumentation LaboratoryItaly) VWF Ag levels in 1 2 1 4 and 1 8 diluted sampleswere estimated by dividing the VWF Ag levels of neat sam-ples by the dilution factors of 2 4 and 8 respectively Latexagglutination test was performed to determine the lowestlevel of VWF Ag that shows positive agglutination reaction
245 Interference Study
(i) Interference fromHemoglobin Interference fromhemoglo-bin is common in coagulation testing and hemolytic speci-men is usually not orminimally affected in latex agglutinationtest The interference study was performed by testing VWFantibody-latex reagent on plasma pools and plasma poolsspiked at different concentrations of hemolysate Hemolysatewas prepared by freezing and thawing the whole bloodfollowed by the osmotic shock protocol [31] Briefly normalsample that was collected in 30mLK
2EDTA tube was
centrifuged at 1000 g for 10 minutes Plasma was removedand replaced with an equal volume of isotonic saline Cells
were resuspended and the suspension was centrifuged againat 1000 g for 10 minutes The saline wash was repeated threetimes In the final wash saline was replaced with distilledwater and the suspension was stored at minus20∘C overnightFrozen cells were thawed mixed and centrifuged at 2000 gfor 30 minutes to remove cell debris
Hemolysate was transferred to a clean tube and hemoglo-bin level wasmeasuredwith the SysmexXE 5000Hematologyanalyzer (Sysmex Corporation Kobe Japan) Stock solutionsof hemolysate in normal saline were prepared and each ofthe stock solutions was used to spike plasma pools with highand low VWF Ag levels In the present study hemolysatewith hemoglobin level of 120 gL was used to prepare stocksolutions of hemolysateThe hemolysate was diluted 1 2 1 41 8 1 20 and 1 40 with normal saline to obtain the stocksolutions with hemoglobin levels of 60 30 15 6 and 3 gLrespectively One hundred microlitres of each stock solutionwere added to 900 120583L of plasma pools to obtain plasma poolswith a final hemoglobin level of 6 3 15 06 and 03mgmL
(ii) Interference from Rheumatoid Factor Rheumatoid factor(RF) is one of the most common interferences in latex-basedmethods It may bind and cross-link antibodies attached tothe latex beads leading to a false-positive result or falselyelevated levels of VWF in a sample [32] The effect of RF onagglutination reaction was studied by testing VWF antibody-latex reagent on plasma pools with high and low VWF Aglevels The plasma pools were spiked at different concen-trations of RF using rheumatoid factor control serum fromMyBioSource USA (1030 IUmL)
The RF control serum was diluted 1 8 1 7 and 1 6 withhigh plasma pools to obtain plasma pools spiked with RFat a concentration of 129 147 and 172 IUmL respectivelyFor the preparation of low plasma pools spiked with RF at aconcentration of 10 15 52 103 and 129 IUmL the RF controlserum was diluted 1 100 1 70 1 20 1 10 and 1 8 respec-tively with low plasma pools
246MethodComparisonwith the ReferenceMethodMethodcomparison study between latex agglutination test and thereference method (immunoturbidimetric assay) was con-ducted by parallel analysis of 132 plasma samples of differentlevels of VWF Ag The samples were obtained from 40healthy individuals 40 patients with cardiovascular diseasesand 32 patients with other diseases including malignant andinflammatory disorders The remaining 20 samples werecryosupernatant samples (ie samples with low levels ofVWF Ag) that were obtained from Blood Transfusion Ser-vices All specimens were tested in ten days and the testingwas spread over ten months
Qualitative and semiquantitative latex agglutination testswere performed using the VWF antibody-latex reagentsThe immunoturbidimetric assay was performed on ACLElite Pro Coagulation analyser (Instrumentation LaboratoryItaly) using Hemosil von Willebrand Factor Antigen kitfrom Instrumentation Laboratory Italy The procedure forVWF Ag by immunoturbidimetric assay was performedaccording to the manufacturerrsquos instructions and adhered to
Journal of Immunology Research 5
Table 2 Estimated concentration of adsorbed antibody in the VWF antibody-latex reagents
the CLSI Document H21-A5 on the specimen collection andprocessing of blood specimens for testing the plasma-basedcoagulation assays Normal reference range for VWF Aglevels by immunoturbidimetric assay in our laboratory is 50ndash150
25 Statistical Analysis Statistical analyses were conductedusing the SPSS software version 20 (SPSS Inc Chicago ILUSA) Linear regression analysis was performed to determinethe linearity of the semiquantitative latex agglutination testThe degree of agreement between qualitative latex aggluti-nation test and immunoturbidimetric assay was estimatedby calculating the kappa value (120581) 081ndash099 almost per-fect agreement 061ndash080 substantial agreement 041ndash060moderate agreement 021ndash040 fair agreement 001ndash020slight agreement and lt0 less than chance agreement [33]Spearmanrsquos rho correlation analysiswas used to determine thedegree of relationship between semiquantitative latex agglu-tination test and immunoturbidimetric assay The strengthof the relationship is showed by the correlation coefficientvalue (119903) at least 08 very strong 06 up to 08 moderatelystrong 03 to 05 fair and less than 03 poor [34] Two-tailed probability (119875) value of less than 005 was consideredstatistically significant
3 Results
31 Preparation of VWF Antibody-Latex Reagent Table 2shows the estimated concentration of adsorbed antibody onthe latex beads in the VWF antibody-latex reagents that wereprepared using different concentrations of the VWF anti-body The results showed that the concentration of adsorbedantibody increased as the initial concentration of the VWFantibody increased However the percentage of adsorbedantibody that is 70 and 74 was not much different whenadsorption method was performed using antibody concen-tration of 620120583g and 700 120583g respectively
Visual observation on the agglutination reaction showedthat less agglutination was observed from latex beadsadsorbed with less than 400 120583g of antibody (ie 118 120583g276120583g and 328 120583g) compared to agglutination reactionfrom latex beads adsorbed with about 400 120583g of antibodyAgglutination reactions from latex beads coated with 520120583gantibody were slightly more compared to agglutination reac-tions from latex beads coated with 436 120583g antibody (data notshown) In other words latex beads with sim400 120583g adsorbed
antibody were sufficient to produce strong agglutinationreaction when mixed with plasma sample Thus 200120583L ofthe VWF antibody with a concentration of 620120583g has beenchosen to be the most appropriate concentration for theadsorption procedure
Autoagglutination was not seen when VWF antibody-latex reagent alone was rotated for three minutes Extendingthe time beyond three minutes resulted in reagent dryingthat might give false-positive result No agglutination wasseen when the control reagent (ie reagent without adsorbedantibody) was tested on plasma sample and commercialcontrols Negative reaction was consistently observed whenthe VWF antibody-latex reagent was tested on normal salineand buffers throughout this study
32 Validation Studies Validation studies were conductedusing VWF antibody-latex reagents that were prepared inseven batches The concentration of adsorbed antibody onthe latex beads ranged from 403 to 447 g (ie 65ndash72 fromthe initial concentration of the VWF antibody used in theadsorption procedure) which showed that the percentage oftheVWFantibody adsorbed on the latex beadswas consistentthroughout this study
321 Reproducibility and Stability Studies In the repro-ducibility study the end-titre of three samples (ie 1 321 64 and 1 128) was consistent when tested three timeswithin a day Similarly the end-titre of two samples (ie 1 16and 1 128) did not change from day 1 to day 21 (betweendays) Testing on the controls showed that the end-titre of lowand normal controls (1 4 and 1 16 resp) was reproduciblefrom day 1 to day 50 In the stability study VWF antibody-latex reagent and plasma sample were found to be stable forqualitative latex agglutination test when stored at 4∘C for atleast 21 days Semiquantitative test on controls showed thatthe VWF antibody-latex reagent was stable for at least 50days
All these findings showed that the semiquantitative latexagglutination test results were reproducible within a day andbetween days and the reagent was found to be stable for atleast 50 days when stored at 4∘C Batch to batch variation wasnot detected
322 Linearity Study In the linearity study a series of knownconcentrations of VWF Ag was established by dilution withfive equally spaced concentrations as shown in Table 3 The
6 Journal of Immunology Research
Table 3 Linearity study on semiquantitative VWF antigen by latex agglutination test
Tube number Sample 1 Sample 2VWF Ag ()a VWF Ag (titre)b VWF Ag ()a VWF Ag (titre)b
1 600 1 128 460 1 642 300 1 64 230 1 323 150 1 32 115 1 164 869 1 16 579 1 85 222 1 8 279 1 46 99 1 4 176 1 2aVWF Ag levels () measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from Instrumentation Laboratory ItalybVWF Ag (titre) estimated by using semiquantitative latex agglutination testVWF von Willebrand factor VWF Ag von Willebrand factor antigen
Table 4 Limit of detection of VWF antigen detected by using latex agglutination test
Samplesample number Neat 1 2 1 4 1 8VWF Ag ()a LATe VWF Ag ()b LATe VWF Ag ()c LATe VWF Ag ()d LATe
1 242 + 121 + 61 + 30 minus
2 202 + 102 + 51 + 25 minus
3 306 + 153 + 69 + 38 minus
4 276 + 138 + 69 + 34 minus
5 269 + 133 + 67 + 34 minus
6 291 + 146 + 73 + 36 minus
7 303 + 152 + 76 + 38 minus
aVWF Ag levels in neat sample that was measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from InstrumentationLaboratory ItalybcdEstimated VWF Ag levels in 1 2 1 4 and 1 8 diluted samples (ie by dividing the VWF Ag levels of neat sample by the dilution factors of 2 4 and 8respectivelyePositive (+) and negative (minus) reactions observed when neat and diluted samples were tested by using latex agglutination testVWF Ag von Willebrand factor antigen LAT latex agglutination test
table shows the latex agglutination test results (titre) and thelevels of VWF Ag in undiluted (tube 1) and diluted samples(tubes 2ndash6) It was shown in the table that the semiquanti-tative latex agglutination test results (titre) were directly pro-portional to the levels of VWF Ag in the plasma sample Lin-earity of the semiquantitative VWF Ag latex agglutinationtest was demonstrated in sample 1 (1199032 = 09985) and sample 2(1199032 = 09999) A regression line that was fit to the pointswas observed in both samples (graphs not shown) Thelinearity was also demonstrated at the titre ranging from 1 2to 1 128 when the results of both samples were combined andanalyzed (1199032 = 09361) All these findings have demonstrateda linear relationship between the observed results (VWF Agtitre) and the true concentrations of VWF Ag in the plasmasamples
323 Determination of Limit of Detection In the limit ofdetection study plasma samples with low levels of VWF Ag(in the range of 202 to 306) were obtained from sevencryosupernatant samples (Table 4) Table 4 shows the agglu-tination reaction observed when latex agglutination test wasperformed on undiluted (neat) samples and diluted samples(at 1 2 1 4 and 1 8 dilutions) As shown in the table thelowest level of VWF Ag that still showed positive reaction is51 (sample number 2 at 1 4 dilution) and the highest level
of VWF Ag that showed negative reaction is 38 (samplenumber 7 at 1 8 dilution) These results showed that thelowest level of VWF Ag that the latex agglutination test candetect to determine the presence (positive result) or absence(negative result) of VWF Ag was 51 Thus the limit ofdetection of VWF Ag by latex agglutination test is approx-imately 5 Besides that reproducibility of the agglutinationreaction at VWF Ag levels of 51 and 38 was also checkedby testing six times the diluted samples of number 2 (at 1 4dilution) and number 7 (at 1 8 dilution) respectively Samplenumber 2 showed positive reactions in all six replicates whilesample number 7 showed negative reaction in all six repli-cates These findings showed that the latex agglutination testreliably produced consistent results near the cutoff concen-tration
324 Interference Study Visible agglutinationwas seenwhenlatex agglutination test was performed on undiluted plasmapools and undiluted plasma pools with a final hemoglobinlevel of 03 06 15 3 6 and 12mgmL The end-titre of1 32 was obtained when semiquantitative latex agglutinationtest was performed on high plasma pool The same end-titre(1 32) was observed when the test was performed on highplasma pools with hemoglobin level of 03 06 15 3 6 and12mgmL Similarly the end-titre of low plasma pools with
Journal of Immunology Research 7
Table 5 Rheumatoid factor interference on high and low plasma pools
Type of the plasma pool Vol of RF control serum(120583L)a
aRheumatoid factor control serum fromMyBioSource USA (1030 IUmL) was used to spike plasma poolsRF rheumatoid factor VWF LAT von Willebrand factor latex agglutination test vol volume conc concentration
Table 6 Crosstabulation of VWF antigen levels and qualitative VWF by latex agglutination test results
Total 6 14 61 51 132VWF von Willebrand factor VWF Ag von Willebrand factor antigen LAT latex agglutination test
hemoglobin level of 03 06 15 3 6 and 12mgmL was thesame as the end-titre of low plasma pool without hemoglobininterference (1 4) These results showed that hemoglobindid not cause interference in latex agglutination test andsemiquantitative test result was not affected by hemoglobinlevel up to 12mgmL
Table 5 shows the end-titre of high and low plasma poolsspiked at different concentrations of RF As shown in thetable interference fromRF on latex agglutination test was notdetected in high plasma pools spiked with RF at concentra-tions up to 129 IUmLNevertheless the interference fromRFwas observed in low plasma pools spiked with RF at concen-trations of 26 52 103 and 129 IUmL The end-titre of highand low plasma pools without interference from RF was 1 64and 1 2 respectivelyThese results showed that RF interferedwith the latex agglutination test result when the test wasperformed on the plasma sample containing low levels ofVWF Ag and high levels of RF (gt26 IUmL) However latexagglutination test result of plasma sample containing highlevels of VWF Ag was not affected by RF at concentrationsof lt130 IUmL
325 Method Comparison with the Reference Method Com-parison study between qualitative latex agglutination testand immunoturbidimetric assay showed a kappa value of077 which indicates a substantial agreement between thetwo tests Table 6 shows the crosstabulation of 132 samplesincluded in this study Semiquantitative latex agglutinationtest was strongly correlated with immunoturbidimetric assay
(Spearmanrsquos rho 119903 = 0946 119875 lt 0001) across a range ofVWF Ag levels from 345 to 870
Based on the results from kappa statistic and correlationstudy the scoring system for agglutination reactions and cor-relation with the VWF Ag levels (using immunoturbidimet-ric assay) is shown in Table 7 The proposed interpretationguide is also put in a footnote
4 Discussion
To the best of our knowledge there has been no reportedstudy or technical description available on the rapid test forVWF Ag utilising latex agglutination method At presentthe application of this method has not been used in clin-ical practice for monitoring of patients with low and highplasma levels of VWF Ag Screening of VWDpatients beforeconfirmation tests and monitoring of patients at high risk ofthrombotic event in the area with low-resource settings or atthe bedside can be performed if rapid test for VWF Ag isavailable Hence there is a need to develop a rapid and simplelatex agglutination test that can reliably detect and estimateVWF Ag in the plasma sample
Latex agglutination test is based on the observation ofvisible clumps that are formed from the reaction of antigen-antibody complexes The antigen-antibody complexes can beprepared by passive adsorption or covalent coupling methodIn the early stage of the present study attempts had beenmadeto couple VWF antibody with the carboxylated latex beads
8 Journal of Immunology Research
Table 7 Correlation of result interpretations by latex agglutination test and immunoturbidimetric assay
Qualitative latex agglutination test (LATscore)
Semiquantitative latex agglutination test(titre)
VWF Ag levels by immunoturbidimetricassay ()
1+ 1 2 lt202+ 1 4 1 8 20ndash503+ 1 16 1 32 50ndash1504+ ge64 gt150The proposed interpretation guide normal level of VWF Ag is indicated by LAT scores of 2+ and 3+ or titres from 1 8 to 1 32 Abnormal low VWF Ag isindicated by LAT scores of 0 1+ and 2+ or titres of 1 2 and 1 4 Abnormal high VWF Ag is indicated by LAT score of 4+ or titres of ge64LAT latex agglutination test VWF Ag von Willebrand factor antigen
by covalent coupling method The coupling procedure wasperformed in MES buffer at pH 60 according to the protocolfrom themanufacturer Unfortunately there was no antibodycoated on the latex beads (data not shown) However theVWF antibody was successfully adsorbed onto the surfaceof chloromethyl latex beads by passive adsorption methodThese findings were similar to the reported study by Garcia etal 2015 [35] In the present study amaximal physical adsorp-tion of the VWF antibody onto chloromethyl latex beads wasobtained at pH 74 a pH that is close to the isoelectric pointof polyclonal immunoglobulin (IgG) antibody [36 37]
Interference by RF in the latex agglutination test for thedetection of VWF Ag was expected RF is known to causeanalytical error in automated immunoturbidimetry assay thatpresented the main limitation of this method Despite thislimitation the findings do nevertheless show that the effect ofthe interference was less prominent in the sample with highlevels of VWF Ag compared to the sample with low levelsof VWF Ag The expected interference is without any doubtdue to the usage of whole IgG in the passive adsorptionmethod It iswell known that rheumatoid factor can bindwiththe Fc region of IgG causing false agglutination thatmay alterthe result of the test In the literature the usage of antibodyfragments such as Fab or F(ab1015840)
2was recommended to elim-
inate the interference from rheumatoid factor [37] Antibodyfragment is suggested in future study and optimization isrequired as the antibody fragment is more acidic than thepolyclonal antibody
In the present study the agglutination observed from thereaction between VWF antibody-latex reagent and positivesample may not be optimum as expected Experiments usinghigher initial concentration of VWF antibody with a moreintensive procedure to ensure maximal bead coating couldbe performed in the future study Not only that but theorientation of adsorbed antibody could also be studied toensure a proper alignment of antibody on latex beads Clariziaet al 2009 [38] have demonstrated a method for detectingthe aligned andmisaligned antibodies on the surface on latexbeads Due to some technical constraint maximal bead coat-ing cannot be ensured and a study on orientation of adsorbedantibody could not be performed Another limitation of thisstudy is that the size of the clumping was only estimatedby visual observation In future study it is recommendedto measure the clumping or macroscopic clusters usingmicroscope or other methods
There is a potential clinical application of this rapid latexagglutination test as a routine test in various premises includ-ing small laboratory and clinic and at the bedsideThe clinicalaspect of low levels of VWF Ag is well recognized but notmany medical personnel realized the implications of highVWF Ag levels in medical practice An inherited bleedingdisorder known as von Willebrand disease (VWD) is asso-ciated with an abnormality in VWF and type 1 VWD is thecommonest form that is associated with low VWF Ag [39]On the other hand high levels of VWF Ag are associatedwith thrombotic risks and had been extensively studied inmany medical conditions particularly coronary heart disease(CHD) and stroke It has been shown that CHD patients withhigh baseline of VWF Ag levels are at risk of acute vascularevents related to thrombosis [40] The current clinical guide-lines do not incorporate the measurement of VWF Ag inthe routine assessment of CHD patients but from variousstudies there is a role in monitoring the levels of VWF AgDetection of persistent high levels or increasing trends ofVWF Ag levels would alert the treating doctor for certainclinical measures to improve the patient care
Latex agglutination test for the detection of VWF Agwasfound to be simple to perform and the results can be obtainedwithin three minutes making it suitable to be used in areaswith limited laboratory facilities The result obtained bythe latex agglutination test is reliable as shown by the findingsfrom reproducibility stability linearity limit of detectionand method comparison studies Prozone effect was notdetected at VWF Ag levels up to 870 The stability of thereagents used for this method was up to 50 days and in thisstudy no stabilizer or preservatives were used The plasmafor VWF Ag estimation was stable for up to 21 days whichmay be related to the protein structure however more testsare needed to confirm the suitable plasma storage recom-mendation for this method All these findings showed theusefulness of this method in detecting and estimating highand low levels of VWF AgThis method might be useful inthe monitoring of VWF Ag in patients that are at a high riskof vascular event related to endothelial dysfunction Similarlythis method could be used to estimate VWF Ag levels inpatients with VWD before confirmation with standard testpanels in the specialized coagulation laboratory
However we only evaluate the latex agglutination testin quantitative terms by using plasma samples containinglow and high levels of VWF Ag This application mainlyaddressed the VWD type 1 which is more common in clinical
Journal of Immunology Research 9
practice In future study plasma samples with qualitativeVWF abnormalities (eg type II VWD or acquired VWDpatients demonstrating abnormal VWF function with vari-ables VWF Ag levels) could be included in the comparisonstudy to confirm the application of the latex agglutination testfor assessing VWF Ag levels in various clinical situations Insummary this test method needs to be refined and comparedwith other latex agglutination tests available in the marketbefore it is used on patient samples
5 Conclusion
Latex agglutination test for VWF Ag is simple rapid andreproducible correlates well with the reference method andis suitable to be used in areaswith limited laboratory facilitiesThe test has commercial potential as a low-cost alternativemethod for the detection and estimation of low levels andhigh levels of VWF Ag that could help in themanagement ofVWDtype 1 patients andpatients at risk of thrombotic eventsrespectively However the testmay not be suitable for patientswith a high level of rheumatoid factor
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by short-term Grant 304PPSP61312085 from Universiti Sains Malaysia Penang MalaysiaThe authors gratefully acknowledge Dr Emmanuel J Faval-oro from Institute of Clinical Pathology and MedicalResearch at Westmead Hospital Australia for his technicaladvice on this work Thanks are due to Dr Lim TheamSoon from Institute for Research in Molecular MedicineUniversiti Sains Malaysia Penang Malaysia for his valuableadvice in the early stage of this work The appreciation alsogoes to the staff of Haematology Department (especially thecoagulation section) BloodTransfusion ServiceDepartmentand Immunology Department Universiti Sains MalaysiaKelantan Malaysia for their continuous support in thiswork
References
[1] Z M Ruggeri ldquoStructure of vonWillebrand factor and its func-tion in platelet adhesion and thrombus formationrdquo Best Practiceand Research Clinical Haematology vol 14 no 2 pp 257ndash2792001
[2] G-P Luo B Ni X Yang and Y-ZWu ldquovonWillebrand factormore than a regulator of hemostasis and thrombosisrdquo ActaHaematologica vol 128 no 3 pp 158ndash169 2012
[3] C V Denis and P J Lenting ldquoVon Willebrand factor at thecrossroads of bleeding and thrombosisrdquo International Journal ofHematology vol 95 no 4 pp 353ndash361 2012
[4] A Alonso W Tang S K Agarwal E Z Soliman A M Cham-berlain and A R Folsom ldquoHemostatic markers are associatedwith the risk and prognosis of atrial fibrillation the ARIC
studyrdquo International Journal of Cardiology vol 155 no 2 pp217ndash222 2012
[5] F Cambronero J A Vilchez A Garcıa-Honrubia et al ldquoPlasmalevels of Von Willebrand factor are increased in patients withhypertrophic cardiomyopathyrdquo Thrombosis Research vol 126no 1 pp e46ndashe50 2010
[6] A M Kucharska-Newton D J Couper J S Pankow et alldquoHemostasis inflammation and fatal and nonfatal coronaryheart disease long-term follow-up of the atherosclerosis risk incommunities (ARIC) cohortrdquo Arteriosclerosis Thrombosis andVascular Biology vol 29 no 12 pp 2182ndash2190 2009
[7] R G Wieberdink M C van Schie P J Koudstaal et al ldquoHighvon Willebrand factor levels increase the risk of stroke theRotterdam studyrdquo Stroke vol 41 no 10 pp 2151ndash2156 2010
[8] P Wennberg F Wensley E Di Angelantonio et al ldquoHaemo-static and inflammatory markers are independently associatedwith myocardial infarction in men and womenrdquo ThrombosisResearch vol 129 no 1 pp 68ndash73 2012
[9] A Hyseni M Roest S L Braun et al ldquoChronic dysfunction ofthe endothelium is associated with mortality in acute coronarysyndrome patientsrdquoThrombosis Research vol 131 no 3 pp 198ndash203 2013
[10] A A Lopes A C Barreto N Y Maeda et al ldquoPlasma vonWillebrand factor as a predictor of survival in pulmonaryarterial hypertension associated with congenital heart diseaserdquoBrazilian Journal of Medical and Biological Research vol 44 no12 pp 1269ndash1275 2011
[11] V Roldan F Marın B Muina et al ldquoPlasma von Willebrandfactor levels are an independent risk factor for adverse eventsincluding mortality and major bleeding in anticoagulated atrialfibrillation patientsrdquo Journal of the American College of Cardiol-ogy vol 57 no 25 pp 2496ndash2504 2011
[12] T A Gaziano A Bitton S Anand S Abrahams-Gessel and AMurphy ldquoGrowing epidemic of coronary heart disease in low-andmiddle-income countriesrdquoCurrent Problems in Cardiologyvol 35 no 2 pp 72ndash115 2010
[13] V L Roger A S Go D M Lloyd-Jones et al ldquoExecutive sum-mary heart disease and stroke statisticsmdash2012 update a reportfrom the American Heart AssociationrdquoCirculation vol 125 no1 pp 188ndash197 2012
