eJIFCC Vol 23 n° 4. 2012 Editor-in-Chief: Gábor L. Kovács, · Béla Nagy Jr, Ildikó Beke...

Aims and Scope

The Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (eJIFCC) is an online journal, published four times a year, on the web site of the IFCC.

The peer-reviewed original articles, posters, case studies and reviews, are focused on the needs of clinical laboratorians worldwide. In addition to the peer-reviewed content, there are also occasional editorials with point-ers to quality resources on the Web.

Also the journal publishes some IFCC news,letters, reviews of books, de-bates and educational material to assist the development of the field of clinical chemistry and laboratory medicine worldwide

eJIFCC Vol 23 n° 4. 2012

Publisher: IFCC

The JOURNAL OF THE INTERNATIONAL FEDERATION OF CLINICAL CHEMISTRY (eJIFCC) is an electronic journal with fre-quent updates on its home page. Our articles, debates, reviews and editorials are addressed to clinical laboratorians. Besides offering original scientific thought in our featured columns, we provide pointers to quality resources on the World Wide Web. The journal will publish general news articles, IFCC publicity/news, educational material and have a letters section.

FLOW CYTOMETRY IN THE CLINICAL LABORATORY, János Kappelmayer

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FOREWORD OF THE EDITOR, Gábor L. Kovács

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FLOW CYTOMETRY IN THE DIAGNOSIS OF MYELODYSPLASTIC SYNDROMES, Bettina Kárai, Eszter Szánthó, János Kappelmayer, Zsuzsa Hevessy

Pages 5-11

PREDICTION OF THERAPY RESPONSE AND PROGNOSIS IN LEUKEMIAS BY FLOW CYTOMETRIC MDR ASSAYS, János Kappelmayer, Zsuzsa Hevessy, András Apjok, Katalin Tauberné Jakab

Pages 12-18

FLOW CYTOMETRIC INVESTIGATION OF CLASSICAL AND ALTERNATIVE PLATELET ACTIVATION MARKERS,Béla Nagy Jr, Ildikó Beke Debreceni, János Kappelmayer

Pages 19-29

MEASUREMENT OF SOLUBLE BIOMARKERS BY FLOW CYTOMETRY,Péter Antal-Szalmás, Béla Nagy Jr, Ildikó Beke Debreceni, János Kappelmayer

Pages 30-37

CALCIUM INFLUX CHARACTERISTICS DURING T LYMPHOCYTE ACTIVATION MEASURED WITH FLOW CYTOMETRY,Enikő Biró, Barna Vásárhelyi, Gergely Toldi

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ADVANCES IN ORAL COAGULANTS,Eleanor S. Pollak

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Editor-in-Chief: Gábor L. Kovács,e-mail:[email protected]

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FLOW CYTOMETRY IN THE CLINICAL LABORATORY

Guest editorJános Kappelmayer MD. PhD. DSc. headDepartment of Laboratory Medicine, University of Debrecen, Hungary

This issue of the eJIFCC is dedicated to highlight some aspects of clinical flow cytometry. In the past 50 years several techniqueshave revolutionized laboratory medicine. Undoubtedly flow cytometry is one of those, with a substantial impact on diagnosingand monitoring diseases in laboratory hematology, hemostasis and immunology. The development in flow technology alsoexerted a considerable effect on the accuracy of testing, as well as on turnaround times and on the objectivity of the reporteddata. Although morphological investigation of peripheral blood and bone marrow smears has remained a gold-standard indiagnosing malignant hematological disorders, recently flow cytometric studies are an absolute requirement in finalizing thediagnosis of de novo leukemias, while in other areas, like minimal residual disease detection it completely replaced morphologyand has become a technique that can reliably identify 1 leukemic cell in 10,000 normal cells. Platelet glycoprotein abnormalities,reticulated platelets, as well as activated platelets are all diagnosed today by flow cytometric techniques. For certain red bloodcell disorders like paroxysmal nocturnal hemoglobinuria and hereditary spherocytosis, flow cytometry is a key technique. Thismethod is required also in the diagnosis of immunodeficencies, autoimmune disorders, antigen specific T-cell responses,allergy-testing and is utilized in transplantation immunology.The wide repertoire of these diagnostic applications was made possible partly by the large arsenal of probes – e.g. directlyconjugated monoclonal antibodies, fluorescent probes for cell function and viability – as well as by the constant improvementof the flow cytometers. Today, CE labeled benchtop analysers are routinely equipped with2-3 lasers and can provide 8-10 color labelings with a high event rate per second, thus enables the acquisition of 0.5-1 millioncells in a reasonable time frame. Along with these developments in hardware and reagent supply, new softwares have beendeveloped. Thus, cytometrists can analyse and provide interpretative report for a large number of clinical samples in a relativelyshort time, not mechanically reporting percent positivities for individual CD markers, but by describing only key phenotypicfindings and corrrelating staining patterns to diseases.Clinical research and laboratory diagnostics can not always be sharply separated in flow cytometry. What is regarded todayas a research tool can soon turn to a diagnostic assay.