[14] G Castaman A Tosetto A Cappelletti et al ldquoValidation ofa rapid test (VWF-LIA) for the quantitative determination ofvonWillebrand factor antigen in type 1 vonWillebrand diseasediagnosis within the European multicenter study MCMDM-1VWDrdquoThrombosis Research vol 126 no 3 pp 227ndash231 2010
[15] A Kappel and M Ehm ldquoImmunoassays for diagnosis of coag-ulation disordersrdquo Hamostaseologie vol 30 no 4 pp 194ndash2012010
[16] R Biswas and S C Parija ldquoA rapid slide agglutination test forthe diagnosis of neurocysticercosis in the rural health set uprdquoTropical Parasitology vol 1 no 2 pp 94ndash98 2011
[17] H Lee Y Park M Kim et al ldquoDevelopment of a latex agglu-tination test for norovirus detectionrdquo Journal of Microbiologyvol 48 no 4 pp 419ndash425 2010
[18] H Y Darani F Ahmadi N Zabardast H A Yousefi and HShirzad ldquoDevelopment of a latex agglutination test as a simpleand rapid method for diagnosis of Trichomonas vaginalisinfectionrdquoAvicenna Journal of Medical Biotechnology vol 2 no1 pp 63ndash66 2010
[19] MMoraveji A Hosseini N Moghaddar M M Namavari andM H Eskandari ldquoDevelopment of latex agglutination test with
10 Journal of Immunology Research
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
laboratory facilities or at the bedside Thus the applicationof rapid simple and less expensive test method for thedetection of VWF might help in improving clinical manage-ment in resource-limited setting In the future estimationof VWF Ag could be possible as a point of care testing inclinical practice routinely
Latex agglutination test has been the method of choice inthe development of a rapid test kit in many fields includingclinical and veterinary medicines [16ndash20] In principle latexagglutination test is based on agglutination reactions betweenantigen and antibody Submicron microspheres or oftencalled ldquolatex beadsrdquo are used as a solid support for the anti-body (or antigen) to be adsorbed onto them The latex beadswith adsorbed antibody (or antigen) are used to detect anti-gen (or antibody) present in biological samples Polystyrenelatex beads are commonly used in the latex agglutinationtest because of their strong hydrophobic characteristic thatis ideal for the adsorption of materials such as proteins by asimple passive adsorption method [21]
The use of latex beads was first described for the detectionof rheumatoid factor by Plotz and Singer in 1956 [22] Sincethen latex tests have been developed to detect specific infec-tious diseases autoimmune diseases hormones drugs andserum proteins The original method for attachment of pro-teins (antibody) to latex beadswas passive adsorption Passiveadsorption using plain polystyrene latex beads has been usedin the development of many latex tests for the detection ofantibody or antigen such as Group D salmonellae [23] CRP[24] lactoferrin [25] and rotavirus [26] Covalent couplingmethod using functionalized latex beads such as carboxylatedpolystyrene latex beads can produce more stabilized protein-latex complex compared to passive adsorption method Ithas then become a method of choice in the development oflatex tests such as for the detection of anti-cysticercus anti-bodies [27] IgM quantification in cerebrospinal fluid [28]and detection of avian influenza virus subtype H5N1 [29]Although covalent coupling methods have many advantagesthe passive adsorption method is still widely used until todaybecause of its simplicity and flexibility
Conventional latex agglutination is not a new methodbut its application is limited in the field of hematology andhemostasisThe latexmethod hasmoved towards automationand currently the antigenic detection has been made easyand specific with this new technology especially using themonoclonal generated antibodies However the conventionalmethod may be useful to be applied as a rapid or screeningtest before running the usual laboratory investigations Thepresent study may provide evidence on the usage of a latexagglutination test for the detection of VWF Ag in the plasmasample that could be commercialized in the future In thisstudy surfactant-free latex beads were selected in order toavoid a variation in protein attachment condition that mightoccur due to the variation in the surfactant purityThe aggre-gation in the latex beads is prevented by the electrical chargethat is built onto the surface of the latex beads during theirsynthesis Chloromethyl latex beads have been selected to beused in the passive adsorptionmethodThe beads have a highdensity of chloromethyl groups attached to the styrenemono-meric unit and these functional groups can react directly
with amino groups in antibodiesThe beads are of hydropho-bic type and can be used at both high and low pH conditionsThe beads are stabilized by negatively charged sulfate groupsthat provide the colloid stability In terms of ease of washingand suitability in simple visible test latex beads with a size of1 120583m were used
This study was conducted with the aims to developand validate a latex agglutination test for the detection ofVWF Ag Method validation is a process to demonstratethe suitability of the test method for its intended purposeMethod validation is performed to determine the perform-ance characteristics of the test method for example repro-ducibility stability linearity limit of detection and compari-son ofmethod and also to estimate certain types of analyticalerrors by performing interference study Reproducibilitystudy estimates imprecision stability study determines sta-bility of the sample and reagent linearity study determinesreportable range detection limit study determines the lowestconcentration of analyte method comparison estimates inac-curacy and interference study estimates error caused by othermaterials that may be present in the specimen
2 Materials and Methods
21 Sample Collection and Plasma Preparation The studyprotocol was approved by the Human Research EthicsCommittee from Universiti Sains Malaysia Health Cam-pus Kubang Kerian Kelantan Malaysia [reference numberUSMKKPPPJEPeM2533(4)] Blood samples for validationstudies were collected into 32 buffered trisodium citrate(Becton Dickinson and Co Plymouth UK) and platelet-poor plasma was prepared by centrifuging the blood at2500 g for 15 minutes at room temperature Following cen-trifugation the top 23 of the plasma layer was transferredinto a plastic tube The collected plasma was recentrifuged at2500 g for 15 minutes to remove any remaining red cells orplatelets After centrifugation the top 23 portion of plasmavolume was transferred into cryogenic vials and the plasmasamples were frozen at minus70∘C until use On the day oftesting plasma samples were thawed at 37∘C for 10 minutesand mixed by gentle inversion prior to analysis which wasperformed within two hours after thawing
22 Preparation of VWF Antibody-Latex Reagent Chloro-methyl latex beads 10120583m in diameter were purchased fromLife Technologies USA Prior to use the latex beads werewashed according to the manufacturerrsquos instructions withsome modifications Briefly 25mL of 4 latex suspen-sion was washed twice in 10mL imidazole buffer pH 74(120mMNaCl 20mM imidazole and 5mM citric acid) bycentrifuging the latex suspension at 3000 g for 20 minutesat room temperature The final pellet was resuspended with5mL imidazole buffer to obtain a suspension of 2
VWF antibody-latex reagent was prepared by passiveadsorption method according to the manufacturerrsquos protocoland themethod described byMina et al 2012 [30] with somemodifications Briefly 10mL of 2 latex suspension was cen-trifuged at 3000 g for 20 minutes at room temperature and
Journal of Immunology Research 3
Table 1 Test score for qualitative latex agglutination test for VWF Ag
Test score Size of the agglutinated beads Appearance of the background Rapidity of the agglutination1+ Small clumps Cloudy gt2min2+ Small clumps Cloudy gt1min3+ Smalllarge clumps Clear gt30 sec4+ Large clumps Clear lt30 secVWF Ag von Willebrand factor antigen
the supernatant was discarded The pellet was resuspendedwith 1mL phosphate-buffered saline pH 74 (135mMNaCl26mMKCl 8mMNa
2HPO4 and 15mMKH
2PO4) that
was added with 200120583L of polyclonal rabbit anti-human vonWillebrand Factor from Dako Denmark (31 gL) The mix-ture was incubated with gentle mixing on a horizontal rotator(Barnstead Thermolyne Labquake Rotator USA) at roomtemperature for 24 hours After incubation the mixture wascentrifuged and the supernatant was kept for determinationof unadsorbed antibody by BCA method using Micro BCAProtein Determination Kit from Pierce Biotechnology USAThe concentration of unadsorbed antibody in the supernatantwas determined according to themanufacturerrsquos instructionsThe concentration of adsorbed antibody was estimated as thedifference between the initial concentration of antibody (ie620120583g) and the concentration of unadsorbed antibody in thesupernatant
Thepellet was thenwashed twice in 4mL imidazole bufferby centrifugation as stated earlier and resuspended with 3mLimidazole buffer containing 1 bovine serum albumin(Sigma-Aldrich USA) The mixture was incubated with gen-tle mixing at room temperature for one hour After incuba-tion the mixture was centrifuged and the pellet was washedtwice in 4mL imidazole buffer by centrifugation as statedearlier The final pellet was resuspended with 2mL imidazolebuffer containing 01 bovine serum albumin to obtain asuspension of 1 and the suspension was stored at 4∘C untiluse
The process described was carried out using differentconcentrations of the VWF antibody to determine the appro-priate concentration of antibody for the adsorption proce-dure As a control reagent the process was conductedwithoutthe antibody All reagents were tested on known negativeand positive samples Normal saline and buffers were usedas negative samples while commercial controls were used aspositive samples
23 Latex Agglutination Test Procedure and Result Interpre-tation VWF antibody-latex reagent was brought to roomtemperature and mixed gently before use The sedimentationof latex beads can occur during storage that can be reversedby pipetting the suspension of latex beads through a fine tippipette until they are returned to a uniform suspension Latexagglutination test for the detection of VWF in a plasma sam-ple was performed on a slide card (Thermo Scientific UK)Using a wooden applicator stick 20 120583L of VWF antibody-latex reagent was mixed with 20120583L of plasma sample on ablack reaction zone of the card The card was rotated on
Figure 1 Positive (number 5) and negative (number 6) reactions ofqualitative latex agglutination test for VWF Ag
a horizontal shaker (Boekel GrantMicroplate Shaker UK) forthreeminutes After threeminutes the agglutination reactionwas observed by visual inspection macroscopically with anaked eye
The detection and estimation of VWF by latex agglutina-tion test can be performed using a qualitative test and semi-quantitative test respectively Qualitative test was performedon undiluted plasma sample and the result was reportedas negative or positive Invisible or hardly visible agglutina-tion was reported as negative and visible agglutination wasreported as positive (Figure 1) For a positive result the degreeof agglutination was scored from 1+ to 4+ based on the sizeof the agglutinated beads the appearance of the backgroundand the rapidity of the agglutination development as shownin Table 1 Semiquantitative test was performed on dilutedplasma sample A positive plasma sample was serially dilutedtwofold with normal saline The highest dilution that stillshows a positive reaction is the end-titre of a sample
24 Validation Studies on VWF Antibody-Latex Reagent
241 Reproducibility Study Reproducibility studies wereperformedwithin a day and between daysWithin-day repro-ducibility study was carried out by testing VWF antibody-latex reagent on three plasma samples and the end-titreof each sample was determined three times Between-dayreproducibility study was conducted by testing the reagentin duplicate on two plasma samples and the end-titre of thesamples was determined on days 1 7 14 and 21 The reagentswere also tested on low and normal controls from Instrumen-tation Laboratories Italy (Hemosil Special Test Control Level2 and Hemosil Normal Control Assayed resp) on days 2 45 7 30 and 50
4 Journal of Immunology Research
242 Stability Study Stability of the reagent was checkedduring storage of VWF antibody-latex reagent at 4∘C for 21days Qualitative test was performed on two plasma sampleson days 1 2 3 4 5 6 7 14 and 21 using aliquots of samplesthat were stored at minus70∘C A new aliquot of sample was usedin each experiment Stability of plasma sample during storagewas also checked by testing VWF antibody-latex reagent(stored at 4∘C) on aliquots of plasma samples which wasstored at 4∘C After 0 (within two hours after blood collec-tion) 1 2 3 4 5 6 7 14 and 21 days the qualitative test wasperformed The reagents were also tested on Hemosil SpecialTest Control Level 2 and Hemosil Normal Control Assayed(Instrumentation Laboratories Italy) Semiquantitative latexagglutination test was performed on these controls for up to50 days Batch to batch variation was tested with the reagentsprepared on two different dates using two different lots of theVWF antibody
243 Linearity Study The linearity of latex agglutinationtest was determined by testing VWF antibody-latex reagenton two patient samples which were serially diluted twofoldThe VWF Ag level in diluted samples was measured byimmunoturbidimetric assay using Hemosil von WillebrandFactor Antigen kit (Instrumentation Laboratory Italy) Semi-quantitative latex agglutination test was performed ondiluted samples and the end-titres were compared with theVWF Ag levels Graphical presentations of the semiquan-titative VWF Ag by latex agglutination test (119884) versus theVWF Ag by immunoturbidimetric assay (119883) were made forindividual sample and also for a combination of the twosamples
244 Determination of Limit of Detection Limit of detectionof latex agglutination test was determined by testing VWFantibody-latex reagent on low-level samples (cryosuper-natant) Seven cryosupernatant samples were serially dilutedtwofold and the VWF Ag levels in the samples (neat) weremeasured by immunoturbidimetric assay using Hemosil vonWillebrand Factor Antigen kit (Instrumentation LaboratoryItaly) VWF Ag levels in 1 2 1 4 and 1 8 diluted sampleswere estimated by dividing the VWF Ag levels of neat sam-ples by the dilution factors of 2 4 and 8 respectively Latexagglutination test was performed to determine the lowestlevel of VWF Ag that shows positive agglutination reaction
245 Interference Study
(i) Interference fromHemoglobin Interference fromhemoglo-bin is common in coagulation testing and hemolytic speci-men is usually not orminimally affected in latex agglutinationtest The interference study was performed by testing VWFantibody-latex reagent on plasma pools and plasma poolsspiked at different concentrations of hemolysate Hemolysatewas prepared by freezing and thawing the whole bloodfollowed by the osmotic shock protocol [31] Briefly normalsample that was collected in 30mLK
2EDTA tube was
centrifuged at 1000 g for 10 minutes Plasma was removedand replaced with an equal volume of isotonic saline Cells
were resuspended and the suspension was centrifuged againat 1000 g for 10 minutes The saline wash was repeated threetimes In the final wash saline was replaced with distilledwater and the suspension was stored at minus20∘C overnightFrozen cells were thawed mixed and centrifuged at 2000 gfor 30 minutes to remove cell debris
Hemolysate was transferred to a clean tube and hemoglo-bin level wasmeasuredwith the SysmexXE 5000Hematologyanalyzer (Sysmex Corporation Kobe Japan) Stock solutionsof hemolysate in normal saline were prepared and each ofthe stock solutions was used to spike plasma pools with highand low VWF Ag levels In the present study hemolysatewith hemoglobin level of 120 gL was used to prepare stocksolutions of hemolysateThe hemolysate was diluted 1 2 1 41 8 1 20 and 1 40 with normal saline to obtain the stocksolutions with hemoglobin levels of 60 30 15 6 and 3 gLrespectively One hundred microlitres of each stock solutionwere added to 900 120583L of plasma pools to obtain plasma poolswith a final hemoglobin level of 6 3 15 06 and 03mgmL
(ii) Interference from Rheumatoid Factor Rheumatoid factor(RF) is one of the most common interferences in latex-basedmethods It may bind and cross-link antibodies attached tothe latex beads leading to a false-positive result or falselyelevated levels of VWF in a sample [32] The effect of RF onagglutination reaction was studied by testing VWF antibody-latex reagent on plasma pools with high and low VWF Aglevels The plasma pools were spiked at different concen-trations of RF using rheumatoid factor control serum fromMyBioSource USA (1030 IUmL)
The RF control serum was diluted 1 8 1 7 and 1 6 withhigh plasma pools to obtain plasma pools spiked with RFat a concentration of 129 147 and 172 IUmL respectivelyFor the preparation of low plasma pools spiked with RF at aconcentration of 10 15 52 103 and 129 IUmL the RF controlserum was diluted 1 100 1 70 1 20 1 10 and 1 8 respec-tively with low plasma pools
246MethodComparisonwith the ReferenceMethodMethodcomparison study between latex agglutination test and thereference method (immunoturbidimetric assay) was con-ducted by parallel analysis of 132 plasma samples of differentlevels of VWF Ag The samples were obtained from 40healthy individuals 40 patients with cardiovascular diseasesand 32 patients with other diseases including malignant andinflammatory disorders The remaining 20 samples werecryosupernatant samples (ie samples with low levels ofVWF Ag) that were obtained from Blood Transfusion Ser-vices All specimens were tested in ten days and the testingwas spread over ten months
Qualitative and semiquantitative latex agglutination testswere performed using the VWF antibody-latex reagentsThe immunoturbidimetric assay was performed on ACLElite Pro Coagulation analyser (Instrumentation LaboratoryItaly) using Hemosil von Willebrand Factor Antigen kitfrom Instrumentation Laboratory Italy The procedure forVWF Ag by immunoturbidimetric assay was performedaccording to the manufacturerrsquos instructions and adhered to
Journal of Immunology Research 5
Table 2 Estimated concentration of adsorbed antibody in the VWF antibody-latex reagents
the CLSI Document H21-A5 on the specimen collection andprocessing of blood specimens for testing the plasma-basedcoagulation assays Normal reference range for VWF Aglevels by immunoturbidimetric assay in our laboratory is 50ndash150
25 Statistical Analysis Statistical analyses were conductedusing the SPSS software version 20 (SPSS Inc Chicago ILUSA) Linear regression analysis was performed to determinethe linearity of the semiquantitative latex agglutination testThe degree of agreement between qualitative latex aggluti-nation test and immunoturbidimetric assay was estimatedby calculating the kappa value (120581) 081ndash099 almost per-fect agreement 061ndash080 substantial agreement 041ndash060moderate agreement 021ndash040 fair agreement 001ndash020slight agreement and lt0 less than chance agreement [33]Spearmanrsquos rho correlation analysiswas used to determine thedegree of relationship between semiquantitative latex agglu-tination test and immunoturbidimetric assay The strengthof the relationship is showed by the correlation coefficientvalue (119903) at least 08 very strong 06 up to 08 moderatelystrong 03 to 05 fair and less than 03 poor [34] Two-tailed probability (119875) value of less than 005 was consideredstatistically significant
3 Results
31 Preparation of VWF Antibody-Latex Reagent Table 2shows the estimated concentration of adsorbed antibody onthe latex beads in the VWF antibody-latex reagents that wereprepared using different concentrations of the VWF anti-body The results showed that the concentration of adsorbedantibody increased as the initial concentration of the VWFantibody increased However the percentage of adsorbedantibody that is 70 and 74 was not much different whenadsorption method was performed using antibody concen-tration of 620120583g and 700 120583g respectively
Visual observation on the agglutination reaction showedthat less agglutination was observed from latex beadsadsorbed with less than 400 120583g of antibody (ie 118 120583g276120583g and 328 120583g) compared to agglutination reactionfrom latex beads adsorbed with about 400 120583g of antibodyAgglutination reactions from latex beads coated with 520120583gantibody were slightly more compared to agglutination reac-tions from latex beads coated with 436 120583g antibody (data notshown) In other words latex beads with sim400 120583g adsorbed
antibody were sufficient to produce strong agglutinationreaction when mixed with plasma sample Thus 200120583L ofthe VWF antibody with a concentration of 620120583g has beenchosen to be the most appropriate concentration for theadsorption procedure
Autoagglutination was not seen when VWF antibody-latex reagent alone was rotated for three minutes Extendingthe time beyond three minutes resulted in reagent dryingthat might give false-positive result No agglutination wasseen when the control reagent (ie reagent without adsorbedantibody) was tested on plasma sample and commercialcontrols Negative reaction was consistently observed whenthe VWF antibody-latex reagent was tested on normal salineand buffers throughout this study
32 Validation Studies Validation studies were conductedusing VWF antibody-latex reagents that were prepared inseven batches The concentration of adsorbed antibody onthe latex beads ranged from 403 to 447 g (ie 65ndash72 fromthe initial concentration of the VWF antibody used in theadsorption procedure) which showed that the percentage oftheVWFantibody adsorbed on the latex beadswas consistentthroughout this study
321 Reproducibility and Stability Studies In the repro-ducibility study the end-titre of three samples (ie 1 321 64 and 1 128) was consistent when tested three timeswithin a day Similarly the end-titre of two samples (ie 1 16and 1 128) did not change from day 1 to day 21 (betweendays) Testing on the controls showed that the end-titre of lowand normal controls (1 4 and 1 16 resp) was reproduciblefrom day 1 to day 50 In the stability study VWF antibody-latex reagent and plasma sample were found to be stable forqualitative latex agglutination test when stored at 4∘C for atleast 21 days Semiquantitative test on controls showed thatthe VWF antibody-latex reagent was stable for at least 50days
All these findings showed that the semiquantitative latexagglutination test results were reproducible within a day andbetween days and the reagent was found to be stable for atleast 50 days when stored at 4∘C Batch to batch variation wasnot detected
322 Linearity Study In the linearity study a series of knownconcentrations of VWF Ag was established by dilution withfive equally spaced concentrations as shown in Table 3 The
6 Journal of Immunology Research
Table 3 Linearity study on semiquantitative VWF antigen by latex agglutination test
Tube number Sample 1 Sample 2VWF Ag ()a VWF Ag (titre)b VWF Ag ()a VWF Ag (titre)b
1 600 1 128 460 1 642 300 1 64 230 1 323 150 1 32 115 1 164 869 1 16 579 1 85 222 1 8 279 1 46 99 1 4 176 1 2aVWF Ag levels () measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from Instrumentation Laboratory ItalybVWF Ag (titre) estimated by using semiquantitative latex agglutination testVWF von Willebrand factor VWF Ag von Willebrand factor antigen
Table 4 Limit of detection of VWF antigen detected by using latex agglutination test
Samplesample number Neat 1 2 1 4 1 8VWF Ag ()a LATe VWF Ag ()b LATe VWF Ag ()c LATe VWF Ag ()d LATe
1 242 + 121 + 61 + 30 minus
2 202 + 102 + 51 + 25 minus
3 306 + 153 + 69 + 38 minus
4 276 + 138 + 69 + 34 minus
5 269 + 133 + 67 + 34 minus
6 291 + 146 + 73 + 36 minus
7 303 + 152 + 76 + 38 minus
aVWF Ag levels in neat sample that was measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from InstrumentationLaboratory ItalybcdEstimated VWF Ag levels in 1 2 1 4 and 1 8 diluted samples (ie by dividing the VWF Ag levels of neat sample by the dilution factors of 2 4 and 8respectivelyePositive (+) and negative (minus) reactions observed when neat and diluted samples were tested by using latex agglutination testVWF Ag von Willebrand factor antigen LAT latex agglutination test
table shows the latex agglutination test results (titre) and thelevels of VWF Ag in undiluted (tube 1) and diluted samples(tubes 2ndash6) It was shown in the table that the semiquanti-tative latex agglutination test results (titre) were directly pro-portional to the levels of VWF Ag in the plasma sample Lin-earity of the semiquantitative VWF Ag latex agglutinationtest was demonstrated in sample 1 (1199032 = 09985) and sample 2(1199032 = 09999) A regression line that was fit to the pointswas observed in both samples (graphs not shown) Thelinearity was also demonstrated at the titre ranging from 1 2to 1 128 when the results of both samples were combined andanalyzed (1199032 = 09361) All these findings have demonstrateda linear relationship between the observed results (VWF Agtitre) and the true concentrations of VWF Ag in the plasmasamples
323 Determination of Limit of Detection In the limit ofdetection study plasma samples with low levels of VWF Ag(in the range of 202 to 306) were obtained from sevencryosupernatant samples (Table 4) Table 4 shows the agglu-tination reaction observed when latex agglutination test wasperformed on undiluted (neat) samples and diluted samples(at 1 2 1 4 and 1 8 dilutions) As shown in the table thelowest level of VWF Ag that still showed positive reaction is51 (sample number 2 at 1 4 dilution) and the highest level
of VWF Ag that showed negative reaction is 38 (samplenumber 7 at 1 8 dilution) These results showed that thelowest level of VWF Ag that the latex agglutination test candetect to determine the presence (positive result) or absence(negative result) of VWF Ag was 51 Thus the limit ofdetection of VWF Ag by latex agglutination test is approx-imately 5 Besides that reproducibility of the agglutinationreaction at VWF Ag levels of 51 and 38 was also checkedby testing six times the diluted samples of number 2 (at 1 4dilution) and number 7 (at 1 8 dilution) respectively Samplenumber 2 showed positive reactions in all six replicates whilesample number 7 showed negative reaction in all six repli-cates These findings showed that the latex agglutination testreliably produced consistent results near the cutoff concen-tration
324 Interference Study Visible agglutinationwas seenwhenlatex agglutination test was performed on undiluted plasmapools and undiluted plasma pools with a final hemoglobinlevel of 03 06 15 3 6 and 12mgmL The end-titre of1 32 was obtained when semiquantitative latex agglutinationtest was performed on high plasma pool The same end-titre(1 32) was observed when the test was performed on highplasma pools with hemoglobin level of 03 06 15 3 6 and12mgmL Similarly the end-titre of low plasma pools with
Journal of Immunology Research 7
Table 5 Rheumatoid factor interference on high and low plasma pools
Type of the plasma pool Vol of RF control serum(120583L)a
aRheumatoid factor control serum fromMyBioSource USA (1030 IUmL) was used to spike plasma poolsRF rheumatoid factor VWF LAT von Willebrand factor latex agglutination test vol volume conc concentration
Table 6 Crosstabulation of VWF antigen levels and qualitative VWF by latex agglutination test results
Total 6 14 61 51 132VWF von Willebrand factor VWF Ag von Willebrand factor antigen LAT latex agglutination test
hemoglobin level of 03 06 15 3 6 and 12mgmL was thesame as the end-titre of low plasma pool without hemoglobininterference (1 4) These results showed that hemoglobindid not cause interference in latex agglutination test andsemiquantitative test result was not affected by hemoglobinlevel up to 12mgmL
Table 5 shows the end-titre of high and low plasma poolsspiked at different concentrations of RF As shown in thetable interference fromRF on latex agglutination test was notdetected in high plasma pools spiked with RF at concentra-tions up to 129 IUmLNevertheless the interference fromRFwas observed in low plasma pools spiked with RF at concen-trations of 26 52 103 and 129 IUmL The end-titre of highand low plasma pools without interference from RF was 1 64and 1 2 respectivelyThese results showed that RF interferedwith the latex agglutination test result when the test wasperformed on the plasma sample containing low levels ofVWF Ag and high levels of RF (gt26 IUmL) However latexagglutination test result of plasma sample containing highlevels of VWF Ag was not affected by RF at concentrationsof lt130 IUmL
325 Method Comparison with the Reference Method Com-parison study between qualitative latex agglutination testand immunoturbidimetric assay showed a kappa value of077 which indicates a substantial agreement between thetwo tests Table 6 shows the crosstabulation of 132 samplesincluded in this study Semiquantitative latex agglutinationtest was strongly correlated with immunoturbidimetric assay
(Spearmanrsquos rho 119903 = 0946 119875 lt 0001) across a range ofVWF Ag levels from 345 to 870
Based on the results from kappa statistic and correlationstudy the scoring system for agglutination reactions and cor-relation with the VWF Ag levels (using immunoturbidimet-ric assay) is shown in Table 7 The proposed interpretationguide is also put in a footnote
4 Discussion
To the best of our knowledge there has been no reportedstudy or technical description available on the rapid test forVWF Ag utilising latex agglutination method At presentthe application of this method has not been used in clin-ical practice for monitoring of patients with low and highplasma levels of VWF Ag Screening of VWDpatients beforeconfirmation tests and monitoring of patients at high risk ofthrombotic event in the area with low-resource settings or atthe bedside can be performed if rapid test for VWF Ag isavailable Hence there is a need to develop a rapid and simplelatex agglutination test that can reliably detect and estimateVWF Ag in the plasma sample
Latex agglutination test is based on the observation ofvisible clumps that are formed from the reaction of antigen-antibody complexes The antigen-antibody complexes can beprepared by passive adsorption or covalent coupling methodIn the early stage of the present study attempts had beenmadeto couple VWF antibody with the carboxylated latex beads
8 Journal of Immunology Research
Table 7 Correlation of result interpretations by latex agglutination test and immunoturbidimetric assay
Qualitative latex agglutination test (LATscore)
Semiquantitative latex agglutination test(titre)
VWF Ag levels by immunoturbidimetricassay ()
1+ 1 2 lt202+ 1 4 1 8 20ndash503+ 1 16 1 32 50ndash1504+ ge64 gt150The proposed interpretation guide normal level of VWF Ag is indicated by LAT scores of 2+ and 3+ or titres from 1 8 to 1 32 Abnormal low VWF Ag isindicated by LAT scores of 0 1+ and 2+ or titres of 1 2 and 1 4 Abnormal high VWF Ag is indicated by LAT score of 4+ or titres of ge64LAT latex agglutination test VWF Ag von Willebrand factor antigen
by covalent coupling method The coupling procedure wasperformed in MES buffer at pH 60 according to the protocolfrom themanufacturer Unfortunately there was no antibodycoated on the latex beads (data not shown) However theVWF antibody was successfully adsorbed onto the surfaceof chloromethyl latex beads by passive adsorption methodThese findings were similar to the reported study by Garcia etal 2015 [35] In the present study amaximal physical adsorp-tion of the VWF antibody onto chloromethyl latex beads wasobtained at pH 74 a pH that is close to the isoelectric pointof polyclonal immunoglobulin (IgG) antibody [36 37]
Interference by RF in the latex agglutination test for thedetection of VWF Ag was expected RF is known to causeanalytical error in automated immunoturbidimetry assay thatpresented the main limitation of this method Despite thislimitation the findings do nevertheless show that the effect ofthe interference was less prominent in the sample with highlevels of VWF Ag compared to the sample with low levelsof VWF Ag The expected interference is without any doubtdue to the usage of whole IgG in the passive adsorptionmethod It iswell known that rheumatoid factor can bindwiththe Fc region of IgG causing false agglutination thatmay alterthe result of the test In the literature the usage of antibodyfragments such as Fab or F(ab1015840)
2was recommended to elim-
inate the interference from rheumatoid factor [37] Antibodyfragment is suggested in future study and optimization isrequired as the antibody fragment is more acidic than thepolyclonal antibody
In the present study the agglutination observed from thereaction between VWF antibody-latex reagent and positivesample may not be optimum as expected Experiments usinghigher initial concentration of VWF antibody with a moreintensive procedure to ensure maximal bead coating couldbe performed in the future study Not only that but theorientation of adsorbed antibody could also be studied toensure a proper alignment of antibody on latex beads Clariziaet al 2009 [38] have demonstrated a method for detectingthe aligned andmisaligned antibodies on the surface on latexbeads Due to some technical constraint maximal bead coat-ing cannot be ensured and a study on orientation of adsorbedantibody could not be performed Another limitation of thisstudy is that the size of the clumping was only estimatedby visual observation In future study it is recommendedto measure the clumping or macroscopic clusters usingmicroscope or other methods
There is a potential clinical application of this rapid latexagglutination test as a routine test in various premises includ-ing small laboratory and clinic and at the bedsideThe clinicalaspect of low levels of VWF Ag is well recognized but notmany medical personnel realized the implications of highVWF Ag levels in medical practice An inherited bleedingdisorder known as von Willebrand disease (VWD) is asso-ciated with an abnormality in VWF and type 1 VWD is thecommonest form that is associated with low VWF Ag [39]On the other hand high levels of VWF Ag are associatedwith thrombotic risks and had been extensively studied inmany medical conditions particularly coronary heart disease(CHD) and stroke It has been shown that CHD patients withhigh baseline of VWF Ag levels are at risk of acute vascularevents related to thrombosis [40] The current clinical guide-lines do not incorporate the measurement of VWF Ag inthe routine assessment of CHD patients but from variousstudies there is a role in monitoring the levels of VWF AgDetection of persistent high levels or increasing trends ofVWF Ag levels would alert the treating doctor for certainclinical measures to improve the patient care
Latex agglutination test for the detection of VWF Agwasfound to be simple to perform and the results can be obtainedwithin three minutes making it suitable to be used in areaswith limited laboratory facilities The result obtained bythe latex agglutination test is reliable as shown by the findingsfrom reproducibility stability linearity limit of detectionand method comparison studies Prozone effect was notdetected at VWF Ag levels up to 870 The stability of thereagents used for this method was up to 50 days and in thisstudy no stabilizer or preservatives were used The plasmafor VWF Ag estimation was stable for up to 21 days whichmay be related to the protein structure however more testsare needed to confirm the suitable plasma storage recom-mendation for this method All these findings showed theusefulness of this method in detecting and estimating highand low levels of VWF AgThis method might be useful inthe monitoring of VWF Ag in patients that are at a high riskof vascular event related to endothelial dysfunction Similarlythis method could be used to estimate VWF Ag levels inpatients with VWD before confirmation with standard testpanels in the specialized coagulation laboratory
However we only evaluate the latex agglutination testin quantitative terms by using plasma samples containinglow and high levels of VWF Ag This application mainlyaddressed the VWD type 1 which is more common in clinical
Journal of Immunology Research 9
practice In future study plasma samples with qualitativeVWF abnormalities (eg type II VWD or acquired VWDpatients demonstrating abnormal VWF function with vari-ables VWF Ag levels) could be included in the comparisonstudy to confirm the application of the latex agglutination testfor assessing VWF Ag levels in various clinical situations Insummary this test method needs to be refined and comparedwith other latex agglutination tests available in the marketbefore it is used on patient samples
5 Conclusion
Latex agglutination test for VWF Ag is simple rapid andreproducible correlates well with the reference method andis suitable to be used in areaswith limited laboratory facilitiesThe test has commercial potential as a low-cost alternativemethod for the detection and estimation of low levels andhigh levels of VWF Ag that could help in themanagement ofVWDtype 1 patients andpatients at risk of thrombotic eventsrespectively However the testmay not be suitable for patientswith a high level of rheumatoid factor
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by short-term Grant 304PPSP61312085 from Universiti Sains Malaysia Penang MalaysiaThe authors gratefully acknowledge Dr Emmanuel J Faval-oro from Institute of Clinical Pathology and MedicalResearch at Westmead Hospital Australia for his technicaladvice on this work Thanks are due to Dr Lim TheamSoon from Institute for Research in Molecular MedicineUniversiti Sains Malaysia Penang Malaysia for his valuableadvice in the early stage of this work The appreciation alsogoes to the staff of Haematology Department (especially thecoagulation section) BloodTransfusion ServiceDepartmentand Immunology Department Universiti Sains MalaysiaKelantan Malaysia for their continuous support in thiswork
References
[1] Z M Ruggeri ldquoStructure of vonWillebrand factor and its func-tion in platelet adhesion and thrombus formationrdquo Best Practiceand Research Clinical Haematology vol 14 no 2 pp 257ndash2792001
[2] G-P Luo B Ni X Yang and Y-ZWu ldquovonWillebrand factormore than a regulator of hemostasis and thrombosisrdquo ActaHaematologica vol 128 no 3 pp 158ndash169 2012
[3] C V Denis and P J Lenting ldquoVon Willebrand factor at thecrossroads of bleeding and thrombosisrdquo International Journal ofHematology vol 95 no 4 pp 353ndash361 2012
[4] A Alonso W Tang S K Agarwal E Z Soliman A M Cham-berlain and A R Folsom ldquoHemostatic markers are associatedwith the risk and prognosis of atrial fibrillation the ARIC
studyrdquo International Journal of Cardiology vol 155 no 2 pp217ndash222 2012
[5] F Cambronero J A Vilchez A Garcıa-Honrubia et al ldquoPlasmalevels of Von Willebrand factor are increased in patients withhypertrophic cardiomyopathyrdquo Thrombosis Research vol 126no 1 pp e46ndashe50 2010
[6] A M Kucharska-Newton D J Couper J S Pankow et alldquoHemostasis inflammation and fatal and nonfatal coronaryheart disease long-term follow-up of the atherosclerosis risk incommunities (ARIC) cohortrdquo Arteriosclerosis Thrombosis andVascular Biology vol 29 no 12 pp 2182ndash2190 2009
[7] R G Wieberdink M C van Schie P J Koudstaal et al ldquoHighvon Willebrand factor levels increase the risk of stroke theRotterdam studyrdquo Stroke vol 41 no 10 pp 2151ndash2156 2010
[8] P Wennberg F Wensley E Di Angelantonio et al ldquoHaemo-static and inflammatory markers are independently associatedwith myocardial infarction in men and womenrdquo ThrombosisResearch vol 129 no 1 pp 68ndash73 2012
[9] A Hyseni M Roest S L Braun et al ldquoChronic dysfunction ofthe endothelium is associated with mortality in acute coronarysyndrome patientsrdquoThrombosis Research vol 131 no 3 pp 198ndash203 2013
[10] A A Lopes A C Barreto N Y Maeda et al ldquoPlasma vonWillebrand factor as a predictor of survival in pulmonaryarterial hypertension associated with congenital heart diseaserdquoBrazilian Journal of Medical and Biological Research vol 44 no12 pp 1269ndash1275 2011
[11] V Roldan F Marın B Muina et al ldquoPlasma von Willebrandfactor levels are an independent risk factor for adverse eventsincluding mortality and major bleeding in anticoagulated atrialfibrillation patientsrdquo Journal of the American College of Cardiol-ogy vol 57 no 25 pp 2496ndash2504 2011
[12] T A Gaziano A Bitton S Anand S Abrahams-Gessel and AMurphy ldquoGrowing epidemic of coronary heart disease in low-andmiddle-income countriesrdquoCurrent Problems in Cardiologyvol 35 no 2 pp 72ndash115 2010
[13] V L Roger A S Go D M Lloyd-Jones et al ldquoExecutive sum-mary heart disease and stroke statisticsmdash2012 update a reportfrom the American Heart AssociationrdquoCirculation vol 125 no1 pp 188ndash197 2012
[14] G Castaman A Tosetto A Cappelletti et al ldquoValidation ofa rapid test (VWF-LIA) for the quantitative determination ofvonWillebrand factor antigen in type 1 vonWillebrand diseasediagnosis within the European multicenter study MCMDM-1VWDrdquoThrombosis Research vol 126 no 3 pp 227ndash231 2010
[15] A Kappel and M Ehm ldquoImmunoassays for diagnosis of coag-ulation disordersrdquo Hamostaseologie vol 30 no 4 pp 194ndash2012010
[16] R Biswas and S C Parija ldquoA rapid slide agglutination test forthe diagnosis of neurocysticercosis in the rural health set uprdquoTropical Parasitology vol 1 no 2 pp 94ndash98 2011
[17] H Lee Y Park M Kim et al ldquoDevelopment of a latex agglu-tination test for norovirus detectionrdquo Journal of Microbiologyvol 48 no 4 pp 419ndash425 2010
[18] H Y Darani F Ahmadi N Zabardast H A Yousefi and HShirzad ldquoDevelopment of a latex agglutination test as a simpleand rapid method for diagnosis of Trichomonas vaginalisinfectionrdquoAvicenna Journal of Medical Biotechnology vol 2 no1 pp 63ndash66 2010
[19] MMoraveji A Hosseini N Moghaddar M M Namavari andM H Eskandari ldquoDevelopment of latex agglutination test with
10 Journal of Immunology Research
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
Table 1 Test score for qualitative latex agglutination test for VWF Ag
Test score Size of the agglutinated beads Appearance of the background Rapidity of the agglutination1+ Small clumps Cloudy gt2min2+ Small clumps Cloudy gt1min3+ Smalllarge clumps Clear gt30 sec4+ Large clumps Clear lt30 secVWF Ag von Willebrand factor antigen
the supernatant was discarded The pellet was resuspendedwith 1mL phosphate-buffered saline pH 74 (135mMNaCl26mMKCl 8mMNa
2HPO4 and 15mMKH
2PO4) that
was added with 200120583L of polyclonal rabbit anti-human vonWillebrand Factor from Dako Denmark (31 gL) The mix-ture was incubated with gentle mixing on a horizontal rotator(Barnstead Thermolyne Labquake Rotator USA) at roomtemperature for 24 hours After incubation the mixture wascentrifuged and the supernatant was kept for determinationof unadsorbed antibody by BCA method using Micro BCAProtein Determination Kit from Pierce Biotechnology USAThe concentration of unadsorbed antibody in the supernatantwas determined according to themanufacturerrsquos instructionsThe concentration of adsorbed antibody was estimated as thedifference between the initial concentration of antibody (ie620120583g) and the concentration of unadsorbed antibody in thesupernatant
Thepellet was thenwashed twice in 4mL imidazole bufferby centrifugation as stated earlier and resuspended with 3mLimidazole buffer containing 1 bovine serum albumin(Sigma-Aldrich USA) The mixture was incubated with gen-tle mixing at room temperature for one hour After incuba-tion the mixture was centrifuged and the pellet was washedtwice in 4mL imidazole buffer by centrifugation as statedearlier The final pellet was resuspended with 2mL imidazolebuffer containing 01 bovine serum albumin to obtain asuspension of 1 and the suspension was stored at 4∘C untiluse
The process described was carried out using differentconcentrations of the VWF antibody to determine the appro-priate concentration of antibody for the adsorption proce-dure As a control reagent the process was conductedwithoutthe antibody All reagents were tested on known negativeand positive samples Normal saline and buffers were usedas negative samples while commercial controls were used aspositive samples
23 Latex Agglutination Test Procedure and Result Interpre-tation VWF antibody-latex reagent was brought to roomtemperature and mixed gently before use The sedimentationof latex beads can occur during storage that can be reversedby pipetting the suspension of latex beads through a fine tippipette until they are returned to a uniform suspension Latexagglutination test for the detection of VWF in a plasma sam-ple was performed on a slide card (Thermo Scientific UK)Using a wooden applicator stick 20 120583L of VWF antibody-latex reagent was mixed with 20120583L of plasma sample on ablack reaction zone of the card The card was rotated on
Figure 1 Positive (number 5) and negative (number 6) reactions ofqualitative latex agglutination test for VWF Ag
a horizontal shaker (Boekel GrantMicroplate Shaker UK) forthreeminutes After threeminutes the agglutination reactionwas observed by visual inspection macroscopically with anaked eye
The detection and estimation of VWF by latex agglutina-tion test can be performed using a qualitative test and semi-quantitative test respectively Qualitative test was performedon undiluted plasma sample and the result was reportedas negative or positive Invisible or hardly visible agglutina-tion was reported as negative and visible agglutination wasreported as positive (Figure 1) For a positive result the degreeof agglutination was scored from 1+ to 4+ based on the sizeof the agglutinated beads the appearance of the backgroundand the rapidity of the agglutination development as shownin Table 1 Semiquantitative test was performed on dilutedplasma sample A positive plasma sample was serially dilutedtwofold with normal saline The highest dilution that stillshows a positive reaction is the end-titre of a sample
24 Validation Studies on VWF Antibody-Latex Reagent
241 Reproducibility Study Reproducibility studies wereperformedwithin a day and between daysWithin-day repro-ducibility study was carried out by testing VWF antibody-latex reagent on three plasma samples and the end-titreof each sample was determined three times Between-dayreproducibility study was conducted by testing the reagentin duplicate on two plasma samples and the end-titre of thesamples was determined on days 1 7 14 and 21 The reagentswere also tested on low and normal controls from Instrumen-tation Laboratories Italy (Hemosil Special Test Control Level2 and Hemosil Normal Control Assayed resp) on days 2 45 7 30 and 50
4 Journal of Immunology Research
242 Stability Study Stability of the reagent was checkedduring storage of VWF antibody-latex reagent at 4∘C for 21days Qualitative test was performed on two plasma sampleson days 1 2 3 4 5 6 7 14 and 21 using aliquots of samplesthat were stored at minus70∘C A new aliquot of sample was usedin each experiment Stability of plasma sample during storagewas also checked by testing VWF antibody-latex reagent(stored at 4∘C) on aliquots of plasma samples which wasstored at 4∘C After 0 (within two hours after blood collec-tion) 1 2 3 4 5 6 7 14 and 21 days the qualitative test wasperformed The reagents were also tested on Hemosil SpecialTest Control Level 2 and Hemosil Normal Control Assayed(Instrumentation Laboratories Italy) Semiquantitative latexagglutination test was performed on these controls for up to50 days Batch to batch variation was tested with the reagentsprepared on two different dates using two different lots of theVWF antibody
243 Linearity Study The linearity of latex agglutinationtest was determined by testing VWF antibody-latex reagenton two patient samples which were serially diluted twofoldThe VWF Ag level in diluted samples was measured byimmunoturbidimetric assay using Hemosil von WillebrandFactor Antigen kit (Instrumentation Laboratory Italy) Semi-quantitative latex agglutination test was performed ondiluted samples and the end-titres were compared with theVWF Ag levels Graphical presentations of the semiquan-titative VWF Ag by latex agglutination test (119884) versus theVWF Ag by immunoturbidimetric assay (119883) were made forindividual sample and also for a combination of the twosamples
244 Determination of Limit of Detection Limit of detectionof latex agglutination test was determined by testing VWFantibody-latex reagent on low-level samples (cryosuper-natant) Seven cryosupernatant samples were serially dilutedtwofold and the VWF Ag levels in the samples (neat) weremeasured by immunoturbidimetric assay using Hemosil vonWillebrand Factor Antigen kit (Instrumentation LaboratoryItaly) VWF Ag levels in 1 2 1 4 and 1 8 diluted sampleswere estimated by dividing the VWF Ag levels of neat sam-ples by the dilution factors of 2 4 and 8 respectively Latexagglutination test was performed to determine the lowestlevel of VWF Ag that shows positive agglutination reaction
245 Interference Study
(i) Interference fromHemoglobin Interference fromhemoglo-bin is common in coagulation testing and hemolytic speci-men is usually not orminimally affected in latex agglutinationtest The interference study was performed by testing VWFantibody-latex reagent on plasma pools and plasma poolsspiked at different concentrations of hemolysate Hemolysatewas prepared by freezing and thawing the whole bloodfollowed by the osmotic shock protocol [31] Briefly normalsample that was collected in 30mLK
2EDTA tube was
centrifuged at 1000 g for 10 minutes Plasma was removedand replaced with an equal volume of isotonic saline Cells
were resuspended and the suspension was centrifuged againat 1000 g for 10 minutes The saline wash was repeated threetimes In the final wash saline was replaced with distilledwater and the suspension was stored at minus20∘C overnightFrozen cells were thawed mixed and centrifuged at 2000 gfor 30 minutes to remove cell debris
Hemolysate was transferred to a clean tube and hemoglo-bin level wasmeasuredwith the SysmexXE 5000Hematologyanalyzer (Sysmex Corporation Kobe Japan) Stock solutionsof hemolysate in normal saline were prepared and each ofthe stock solutions was used to spike plasma pools with highand low VWF Ag levels In the present study hemolysatewith hemoglobin level of 120 gL was used to prepare stocksolutions of hemolysateThe hemolysate was diluted 1 2 1 41 8 1 20 and 1 40 with normal saline to obtain the stocksolutions with hemoglobin levels of 60 30 15 6 and 3 gLrespectively One hundred microlitres of each stock solutionwere added to 900 120583L of plasma pools to obtain plasma poolswith a final hemoglobin level of 6 3 15 06 and 03mgmL
(ii) Interference from Rheumatoid Factor Rheumatoid factor(RF) is one of the most common interferences in latex-basedmethods It may bind and cross-link antibodies attached tothe latex beads leading to a false-positive result or falselyelevated levels of VWF in a sample [32] The effect of RF onagglutination reaction was studied by testing VWF antibody-latex reagent on plasma pools with high and low VWF Aglevels The plasma pools were spiked at different concen-trations of RF using rheumatoid factor control serum fromMyBioSource USA (1030 IUmL)
The RF control serum was diluted 1 8 1 7 and 1 6 withhigh plasma pools to obtain plasma pools spiked with RFat a concentration of 129 147 and 172 IUmL respectivelyFor the preparation of low plasma pools spiked with RF at aconcentration of 10 15 52 103 and 129 IUmL the RF controlserum was diluted 1 100 1 70 1 20 1 10 and 1 8 respec-tively with low plasma pools
246MethodComparisonwith the ReferenceMethodMethodcomparison study between latex agglutination test and thereference method (immunoturbidimetric assay) was con-ducted by parallel analysis of 132 plasma samples of differentlevels of VWF Ag The samples were obtained from 40healthy individuals 40 patients with cardiovascular diseasesand 32 patients with other diseases including malignant andinflammatory disorders The remaining 20 samples werecryosupernatant samples (ie samples with low levels ofVWF Ag) that were obtained from Blood Transfusion Ser-vices All specimens were tested in ten days and the testingwas spread over ten months
Qualitative and semiquantitative latex agglutination testswere performed using the VWF antibody-latex reagentsThe immunoturbidimetric assay was performed on ACLElite Pro Coagulation analyser (Instrumentation LaboratoryItaly) using Hemosil von Willebrand Factor Antigen kitfrom Instrumentation Laboratory Italy The procedure forVWF Ag by immunoturbidimetric assay was performedaccording to the manufacturerrsquos instructions and adhered to
Journal of Immunology Research 5
Table 2 Estimated concentration of adsorbed antibody in the VWF antibody-latex reagents
the CLSI Document H21-A5 on the specimen collection andprocessing of blood specimens for testing the plasma-basedcoagulation assays Normal reference range for VWF Aglevels by immunoturbidimetric assay in our laboratory is 50ndash150