This issue of eJIFCC provides some examples for the versatility of this technology. The first paper describes the state of the artmulticolor flow analysis of myelodysplasia. The second publication is on a functional assay to identify the multidrug resistantphenotype in hematological malignancies. Cardiovascular disorders like myocardial infarction and stroke are exemplified bythe presence of activated platelets. The third article depicts the possibilities to identify activated platelets by flow cytometry.The fourth paper deals with a different approach, where we are analysing soluble plasma proteins by a flow technology thatuses either beads or cells as a reagent. Finally the analysis of intracellular calcium as a cell signalling event as well as a potentialdisease marker is described by flow cytometric methods.I am fascinated how, this ever-growing technology influenced our daily work in the past decades and I am sure that at leasttwo different directions of future developments will prevail. Most likely many assays will be applied to smaller scaleequipments, that will be affordable by more and more laboratories while on the other hand, frontline applied research willgenerate diagnostic tests in many areas of cell biology – apoptosis studies, phosphoprotein and cell cycle analysis - that todayare carried out mostly only in research applications.

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JÁNOS KAPPELMAYER FLOW CYTOMETRY IN THE CLINICAL LABORATORY

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The Journal of the International Federation of Clinical Chemistryand Laboratory Medicine

In this issue: FLOW CYTOMETRY IN THE CLINICAL LABORATORY

1Janos

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KOVÁCS L. GÁBOR FOREWORD OF THE EDITOR

FOREWORD OF THE EDITOR

Janos Kappelmayer was born in Debrecen, Hungary in 1960. In 1985 he received his medical degree from the University ofDebrecen with "summa cum laude". After his residency program in clinical pathology, he obtained his board certification in1989. He received a second board certification in laboratory hematology and immunology in 2003. He defended the PhD thesisin 1994 and D.Sc. degree in 2008. His scientific interest is in laboratory diagnostics, hematology, and thrombosis research. Since2004, he is the director of the Institute of Laboratory Medicine at the University of Debrecen. He spent two postdoctoral yearsat The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia (1990-92) and one year atthe Cardiovascular Biology Program, Oklahoma Medical Research Foundation as a Greenberg Scholar (2001). Dr. Kappelmayerhas several ongoing international research collaborations, e.g. with the Department of Medicine and Biochemistry at theOklahoma Health Sciences Center and the Cytometry Services Department of Hematology, University of Salamanca, Spain. Hewas the tutor of 4 graduated PhD students in the areas of flow cytometry, leukemia diagnostics and thrombosis research. Hewas invited speaker at several international meetings, e.g. Thrombosis research (ISTH, SSC meetings): Venice 2004, Oslo 2006;Flow cytometry: Odense 1999, Antalya 2010; Laboratory medicine: Belgrade 2003, Berlin 2011, Istanbul 2014. He published 143original papers, 93 of them in highly ranked international journals. His cumulative impact factor is 290, with 1540 independentcitations in the literature. In 2009-2011 he was the president of the Hungarian Society of Laboratory Medicine and a boardmember of the European Society of Clinical Cell Analysis (ESCCA). Since 2003, he is the treasurer of the Hungarian Society ofThrombosis and Hemostasis.