25 Statistical Analysis Statistical analyses were conductedusing the SPSS software version 20 (SPSS Inc Chicago ILUSA) Linear regression analysis was performed to determinethe linearity of the semiquantitative latex agglutination testThe degree of agreement between qualitative latex aggluti-nation test and immunoturbidimetric assay was estimatedby calculating the kappa value (120581) 081ndash099 almost per-fect agreement 061ndash080 substantial agreement 041ndash060moderate agreement 021ndash040 fair agreement 001ndash020slight agreement and lt0 less than chance agreement [33]Spearmanrsquos rho correlation analysiswas used to determine thedegree of relationship between semiquantitative latex agglu-tination test and immunoturbidimetric assay The strengthof the relationship is showed by the correlation coefficientvalue (119903) at least 08 very strong 06 up to 08 moderatelystrong 03 to 05 fair and less than 03 poor [34] Two-tailed probability (119875) value of less than 005 was consideredstatistically significant
3 Results
31 Preparation of VWF Antibody-Latex Reagent Table 2shows the estimated concentration of adsorbed antibody onthe latex beads in the VWF antibody-latex reagents that wereprepared using different concentrations of the VWF anti-body The results showed that the concentration of adsorbedantibody increased as the initial concentration of the VWFantibody increased However the percentage of adsorbedantibody that is 70 and 74 was not much different whenadsorption method was performed using antibody concen-tration of 620120583g and 700 120583g respectively
Visual observation on the agglutination reaction showedthat less agglutination was observed from latex beadsadsorbed with less than 400 120583g of antibody (ie 118 120583g276120583g and 328 120583g) compared to agglutination reactionfrom latex beads adsorbed with about 400 120583g of antibodyAgglutination reactions from latex beads coated with 520120583gantibody were slightly more compared to agglutination reac-tions from latex beads coated with 436 120583g antibody (data notshown) In other words latex beads with sim400 120583g adsorbed
antibody were sufficient to produce strong agglutinationreaction when mixed with plasma sample Thus 200120583L ofthe VWF antibody with a concentration of 620120583g has beenchosen to be the most appropriate concentration for theadsorption procedure
Autoagglutination was not seen when VWF antibody-latex reagent alone was rotated for three minutes Extendingthe time beyond three minutes resulted in reagent dryingthat might give false-positive result No agglutination wasseen when the control reagent (ie reagent without adsorbedantibody) was tested on plasma sample and commercialcontrols Negative reaction was consistently observed whenthe VWF antibody-latex reagent was tested on normal salineand buffers throughout this study
32 Validation Studies Validation studies were conductedusing VWF antibody-latex reagents that were prepared inseven batches The concentration of adsorbed antibody onthe latex beads ranged from 403 to 447 g (ie 65ndash72 fromthe initial concentration of the VWF antibody used in theadsorption procedure) which showed that the percentage oftheVWFantibody adsorbed on the latex beadswas consistentthroughout this study
321 Reproducibility and Stability Studies In the repro-ducibility study the end-titre of three samples (ie 1 321 64 and 1 128) was consistent when tested three timeswithin a day Similarly the end-titre of two samples (ie 1 16and 1 128) did not change from day 1 to day 21 (betweendays) Testing on the controls showed that the end-titre of lowand normal controls (1 4 and 1 16 resp) was reproduciblefrom day 1 to day 50 In the stability study VWF antibody-latex reagent and plasma sample were found to be stable forqualitative latex agglutination test when stored at 4∘C for atleast 21 days Semiquantitative test on controls showed thatthe VWF antibody-latex reagent was stable for at least 50days
All these findings showed that the semiquantitative latexagglutination test results were reproducible within a day andbetween days and the reagent was found to be stable for atleast 50 days when stored at 4∘C Batch to batch variation wasnot detected
322 Linearity Study In the linearity study a series of knownconcentrations of VWF Ag was established by dilution withfive equally spaced concentrations as shown in Table 3 The
6 Journal of Immunology Research
Table 3 Linearity study on semiquantitative VWF antigen by latex agglutination test
Tube number Sample 1 Sample 2VWF Ag ()a VWF Ag (titre)b VWF Ag ()a VWF Ag (titre)b
1 600 1 128 460 1 642 300 1 64 230 1 323 150 1 32 115 1 164 869 1 16 579 1 85 222 1 8 279 1 46 99 1 4 176 1 2aVWF Ag levels () measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from Instrumentation Laboratory ItalybVWF Ag (titre) estimated by using semiquantitative latex agglutination testVWF von Willebrand factor VWF Ag von Willebrand factor antigen
Table 4 Limit of detection of VWF antigen detected by using latex agglutination test
Samplesample number Neat 1 2 1 4 1 8VWF Ag ()a LATe VWF Ag ()b LATe VWF Ag ()c LATe VWF Ag ()d LATe
1 242 + 121 + 61 + 30 minus
2 202 + 102 + 51 + 25 minus
3 306 + 153 + 69 + 38 minus
4 276 + 138 + 69 + 34 minus
5 269 + 133 + 67 + 34 minus
6 291 + 146 + 73 + 36 minus
7 303 + 152 + 76 + 38 minus
aVWF Ag levels in neat sample that was measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from InstrumentationLaboratory ItalybcdEstimated VWF Ag levels in 1 2 1 4 and 1 8 diluted samples (ie by dividing the VWF Ag levels of neat sample by the dilution factors of 2 4 and 8respectivelyePositive (+) and negative (minus) reactions observed when neat and diluted samples were tested by using latex agglutination testVWF Ag von Willebrand factor antigen LAT latex agglutination test
table shows the latex agglutination test results (titre) and thelevels of VWF Ag in undiluted (tube 1) and diluted samples(tubes 2ndash6) It was shown in the table that the semiquanti-tative latex agglutination test results (titre) were directly pro-portional to the levels of VWF Ag in the plasma sample Lin-earity of the semiquantitative VWF Ag latex agglutinationtest was demonstrated in sample 1 (1199032 = 09985) and sample 2(1199032 = 09999) A regression line that was fit to the pointswas observed in both samples (graphs not shown) Thelinearity was also demonstrated at the titre ranging from 1 2to 1 128 when the results of both samples were combined andanalyzed (1199032 = 09361) All these findings have demonstrateda linear relationship between the observed results (VWF Agtitre) and the true concentrations of VWF Ag in the plasmasamples
323 Determination of Limit of Detection In the limit ofdetection study plasma samples with low levels of VWF Ag(in the range of 202 to 306) were obtained from sevencryosupernatant samples (Table 4) Table 4 shows the agglu-tination reaction observed when latex agglutination test wasperformed on undiluted (neat) samples and diluted samples(at 1 2 1 4 and 1 8 dilutions) As shown in the table thelowest level of VWF Ag that still showed positive reaction is51 (sample number 2 at 1 4 dilution) and the highest level
of VWF Ag that showed negative reaction is 38 (samplenumber 7 at 1 8 dilution) These results showed that thelowest level of VWF Ag that the latex agglutination test candetect to determine the presence (positive result) or absence(negative result) of VWF Ag was 51 Thus the limit ofdetection of VWF Ag by latex agglutination test is approx-imately 5 Besides that reproducibility of the agglutinationreaction at VWF Ag levels of 51 and 38 was also checkedby testing six times the diluted samples of number 2 (at 1 4dilution) and number 7 (at 1 8 dilution) respectively Samplenumber 2 showed positive reactions in all six replicates whilesample number 7 showed negative reaction in all six repli-cates These findings showed that the latex agglutination testreliably produced consistent results near the cutoff concen-tration
324 Interference Study Visible agglutinationwas seenwhenlatex agglutination test was performed on undiluted plasmapools and undiluted plasma pools with a final hemoglobinlevel of 03 06 15 3 6 and 12mgmL The end-titre of1 32 was obtained when semiquantitative latex agglutinationtest was performed on high plasma pool The same end-titre(1 32) was observed when the test was performed on highplasma pools with hemoglobin level of 03 06 15 3 6 and12mgmL Similarly the end-titre of low plasma pools with
Journal of Immunology Research 7
Table 5 Rheumatoid factor interference on high and low plasma pools
Type of the plasma pool Vol of RF control serum(120583L)a
aRheumatoid factor control serum fromMyBioSource USA (1030 IUmL) was used to spike plasma poolsRF rheumatoid factor VWF LAT von Willebrand factor latex agglutination test vol volume conc concentration
Table 6 Crosstabulation of VWF antigen levels and qualitative VWF by latex agglutination test results
Total 6 14 61 51 132VWF von Willebrand factor VWF Ag von Willebrand factor antigen LAT latex agglutination test
hemoglobin level of 03 06 15 3 6 and 12mgmL was thesame as the end-titre of low plasma pool without hemoglobininterference (1 4) These results showed that hemoglobindid not cause interference in latex agglutination test andsemiquantitative test result was not affected by hemoglobinlevel up to 12mgmL
Table 5 shows the end-titre of high and low plasma poolsspiked at different concentrations of RF As shown in thetable interference fromRF on latex agglutination test was notdetected in high plasma pools spiked with RF at concentra-tions up to 129 IUmLNevertheless the interference fromRFwas observed in low plasma pools spiked with RF at concen-trations of 26 52 103 and 129 IUmL The end-titre of highand low plasma pools without interference from RF was 1 64and 1 2 respectivelyThese results showed that RF interferedwith the latex agglutination test result when the test wasperformed on the plasma sample containing low levels ofVWF Ag and high levels of RF (gt26 IUmL) However latexagglutination test result of plasma sample containing highlevels of VWF Ag was not affected by RF at concentrationsof lt130 IUmL
325 Method Comparison with the Reference Method Com-parison study between qualitative latex agglutination testand immunoturbidimetric assay showed a kappa value of077 which indicates a substantial agreement between thetwo tests Table 6 shows the crosstabulation of 132 samplesincluded in this study Semiquantitative latex agglutinationtest was strongly correlated with immunoturbidimetric assay
(Spearmanrsquos rho 119903 = 0946 119875 lt 0001) across a range ofVWF Ag levels from 345 to 870
Based on the results from kappa statistic and correlationstudy the scoring system for agglutination reactions and cor-relation with the VWF Ag levels (using immunoturbidimet-ric assay) is shown in Table 7 The proposed interpretationguide is also put in a footnote
4 Discussion
To the best of our knowledge there has been no reportedstudy or technical description available on the rapid test forVWF Ag utilising latex agglutination method At presentthe application of this method has not been used in clin-ical practice for monitoring of patients with low and highplasma levels of VWF Ag Screening of VWDpatients beforeconfirmation tests and monitoring of patients at high risk ofthrombotic event in the area with low-resource settings or atthe bedside can be performed if rapid test for VWF Ag isavailable Hence there is a need to develop a rapid and simplelatex agglutination test that can reliably detect and estimateVWF Ag in the plasma sample
Latex agglutination test is based on the observation ofvisible clumps that are formed from the reaction of antigen-antibody complexes The antigen-antibody complexes can beprepared by passive adsorption or covalent coupling methodIn the early stage of the present study attempts had beenmadeto couple VWF antibody with the carboxylated latex beads
8 Journal of Immunology Research
Table 7 Correlation of result interpretations by latex agglutination test and immunoturbidimetric assay
Qualitative latex agglutination test (LATscore)
Semiquantitative latex agglutination test(titre)
VWF Ag levels by immunoturbidimetricassay ()
1+ 1 2 lt202+ 1 4 1 8 20ndash503+ 1 16 1 32 50ndash1504+ ge64 gt150The proposed interpretation guide normal level of VWF Ag is indicated by LAT scores of 2+ and 3+ or titres from 1 8 to 1 32 Abnormal low VWF Ag isindicated by LAT scores of 0 1+ and 2+ or titres of 1 2 and 1 4 Abnormal high VWF Ag is indicated by LAT score of 4+ or titres of ge64LAT latex agglutination test VWF Ag von Willebrand factor antigen
by covalent coupling method The coupling procedure wasperformed in MES buffer at pH 60 according to the protocolfrom themanufacturer Unfortunately there was no antibodycoated on the latex beads (data not shown) However theVWF antibody was successfully adsorbed onto the surfaceof chloromethyl latex beads by passive adsorption methodThese findings were similar to the reported study by Garcia etal 2015 [35] In the present study amaximal physical adsorp-tion of the VWF antibody onto chloromethyl latex beads wasobtained at pH 74 a pH that is close to the isoelectric pointof polyclonal immunoglobulin (IgG) antibody [36 37]
Interference by RF in the latex agglutination test for thedetection of VWF Ag was expected RF is known to causeanalytical error in automated immunoturbidimetry assay thatpresented the main limitation of this method Despite thislimitation the findings do nevertheless show that the effect ofthe interference was less prominent in the sample with highlevels of VWF Ag compared to the sample with low levelsof VWF Ag The expected interference is without any doubtdue to the usage of whole IgG in the passive adsorptionmethod It iswell known that rheumatoid factor can bindwiththe Fc region of IgG causing false agglutination thatmay alterthe result of the test In the literature the usage of antibodyfragments such as Fab or F(ab1015840)
2was recommended to elim-
inate the interference from rheumatoid factor [37] Antibodyfragment is suggested in future study and optimization isrequired as the antibody fragment is more acidic than thepolyclonal antibody
In the present study the agglutination observed from thereaction between VWF antibody-latex reagent and positivesample may not be optimum as expected Experiments usinghigher initial concentration of VWF antibody with a moreintensive procedure to ensure maximal bead coating couldbe performed in the future study Not only that but theorientation of adsorbed antibody could also be studied toensure a proper alignment of antibody on latex beads Clariziaet al 2009 [38] have demonstrated a method for detectingthe aligned andmisaligned antibodies on the surface on latexbeads Due to some technical constraint maximal bead coat-ing cannot be ensured and a study on orientation of adsorbedantibody could not be performed Another limitation of thisstudy is that the size of the clumping was only estimatedby visual observation In future study it is recommendedto measure the clumping or macroscopic clusters usingmicroscope or other methods
There is a potential clinical application of this rapid latexagglutination test as a routine test in various premises includ-ing small laboratory and clinic and at the bedsideThe clinicalaspect of low levels of VWF Ag is well recognized but notmany medical personnel realized the implications of highVWF Ag levels in medical practice An inherited bleedingdisorder known as von Willebrand disease (VWD) is asso-ciated with an abnormality in VWF and type 1 VWD is thecommonest form that is associated with low VWF Ag [39]On the other hand high levels of VWF Ag are associatedwith thrombotic risks and had been extensively studied inmany medical conditions particularly coronary heart disease(CHD) and stroke It has been shown that CHD patients withhigh baseline of VWF Ag levels are at risk of acute vascularevents related to thrombosis [40] The current clinical guide-lines do not incorporate the measurement of VWF Ag inthe routine assessment of CHD patients but from variousstudies there is a role in monitoring the levels of VWF AgDetection of persistent high levels or increasing trends ofVWF Ag levels would alert the treating doctor for certainclinical measures to improve the patient care
Latex agglutination test for the detection of VWF Agwasfound to be simple to perform and the results can be obtainedwithin three minutes making it suitable to be used in areaswith limited laboratory facilities The result obtained bythe latex agglutination test is reliable as shown by the findingsfrom reproducibility stability linearity limit of detectionand method comparison studies Prozone effect was notdetected at VWF Ag levels up to 870 The stability of thereagents used for this method was up to 50 days and in thisstudy no stabilizer or preservatives were used The plasmafor VWF Ag estimation was stable for up to 21 days whichmay be related to the protein structure however more testsare needed to confirm the suitable plasma storage recom-mendation for this method All these findings showed theusefulness of this method in detecting and estimating highand low levels of VWF AgThis method might be useful inthe monitoring of VWF Ag in patients that are at a high riskof vascular event related to endothelial dysfunction Similarlythis method could be used to estimate VWF Ag levels inpatients with VWD before confirmation with standard testpanels in the specialized coagulation laboratory
However we only evaluate the latex agglutination testin quantitative terms by using plasma samples containinglow and high levels of VWF Ag This application mainlyaddressed the VWD type 1 which is more common in clinical
Journal of Immunology Research 9
practice In future study plasma samples with qualitativeVWF abnormalities (eg type II VWD or acquired VWDpatients demonstrating abnormal VWF function with vari-ables VWF Ag levels) could be included in the comparisonstudy to confirm the application of the latex agglutination testfor assessing VWF Ag levels in various clinical situations Insummary this test method needs to be refined and comparedwith other latex agglutination tests available in the marketbefore it is used on patient samples
5 Conclusion
Latex agglutination test for VWF Ag is simple rapid andreproducible correlates well with the reference method andis suitable to be used in areaswith limited laboratory facilitiesThe test has commercial potential as a low-cost alternativemethod for the detection and estimation of low levels andhigh levels of VWF Ag that could help in themanagement ofVWDtype 1 patients andpatients at risk of thrombotic eventsrespectively However the testmay not be suitable for patientswith a high level of rheumatoid factor
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by short-term Grant 304PPSP61312085 from Universiti Sains Malaysia Penang MalaysiaThe authors gratefully acknowledge Dr Emmanuel J Faval-oro from Institute of Clinical Pathology and MedicalResearch at Westmead Hospital Australia for his technicaladvice on this work Thanks are due to Dr Lim TheamSoon from Institute for Research in Molecular MedicineUniversiti Sains Malaysia Penang Malaysia for his valuableadvice in the early stage of this work The appreciation alsogoes to the staff of Haematology Department (especially thecoagulation section) BloodTransfusion ServiceDepartmentand Immunology Department Universiti Sains MalaysiaKelantan Malaysia for their continuous support in thiswork
References
[1] Z M Ruggeri ldquoStructure of vonWillebrand factor and its func-tion in platelet adhesion and thrombus formationrdquo Best Practiceand Research Clinical Haematology vol 14 no 2 pp 257ndash2792001
[2] G-P Luo B Ni X Yang and Y-ZWu ldquovonWillebrand factormore than a regulator of hemostasis and thrombosisrdquo ActaHaematologica vol 128 no 3 pp 158ndash169 2012
[3] C V Denis and P J Lenting ldquoVon Willebrand factor at thecrossroads of bleeding and thrombosisrdquo International Journal ofHematology vol 95 no 4 pp 353ndash361 2012
[4] A Alonso W Tang S K Agarwal E Z Soliman A M Cham-berlain and A R Folsom ldquoHemostatic markers are associatedwith the risk and prognosis of atrial fibrillation the ARIC
studyrdquo International Journal of Cardiology vol 155 no 2 pp217ndash222 2012
[5] F Cambronero J A Vilchez A Garcıa-Honrubia et al ldquoPlasmalevels of Von Willebrand factor are increased in patients withhypertrophic cardiomyopathyrdquo Thrombosis Research vol 126no 1 pp e46ndashe50 2010
[6] A M Kucharska-Newton D J Couper J S Pankow et alldquoHemostasis inflammation and fatal and nonfatal coronaryheart disease long-term follow-up of the atherosclerosis risk incommunities (ARIC) cohortrdquo Arteriosclerosis Thrombosis andVascular Biology vol 29 no 12 pp 2182ndash2190 2009
[7] R G Wieberdink M C van Schie P J Koudstaal et al ldquoHighvon Willebrand factor levels increase the risk of stroke theRotterdam studyrdquo Stroke vol 41 no 10 pp 2151ndash2156 2010
[8] P Wennberg F Wensley E Di Angelantonio et al ldquoHaemo-static and inflammatory markers are independently associatedwith myocardial infarction in men and womenrdquo ThrombosisResearch vol 129 no 1 pp 68ndash73 2012
[9] A Hyseni M Roest S L Braun et al ldquoChronic dysfunction ofthe endothelium is associated with mortality in acute coronarysyndrome patientsrdquoThrombosis Research vol 131 no 3 pp 198ndash203 2013
[10] A A Lopes A C Barreto N Y Maeda et al ldquoPlasma vonWillebrand factor as a predictor of survival in pulmonaryarterial hypertension associated with congenital heart diseaserdquoBrazilian Journal of Medical and Biological Research vol 44 no12 pp 1269ndash1275 2011
[11] V Roldan F Marın B Muina et al ldquoPlasma von Willebrandfactor levels are an independent risk factor for adverse eventsincluding mortality and major bleeding in anticoagulated atrialfibrillation patientsrdquo Journal of the American College of Cardiol-ogy vol 57 no 25 pp 2496ndash2504 2011
[12] T A Gaziano A Bitton S Anand S Abrahams-Gessel and AMurphy ldquoGrowing epidemic of coronary heart disease in low-andmiddle-income countriesrdquoCurrent Problems in Cardiologyvol 35 no 2 pp 72ndash115 2010
[13] V L Roger A S Go D M Lloyd-Jones et al ldquoExecutive sum-mary heart disease and stroke statisticsmdash2012 update a reportfrom the American Heart AssociationrdquoCirculation vol 125 no1 pp 188ndash197 2012
[14] G Castaman A Tosetto A Cappelletti et al ldquoValidation ofa rapid test (VWF-LIA) for the quantitative determination ofvonWillebrand factor antigen in type 1 vonWillebrand diseasediagnosis within the European multicenter study MCMDM-1VWDrdquoThrombosis Research vol 126 no 3 pp 227ndash231 2010
[15] A Kappel and M Ehm ldquoImmunoassays for diagnosis of coag-ulation disordersrdquo Hamostaseologie vol 30 no 4 pp 194ndash2012010
[16] R Biswas and S C Parija ldquoA rapid slide agglutination test forthe diagnosis of neurocysticercosis in the rural health set uprdquoTropical Parasitology vol 1 no 2 pp 94ndash98 2011
[17] H Lee Y Park M Kim et al ldquoDevelopment of a latex agglu-tination test for norovirus detectionrdquo Journal of Microbiologyvol 48 no 4 pp 419ndash425 2010
[18] H Y Darani F Ahmadi N Zabardast H A Yousefi and HShirzad ldquoDevelopment of a latex agglutination test as a simpleand rapid method for diagnosis of Trichomonas vaginalisinfectionrdquoAvicenna Journal of Medical Biotechnology vol 2 no1 pp 63ndash66 2010
[19] MMoraveji A Hosseini N Moghaddar M M Namavari andM H Eskandari ldquoDevelopment of latex agglutination test with
10 Journal of Immunology Research
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
242 Stability Study Stability of the reagent was checkedduring storage of VWF antibody-latex reagent at 4∘C for 21days Qualitative test was performed on two plasma sampleson days 1 2 3 4 5 6 7 14 and 21 using aliquots of samplesthat were stored at minus70∘C A new aliquot of sample was usedin each experiment Stability of plasma sample during storagewas also checked by testing VWF antibody-latex reagent(stored at 4∘C) on aliquots of plasma samples which wasstored at 4∘C After 0 (within two hours after blood collec-tion) 1 2 3 4 5 6 7 14 and 21 days the qualitative test wasperformed The reagents were also tested on Hemosil SpecialTest Control Level 2 and Hemosil Normal Control Assayed(Instrumentation Laboratories Italy) Semiquantitative latexagglutination test was performed on these controls for up to50 days Batch to batch variation was tested with the reagentsprepared on two different dates using two different lots of theVWF antibody
243 Linearity Study The linearity of latex agglutinationtest was determined by testing VWF antibody-latex reagenton two patient samples which were serially diluted twofoldThe VWF Ag level in diluted samples was measured byimmunoturbidimetric assay using Hemosil von WillebrandFactor Antigen kit (Instrumentation Laboratory Italy) Semi-quantitative latex agglutination test was performed ondiluted samples and the end-titres were compared with theVWF Ag levels Graphical presentations of the semiquan-titative VWF Ag by latex agglutination test (119884) versus theVWF Ag by immunoturbidimetric assay (119883) were made forindividual sample and also for a combination of the twosamples
244 Determination of Limit of Detection Limit of detectionof latex agglutination test was determined by testing VWFantibody-latex reagent on low-level samples (cryosuper-natant) Seven cryosupernatant samples were serially dilutedtwofold and the VWF Ag levels in the samples (neat) weremeasured by immunoturbidimetric assay using Hemosil vonWillebrand Factor Antigen kit (Instrumentation LaboratoryItaly) VWF Ag levels in 1 2 1 4 and 1 8 diluted sampleswere estimated by dividing the VWF Ag levels of neat sam-ples by the dilution factors of 2 4 and 8 respectively Latexagglutination test was performed to determine the lowestlevel of VWF Ag that shows positive agglutination reaction
245 Interference Study
(i) Interference fromHemoglobin Interference fromhemoglo-bin is common in coagulation testing and hemolytic speci-men is usually not orminimally affected in latex agglutinationtest The interference study was performed by testing VWFantibody-latex reagent on plasma pools and plasma poolsspiked at different concentrations of hemolysate Hemolysatewas prepared by freezing and thawing the whole bloodfollowed by the osmotic shock protocol [31] Briefly normalsample that was collected in 30mLK
2EDTA tube was
centrifuged at 1000 g for 10 minutes Plasma was removedand replaced with an equal volume of isotonic saline Cells
were resuspended and the suspension was centrifuged againat 1000 g for 10 minutes The saline wash was repeated threetimes In the final wash saline was replaced with distilledwater and the suspension was stored at minus20∘C overnightFrozen cells were thawed mixed and centrifuged at 2000 gfor 30 minutes to remove cell debris
Hemolysate was transferred to a clean tube and hemoglo-bin level wasmeasuredwith the SysmexXE 5000Hematologyanalyzer (Sysmex Corporation Kobe Japan) Stock solutionsof hemolysate in normal saline were prepared and each ofthe stock solutions was used to spike plasma pools with highand low VWF Ag levels In the present study hemolysatewith hemoglobin level of 120 gL was used to prepare stocksolutions of hemolysateThe hemolysate was diluted 1 2 1 41 8 1 20 and 1 40 with normal saline to obtain the stocksolutions with hemoglobin levels of 60 30 15 6 and 3 gLrespectively One hundred microlitres of each stock solutionwere added to 900 120583L of plasma pools to obtain plasma poolswith a final hemoglobin level of 6 3 15 06 and 03mgmL
(ii) Interference from Rheumatoid Factor Rheumatoid factor(RF) is one of the most common interferences in latex-basedmethods It may bind and cross-link antibodies attached tothe latex beads leading to a false-positive result or falselyelevated levels of VWF in a sample [32] The effect of RF onagglutination reaction was studied by testing VWF antibody-latex reagent on plasma pools with high and low VWF Aglevels The plasma pools were spiked at different concen-trations of RF using rheumatoid factor control serum fromMyBioSource USA (1030 IUmL)
The RF control serum was diluted 1 8 1 7 and 1 6 withhigh plasma pools to obtain plasma pools spiked with RFat a concentration of 129 147 and 172 IUmL respectivelyFor the preparation of low plasma pools spiked with RF at aconcentration of 10 15 52 103 and 129 IUmL the RF controlserum was diluted 1 100 1 70 1 20 1 10 and 1 8 respec-tively with low plasma pools
246MethodComparisonwith the ReferenceMethodMethodcomparison study between latex agglutination test and thereference method (immunoturbidimetric assay) was con-ducted by parallel analysis of 132 plasma samples of differentlevels of VWF Ag The samples were obtained from 40healthy individuals 40 patients with cardiovascular diseasesand 32 patients with other diseases including malignant andinflammatory disorders The remaining 20 samples werecryosupernatant samples (ie samples with low levels ofVWF Ag) that were obtained from Blood Transfusion Ser-vices All specimens were tested in ten days and the testingwas spread over ten months
Qualitative and semiquantitative latex agglutination testswere performed using the VWF antibody-latex reagentsThe immunoturbidimetric assay was performed on ACLElite Pro Coagulation analyser (Instrumentation LaboratoryItaly) using Hemosil von Willebrand Factor Antigen kitfrom Instrumentation Laboratory Italy The procedure forVWF Ag by immunoturbidimetric assay was performedaccording to the manufacturerrsquos instructions and adhered to
Journal of Immunology Research 5
Table 2 Estimated concentration of adsorbed antibody in the VWF antibody-latex reagents
the CLSI Document H21-A5 on the specimen collection andprocessing of blood specimens for testing the plasma-basedcoagulation assays Normal reference range for VWF Aglevels by immunoturbidimetric assay in our laboratory is 50ndash150
25 Statistical Analysis Statistical analyses were conductedusing the SPSS software version 20 (SPSS Inc Chicago ILUSA) Linear regression analysis was performed to determinethe linearity of the semiquantitative latex agglutination testThe degree of agreement between qualitative latex aggluti-nation test and immunoturbidimetric assay was estimatedby calculating the kappa value (120581) 081ndash099 almost per-fect agreement 061ndash080 substantial agreement 041ndash060moderate agreement 021ndash040 fair agreement 001ndash020slight agreement and lt0 less than chance agreement [33]Spearmanrsquos rho correlation analysiswas used to determine thedegree of relationship between semiquantitative latex agglu-tination test and immunoturbidimetric assay The strengthof the relationship is showed by the correlation coefficientvalue (119903) at least 08 very strong 06 up to 08 moderatelystrong 03 to 05 fair and less than 03 poor [34] Two-tailed probability (119875) value of less than 005 was consideredstatistically significant
3 Results
31 Preparation of VWF Antibody-Latex Reagent Table 2shows the estimated concentration of adsorbed antibody onthe latex beads in the VWF antibody-latex reagents that wereprepared using different concentrations of the VWF anti-body The results showed that the concentration of adsorbedantibody increased as the initial concentration of the VWFantibody increased However the percentage of adsorbedantibody that is 70 and 74 was not much different whenadsorption method was performed using antibody concen-tration of 620120583g and 700 120583g respectively
Visual observation on the agglutination reaction showedthat less agglutination was observed from latex beadsadsorbed with less than 400 120583g of antibody (ie 118 120583g276120583g and 328 120583g) compared to agglutination reactionfrom latex beads adsorbed with about 400 120583g of antibodyAgglutination reactions from latex beads coated with 520120583gantibody were slightly more compared to agglutination reac-tions from latex beads coated with 436 120583g antibody (data notshown) In other words latex beads with sim400 120583g adsorbed
antibody were sufficient to produce strong agglutinationreaction when mixed with plasma sample Thus 200120583L ofthe VWF antibody with a concentration of 620120583g has beenchosen to be the most appropriate concentration for theadsorption procedure
Autoagglutination was not seen when VWF antibody-latex reagent alone was rotated for three minutes Extendingthe time beyond three minutes resulted in reagent dryingthat might give false-positive result No agglutination wasseen when the control reagent (ie reagent without adsorbedantibody) was tested on plasma sample and commercialcontrols Negative reaction was consistently observed whenthe VWF antibody-latex reagent was tested on normal salineand buffers throughout this study
32 Validation Studies Validation studies were conductedusing VWF antibody-latex reagents that were prepared inseven batches The concentration of adsorbed antibody onthe latex beads ranged from 403 to 447 g (ie 65ndash72 fromthe initial concentration of the VWF antibody used in theadsorption procedure) which showed that the percentage oftheVWFantibody adsorbed on the latex beadswas consistentthroughout this study
321 Reproducibility and Stability Studies In the repro-ducibility study the end-titre of three samples (ie 1 321 64 and 1 128) was consistent when tested three timeswithin a day Similarly the end-titre of two samples (ie 1 16and 1 128) did not change from day 1 to day 21 (betweendays) Testing on the controls showed that the end-titre of lowand normal controls (1 4 and 1 16 resp) was reproduciblefrom day 1 to day 50 In the stability study VWF antibody-latex reagent and plasma sample were found to be stable forqualitative latex agglutination test when stored at 4∘C for atleast 21 days Semiquantitative test on controls showed thatthe VWF antibody-latex reagent was stable for at least 50days
All these findings showed that the semiquantitative latexagglutination test results were reproducible within a day andbetween days and the reagent was found to be stable for atleast 50 days when stored at 4∘C Batch to batch variation wasnot detected
322 Linearity Study In the linearity study a series of knownconcentrations of VWF Ag was established by dilution withfive equally spaced concentrations as shown in Table 3 The
6 Journal of Immunology Research
Table 3 Linearity study on semiquantitative VWF antigen by latex agglutination test
Tube number Sample 1 Sample 2VWF Ag ()a VWF Ag (titre)b VWF Ag ()a VWF Ag (titre)b
1 600 1 128 460 1 642 300 1 64 230 1 323 150 1 32 115 1 164 869 1 16 579 1 85 222 1 8 279 1 46 99 1 4 176 1 2aVWF Ag levels () measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from Instrumentation Laboratory ItalybVWF Ag (titre) estimated by using semiquantitative latex agglutination testVWF von Willebrand factor VWF Ag von Willebrand factor antigen
Table 4 Limit of detection of VWF antigen detected by using latex agglutination test
Samplesample number Neat 1 2 1 4 1 8VWF Ag ()a LATe VWF Ag ()b LATe VWF Ag ()c LATe VWF Ag ()d LATe
1 242 + 121 + 61 + 30 minus
2 202 + 102 + 51 + 25 minus
3 306 + 153 + 69 + 38 minus
4 276 + 138 + 69 + 34 minus
5 269 + 133 + 67 + 34 minus
6 291 + 146 + 73 + 36 minus
7 303 + 152 + 76 + 38 minus
aVWF Ag levels in neat sample that was measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from InstrumentationLaboratory ItalybcdEstimated VWF Ag levels in 1 2 1 4 and 1 8 diluted samples (ie by dividing the VWF Ag levels of neat sample by the dilution factors of 2 4 and 8respectivelyePositive (+) and negative (minus) reactions observed when neat and diluted samples were tested by using latex agglutination testVWF Ag von Willebrand factor antigen LAT latex agglutination test
table shows the latex agglutination test results (titre) and thelevels of VWF Ag in undiluted (tube 1) and diluted samples(tubes 2ndash6) It was shown in the table that the semiquanti-tative latex agglutination test results (titre) were directly pro-portional to the levels of VWF Ag in the plasma sample Lin-earity of the semiquantitative VWF Ag latex agglutinationtest was demonstrated in sample 1 (1199032 = 09985) and sample 2(1199032 = 09999) A regression line that was fit to the pointswas observed in both samples (graphs not shown) Thelinearity was also demonstrated at the titre ranging from 1 2to 1 128 when the results of both samples were combined andanalyzed (1199032 = 09361) All these findings have demonstrateda linear relationship between the observed results (VWF Agtitre) and the true concentrations of VWF Ag in the plasmasamples
323 Determination of Limit of Detection In the limit ofdetection study plasma samples with low levels of VWF Ag(in the range of 202 to 306) were obtained from sevencryosupernatant samples (Table 4) Table 4 shows the agglu-tination reaction observed when latex agglutination test wasperformed on undiluted (neat) samples and diluted samples(at 1 2 1 4 and 1 8 dilutions) As shown in the table thelowest level of VWF Ag that still showed positive reaction is51 (sample number 2 at 1 4 dilution) and the highest level
of VWF Ag that showed negative reaction is 38 (samplenumber 7 at 1 8 dilution) These results showed that thelowest level of VWF Ag that the latex agglutination test candetect to determine the presence (positive result) or absence(negative result) of VWF Ag was 51 Thus the limit ofdetection of VWF Ag by latex agglutination test is approx-imately 5 Besides that reproducibility of the agglutinationreaction at VWF Ag levels of 51 and 38 was also checkedby testing six times the diluted samples of number 2 (at 1 4dilution) and number 7 (at 1 8 dilution) respectively Samplenumber 2 showed positive reactions in all six replicates whilesample number 7 showed negative reaction in all six repli-cates These findings showed that the latex agglutination testreliably produced consistent results near the cutoff concen-tration
324 Interference Study Visible agglutinationwas seenwhenlatex agglutination test was performed on undiluted plasmapools and undiluted plasma pools with a final hemoglobinlevel of 03 06 15 3 6 and 12mgmL The end-titre of1 32 was obtained when semiquantitative latex agglutinationtest was performed on high plasma pool The same end-titre(1 32) was observed when the test was performed on highplasma pools with hemoglobin level of 03 06 15 3 6 and12mgmL Similarly the end-titre of low plasma pools with
Journal of Immunology Research 7
Table 5 Rheumatoid factor interference on high and low plasma pools
Type of the plasma pool Vol of RF control serum(120583L)a
aRheumatoid factor control serum fromMyBioSource USA (1030 IUmL) was used to spike plasma poolsRF rheumatoid factor VWF LAT von Willebrand factor latex agglutination test vol volume conc concentration
Table 6 Crosstabulation of VWF antigen levels and qualitative VWF by latex agglutination test results
Total 6 14 61 51 132VWF von Willebrand factor VWF Ag von Willebrand factor antigen LAT latex agglutination test
hemoglobin level of 03 06 15 3 6 and 12mgmL was thesame as the end-titre of low plasma pool without hemoglobininterference (1 4) These results showed that hemoglobindid not cause interference in latex agglutination test andsemiquantitative test result was not affected by hemoglobinlevel up to 12mgmL
Table 5 shows the end-titre of high and low plasma poolsspiked at different concentrations of RF As shown in thetable interference fromRF on latex agglutination test was notdetected in high plasma pools spiked with RF at concentra-tions up to 129 IUmLNevertheless the interference fromRFwas observed in low plasma pools spiked with RF at concen-trations of 26 52 103 and 129 IUmL The end-titre of highand low plasma pools without interference from RF was 1 64and 1 2 respectivelyThese results showed that RF interferedwith the latex agglutination test result when the test wasperformed on the plasma sample containing low levels ofVWF Ag and high levels of RF (gt26 IUmL) However latexagglutination test result of plasma sample containing highlevels of VWF Ag was not affected by RF at concentrationsof lt130 IUmL
325 Method Comparison with the Reference Method Com-parison study between qualitative latex agglutination testand immunoturbidimetric assay showed a kappa value of077 which indicates a substantial agreement between thetwo tests Table 6 shows the crosstabulation of 132 samplesincluded in this study Semiquantitative latex agglutinationtest was strongly correlated with immunoturbidimetric assay
(Spearmanrsquos rho 119903 = 0946 119875 lt 0001) across a range ofVWF Ag levels from 345 to 870
Based on the results from kappa statistic and correlationstudy the scoring system for agglutination reactions and cor-relation with the VWF Ag levels (using immunoturbidimet-ric assay) is shown in Table 7 The proposed interpretationguide is also put in a footnote
4 Discussion
To the best of our knowledge there has been no reportedstudy or technical description available on the rapid test forVWF Ag utilising latex agglutination method At presentthe application of this method has not been used in clin-ical practice for monitoring of patients with low and highplasma levels of VWF Ag Screening of VWDpatients beforeconfirmation tests and monitoring of patients at high risk ofthrombotic event in the area with low-resource settings or atthe bedside can be performed if rapid test for VWF Ag isavailable Hence there is a need to develop a rapid and simplelatex agglutination test that can reliably detect and estimateVWF Ag in the plasma sample
Latex agglutination test is based on the observation ofvisible clumps that are formed from the reaction of antigen-antibody complexes The antigen-antibody complexes can beprepared by passive adsorption or covalent coupling methodIn the early stage of the present study attempts had beenmadeto couple VWF antibody with the carboxylated latex beads
8 Journal of Immunology Research
Table 7 Correlation of result interpretations by latex agglutination test and immunoturbidimetric assay
Qualitative latex agglutination test (LATscore)
Semiquantitative latex agglutination test(titre)
VWF Ag levels by immunoturbidimetricassay ()
1+ 1 2 lt202+ 1 4 1 8 20ndash503+ 1 16 1 32 50ndash1504+ ge64 gt150The proposed interpretation guide normal level of VWF Ag is indicated by LAT scores of 2+ and 3+ or titres from 1 8 to 1 32 Abnormal low VWF Ag isindicated by LAT scores of 0 1+ and 2+ or titres of 1 2 and 1 4 Abnormal high VWF Ag is indicated by LAT score of 4+ or titres of ge64LAT latex agglutination test VWF Ag von Willebrand factor antigen
by covalent coupling method The coupling procedure wasperformed in MES buffer at pH 60 according to the protocolfrom themanufacturer Unfortunately there was no antibodycoated on the latex beads (data not shown) However theVWF antibody was successfully adsorbed onto the surfaceof chloromethyl latex beads by passive adsorption methodThese findings were similar to the reported study by Garcia etal 2015 [35] In the present study amaximal physical adsorp-tion of the VWF antibody onto chloromethyl latex beads wasobtained at pH 74 a pH that is close to the isoelectric pointof polyclonal immunoglobulin (IgG) antibody [36 37]
Interference by RF in the latex agglutination test for thedetection of VWF Ag was expected RF is known to causeanalytical error in automated immunoturbidimetry assay thatpresented the main limitation of this method Despite thislimitation the findings do nevertheless show that the effect ofthe interference was less prominent in the sample with highlevels of VWF Ag compared to the sample with low levelsof VWF Ag The expected interference is without any doubtdue to the usage of whole IgG in the passive adsorptionmethod It iswell known that rheumatoid factor can bindwiththe Fc region of IgG causing false agglutination thatmay alterthe result of the test In the literature the usage of antibodyfragments such as Fab or F(ab1015840)
2was recommended to elim-
inate the interference from rheumatoid factor [37] Antibodyfragment is suggested in future study and optimization isrequired as the antibody fragment is more acidic than thepolyclonal antibody
In the present study the agglutination observed from thereaction between VWF antibody-latex reagent and positivesample may not be optimum as expected Experiments usinghigher initial concentration of VWF antibody with a moreintensive procedure to ensure maximal bead coating couldbe performed in the future study Not only that but theorientation of adsorbed antibody could also be studied toensure a proper alignment of antibody on latex beads Clariziaet al 2009 [38] have demonstrated a method for detectingthe aligned andmisaligned antibodies on the surface on latexbeads Due to some technical constraint maximal bead coat-ing cannot be ensured and a study on orientation of adsorbedantibody could not be performed Another limitation of thisstudy is that the size of the clumping was only estimatedby visual observation In future study it is recommendedto measure the clumping or macroscopic clusters usingmicroscope or other methods
There is a potential clinical application of this rapid latexagglutination test as a routine test in various premises includ-ing small laboratory and clinic and at the bedsideThe clinicalaspect of low levels of VWF Ag is well recognized but notmany medical personnel realized the implications of highVWF Ag levels in medical practice An inherited bleedingdisorder known as von Willebrand disease (VWD) is asso-ciated with an abnormality in VWF and type 1 VWD is thecommonest form that is associated with low VWF Ag [39]On the other hand high levels of VWF Ag are associatedwith thrombotic risks and had been extensively studied inmany medical conditions particularly coronary heart disease(CHD) and stroke It has been shown that CHD patients withhigh baseline of VWF Ag levels are at risk of acute vascularevents related to thrombosis [40] The current clinical guide-lines do not incorporate the measurement of VWF Ag inthe routine assessment of CHD patients but from variousstudies there is a role in monitoring the levels of VWF AgDetection of persistent high levels or increasing trends ofVWF Ag levels would alert the treating doctor for certainclinical measures to improve the patient care
Latex agglutination test for the detection of VWF Agwasfound to be simple to perform and the results can be obtainedwithin three minutes making it suitable to be used in areaswith limited laboratory facilities The result obtained bythe latex agglutination test is reliable as shown by the findingsfrom reproducibility stability linearity limit of detectionand method comparison studies Prozone effect was notdetected at VWF Ag levels up to 870 The stability of thereagents used for this method was up to 50 days and in thisstudy no stabilizer or preservatives were used The plasmafor VWF Ag estimation was stable for up to 21 days whichmay be related to the protein structure however more testsare needed to confirm the suitable plasma storage recom-mendation for this method All these findings showed theusefulness of this method in detecting and estimating highand low levels of VWF AgThis method might be useful inthe monitoring of VWF Ag in patients that are at a high riskof vascular event related to endothelial dysfunction Similarlythis method could be used to estimate VWF Ag levels inpatients with VWD before confirmation with standard testpanels in the specialized coagulation laboratory
However we only evaluate the latex agglutination testin quantitative terms by using plasma samples containinglow and high levels of VWF Ag This application mainlyaddressed the VWD type 1 which is more common in clinical
Journal of Immunology Research 9
practice In future study plasma samples with qualitativeVWF abnormalities (eg type II VWD or acquired VWDpatients demonstrating abnormal VWF function with vari-ables VWF Ag levels) could be included in the comparisonstudy to confirm the application of the latex agglutination testfor assessing VWF Ag levels in various clinical situations Insummary this test method needs to be refined and comparedwith other latex agglutination tests available in the marketbefore it is used on patient samples
5 Conclusion
Latex agglutination test for VWF Ag is simple rapid andreproducible correlates well with the reference method andis suitable to be used in areaswith limited laboratory facilitiesThe test has commercial potential as a low-cost alternativemethod for the detection and estimation of low levels andhigh levels of VWF Ag that could help in themanagement ofVWDtype 1 patients andpatients at risk of thrombotic eventsrespectively However the testmay not be suitable for patientswith a high level of rheumatoid factor
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by short-term Grant 304PPSP61312085 from Universiti Sains Malaysia Penang MalaysiaThe authors gratefully acknowledge Dr Emmanuel J Faval-oro from Institute of Clinical Pathology and MedicalResearch at Westmead Hospital Australia for his technicaladvice on this work Thanks are due to Dr Lim TheamSoon from Institute for Research in Molecular MedicineUniversiti Sains Malaysia Penang Malaysia for his valuableadvice in the early stage of this work The appreciation alsogoes to the staff of Haematology Department (especially thecoagulation section) BloodTransfusion ServiceDepartmentand Immunology Department Universiti Sains MalaysiaKelantan Malaysia for their continuous support in thiswork
References
[1] Z M Ruggeri ldquoStructure of vonWillebrand factor and its func-tion in platelet adhesion and thrombus formationrdquo Best Practiceand Research Clinical Haematology vol 14 no 2 pp 257ndash2792001
[2] G-P Luo B Ni X Yang and Y-ZWu ldquovonWillebrand factormore than a regulator of hemostasis and thrombosisrdquo ActaHaematologica vol 128 no 3 pp 158ndash169 2012
[3] C V Denis and P J Lenting ldquoVon Willebrand factor at thecrossroads of bleeding and thrombosisrdquo International Journal ofHematology vol 95 no 4 pp 353ndash361 2012
[4] A Alonso W Tang S K Agarwal E Z Soliman A M Cham-berlain and A R Folsom ldquoHemostatic markers are associatedwith the risk and prognosis of atrial fibrillation the ARIC
studyrdquo International Journal of Cardiology vol 155 no 2 pp217ndash222 2012
[5] F Cambronero J A Vilchez A Garcıa-Honrubia et al ldquoPlasmalevels of Von Willebrand factor are increased in patients withhypertrophic cardiomyopathyrdquo Thrombosis Research vol 126no 1 pp e46ndashe50 2010
[6] A M Kucharska-Newton D J Couper J S Pankow et alldquoHemostasis inflammation and fatal and nonfatal coronaryheart disease long-term follow-up of the atherosclerosis risk incommunities (ARIC) cohortrdquo Arteriosclerosis Thrombosis andVascular Biology vol 29 no 12 pp 2182ndash2190 2009
[7] R G Wieberdink M C van Schie P J Koudstaal et al ldquoHighvon Willebrand factor levels increase the risk of stroke theRotterdam studyrdquo Stroke vol 41 no 10 pp 2151ndash2156 2010
[8] P Wennberg F Wensley E Di Angelantonio et al ldquoHaemo-static and inflammatory markers are independently associatedwith myocardial infarction in men and womenrdquo ThrombosisResearch vol 129 no 1 pp 68ndash73 2012
[9] A Hyseni M Roest S L Braun et al ldquoChronic dysfunction ofthe endothelium is associated with mortality in acute coronarysyndrome patientsrdquoThrombosis Research vol 131 no 3 pp 198ndash203 2013
[10] A A Lopes A C Barreto N Y Maeda et al ldquoPlasma vonWillebrand factor as a predictor of survival in pulmonaryarterial hypertension associated with congenital heart diseaserdquoBrazilian Journal of Medical and Biological Research vol 44 no12 pp 1269ndash1275 2011
[11] V Roldan F Marın B Muina et al ldquoPlasma von Willebrandfactor levels are an independent risk factor for adverse eventsincluding mortality and major bleeding in anticoagulated atrialfibrillation patientsrdquo Journal of the American College of Cardiol-ogy vol 57 no 25 pp 2496ndash2504 2011
[12] T A Gaziano A Bitton S Anand S Abrahams-Gessel and AMurphy ldquoGrowing epidemic of coronary heart disease in low-andmiddle-income countriesrdquoCurrent Problems in Cardiologyvol 35 no 2 pp 72ndash115 2010
[13] V L Roger A S Go D M Lloyd-Jones et al ldquoExecutive sum-mary heart disease and stroke statisticsmdash2012 update a reportfrom the American Heart AssociationrdquoCirculation vol 125 no1 pp 188ndash197 2012
[14] G Castaman A Tosetto A Cappelletti et al ldquoValidation ofa rapid test (VWF-LIA) for the quantitative determination ofvonWillebrand factor antigen in type 1 vonWillebrand diseasediagnosis within the European multicenter study MCMDM-1VWDrdquoThrombosis Research vol 126 no 3 pp 227ndash231 2010
[15] A Kappel and M Ehm ldquoImmunoassays for diagnosis of coag-ulation disordersrdquo Hamostaseologie vol 30 no 4 pp 194ndash2012010
[16] R Biswas and S C Parija ldquoA rapid slide agglutination test forthe diagnosis of neurocysticercosis in the rural health set uprdquoTropical Parasitology vol 1 no 2 pp 94ndash98 2011
[17] H Lee Y Park M Kim et al ldquoDevelopment of a latex agglu-tination test for norovirus detectionrdquo Journal of Microbiologyvol 48 no 4 pp 419ndash425 2010
[18] H Y Darani F Ahmadi N Zabardast H A Yousefi and HShirzad ldquoDevelopment of a latex agglutination test as a simpleand rapid method for diagnosis of Trichomonas vaginalisinfectionrdquoAvicenna Journal of Medical Biotechnology vol 2 no1 pp 63ndash66 2010
[19] MMoraveji A Hosseini N Moghaddar M M Namavari andM H Eskandari ldquoDevelopment of latex agglutination test with
10 Journal of Immunology Research
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