Kovács L. Gábor MD, PhD, DSc.eJournal IFCC Editor-in-chief

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21KOvacs

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FLOW CYTOMETRY IN THE DIAGNOSIS OF MYELODYSPLASTIC SYNDROMES

Bettina Kárai, Eszter Szánthó, János Kappelmayer, Zsuzsa HevessyDepartment of Laboratory Medicine, University of Debrecen, Debrecen H-4032, Hungary

Corresponding Author:

Bettina KáraiDepartment of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98,Debrecen H-4032, HungaryTel: +36 30 386 2672Fax: +36 52 417 631e-mail: [email protected]

Key words: myelodysplastic syndromes, flow cytometry, classification system, prognostic scoring system

ABSTRACT

Myelodysplastic syndromes are clonal hematopoietic stem cell disorders. Their exact etiology is unknown. Myelodysplasticsyndromes cause progressive bone marrow failure resulting in pancytopenia and refractory, transfusion-dependent anemia.One can observe typical morphological alterations in the erythroid, myeloid and/or megakaryocytic cell lineage. Blast countsmay also be increased. The pathologic cells are genetically unstable, and a myelodysplastic syndrome might transform into acutemyeloid leukemia. The overall survival of these diseases range between few months to around ten years. Correct diagnosis andaccurate prognostic classification is essential. In the past decades several scoring systems were established beginning with theFrench-American-British classification to the most recent Revised International Prognostic Scoring System. In all of theseclassifications bone marrow morphology is still the most important factor, though nowadays the genetic aberrations and flowcytometry findings are also included. The diagnosis and prognostic classification of myelodysplastic syndromes remain a greatchallenge for hematologists.

INTRODUCTION

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem-cell disorders. The incidence of MDS is 3.4 per 100,000/peryear in the United States, which increases with age. The median age at diagnosis is 76 years in the U.S. and 74 years in Europe.The incidence is slightly higher in men than in women [1, 2].

The exact etiology of MDS is unknown. MDS have two subtypes according to their etiology, a primary (de novo) and a secondaryone. The development of the second type of MDS occurs more frequently after some environmental mutagenic event, such asthe effect of toxic chemicals, e.g. benzene, or treatment of malignant tumor with radiation and/or chemotherapy. Several studieshave examined the causes of MDS, which include environmental exposures, cytogenetic and epigenetic changes in stem cellsand progenitors, altered bone marrow microenvironment, immune dysregulation, and abnormal cell cycle regulation anddifferentiation (Figure 1). Thus it has become commonly accepted that MDS is the result of a complex process [3].

Although MDS are a heterogeneous diseases group, there are some common characteristics to these pathological conditions.One of these is the progressive bone marrow failure, which manifests in peripheral cytopenia due to ineffective hematopoesis.In patient histories we often encounter anemia resistant to treatment (refractory anemia), while the bone marrow is hypercellularand erythroid hyperplasia can be detected. When examining these peripheral and bone marrow samples, typical morphological

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B. KÁRAI, E. SZÁNTHÓ, J. KAPPELMAYER, Z. HEVESSY FLOW CYTOMETRY IN THE DIAGNOSIS OFMYELODYSPLASTIC SYNDROMES

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3karai

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alterations – dysplastic features – can be observed, which might affect the erythroid, myeloid and megakaryocytic cell lineages.In addition, blast counts might also be increased in severe cases. Another common characteristic is the genetic instability of thepathological cells, which results in an enhanced risk of MDS transforming into acute myeloid leukemia (AML). This transformationoccurs in approximately 30 percent of the cases, and it is one of the most important causes of mortality of MDS. Further causesof mortality may include consequences of ineffective haematopiesis and the complications of cytopenia (e.g. infections,bleeding).

Overall survival time in MDS has a large interval from some months up to more than ten years, therefore correct diagnosis andaccurate prognostic classification are essential for theoptimal treatment [4, 5].

CLASSIFICATION SYSTEM, PROGNOSTIC SCORING SYSTEM

In the past 30 years, several classification and prognostic scoring systems have been developed. The first widespreadclassification system was the French-American-British (FAB), which assigned patients to five categories: refractory anemia(RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), refractory anemia withexcess blasts in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML) [6]. This classification system isbased on the histopathological examination of peripheral and bone marrow specimens (Table 1), where the percentage ofsideroblasts and blasts are taken into consideration along with the morphologic features.

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FLOW CYTOMETRY IN THE DIAGNOSIS OFMYELODYSPLASTIC SYNDROMES

B. KÁRAI, E. SZÁNTHÓ, J. KAPPELMAYER, Z. HEVESSY

Figure 1Theories of pathophysiology involved in MDS development.