the CLSI Document H21-A5 on the specimen collection andprocessing of blood specimens for testing the plasma-basedcoagulation assays Normal reference range for VWF Aglevels by immunoturbidimetric assay in our laboratory is 50ndash150
25 Statistical Analysis Statistical analyses were conductedusing the SPSS software version 20 (SPSS Inc Chicago ILUSA) Linear regression analysis was performed to determinethe linearity of the semiquantitative latex agglutination testThe degree of agreement between qualitative latex aggluti-nation test and immunoturbidimetric assay was estimatedby calculating the kappa value (120581) 081ndash099 almost per-fect agreement 061ndash080 substantial agreement 041ndash060moderate agreement 021ndash040 fair agreement 001ndash020slight agreement and lt0 less than chance agreement [33]Spearmanrsquos rho correlation analysiswas used to determine thedegree of relationship between semiquantitative latex agglu-tination test and immunoturbidimetric assay The strengthof the relationship is showed by the correlation coefficientvalue (119903) at least 08 very strong 06 up to 08 moderatelystrong 03 to 05 fair and less than 03 poor [34] Two-tailed probability (119875) value of less than 005 was consideredstatistically significant
3 Results
31 Preparation of VWF Antibody-Latex Reagent Table 2shows the estimated concentration of adsorbed antibody onthe latex beads in the VWF antibody-latex reagents that wereprepared using different concentrations of the VWF anti-body The results showed that the concentration of adsorbedantibody increased as the initial concentration of the VWFantibody increased However the percentage of adsorbedantibody that is 70 and 74 was not much different whenadsorption method was performed using antibody concen-tration of 620120583g and 700 120583g respectively
Visual observation on the agglutination reaction showedthat less agglutination was observed from latex beadsadsorbed with less than 400 120583g of antibody (ie 118 120583g276120583g and 328 120583g) compared to agglutination reactionfrom latex beads adsorbed with about 400 120583g of antibodyAgglutination reactions from latex beads coated with 520120583gantibody were slightly more compared to agglutination reac-tions from latex beads coated with 436 120583g antibody (data notshown) In other words latex beads with sim400 120583g adsorbed
antibody were sufficient to produce strong agglutinationreaction when mixed with plasma sample Thus 200120583L ofthe VWF antibody with a concentration of 620120583g has beenchosen to be the most appropriate concentration for theadsorption procedure
Autoagglutination was not seen when VWF antibody-latex reagent alone was rotated for three minutes Extendingthe time beyond three minutes resulted in reagent dryingthat might give false-positive result No agglutination wasseen when the control reagent (ie reagent without adsorbedantibody) was tested on plasma sample and commercialcontrols Negative reaction was consistently observed whenthe VWF antibody-latex reagent was tested on normal salineand buffers throughout this study
32 Validation Studies Validation studies were conductedusing VWF antibody-latex reagents that were prepared inseven batches The concentration of adsorbed antibody onthe latex beads ranged from 403 to 447 g (ie 65ndash72 fromthe initial concentration of the VWF antibody used in theadsorption procedure) which showed that the percentage oftheVWFantibody adsorbed on the latex beadswas consistentthroughout this study
321 Reproducibility and Stability Studies In the repro-ducibility study the end-titre of three samples (ie 1 321 64 and 1 128) was consistent when tested three timeswithin a day Similarly the end-titre of two samples (ie 1 16and 1 128) did not change from day 1 to day 21 (betweendays) Testing on the controls showed that the end-titre of lowand normal controls (1 4 and 1 16 resp) was reproduciblefrom day 1 to day 50 In the stability study VWF antibody-latex reagent and plasma sample were found to be stable forqualitative latex agglutination test when stored at 4∘C for atleast 21 days Semiquantitative test on controls showed thatthe VWF antibody-latex reagent was stable for at least 50days
All these findings showed that the semiquantitative latexagglutination test results were reproducible within a day andbetween days and the reagent was found to be stable for atleast 50 days when stored at 4∘C Batch to batch variation wasnot detected
322 Linearity Study In the linearity study a series of knownconcentrations of VWF Ag was established by dilution withfive equally spaced concentrations as shown in Table 3 The
6 Journal of Immunology Research
Table 3 Linearity study on semiquantitative VWF antigen by latex agglutination test
Tube number Sample 1 Sample 2VWF Ag ()a VWF Ag (titre)b VWF Ag ()a VWF Ag (titre)b
1 600 1 128 460 1 642 300 1 64 230 1 323 150 1 32 115 1 164 869 1 16 579 1 85 222 1 8 279 1 46 99 1 4 176 1 2aVWF Ag levels () measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from Instrumentation Laboratory ItalybVWF Ag (titre) estimated by using semiquantitative latex agglutination testVWF von Willebrand factor VWF Ag von Willebrand factor antigen
Table 4 Limit of detection of VWF antigen detected by using latex agglutination test
Samplesample number Neat 1 2 1 4 1 8VWF Ag ()a LATe VWF Ag ()b LATe VWF Ag ()c LATe VWF Ag ()d LATe
1 242 + 121 + 61 + 30 minus
2 202 + 102 + 51 + 25 minus
3 306 + 153 + 69 + 38 minus
4 276 + 138 + 69 + 34 minus
5 269 + 133 + 67 + 34 minus
6 291 + 146 + 73 + 36 minus
7 303 + 152 + 76 + 38 minus
aVWF Ag levels in neat sample that was measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from InstrumentationLaboratory ItalybcdEstimated VWF Ag levels in 1 2 1 4 and 1 8 diluted samples (ie by dividing the VWF Ag levels of neat sample by the dilution factors of 2 4 and 8respectivelyePositive (+) and negative (minus) reactions observed when neat and diluted samples were tested by using latex agglutination testVWF Ag von Willebrand factor antigen LAT latex agglutination test
table shows the latex agglutination test results (titre) and thelevels of VWF Ag in undiluted (tube 1) and diluted samples(tubes 2ndash6) It was shown in the table that the semiquanti-tative latex agglutination test results (titre) were directly pro-portional to the levels of VWF Ag in the plasma sample Lin-earity of the semiquantitative VWF Ag latex agglutinationtest was demonstrated in sample 1 (1199032 = 09985) and sample 2(1199032 = 09999) A regression line that was fit to the pointswas observed in both samples (graphs not shown) Thelinearity was also demonstrated at the titre ranging from 1 2to 1 128 when the results of both samples were combined andanalyzed (1199032 = 09361) All these findings have demonstrateda linear relationship between the observed results (VWF Agtitre) and the true concentrations of VWF Ag in the plasmasamples
323 Determination of Limit of Detection In the limit ofdetection study plasma samples with low levels of VWF Ag(in the range of 202 to 306) were obtained from sevencryosupernatant samples (Table 4) Table 4 shows the agglu-tination reaction observed when latex agglutination test wasperformed on undiluted (neat) samples and diluted samples(at 1 2 1 4 and 1 8 dilutions) As shown in the table thelowest level of VWF Ag that still showed positive reaction is51 (sample number 2 at 1 4 dilution) and the highest level
of VWF Ag that showed negative reaction is 38 (samplenumber 7 at 1 8 dilution) These results showed that thelowest level of VWF Ag that the latex agglutination test candetect to determine the presence (positive result) or absence(negative result) of VWF Ag was 51 Thus the limit ofdetection of VWF Ag by latex agglutination test is approx-imately 5 Besides that reproducibility of the agglutinationreaction at VWF Ag levels of 51 and 38 was also checkedby testing six times the diluted samples of number 2 (at 1 4dilution) and number 7 (at 1 8 dilution) respectively Samplenumber 2 showed positive reactions in all six replicates whilesample number 7 showed negative reaction in all six repli-cates These findings showed that the latex agglutination testreliably produced consistent results near the cutoff concen-tration
324 Interference Study Visible agglutinationwas seenwhenlatex agglutination test was performed on undiluted plasmapools and undiluted plasma pools with a final hemoglobinlevel of 03 06 15 3 6 and 12mgmL The end-titre of1 32 was obtained when semiquantitative latex agglutinationtest was performed on high plasma pool The same end-titre(1 32) was observed when the test was performed on highplasma pools with hemoglobin level of 03 06 15 3 6 and12mgmL Similarly the end-titre of low plasma pools with
Journal of Immunology Research 7
Table 5 Rheumatoid factor interference on high and low plasma pools
Type of the plasma pool Vol of RF control serum(120583L)a
aRheumatoid factor control serum fromMyBioSource USA (1030 IUmL) was used to spike plasma poolsRF rheumatoid factor VWF LAT von Willebrand factor latex agglutination test vol volume conc concentration
Table 6 Crosstabulation of VWF antigen levels and qualitative VWF by latex agglutination test results
Total 6 14 61 51 132VWF von Willebrand factor VWF Ag von Willebrand factor antigen LAT latex agglutination test
hemoglobin level of 03 06 15 3 6 and 12mgmL was thesame as the end-titre of low plasma pool without hemoglobininterference (1 4) These results showed that hemoglobindid not cause interference in latex agglutination test andsemiquantitative test result was not affected by hemoglobinlevel up to 12mgmL
Table 5 shows the end-titre of high and low plasma poolsspiked at different concentrations of RF As shown in thetable interference fromRF on latex agglutination test was notdetected in high plasma pools spiked with RF at concentra-tions up to 129 IUmLNevertheless the interference fromRFwas observed in low plasma pools spiked with RF at concen-trations of 26 52 103 and 129 IUmL The end-titre of highand low plasma pools without interference from RF was 1 64and 1 2 respectivelyThese results showed that RF interferedwith the latex agglutination test result when the test wasperformed on the plasma sample containing low levels ofVWF Ag and high levels of RF (gt26 IUmL) However latexagglutination test result of plasma sample containing highlevels of VWF Ag was not affected by RF at concentrationsof lt130 IUmL
325 Method Comparison with the Reference Method Com-parison study between qualitative latex agglutination testand immunoturbidimetric assay showed a kappa value of077 which indicates a substantial agreement between thetwo tests Table 6 shows the crosstabulation of 132 samplesincluded in this study Semiquantitative latex agglutinationtest was strongly correlated with immunoturbidimetric assay
(Spearmanrsquos rho 119903 = 0946 119875 lt 0001) across a range ofVWF Ag levels from 345 to 870
Based on the results from kappa statistic and correlationstudy the scoring system for agglutination reactions and cor-relation with the VWF Ag levels (using immunoturbidimet-ric assay) is shown in Table 7 The proposed interpretationguide is also put in a footnote
4 Discussion
To the best of our knowledge there has been no reportedstudy or technical description available on the rapid test forVWF Ag utilising latex agglutination method At presentthe application of this method has not been used in clin-ical practice for monitoring of patients with low and highplasma levels of VWF Ag Screening of VWDpatients beforeconfirmation tests and monitoring of patients at high risk ofthrombotic event in the area with low-resource settings or atthe bedside can be performed if rapid test for VWF Ag isavailable Hence there is a need to develop a rapid and simplelatex agglutination test that can reliably detect and estimateVWF Ag in the plasma sample
Latex agglutination test is based on the observation ofvisible clumps that are formed from the reaction of antigen-antibody complexes The antigen-antibody complexes can beprepared by passive adsorption or covalent coupling methodIn the early stage of the present study attempts had beenmadeto couple VWF antibody with the carboxylated latex beads
8 Journal of Immunology Research
Table 7 Correlation of result interpretations by latex agglutination test and immunoturbidimetric assay
Qualitative latex agglutination test (LATscore)
Semiquantitative latex agglutination test(titre)
VWF Ag levels by immunoturbidimetricassay ()
1+ 1 2 lt202+ 1 4 1 8 20ndash503+ 1 16 1 32 50ndash1504+ ge64 gt150The proposed interpretation guide normal level of VWF Ag is indicated by LAT scores of 2+ and 3+ or titres from 1 8 to 1 32 Abnormal low VWF Ag isindicated by LAT scores of 0 1+ and 2+ or titres of 1 2 and 1 4 Abnormal high VWF Ag is indicated by LAT score of 4+ or titres of ge64LAT latex agglutination test VWF Ag von Willebrand factor antigen
by covalent coupling method The coupling procedure wasperformed in MES buffer at pH 60 according to the protocolfrom themanufacturer Unfortunately there was no antibodycoated on the latex beads (data not shown) However theVWF antibody was successfully adsorbed onto the surfaceof chloromethyl latex beads by passive adsorption methodThese findings were similar to the reported study by Garcia etal 2015 [35] In the present study amaximal physical adsorp-tion of the VWF antibody onto chloromethyl latex beads wasobtained at pH 74 a pH that is close to the isoelectric pointof polyclonal immunoglobulin (IgG) antibody [36 37]
Interference by RF in the latex agglutination test for thedetection of VWF Ag was expected RF is known to causeanalytical error in automated immunoturbidimetry assay thatpresented the main limitation of this method Despite thislimitation the findings do nevertheless show that the effect ofthe interference was less prominent in the sample with highlevels of VWF Ag compared to the sample with low levelsof VWF Ag The expected interference is without any doubtdue to the usage of whole IgG in the passive adsorptionmethod It iswell known that rheumatoid factor can bindwiththe Fc region of IgG causing false agglutination thatmay alterthe result of the test In the literature the usage of antibodyfragments such as Fab or F(ab1015840)
2was recommended to elim-
inate the interference from rheumatoid factor [37] Antibodyfragment is suggested in future study and optimization isrequired as the antibody fragment is more acidic than thepolyclonal antibody
In the present study the agglutination observed from thereaction between VWF antibody-latex reagent and positivesample may not be optimum as expected Experiments usinghigher initial concentration of VWF antibody with a moreintensive procedure to ensure maximal bead coating couldbe performed in the future study Not only that but theorientation of adsorbed antibody could also be studied toensure a proper alignment of antibody on latex beads Clariziaet al 2009 [38] have demonstrated a method for detectingthe aligned andmisaligned antibodies on the surface on latexbeads Due to some technical constraint maximal bead coat-ing cannot be ensured and a study on orientation of adsorbedantibody could not be performed Another limitation of thisstudy is that the size of the clumping was only estimatedby visual observation In future study it is recommendedto measure the clumping or macroscopic clusters usingmicroscope or other methods
There is a potential clinical application of this rapid latexagglutination test as a routine test in various premises includ-ing small laboratory and clinic and at the bedsideThe clinicalaspect of low levels of VWF Ag is well recognized but notmany medical personnel realized the implications of highVWF Ag levels in medical practice An inherited bleedingdisorder known as von Willebrand disease (VWD) is asso-ciated with an abnormality in VWF and type 1 VWD is thecommonest form that is associated with low VWF Ag [39]On the other hand high levels of VWF Ag are associatedwith thrombotic risks and had been extensively studied inmany medical conditions particularly coronary heart disease(CHD) and stroke It has been shown that CHD patients withhigh baseline of VWF Ag levels are at risk of acute vascularevents related to thrombosis [40] The current clinical guide-lines do not incorporate the measurement of VWF Ag inthe routine assessment of CHD patients but from variousstudies there is a role in monitoring the levels of VWF AgDetection of persistent high levels or increasing trends ofVWF Ag levels would alert the treating doctor for certainclinical measures to improve the patient care
Latex agglutination test for the detection of VWF Agwasfound to be simple to perform and the results can be obtainedwithin three minutes making it suitable to be used in areaswith limited laboratory facilities The result obtained bythe latex agglutination test is reliable as shown by the findingsfrom reproducibility stability linearity limit of detectionand method comparison studies Prozone effect was notdetected at VWF Ag levels up to 870 The stability of thereagents used for this method was up to 50 days and in thisstudy no stabilizer or preservatives were used The plasmafor VWF Ag estimation was stable for up to 21 days whichmay be related to the protein structure however more testsare needed to confirm the suitable plasma storage recom-mendation for this method All these findings showed theusefulness of this method in detecting and estimating highand low levels of VWF AgThis method might be useful inthe monitoring of VWF Ag in patients that are at a high riskof vascular event related to endothelial dysfunction Similarlythis method could be used to estimate VWF Ag levels inpatients with VWD before confirmation with standard testpanels in the specialized coagulation laboratory
However we only evaluate the latex agglutination testin quantitative terms by using plasma samples containinglow and high levels of VWF Ag This application mainlyaddressed the VWD type 1 which is more common in clinical
Journal of Immunology Research 9
practice In future study plasma samples with qualitativeVWF abnormalities (eg type II VWD or acquired VWDpatients demonstrating abnormal VWF function with vari-ables VWF Ag levels) could be included in the comparisonstudy to confirm the application of the latex agglutination testfor assessing VWF Ag levels in various clinical situations Insummary this test method needs to be refined and comparedwith other latex agglutination tests available in the marketbefore it is used on patient samples
5 Conclusion
Latex agglutination test for VWF Ag is simple rapid andreproducible correlates well with the reference method andis suitable to be used in areaswith limited laboratory facilitiesThe test has commercial potential as a low-cost alternativemethod for the detection and estimation of low levels andhigh levels of VWF Ag that could help in themanagement ofVWDtype 1 patients andpatients at risk of thrombotic eventsrespectively However the testmay not be suitable for patientswith a high level of rheumatoid factor
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by short-term Grant 304PPSP61312085 from Universiti Sains Malaysia Penang MalaysiaThe authors gratefully acknowledge Dr Emmanuel J Faval-oro from Institute of Clinical Pathology and MedicalResearch at Westmead Hospital Australia for his technicaladvice on this work Thanks are due to Dr Lim TheamSoon from Institute for Research in Molecular MedicineUniversiti Sains Malaysia Penang Malaysia for his valuableadvice in the early stage of this work The appreciation alsogoes to the staff of Haematology Department (especially thecoagulation section) BloodTransfusion ServiceDepartmentand Immunology Department Universiti Sains MalaysiaKelantan Malaysia for their continuous support in thiswork
References
[1] Z M Ruggeri ldquoStructure of vonWillebrand factor and its func-tion in platelet adhesion and thrombus formationrdquo Best Practiceand Research Clinical Haematology vol 14 no 2 pp 257ndash2792001
[2] G-P Luo B Ni X Yang and Y-ZWu ldquovonWillebrand factormore than a regulator of hemostasis and thrombosisrdquo ActaHaematologica vol 128 no 3 pp 158ndash169 2012
[3] C V Denis and P J Lenting ldquoVon Willebrand factor at thecrossroads of bleeding and thrombosisrdquo International Journal ofHematology vol 95 no 4 pp 353ndash361 2012
[4] A Alonso W Tang S K Agarwal E Z Soliman A M Cham-berlain and A R Folsom ldquoHemostatic markers are associatedwith the risk and prognosis of atrial fibrillation the ARIC
studyrdquo International Journal of Cardiology vol 155 no 2 pp217ndash222 2012
[5] F Cambronero J A Vilchez A Garcıa-Honrubia et al ldquoPlasmalevels of Von Willebrand factor are increased in patients withhypertrophic cardiomyopathyrdquo Thrombosis Research vol 126no 1 pp e46ndashe50 2010
[6] A M Kucharska-Newton D J Couper J S Pankow et alldquoHemostasis inflammation and fatal and nonfatal coronaryheart disease long-term follow-up of the atherosclerosis risk incommunities (ARIC) cohortrdquo Arteriosclerosis Thrombosis andVascular Biology vol 29 no 12 pp 2182ndash2190 2009
[7] R G Wieberdink M C van Schie P J Koudstaal et al ldquoHighvon Willebrand factor levels increase the risk of stroke theRotterdam studyrdquo Stroke vol 41 no 10 pp 2151ndash2156 2010
[8] P Wennberg F Wensley E Di Angelantonio et al ldquoHaemo-static and inflammatory markers are independently associatedwith myocardial infarction in men and womenrdquo ThrombosisResearch vol 129 no 1 pp 68ndash73 2012
[9] A Hyseni M Roest S L Braun et al ldquoChronic dysfunction ofthe endothelium is associated with mortality in acute coronarysyndrome patientsrdquoThrombosis Research vol 131 no 3 pp 198ndash203 2013
[10] A A Lopes A C Barreto N Y Maeda et al ldquoPlasma vonWillebrand factor as a predictor of survival in pulmonaryarterial hypertension associated with congenital heart diseaserdquoBrazilian Journal of Medical and Biological Research vol 44 no12 pp 1269ndash1275 2011
[11] V Roldan F Marın B Muina et al ldquoPlasma von Willebrandfactor levels are an independent risk factor for adverse eventsincluding mortality and major bleeding in anticoagulated atrialfibrillation patientsrdquo Journal of the American College of Cardiol-ogy vol 57 no 25 pp 2496ndash2504 2011
[12] T A Gaziano A Bitton S Anand S Abrahams-Gessel and AMurphy ldquoGrowing epidemic of coronary heart disease in low-andmiddle-income countriesrdquoCurrent Problems in Cardiologyvol 35 no 2 pp 72ndash115 2010
[13] V L Roger A S Go D M Lloyd-Jones et al ldquoExecutive sum-mary heart disease and stroke statisticsmdash2012 update a reportfrom the American Heart AssociationrdquoCirculation vol 125 no1 pp 188ndash197 2012
[14] G Castaman A Tosetto A Cappelletti et al ldquoValidation ofa rapid test (VWF-LIA) for the quantitative determination ofvonWillebrand factor antigen in type 1 vonWillebrand diseasediagnosis within the European multicenter study MCMDM-1VWDrdquoThrombosis Research vol 126 no 3 pp 227ndash231 2010
[15] A Kappel and M Ehm ldquoImmunoassays for diagnosis of coag-ulation disordersrdquo Hamostaseologie vol 30 no 4 pp 194ndash2012010
[16] R Biswas and S C Parija ldquoA rapid slide agglutination test forthe diagnosis of neurocysticercosis in the rural health set uprdquoTropical Parasitology vol 1 no 2 pp 94ndash98 2011
[17] H Lee Y Park M Kim et al ldquoDevelopment of a latex agglu-tination test for norovirus detectionrdquo Journal of Microbiologyvol 48 no 4 pp 419ndash425 2010
[18] H Y Darani F Ahmadi N Zabardast H A Yousefi and HShirzad ldquoDevelopment of a latex agglutination test as a simpleand rapid method for diagnosis of Trichomonas vaginalisinfectionrdquoAvicenna Journal of Medical Biotechnology vol 2 no1 pp 63ndash66 2010
[19] MMoraveji A Hosseini N Moghaddar M M Namavari andM H Eskandari ldquoDevelopment of latex agglutination test with
10 Journal of Immunology Research
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
Table 3 Linearity study on semiquantitative VWF antigen by latex agglutination test
Tube number Sample 1 Sample 2VWF Ag ()a VWF Ag (titre)b VWF Ag ()a VWF Ag (titre)b
1 600 1 128 460 1 642 300 1 64 230 1 323 150 1 32 115 1 164 869 1 16 579 1 85 222 1 8 279 1 46 99 1 4 176 1 2aVWF Ag levels () measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from Instrumentation Laboratory ItalybVWF Ag (titre) estimated by using semiquantitative latex agglutination testVWF von Willebrand factor VWF Ag von Willebrand factor antigen
Table 4 Limit of detection of VWF antigen detected by using latex agglutination test
Samplesample number Neat 1 2 1 4 1 8VWF Ag ()a LATe VWF Ag ()b LATe VWF Ag ()c LATe VWF Ag ()d LATe
1 242 + 121 + 61 + 30 minus
2 202 + 102 + 51 + 25 minus
3 306 + 153 + 69 + 38 minus
4 276 + 138 + 69 + 34 minus
5 269 + 133 + 67 + 34 minus
6 291 + 146 + 73 + 36 minus
7 303 + 152 + 76 + 38 minus
aVWF Ag levels in neat sample that was measured by immunoturbidimetric assay using Hemosil von Willebrand Factor Antigen kit from InstrumentationLaboratory ItalybcdEstimated VWF Ag levels in 1 2 1 4 and 1 8 diluted samples (ie by dividing the VWF Ag levels of neat sample by the dilution factors of 2 4 and 8respectivelyePositive (+) and negative (minus) reactions observed when neat and diluted samples were tested by using latex agglutination testVWF Ag von Willebrand factor antigen LAT latex agglutination test
table shows the latex agglutination test results (titre) and thelevels of VWF Ag in undiluted (tube 1) and diluted samples(tubes 2ndash6) It was shown in the table that the semiquanti-tative latex agglutination test results (titre) were directly pro-portional to the levels of VWF Ag in the plasma sample Lin-earity of the semiquantitative VWF Ag latex agglutinationtest was demonstrated in sample 1 (1199032 = 09985) and sample 2(1199032 = 09999) A regression line that was fit to the pointswas observed in both samples (graphs not shown) Thelinearity was also demonstrated at the titre ranging from 1 2to 1 128 when the results of both samples were combined andanalyzed (1199032 = 09361) All these findings have demonstrateda linear relationship between the observed results (VWF Agtitre) and the true concentrations of VWF Ag in the plasmasamples
323 Determination of Limit of Detection In the limit ofdetection study plasma samples with low levels of VWF Ag(in the range of 202 to 306) were obtained from sevencryosupernatant samples (Table 4) Table 4 shows the agglu-tination reaction observed when latex agglutination test wasperformed on undiluted (neat) samples and diluted samples(at 1 2 1 4 and 1 8 dilutions) As shown in the table thelowest level of VWF Ag that still showed positive reaction is51 (sample number 2 at 1 4 dilution) and the highest level
of VWF Ag that showed negative reaction is 38 (samplenumber 7 at 1 8 dilution) These results showed that thelowest level of VWF Ag that the latex agglutination test candetect to determine the presence (positive result) or absence(negative result) of VWF Ag was 51 Thus the limit ofdetection of VWF Ag by latex agglutination test is approx-imately 5 Besides that reproducibility of the agglutinationreaction at VWF Ag levels of 51 and 38 was also checkedby testing six times the diluted samples of number 2 (at 1 4dilution) and number 7 (at 1 8 dilution) respectively Samplenumber 2 showed positive reactions in all six replicates whilesample number 7 showed negative reaction in all six repli-cates These findings showed that the latex agglutination testreliably produced consistent results near the cutoff concen-tration
324 Interference Study Visible agglutinationwas seenwhenlatex agglutination test was performed on undiluted plasmapools and undiluted plasma pools with a final hemoglobinlevel of 03 06 15 3 6 and 12mgmL The end-titre of1 32 was obtained when semiquantitative latex agglutinationtest was performed on high plasma pool The same end-titre(1 32) was observed when the test was performed on highplasma pools with hemoglobin level of 03 06 15 3 6 and12mgmL Similarly the end-titre of low plasma pools with
Journal of Immunology Research 7
Table 5 Rheumatoid factor interference on high and low plasma pools
Type of the plasma pool Vol of RF control serum(120583L)a
aRheumatoid factor control serum fromMyBioSource USA (1030 IUmL) was used to spike plasma poolsRF rheumatoid factor VWF LAT von Willebrand factor latex agglutination test vol volume conc concentration
Table 6 Crosstabulation of VWF antigen levels and qualitative VWF by latex agglutination test results
Total 6 14 61 51 132VWF von Willebrand factor VWF Ag von Willebrand factor antigen LAT latex agglutination test
hemoglobin level of 03 06 15 3 6 and 12mgmL was thesame as the end-titre of low plasma pool without hemoglobininterference (1 4) These results showed that hemoglobindid not cause interference in latex agglutination test andsemiquantitative test result was not affected by hemoglobinlevel up to 12mgmL
Table 5 shows the end-titre of high and low plasma poolsspiked at different concentrations of RF As shown in thetable interference fromRF on latex agglutination test was notdetected in high plasma pools spiked with RF at concentra-tions up to 129 IUmLNevertheless the interference fromRFwas observed in low plasma pools spiked with RF at concen-trations of 26 52 103 and 129 IUmL The end-titre of highand low plasma pools without interference from RF was 1 64and 1 2 respectivelyThese results showed that RF interferedwith the latex agglutination test result when the test wasperformed on the plasma sample containing low levels ofVWF Ag and high levels of RF (gt26 IUmL) However latexagglutination test result of plasma sample containing highlevels of VWF Ag was not affected by RF at concentrationsof lt130 IUmL
325 Method Comparison with the Reference Method Com-parison study between qualitative latex agglutination testand immunoturbidimetric assay showed a kappa value of077 which indicates a substantial agreement between thetwo tests Table 6 shows the crosstabulation of 132 samplesincluded in this study Semiquantitative latex agglutinationtest was strongly correlated with immunoturbidimetric assay
(Spearmanrsquos rho 119903 = 0946 119875 lt 0001) across a range ofVWF Ag levels from 345 to 870
Based on the results from kappa statistic and correlationstudy the scoring system for agglutination reactions and cor-relation with the VWF Ag levels (using immunoturbidimet-ric assay) is shown in Table 7 The proposed interpretationguide is also put in a footnote
4 Discussion
To the best of our knowledge there has been no reportedstudy or technical description available on the rapid test forVWF Ag utilising latex agglutination method At presentthe application of this method has not been used in clin-ical practice for monitoring of patients with low and highplasma levels of VWF Ag Screening of VWDpatients beforeconfirmation tests and monitoring of patients at high risk ofthrombotic event in the area with low-resource settings or atthe bedside can be performed if rapid test for VWF Ag isavailable Hence there is a need to develop a rapid and simplelatex agglutination test that can reliably detect and estimateVWF Ag in the plasma sample
Latex agglutination test is based on the observation ofvisible clumps that are formed from the reaction of antigen-antibody complexes The antigen-antibody complexes can beprepared by passive adsorption or covalent coupling methodIn the early stage of the present study attempts had beenmadeto couple VWF antibody with the carboxylated latex beads
8 Journal of Immunology Research
Table 7 Correlation of result interpretations by latex agglutination test and immunoturbidimetric assay
Qualitative latex agglutination test (LATscore)
Semiquantitative latex agglutination test(titre)
VWF Ag levels by immunoturbidimetricassay ()
1+ 1 2 lt202+ 1 4 1 8 20ndash503+ 1 16 1 32 50ndash1504+ ge64 gt150The proposed interpretation guide normal level of VWF Ag is indicated by LAT scores of 2+ and 3+ or titres from 1 8 to 1 32 Abnormal low VWF Ag isindicated by LAT scores of 0 1+ and 2+ or titres of 1 2 and 1 4 Abnormal high VWF Ag is indicated by LAT score of 4+ or titres of ge64LAT latex agglutination test VWF Ag von Willebrand factor antigen
by covalent coupling method The coupling procedure wasperformed in MES buffer at pH 60 according to the protocolfrom themanufacturer Unfortunately there was no antibodycoated on the latex beads (data not shown) However theVWF antibody was successfully adsorbed onto the surfaceof chloromethyl latex beads by passive adsorption methodThese findings were similar to the reported study by Garcia etal 2015 [35] In the present study amaximal physical adsorp-tion of the VWF antibody onto chloromethyl latex beads wasobtained at pH 74 a pH that is close to the isoelectric pointof polyclonal immunoglobulin (IgG) antibody [36 37]
Interference by RF in the latex agglutination test for thedetection of VWF Ag was expected RF is known to causeanalytical error in automated immunoturbidimetry assay thatpresented the main limitation of this method Despite thislimitation the findings do nevertheless show that the effect ofthe interference was less prominent in the sample with highlevels of VWF Ag compared to the sample with low levelsof VWF Ag The expected interference is without any doubtdue to the usage of whole IgG in the passive adsorptionmethod It iswell known that rheumatoid factor can bindwiththe Fc region of IgG causing false agglutination thatmay alterthe result of the test In the literature the usage of antibodyfragments such as Fab or F(ab1015840)
2was recommended to elim-
inate the interference from rheumatoid factor [37] Antibodyfragment is suggested in future study and optimization isrequired as the antibody fragment is more acidic than thepolyclonal antibody
In the present study the agglutination observed from thereaction between VWF antibody-latex reagent and positivesample may not be optimum as expected Experiments usinghigher initial concentration of VWF antibody with a moreintensive procedure to ensure maximal bead coating couldbe performed in the future study Not only that but theorientation of adsorbed antibody could also be studied toensure a proper alignment of antibody on latex beads Clariziaet al 2009 [38] have demonstrated a method for detectingthe aligned andmisaligned antibodies on the surface on latexbeads Due to some technical constraint maximal bead coat-ing cannot be ensured and a study on orientation of adsorbedantibody could not be performed Another limitation of thisstudy is that the size of the clumping was only estimatedby visual observation In future study it is recommendedto measure the clumping or macroscopic clusters usingmicroscope or other methods
There is a potential clinical application of this rapid latexagglutination test as a routine test in various premises includ-ing small laboratory and clinic and at the bedsideThe clinicalaspect of low levels of VWF Ag is well recognized but notmany medical personnel realized the implications of highVWF Ag levels in medical practice An inherited bleedingdisorder known as von Willebrand disease (VWD) is asso-ciated with an abnormality in VWF and type 1 VWD is thecommonest form that is associated with low VWF Ag [39]On the other hand high levels of VWF Ag are associatedwith thrombotic risks and had been extensively studied inmany medical conditions particularly coronary heart disease(CHD) and stroke It has been shown that CHD patients withhigh baseline of VWF Ag levels are at risk of acute vascularevents related to thrombosis [40] The current clinical guide-lines do not incorporate the measurement of VWF Ag inthe routine assessment of CHD patients but from variousstudies there is a role in monitoring the levels of VWF AgDetection of persistent high levels or increasing trends ofVWF Ag levels would alert the treating doctor for certainclinical measures to improve the patient care
Latex agglutination test for the detection of VWF Agwasfound to be simple to perform and the results can be obtainedwithin three minutes making it suitable to be used in areaswith limited laboratory facilities The result obtained bythe latex agglutination test is reliable as shown by the findingsfrom reproducibility stability linearity limit of detectionand method comparison studies Prozone effect was notdetected at VWF Ag levels up to 870 The stability of thereagents used for this method was up to 50 days and in thisstudy no stabilizer or preservatives were used The plasmafor VWF Ag estimation was stable for up to 21 days whichmay be related to the protein structure however more testsare needed to confirm the suitable plasma storage recom-mendation for this method All these findings showed theusefulness of this method in detecting and estimating highand low levels of VWF AgThis method might be useful inthe monitoring of VWF Ag in patients that are at a high riskof vascular event related to endothelial dysfunction Similarlythis method could be used to estimate VWF Ag levels inpatients with VWD before confirmation with standard testpanels in the specialized coagulation laboratory
However we only evaluate the latex agglutination testin quantitative terms by using plasma samples containinglow and high levels of VWF Ag This application mainlyaddressed the VWD type 1 which is more common in clinical
Journal of Immunology Research 9
practice In future study plasma samples with qualitativeVWF abnormalities (eg type II VWD or acquired VWDpatients demonstrating abnormal VWF function with vari-ables VWF Ag levels) could be included in the comparisonstudy to confirm the application of the latex agglutination testfor assessing VWF Ag levels in various clinical situations Insummary this test method needs to be refined and comparedwith other latex agglutination tests available in the marketbefore it is used on patient samples
5 Conclusion
Latex agglutination test for VWF Ag is simple rapid andreproducible correlates well with the reference method andis suitable to be used in areaswith limited laboratory facilitiesThe test has commercial potential as a low-cost alternativemethod for the detection and estimation of low levels andhigh levels of VWF Ag that could help in themanagement ofVWDtype 1 patients andpatients at risk of thrombotic eventsrespectively However the testmay not be suitable for patientswith a high level of rheumatoid factor
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by short-term Grant 304PPSP61312085 from Universiti Sains Malaysia Penang MalaysiaThe authors gratefully acknowledge Dr Emmanuel J Faval-oro from Institute of Clinical Pathology and MedicalResearch at Westmead Hospital Australia for his technicaladvice on this work Thanks are due to Dr Lim TheamSoon from Institute for Research in Molecular MedicineUniversiti Sains Malaysia Penang Malaysia for his valuableadvice in the early stage of this work The appreciation alsogoes to the staff of Haematology Department (especially thecoagulation section) BloodTransfusion ServiceDepartmentand Immunology Department Universiti Sains MalaysiaKelantan Malaysia for their continuous support in thiswork
References
[1] Z M Ruggeri ldquoStructure of vonWillebrand factor and its func-tion in platelet adhesion and thrombus formationrdquo Best Practiceand Research Clinical Haematology vol 14 no 2 pp 257ndash2792001
[2] G-P Luo B Ni X Yang and Y-ZWu ldquovonWillebrand factormore than a regulator of hemostasis and thrombosisrdquo ActaHaematologica vol 128 no 3 pp 158ndash169 2012
[3] C V Denis and P J Lenting ldquoVon Willebrand factor at thecrossroads of bleeding and thrombosisrdquo International Journal ofHematology vol 95 no 4 pp 353ndash361 2012
[4] A Alonso W Tang S K Agarwal E Z Soliman A M Cham-berlain and A R Folsom ldquoHemostatic markers are associatedwith the risk and prognosis of atrial fibrillation the ARIC
studyrdquo International Journal of Cardiology vol 155 no 2 pp217ndash222 2012
[5] F Cambronero J A Vilchez A Garcıa-Honrubia et al ldquoPlasmalevels of Von Willebrand factor are increased in patients withhypertrophic cardiomyopathyrdquo Thrombosis Research vol 126no 1 pp e46ndashe50 2010
[6] A M Kucharska-Newton D J Couper J S Pankow et alldquoHemostasis inflammation and fatal and nonfatal coronaryheart disease long-term follow-up of the atherosclerosis risk incommunities (ARIC) cohortrdquo Arteriosclerosis Thrombosis andVascular Biology vol 29 no 12 pp 2182ndash2190 2009
[7] R G Wieberdink M C van Schie P J Koudstaal et al ldquoHighvon Willebrand factor levels increase the risk of stroke theRotterdam studyrdquo Stroke vol 41 no 10 pp 2151ndash2156 2010
[8] P Wennberg F Wensley E Di Angelantonio et al ldquoHaemo-static and inflammatory markers are independently associatedwith myocardial infarction in men and womenrdquo ThrombosisResearch vol 129 no 1 pp 68ndash73 2012
[9] A Hyseni M Roest S L Braun et al ldquoChronic dysfunction ofthe endothelium is associated with mortality in acute coronarysyndrome patientsrdquoThrombosis Research vol 131 no 3 pp 198ndash203 2013
[10] A A Lopes A C Barreto N Y Maeda et al ldquoPlasma vonWillebrand factor as a predictor of survival in pulmonaryarterial hypertension associated with congenital heart diseaserdquoBrazilian Journal of Medical and Biological Research vol 44 no12 pp 1269ndash1275 2011
[11] V Roldan F Marın B Muina et al ldquoPlasma von Willebrandfactor levels are an independent risk factor for adverse eventsincluding mortality and major bleeding in anticoagulated atrialfibrillation patientsrdquo Journal of the American College of Cardiol-ogy vol 57 no 25 pp 2496ndash2504 2011
[12] T A Gaziano A Bitton S Anand S Abrahams-Gessel and AMurphy ldquoGrowing epidemic of coronary heart disease in low-andmiddle-income countriesrdquoCurrent Problems in Cardiologyvol 35 no 2 pp 72ndash115 2010
[13] V L Roger A S Go D M Lloyd-Jones et al ldquoExecutive sum-mary heart disease and stroke statisticsmdash2012 update a reportfrom the American Heart AssociationrdquoCirculation vol 125 no1 pp 188ndash197 2012
[14] G Castaman A Tosetto A Cappelletti et al ldquoValidation ofa rapid test (VWF-LIA) for the quantitative determination ofvonWillebrand factor antigen in type 1 vonWillebrand diseasediagnosis within the European multicenter study MCMDM-1VWDrdquoThrombosis Research vol 126 no 3 pp 227ndash231 2010
[15] A Kappel and M Ehm ldquoImmunoassays for diagnosis of coag-ulation disordersrdquo Hamostaseologie vol 30 no 4 pp 194ndash2012010
[16] R Biswas and S C Parija ldquoA rapid slide agglutination test forthe diagnosis of neurocysticercosis in the rural health set uprdquoTropical Parasitology vol 1 no 2 pp 94ndash98 2011
[17] H Lee Y Park M Kim et al ldquoDevelopment of a latex agglu-tination test for norovirus detectionrdquo Journal of Microbiologyvol 48 no 4 pp 419ndash425 2010
[18] H Y Darani F Ahmadi N Zabardast H A Yousefi and HShirzad ldquoDevelopment of a latex agglutination test as a simpleand rapid method for diagnosis of Trichomonas vaginalisinfectionrdquoAvicenna Journal of Medical Biotechnology vol 2 no1 pp 63ndash66 2010
[19] MMoraveji A Hosseini N Moghaddar M M Namavari andM H Eskandari ldquoDevelopment of latex agglutination test with
10 Journal of Immunology Research
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
aRheumatoid factor control serum fromMyBioSource USA (1030 IUmL) was used to spike plasma poolsRF rheumatoid factor VWF LAT von Willebrand factor latex agglutination test vol volume conc concentration
Table 6 Crosstabulation of VWF antigen levels and qualitative VWF by latex agglutination test results
Total 6 14 61 51 132VWF von Willebrand factor VWF Ag von Willebrand factor antigen LAT latex agglutination test
hemoglobin level of 03 06 15 3 6 and 12mgmL was thesame as the end-titre of low plasma pool without hemoglobininterference (1 4) These results showed that hemoglobindid not cause interference in latex agglutination test andsemiquantitative test result was not affected by hemoglobinlevel up to 12mgmL
Table 5 shows the end-titre of high and low plasma poolsspiked at different concentrations of RF As shown in thetable interference fromRF on latex agglutination test was notdetected in high plasma pools spiked with RF at concentra-tions up to 129 IUmLNevertheless the interference fromRFwas observed in low plasma pools spiked with RF at concen-trations of 26 52 103 and 129 IUmL The end-titre of highand low plasma pools without interference from RF was 1 64and 1 2 respectivelyThese results showed that RF interferedwith the latex agglutination test result when the test wasperformed on the plasma sample containing low levels ofVWF Ag and high levels of RF (gt26 IUmL) However latexagglutination test result of plasma sample containing highlevels of VWF Ag was not affected by RF at concentrationsof lt130 IUmL
325 Method Comparison with the Reference Method Com-parison study between qualitative latex agglutination testand immunoturbidimetric assay showed a kappa value of077 which indicates a substantial agreement between thetwo tests Table 6 shows the crosstabulation of 132 samplesincluded in this study Semiquantitative latex agglutinationtest was strongly correlated with immunoturbidimetric assay
(Spearmanrsquos rho 119903 = 0946 119875 lt 0001) across a range ofVWF Ag levels from 345 to 870
Based on the results from kappa statistic and correlationstudy the scoring system for agglutination reactions and cor-relation with the VWF Ag levels (using immunoturbidimet-ric assay) is shown in Table 7 The proposed interpretationguide is also put in a footnote
4 Discussion
To the best of our knowledge there has been no reportedstudy or technical description available on the rapid test forVWF Ag utilising latex agglutination method At presentthe application of this method has not been used in clin-ical practice for monitoring of patients with low and highplasma levels of VWF Ag Screening of VWDpatients beforeconfirmation tests and monitoring of patients at high risk ofthrombotic event in the area with low-resource settings or atthe bedside can be performed if rapid test for VWF Ag isavailable Hence there is a need to develop a rapid and simplelatex agglutination test that can reliably detect and estimateVWF Ag in the plasma sample
Latex agglutination test is based on the observation ofvisible clumps that are formed from the reaction of antigen-antibody complexes The antigen-antibody complexes can beprepared by passive adsorption or covalent coupling methodIn the early stage of the present study attempts had beenmadeto couple VWF antibody with the carboxylated latex beads
8 Journal of Immunology Research
Table 7 Correlation of result interpretations by latex agglutination test and immunoturbidimetric assay
Qualitative latex agglutination test (LATscore)
Semiquantitative latex agglutination test(titre)
VWF Ag levels by immunoturbidimetricassay ()
1+ 1 2 lt202+ 1 4 1 8 20ndash503+ 1 16 1 32 50ndash1504+ ge64 gt150The proposed interpretation guide normal level of VWF Ag is indicated by LAT scores of 2+ and 3+ or titres from 1 8 to 1 32 Abnormal low VWF Ag isindicated by LAT scores of 0 1+ and 2+ or titres of 1 2 and 1 4 Abnormal high VWF Ag is indicated by LAT score of 4+ or titres of ge64LAT latex agglutination test VWF Ag von Willebrand factor antigen
by covalent coupling method The coupling procedure wasperformed in MES buffer at pH 60 according to the protocolfrom themanufacturer Unfortunately there was no antibodycoated on the latex beads (data not shown) However theVWF antibody was successfully adsorbed onto the surfaceof chloromethyl latex beads by passive adsorption methodThese findings were similar to the reported study by Garcia etal 2015 [35] In the present study amaximal physical adsorp-tion of the VWF antibody onto chloromethyl latex beads wasobtained at pH 74 a pH that is close to the isoelectric pointof polyclonal immunoglobulin (IgG) antibody [36 37]
Interference by RF in the latex agglutination test for thedetection of VWF Ag was expected RF is known to causeanalytical error in automated immunoturbidimetry assay thatpresented the main limitation of this method Despite thislimitation the findings do nevertheless show that the effect ofthe interference was less prominent in the sample with highlevels of VWF Ag compared to the sample with low levelsof VWF Ag The expected interference is without any doubtdue to the usage of whole IgG in the passive adsorptionmethod It iswell known that rheumatoid factor can bindwiththe Fc region of IgG causing false agglutination thatmay alterthe result of the test In the literature the usage of antibodyfragments such as Fab or F(ab1015840)
2was recommended to elim-
inate the interference from rheumatoid factor [37] Antibodyfragment is suggested in future study and optimization isrequired as the antibody fragment is more acidic than thepolyclonal antibody
In the present study the agglutination observed from thereaction between VWF antibody-latex reagent and positivesample may not be optimum as expected Experiments usinghigher initial concentration of VWF antibody with a moreintensive procedure to ensure maximal bead coating couldbe performed in the future study Not only that but theorientation of adsorbed antibody could also be studied toensure a proper alignment of antibody on latex beads Clariziaet al 2009 [38] have demonstrated a method for detectingthe aligned andmisaligned antibodies on the surface on latexbeads Due to some technical constraint maximal bead coat-ing cannot be ensured and a study on orientation of adsorbedantibody could not be performed Another limitation of thisstudy is that the size of the clumping was only estimatedby visual observation In future study it is recommendedto measure the clumping or macroscopic clusters usingmicroscope or other methods
There is a potential clinical application of this rapid latexagglutination test as a routine test in various premises includ-ing small laboratory and clinic and at the bedsideThe clinicalaspect of low levels of VWF Ag is well recognized but notmany medical personnel realized the implications of highVWF Ag levels in medical practice An inherited bleedingdisorder known as von Willebrand disease (VWD) is asso-ciated with an abnormality in VWF and type 1 VWD is thecommonest form that is associated with low VWF Ag [39]On the other hand high levels of VWF Ag are associatedwith thrombotic risks and had been extensively studied inmany medical conditions particularly coronary heart disease(CHD) and stroke It has been shown that CHD patients withhigh baseline of VWF Ag levels are at risk of acute vascularevents related to thrombosis [40] The current clinical guide-lines do not incorporate the measurement of VWF Ag inthe routine assessment of CHD patients but from variousstudies there is a role in monitoring the levels of VWF AgDetection of persistent high levels or increasing trends ofVWF Ag levels would alert the treating doctor for certainclinical measures to improve the patient care
Latex agglutination test for the detection of VWF Agwasfound to be simple to perform and the results can be obtainedwithin three minutes making it suitable to be used in areaswith limited laboratory facilities The result obtained bythe latex agglutination test is reliable as shown by the findingsfrom reproducibility stability linearity limit of detectionand method comparison studies Prozone effect was notdetected at VWF Ag levels up to 870 The stability of thereagents used for this method was up to 50 days and in thisstudy no stabilizer or preservatives were used The plasmafor VWF Ag estimation was stable for up to 21 days whichmay be related to the protein structure however more testsare needed to confirm the suitable plasma storage recom-mendation for this method All these findings showed theusefulness of this method in detecting and estimating highand low levels of VWF AgThis method might be useful inthe monitoring of VWF Ag in patients that are at a high riskof vascular event related to endothelial dysfunction Similarlythis method could be used to estimate VWF Ag levels inpatients with VWD before confirmation with standard testpanels in the specialized coagulation laboratory
However we only evaluate the latex agglutination testin quantitative terms by using plasma samples containinglow and high levels of VWF Ag This application mainlyaddressed the VWD type 1 which is more common in clinical
Journal of Immunology Research 9
practice In future study plasma samples with qualitativeVWF abnormalities (eg type II VWD or acquired VWDpatients demonstrating abnormal VWF function with vari-ables VWF Ag levels) could be included in the comparisonstudy to confirm the application of the latex agglutination testfor assessing VWF Ag levels in various clinical situations Insummary this test method needs to be refined and comparedwith other latex agglutination tests available in the marketbefore it is used on patient samples
5 Conclusion
Latex agglutination test for VWF Ag is simple rapid andreproducible correlates well with the reference method andis suitable to be used in areaswith limited laboratory facilitiesThe test has commercial potential as a low-cost alternativemethod for the detection and estimation of low levels andhigh levels of VWF Ag that could help in themanagement ofVWDtype 1 patients andpatients at risk of thrombotic eventsrespectively However the testmay not be suitable for patientswith a high level of rheumatoid factor