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The International Prognostic Scoring System (IPSS), published in 1997, was based on at least seven previous risk assessments,including the FAB classification as the most dominant source. In this study 816 primary MDS patients were examined in termsof survival and AML evolution, respectively. Patients who had previously received intensive chemotherapy and those with CMML(proliferative subtype) who had a higher white blood cell count (WBC) than 12000/µL were excluded from the analysis. Allvariables (blast percentage, peripheral cytopenia, cytogenetic abnormalities, age and gender) were weighed according to theirstatistical power. Finally three prognostic parameters – percentage of blasts, cytogenetic alteration, and the degree of peripheralcytopenia – were selected to develop a new prognostic scoring system that assigned patients into one of four risk groups: low,intermediate-1, intermediate-2, high (Table 2). There is significant difference between these groups in overall survival and inthe probability of AML evolution. Patients older than 60 and assigned to the low and intermediate-1 groups exhibited significantlyreduced overall survival [7].

Based on the results of IPSS the World Health Organization (WHO) made several changes to the FAB classification andintroduced a new system. One of the major alterations concerned the criteria of AML. While the FAB classification establishedthe diagnosis of AML when the blast percentage reached 30% in peripheral blood or bone marrow, the WHO reduced thisthreshold to 20%; furthermore, it established a new category within AML, namely, AML transformed from MDS.Consequently the former RAEB-T group is absent from the WHO classification. On the other hand, new groups were also created,such as MDS with isolated 5q deletion – MDS del(5q); refractory cytopenia with multiple cell lineage dysplasia (RCMD), andunclassified MDS – the RAEB group was also split on the basis of blast percentage (RAEB-1 and RAEB-2). The creation of theMDS del(5q) group is justified by the different therapy requirements, especially good prognosis and idiosyncratic clinicalsymptoms (anemia, normal or increased platelet count in the peripheral bloody, and increased count of hypolobulatedmegakaryocytes in the bone marrow) of these patients. According to the most recent (2008) WHO recommendations, theunclassified MDS group consists of patients with cytopenia and blast count under 1% in the peripheral blood and under 5% inthe bone marrow, while upon analyzing the latter, no cell lineage can be declared dysplastic, yet characteristic cytogeneticalterations of MDS can be detected (Table 2). A cell lineage is dysplastic if clear dysplastic features are observed in at least 10%of its cells. Beyond these morphological criteria, factors causing secondary dysplasia must also be excluded (iron-, B12-, folicacid-, or copper-deficiency; infection (HIV), autoimmune disorders. [3,8,9,10,11].

The WHO Classification-Based Prognostic Scoring System (WPSS) was published in 2007, the advantage of which over IPSS isthe exclusion of FAB RAEB-T- and CMML patients . These patients are currently classified in the AML and MDS/MPN (MPN:Myelo- Proliferative Neoplasm) category. Another advantage of WPSS is that it is a dynamic system that can be appliedthroughout the course of the illness. This is because while in the IPSS study patients were examined only at diagnosis, participantsof the WPSS monitoring were repeatedly checked and re-classified if necessary. Furthermore, in addition to the WHOclassification and the karyotype, the WPSS incorporated a new, independent prognostic factor that is transfusion dependency(Table 2) [12].

The above data demonstrate that morphology remains the basis for both diagnosis and prognostic classification but the currentWHO recommendations (2008) and the WPSS also considers the cytogenetic and clinical features. Even if the quality of thesample is appropriate the examiners face a difficult task when looking for the minimum morphological criteria determined bythe WHO and the International Working Group on Morphology of Myelodysplastic Syndrome (IWGM-MDS) (Figure 2) [13]. In

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Table 1Typical morphologic alteration in MDS

dyserythropoiesis dysgranulopoiesis dysmegakaryocytopoiesis

anisocytosis nuclear/cytoplasmic asynchrony large megakaryocytes with unsegmented nuclei

poikilocytosis hypogranulation micromegakaryocytes

macrocytosis nuclear hyposegmentation pseudo Pelger- Huet cells megakaryocytes with two or more small, unconnected nuclei

increased dacryocytes giant hypogranular platelets

basophil stippling

increased

nucleated red blood cell

nuclear fragmentation (karyiorrhexis)

nuclear budding (bridging)

ring sideroblasts

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Table 2Prognostic scoring systems in MDS