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by short-term Grant 304PPSP61312085 from Universiti Sains Malaysia Penang MalaysiaThe authors gratefully acknowledge Dr Emmanuel J Faval-oro from Institute of Clinical Pathology and MedicalResearch at Westmead Hospital Australia for his technicaladvice on this work Thanks are due to Dr Lim TheamSoon from Institute for Research in Molecular MedicineUniversiti Sains Malaysia Penang Malaysia for his valuableadvice in the early stage of this work The appreciation alsogoes to the staff of Haematology Department (especially thecoagulation section) BloodTransfusion ServiceDepartmentand Immunology Department Universiti Sains MalaysiaKelantan Malaysia for their continuous support in thiswork
References
[1] Z M Ruggeri ldquoStructure of vonWillebrand factor and its func-tion in platelet adhesion and thrombus formationrdquo Best Practiceand Research Clinical Haematology vol 14 no 2 pp 257ndash2792001
[2] G-P Luo B Ni X Yang and Y-ZWu ldquovonWillebrand factormore than a regulator of hemostasis and thrombosisrdquo ActaHaematologica vol 128 no 3 pp 158ndash169 2012
[3] C V Denis and P J Lenting ldquoVon Willebrand factor at thecrossroads of bleeding and thrombosisrdquo International Journal ofHematology vol 95 no 4 pp 353ndash361 2012
[4] A Alonso W Tang S K Agarwal E Z Soliman A M Cham-berlain and A R Folsom ldquoHemostatic markers are associatedwith the risk and prognosis of atrial fibrillation the ARIC
studyrdquo International Journal of Cardiology vol 155 no 2 pp217ndash222 2012
[5] F Cambronero J A Vilchez A Garcıa-Honrubia et al ldquoPlasmalevels of Von Willebrand factor are increased in patients withhypertrophic cardiomyopathyrdquo Thrombosis Research vol 126no 1 pp e46ndashe50 2010
[6] A M Kucharska-Newton D J Couper J S Pankow et alldquoHemostasis inflammation and fatal and nonfatal coronaryheart disease long-term follow-up of the atherosclerosis risk incommunities (ARIC) cohortrdquo Arteriosclerosis Thrombosis andVascular Biology vol 29 no 12 pp 2182ndash2190 2009
[7] R G Wieberdink M C van Schie P J Koudstaal et al ldquoHighvon Willebrand factor levels increase the risk of stroke theRotterdam studyrdquo Stroke vol 41 no 10 pp 2151ndash2156 2010
[8] P Wennberg F Wensley E Di Angelantonio et al ldquoHaemo-static and inflammatory markers are independently associatedwith myocardial infarction in men and womenrdquo ThrombosisResearch vol 129 no 1 pp 68ndash73 2012
[9] A Hyseni M Roest S L Braun et al ldquoChronic dysfunction ofthe endothelium is associated with mortality in acute coronarysyndrome patientsrdquoThrombosis Research vol 131 no 3 pp 198ndash203 2013
[10] A A Lopes A C Barreto N Y Maeda et al ldquoPlasma vonWillebrand factor as a predictor of survival in pulmonaryarterial hypertension associated with congenital heart diseaserdquoBrazilian Journal of Medical and Biological Research vol 44 no12 pp 1269ndash1275 2011
[11] V Roldan F Marın B Muina et al ldquoPlasma von Willebrandfactor levels are an independent risk factor for adverse eventsincluding mortality and major bleeding in anticoagulated atrialfibrillation patientsrdquo Journal of the American College of Cardiol-ogy vol 57 no 25 pp 2496ndash2504 2011
[12] T A Gaziano A Bitton S Anand S Abrahams-Gessel and AMurphy ldquoGrowing epidemic of coronary heart disease in low-andmiddle-income countriesrdquoCurrent Problems in Cardiologyvol 35 no 2 pp 72ndash115 2010
[13] V L Roger A S Go D M Lloyd-Jones et al ldquoExecutive sum-mary heart disease and stroke statisticsmdash2012 update a reportfrom the American Heart AssociationrdquoCirculation vol 125 no1 pp 188ndash197 2012
[14] G Castaman A Tosetto A Cappelletti et al ldquoValidation ofa rapid test (VWF-LIA) for the quantitative determination ofvonWillebrand factor antigen in type 1 vonWillebrand diseasediagnosis within the European multicenter study MCMDM-1VWDrdquoThrombosis Research vol 126 no 3 pp 227ndash231 2010
[15] A Kappel and M Ehm ldquoImmunoassays for diagnosis of coag-ulation disordersrdquo Hamostaseologie vol 30 no 4 pp 194ndash2012010
[16] R Biswas and S C Parija ldquoA rapid slide agglutination test forthe diagnosis of neurocysticercosis in the rural health set uprdquoTropical Parasitology vol 1 no 2 pp 94ndash98 2011
[17] H Lee Y Park M Kim et al ldquoDevelopment of a latex agglu-tination test for norovirus detectionrdquo Journal of Microbiologyvol 48 no 4 pp 419ndash425 2010
[18] H Y Darani F Ahmadi N Zabardast H A Yousefi and HShirzad ldquoDevelopment of a latex agglutination test as a simpleand rapid method for diagnosis of Trichomonas vaginalisinfectionrdquoAvicenna Journal of Medical Biotechnology vol 2 no1 pp 63ndash66 2010
[19] MMoraveji A Hosseini N Moghaddar M M Namavari andM H Eskandari ldquoDevelopment of latex agglutination test with
10 Journal of Immunology Research
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
Table 7 Correlation of result interpretations by latex agglutination test and immunoturbidimetric assay
Qualitative latex agglutination test (LATscore)
Semiquantitative latex agglutination test(titre)
VWF Ag levels by immunoturbidimetricassay ()
1+ 1 2 lt202+ 1 4 1 8 20ndash503+ 1 16 1 32 50ndash1504+ ge64 gt150The proposed interpretation guide normal level of VWF Ag is indicated by LAT scores of 2+ and 3+ or titres from 1 8 to 1 32 Abnormal low VWF Ag isindicated by LAT scores of 0 1+ and 2+ or titres of 1 2 and 1 4 Abnormal high VWF Ag is indicated by LAT score of 4+ or titres of ge64LAT latex agglutination test VWF Ag von Willebrand factor antigen
by covalent coupling method The coupling procedure wasperformed in MES buffer at pH 60 according to the protocolfrom themanufacturer Unfortunately there was no antibodycoated on the latex beads (data not shown) However theVWF antibody was successfully adsorbed onto the surfaceof chloromethyl latex beads by passive adsorption methodThese findings were similar to the reported study by Garcia etal 2015 [35] In the present study amaximal physical adsorp-tion of the VWF antibody onto chloromethyl latex beads wasobtained at pH 74 a pH that is close to the isoelectric pointof polyclonal immunoglobulin (IgG) antibody [36 37]
Interference by RF in the latex agglutination test for thedetection of VWF Ag was expected RF is known to causeanalytical error in automated immunoturbidimetry assay thatpresented the main limitation of this method Despite thislimitation the findings do nevertheless show that the effect ofthe interference was less prominent in the sample with highlevels of VWF Ag compared to the sample with low levelsof VWF Ag The expected interference is without any doubtdue to the usage of whole IgG in the passive adsorptionmethod It iswell known that rheumatoid factor can bindwiththe Fc region of IgG causing false agglutination thatmay alterthe result of the test In the literature the usage of antibodyfragments such as Fab or F(ab1015840)
2was recommended to elim-
inate the interference from rheumatoid factor [37] Antibodyfragment is suggested in future study and optimization isrequired as the antibody fragment is more acidic than thepolyclonal antibody
In the present study the agglutination observed from thereaction between VWF antibody-latex reagent and positivesample may not be optimum as expected Experiments usinghigher initial concentration of VWF antibody with a moreintensive procedure to ensure maximal bead coating couldbe performed in the future study Not only that but theorientation of adsorbed antibody could also be studied toensure a proper alignment of antibody on latex beads Clariziaet al 2009 [38] have demonstrated a method for detectingthe aligned andmisaligned antibodies on the surface on latexbeads Due to some technical constraint maximal bead coat-ing cannot be ensured and a study on orientation of adsorbedantibody could not be performed Another limitation of thisstudy is that the size of the clumping was only estimatedby visual observation In future study it is recommendedto measure the clumping or macroscopic clusters usingmicroscope or other methods
There is a potential clinical application of this rapid latexagglutination test as a routine test in various premises includ-ing small laboratory and clinic and at the bedsideThe clinicalaspect of low levels of VWF Ag is well recognized but notmany medical personnel realized the implications of highVWF Ag levels in medical practice An inherited bleedingdisorder known as von Willebrand disease (VWD) is asso-ciated with an abnormality in VWF and type 1 VWD is thecommonest form that is associated with low VWF Ag [39]On the other hand high levels of VWF Ag are associatedwith thrombotic risks and had been extensively studied inmany medical conditions particularly coronary heart disease(CHD) and stroke It has been shown that CHD patients withhigh baseline of VWF Ag levels are at risk of acute vascularevents related to thrombosis [40] The current clinical guide-lines do not incorporate the measurement of VWF Ag inthe routine assessment of CHD patients but from variousstudies there is a role in monitoring the levels of VWF AgDetection of persistent high levels or increasing trends ofVWF Ag levels would alert the treating doctor for certainclinical measures to improve the patient care
Latex agglutination test for the detection of VWF Agwasfound to be simple to perform and the results can be obtainedwithin three minutes making it suitable to be used in areaswith limited laboratory facilities The result obtained bythe latex agglutination test is reliable as shown by the findingsfrom reproducibility stability linearity limit of detectionand method comparison studies Prozone effect was notdetected at VWF Ag levels up to 870 The stability of thereagents used for this method was up to 50 days and in thisstudy no stabilizer or preservatives were used The plasmafor VWF Ag estimation was stable for up to 21 days whichmay be related to the protein structure however more testsare needed to confirm the suitable plasma storage recom-mendation for this method All these findings showed theusefulness of this method in detecting and estimating highand low levels of VWF AgThis method might be useful inthe monitoring of VWF Ag in patients that are at a high riskof vascular event related to endothelial dysfunction Similarlythis method could be used to estimate VWF Ag levels inpatients with VWD before confirmation with standard testpanels in the specialized coagulation laboratory
However we only evaluate the latex agglutination testin quantitative terms by using plasma samples containinglow and high levels of VWF Ag This application mainlyaddressed the VWD type 1 which is more common in clinical
Journal of Immunology Research 9
practice In future study plasma samples with qualitativeVWF abnormalities (eg type II VWD or acquired VWDpatients demonstrating abnormal VWF function with vari-ables VWF Ag levels) could be included in the comparisonstudy to confirm the application of the latex agglutination testfor assessing VWF Ag levels in various clinical situations Insummary this test method needs to be refined and comparedwith other latex agglutination tests available in the marketbefore it is used on patient samples
5 Conclusion
Latex agglutination test for VWF Ag is simple rapid andreproducible correlates well with the reference method andis suitable to be used in areaswith limited laboratory facilitiesThe test has commercial potential as a low-cost alternativemethod for the detection and estimation of low levels andhigh levels of VWF Ag that could help in themanagement ofVWDtype 1 patients andpatients at risk of thrombotic eventsrespectively However the testmay not be suitable for patientswith a high level of rheumatoid factor
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by short-term Grant 304PPSP61312085 from Universiti Sains Malaysia Penang MalaysiaThe authors gratefully acknowledge Dr Emmanuel J Faval-oro from Institute of Clinical Pathology and MedicalResearch at Westmead Hospital Australia for his technicaladvice on this work Thanks are due to Dr Lim TheamSoon from Institute for Research in Molecular MedicineUniversiti Sains Malaysia Penang Malaysia for his valuableadvice in the early stage of this work The appreciation alsogoes to the staff of Haematology Department (especially thecoagulation section) BloodTransfusion ServiceDepartmentand Immunology Department Universiti Sains MalaysiaKelantan Malaysia for their continuous support in thiswork
References
[1] Z M Ruggeri ldquoStructure of vonWillebrand factor and its func-tion in platelet adhesion and thrombus formationrdquo Best Practiceand Research Clinical Haematology vol 14 no 2 pp 257ndash2792001
[2] G-P Luo B Ni X Yang and Y-ZWu ldquovonWillebrand factormore than a regulator of hemostasis and thrombosisrdquo ActaHaematologica vol 128 no 3 pp 158ndash169 2012
[3] C V Denis and P J Lenting ldquoVon Willebrand factor at thecrossroads of bleeding and thrombosisrdquo International Journal ofHematology vol 95 no 4 pp 353ndash361 2012
[4] A Alonso W Tang S K Agarwal E Z Soliman A M Cham-berlain and A R Folsom ldquoHemostatic markers are associatedwith the risk and prognosis of atrial fibrillation the ARIC
studyrdquo International Journal of Cardiology vol 155 no 2 pp217ndash222 2012
[5] F Cambronero J A Vilchez A Garcıa-Honrubia et al ldquoPlasmalevels of Von Willebrand factor are increased in patients withhypertrophic cardiomyopathyrdquo Thrombosis Research vol 126no 1 pp e46ndashe50 2010
[6] A M Kucharska-Newton D J Couper J S Pankow et alldquoHemostasis inflammation and fatal and nonfatal coronaryheart disease long-term follow-up of the atherosclerosis risk incommunities (ARIC) cohortrdquo Arteriosclerosis Thrombosis andVascular Biology vol 29 no 12 pp 2182ndash2190 2009
[7] R G Wieberdink M C van Schie P J Koudstaal et al ldquoHighvon Willebrand factor levels increase the risk of stroke theRotterdam studyrdquo Stroke vol 41 no 10 pp 2151ndash2156 2010
[8] P Wennberg F Wensley E Di Angelantonio et al ldquoHaemo-static and inflammatory markers are independently associatedwith myocardial infarction in men and womenrdquo ThrombosisResearch vol 129 no 1 pp 68ndash73 2012
[9] A Hyseni M Roest S L Braun et al ldquoChronic dysfunction ofthe endothelium is associated with mortality in acute coronarysyndrome patientsrdquoThrombosis Research vol 131 no 3 pp 198ndash203 2013
[10] A A Lopes A C Barreto N Y Maeda et al ldquoPlasma vonWillebrand factor as a predictor of survival in pulmonaryarterial hypertension associated with congenital heart diseaserdquoBrazilian Journal of Medical and Biological Research vol 44 no12 pp 1269ndash1275 2011
[11] V Roldan F Marın B Muina et al ldquoPlasma von Willebrandfactor levels are an independent risk factor for adverse eventsincluding mortality and major bleeding in anticoagulated atrialfibrillation patientsrdquo Journal of the American College of Cardiol-ogy vol 57 no 25 pp 2496ndash2504 2011
[12] T A Gaziano A Bitton S Anand S Abrahams-Gessel and AMurphy ldquoGrowing epidemic of coronary heart disease in low-andmiddle-income countriesrdquoCurrent Problems in Cardiologyvol 35 no 2 pp 72ndash115 2010
[13] V L Roger A S Go D M Lloyd-Jones et al ldquoExecutive sum-mary heart disease and stroke statisticsmdash2012 update a reportfrom the American Heart AssociationrdquoCirculation vol 125 no1 pp 188ndash197 2012
[14] G Castaman A Tosetto A Cappelletti et al ldquoValidation ofa rapid test (VWF-LIA) for the quantitative determination ofvonWillebrand factor antigen in type 1 vonWillebrand diseasediagnosis within the European multicenter study MCMDM-1VWDrdquoThrombosis Research vol 126 no 3 pp 227ndash231 2010
[15] A Kappel and M Ehm ldquoImmunoassays for diagnosis of coag-ulation disordersrdquo Hamostaseologie vol 30 no 4 pp 194ndash2012010
[16] R Biswas and S C Parija ldquoA rapid slide agglutination test forthe diagnosis of neurocysticercosis in the rural health set uprdquoTropical Parasitology vol 1 no 2 pp 94ndash98 2011
[17] H Lee Y Park M Kim et al ldquoDevelopment of a latex agglu-tination test for norovirus detectionrdquo Journal of Microbiologyvol 48 no 4 pp 419ndash425 2010
[18] H Y Darani F Ahmadi N Zabardast H A Yousefi and HShirzad ldquoDevelopment of a latex agglutination test as a simpleand rapid method for diagnosis of Trichomonas vaginalisinfectionrdquoAvicenna Journal of Medical Biotechnology vol 2 no1 pp 63ndash66 2010
[19] MMoraveji A Hosseini N Moghaddar M M Namavari andM H Eskandari ldquoDevelopment of latex agglutination test with
10 Journal of Immunology Research
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
practice In future study plasma samples with qualitativeVWF abnormalities (eg type II VWD or acquired VWDpatients demonstrating abnormal VWF function with vari-ables VWF Ag levels) could be included in the comparisonstudy to confirm the application of the latex agglutination testfor assessing VWF Ag levels in various clinical situations Insummary this test method needs to be refined and comparedwith other latex agglutination tests available in the marketbefore it is used on patient samples
5 Conclusion
Latex agglutination test for VWF Ag is simple rapid andreproducible correlates well with the reference method andis suitable to be used in areaswith limited laboratory facilitiesThe test has commercial potential as a low-cost alternativemethod for the detection and estimation of low levels andhigh levels of VWF Ag that could help in themanagement ofVWDtype 1 patients andpatients at risk of thrombotic eventsrespectively However the testmay not be suitable for patientswith a high level of rheumatoid factor
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by short-term Grant 304PPSP61312085 from Universiti Sains Malaysia Penang MalaysiaThe authors gratefully acknowledge Dr Emmanuel J Faval-oro from Institute of Clinical Pathology and MedicalResearch at Westmead Hospital Australia for his technicaladvice on this work Thanks are due to Dr Lim TheamSoon from Institute for Research in Molecular MedicineUniversiti Sains Malaysia Penang Malaysia for his valuableadvice in the early stage of this work The appreciation alsogoes to the staff of Haematology Department (especially thecoagulation section) BloodTransfusion ServiceDepartmentand Immunology Department Universiti Sains MalaysiaKelantan Malaysia for their continuous support in thiswork
References
[1] Z M Ruggeri ldquoStructure of vonWillebrand factor and its func-tion in platelet adhesion and thrombus formationrdquo Best Practiceand Research Clinical Haematology vol 14 no 2 pp 257ndash2792001
[2] G-P Luo B Ni X Yang and Y-ZWu ldquovonWillebrand factormore than a regulator of hemostasis and thrombosisrdquo ActaHaematologica vol 128 no 3 pp 158ndash169 2012
[3] C V Denis and P J Lenting ldquoVon Willebrand factor at thecrossroads of bleeding and thrombosisrdquo International Journal ofHematology vol 95 no 4 pp 353ndash361 2012
[4] A Alonso W Tang S K Agarwal E Z Soliman A M Cham-berlain and A R Folsom ldquoHemostatic markers are associatedwith the risk and prognosis of atrial fibrillation the ARIC
studyrdquo International Journal of Cardiology vol 155 no 2 pp217ndash222 2012
[5] F Cambronero J A Vilchez A Garcıa-Honrubia et al ldquoPlasmalevels of Von Willebrand factor are increased in patients withhypertrophic cardiomyopathyrdquo Thrombosis Research vol 126no 1 pp e46ndashe50 2010
[6] A M Kucharska-Newton D J Couper J S Pankow et alldquoHemostasis inflammation and fatal and nonfatal coronaryheart disease long-term follow-up of the atherosclerosis risk incommunities (ARIC) cohortrdquo Arteriosclerosis Thrombosis andVascular Biology vol 29 no 12 pp 2182ndash2190 2009
[7] R G Wieberdink M C van Schie P J Koudstaal et al ldquoHighvon Willebrand factor levels increase the risk of stroke theRotterdam studyrdquo Stroke vol 41 no 10 pp 2151ndash2156 2010
[8] P Wennberg F Wensley E Di Angelantonio et al ldquoHaemo-static and inflammatory markers are independently associatedwith myocardial infarction in men and womenrdquo ThrombosisResearch vol 129 no 1 pp 68ndash73 2012
[9] A Hyseni M Roest S L Braun et al ldquoChronic dysfunction ofthe endothelium is associated with mortality in acute coronarysyndrome patientsrdquoThrombosis Research vol 131 no 3 pp 198ndash203 2013
[10] A A Lopes A C Barreto N Y Maeda et al ldquoPlasma vonWillebrand factor as a predictor of survival in pulmonaryarterial hypertension associated with congenital heart diseaserdquoBrazilian Journal of Medical and Biological Research vol 44 no12 pp 1269ndash1275 2011
[11] V Roldan F Marın B Muina et al ldquoPlasma von Willebrandfactor levels are an independent risk factor for adverse eventsincluding mortality and major bleeding in anticoagulated atrialfibrillation patientsrdquo Journal of the American College of Cardiol-ogy vol 57 no 25 pp 2496ndash2504 2011
[12] T A Gaziano A Bitton S Anand S Abrahams-Gessel and AMurphy ldquoGrowing epidemic of coronary heart disease in low-andmiddle-income countriesrdquoCurrent Problems in Cardiologyvol 35 no 2 pp 72ndash115 2010
[13] V L Roger A S Go D M Lloyd-Jones et al ldquoExecutive sum-mary heart disease and stroke statisticsmdash2012 update a reportfrom the American Heart AssociationrdquoCirculation vol 125 no1 pp 188ndash197 2012
[14] G Castaman A Tosetto A Cappelletti et al ldquoValidation ofa rapid test (VWF-LIA) for the quantitative determination ofvonWillebrand factor antigen in type 1 vonWillebrand diseasediagnosis within the European multicenter study MCMDM-1VWDrdquoThrombosis Research vol 126 no 3 pp 227ndash231 2010
[15] A Kappel and M Ehm ldquoImmunoassays for diagnosis of coag-ulation disordersrdquo Hamostaseologie vol 30 no 4 pp 194ndash2012010
[16] R Biswas and S C Parija ldquoA rapid slide agglutination test forthe diagnosis of neurocysticercosis in the rural health set uprdquoTropical Parasitology vol 1 no 2 pp 94ndash98 2011
[17] H Lee Y Park M Kim et al ldquoDevelopment of a latex agglu-tination test for norovirus detectionrdquo Journal of Microbiologyvol 48 no 4 pp 419ndash425 2010
[18] H Y Darani F Ahmadi N Zabardast H A Yousefi and HShirzad ldquoDevelopment of a latex agglutination test as a simpleand rapid method for diagnosis of Trichomonas vaginalisinfectionrdquoAvicenna Journal of Medical Biotechnology vol 2 no1 pp 63ndash66 2010
[19] MMoraveji A Hosseini N Moghaddar M M Namavari andM H Eskandari ldquoDevelopment of latex agglutination test with
10 Journal of Immunology Research
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
recombinant NcSAG1 for the rapid detection of antibodies toNeospora caninum in cattlerdquo Veterinary Parasitology vol 189no 2ndash4 pp 211ndash217 2012
[20] K P Shyma S K Gupta A Singh S S Chaudhary and JGupta ldquoMonoclonal antibody based latex agglutination test forthe diagnosis of trypanosomosis in cattlerdquo Journal of AdvancedVeterinary Research vol 2 no 1ndash4 pp 1ndash4 2012
[21] J A Molina-Bolıvar and F Galisteo-Gonzalez ldquoLatex immuno-agglutination assaysrdquo Journal of Macromolecular SciencemdashPoly-mer Reviews vol 45 no 1 pp 59ndash98 2005
[22] C M Plotz and J M Singer ldquoThe latex fixation test I Appli-cation to the serologic diagnosis of rheumatoid arthritisrdquo TheAmerican Journal of Medicine vol 21 no 6 pp 888ndash892 1956
[23] P-L Lim and Y-P Fok ldquoDetection of group D Salmonellae inblood culture broth and of soluble antigen by tube agglutinationusing an O-9 monoclonal antibody latex conjugaterdquo Journal ofClinical Microbiology vol 25 no 7 pp 1165ndash1168 1987
[24] M Sarikaputi M Morimatsu S Yamamoto B Syuto M Saitoand M Naiki ldquoLatex agglutination test a simple rapid andpractical method for bovine serum CRP determinationrdquo TheJapanese Journal of Veterinary Research vol 40 no 1-2 pp 1ndash12 1992
[25] S Yamamoto K Tagata Y Ishikawa et al ldquoPreparation of latexsensitized with rabbit IgG antibody for slide reversed passiveagglutinationrdquoVeterinary Research Communications vol 16 no4 pp 265ndash272 1992
[26] K L Yap ldquoDevelopment of a slide latex agglutination test forrotavirus antigen detectionrdquo The Malaysian Journal of Pathol-ogy vol 16 no 1 pp 49ndash56 1994
[27] S M Rocha L A Suzuki A D T da Silva G C Arruda andC L Rossi ldquoA rapid latex agglutination test for the detectionof anti-cysticercus antibodies in cerebrospinal fluid (CSF)rdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 44no 1 pp 57ndash58 2002
[28] V Lejon D Legros M Richer et al ldquoIgM quantification in thecerebrospinal fluid of sleeping sickness patients by a latex cardagglutination testrdquo Tropical Medicine and International Healthvol 7 no 8 pp 685ndash692 2002
[29] J Chen M Jin Z Yu et al ldquoA Latex agglutination test for therapid detection of avian influenza virus subtype H5N1 and itsclinical applicationrdquo Journal of Veterinary Diagnostic Investiga-tion vol 19 no 2 pp 155ndash160 2007
[30] A Mina E J Favaloro and J Koutts ldquoA novel flow cytometrysingle tube bead assay for quantitation of vonWillebrand factorantigen and collagen-bindingrdquo Thrombosis and Haemostasisvol 108 no 5 pp 999ndash1005 2012
[31] G Dimeski ldquoInterference testingrdquo The Clinical BiochemistReviews vol 29 supplement 1 pp S43ndashS48 2008
[32] J Tate and G Ward ldquoInterferences in Immunoassayrdquo TheClinical Biochemist Reviews vol 25 no 2 pp 105ndash120 2004
[33] A J Viera and J M Garrett ldquoUnderstanding interobserveragreement the kappa statisticrdquo Family Medicine vol 37 no 5pp 360ndash363 2005
[34] Y H Chan ldquoBiostatistics 104 correlational Analysisrdquo SingaporeMedical Journal vol 44 no 12 pp 614ndash619 2003
[35] V S Garcia V D G Gonzalez P C Caudana J R Vega IS Marcipar and L M Gugliotta ldquoSynthesis of latex-antigencomplexes from single and multiepitope recombinant proteinsApplication in immunoagglutination assays for the diagnosisof Trypanosoma cruzi infectionrdquo Colloids and Surfaces BBiointerfaces vol 101 pp 384ndash391 2013
[36] J L Ortega-Vinuesa R Hidalgo-Alvarez F J De Las NievesC L Davey D J Newman and C P Price ldquoCharacterizationof immunoglobulin G bound to latex particles using surfaceplasmon resonance and electrophoretic mobilityrdquo Journal ofColloid and Interface Science vol 204 no 2 pp 300ndash311 1998
[37] J L Ortega-Vinuesa and D Bastos-Gonzalez ldquoA review offactors affecting the performances of latex agglutination testsrdquoJournal of Biomaterials Science vol 12 no 4 pp 379ndash408 2001
[38] L-J A Clarizia D Sok M Wei J Mead C Barry andM J McDonald ldquoAntibody orientation enhanced by selectivepolymer-protein noncovalent interactionsrdquo Analytical and Bio-analytical Chemistry vol 393 no 5 pp 1531ndash1538 2009
[39] D Lillicrap ldquoVonWillebrand disease advances in pathogeneticunderstanding diagnosis and therapyrdquo Blood vol 122 no 23pp 3735ndash3740 2013
[40] P E Morange C Simon M C Alessi et al ldquoEndothelial cellmarkers and the risk of coronary heart disease the Prospec-tive Epidemiological Study of Myocardial Infarction (PRIME)studyrdquo Circulation vol 109 no 11 pp 1343ndash1348 2004