IPSS WPSS R…IPSS

prognostic variable

BM blast(% ) point WHO category point BM blast % point

• <5 0 • RA, RARS, 5q… 0 • ≤2 0

• 5…10 0,5 • RCMD, RCMD…RS 1 • >2…<5 1

• 11…20 1,5 • RAEB…1 2 • 5…10 2

• 21…30 2 • RAEB…2 3 • >10 3

karyotype* point karyotype* point karyotype* point

• good 0 • good 0 • very good 0

• intermediate 0,5 • intermediate 1 • good 1

• poor 1 • poor 2 • intermediate 2

cytopenias point transfusion requirement point • poor 3

• 0/1 0 • no 0 • very poor 4

• 2/3 0,5 • regular 1 hemoglobin (g/dl) point

• ≥10 0

• 8…<10 1

• <8 1,5

platelets (G/L) point

• ≥100 0

• 50- -<100 0,5

• <50 1

ANC (G/L) point

• ≥0,8 0

• <8 0,5

risk groups

risk score risk score risk score

low 0 very low 0 very low ≤1,5

intermediate-1 0,5-1 low 1 low >1,5-3

intermediate-2 1,5-2 intermediate 2 intermediate >3-4,5

high ≥2 high 3-4 high >4,5-6

very high 5-6 very high >6

very good • -Y alone

• del(11q)

good • normal• -Y alone• del(5q) alone• del(20q)alone

good • normal• -Y alone• del(5q) alone• del(20q) alone

good • normal• del(5q)• del(20q)• del(12p)• double including del(5q)

karyotype*

intermediate • +8• single miscellaneous• doubleabnormalities

intermediate • +8• single miscellaneous• doubleabnormalities

intermediate • del(7q)• +8• +19• i(17q)• any other single/doubleindependent clones

poor • ≥3 abnormalities• chrom. 7 anomalies

poor • ≥3 abnormalities• chrom. 7 anomalies

poor • -7• inv(3)/t(3q)/del(3q)• double including-7/del(7q)• complex 3 abnormalities

very poor • complex >3 abnormalities

Based on• Greenberg P. et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997;89:2079-2088.• Malcovati L. et al. Time-dependent prognostic scoring system for predicting survival and leukemic evolution in myelodysplastic syndromes. Journal of Clicinical

Oncology 2007; 25:3503-3510. • Greenberg PL et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012; 120:2454-2465.

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an attempt to provide an objective diagnosis and prognostic classification of MDS, in the last two decades several working groupshave been trying to introduce new technologies and to establish a new system of criteria.One of the most documented methods is the application of flow cytometry [14] (Figure 3). comparing the antigen-expressionpatterns of normal hematopoietic cells and of those taken from MDS patients reveals several characteristic distinctions on theblasts [15,16] as well as on cells of the myeloid [17,18], erythroid [19], and megakaryocytic lineage [20,21]. The most importantare the followings: abnormal CD45 expression on the granulocytes and blast cells, decreased CD11b, HLA-DR, CD13, CD33,CD14 expression on the monocytes; attenuation or complete loss of CD11b, CD13, CD16, CD33 on the granulocytes; appearanceof lymphoid markers (CD7, CD56) on granulocytes (Figure 4).On this basis a flow-cytometric scoring system was created in 2003 (Flow Cytometric Scoring System, FCSS). The bone marrowpatterns of 115 MDS and 104 other patients along with 25 healthy individuals were examined with three-color flow cytometricanalysis. According to the pathological differences in the antigen-expression of the cells of the myeloid line, the intensity of theside-scatter, the myeloid-lymphoid ratio, and the blast percentage MDS patients were classified in three groups (mild, moderate,severe). Significant differences were found between the groups in terms of mean overall survival and relapse potential followingallogeneic bone marrow transplantation (111 patients). Comparing the FCSS and IPSS results of MDS patients, the two systemsshowed good correlation, and the FCSS can offer extra information in the case of the IPSS intermedier-1 group, which facilitatesprognostic stratification [17].In the minimum diagnostic criteria system based on the agreements of the 2006 MDS conference, flow cytometry figures as aco-criterion. This way flow cytometry is indicated as a useful tool in cases where an unequivocal MDS diagnosis cannot beestablished on the basis of clinical data, morphology, and cytogenetics. Two such conditions are known today, namely,idiopathic cytopenia of undetermined significance (ICUS) and idiopathic dysplasia of uncertain significance (IDUS). In both

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Figure 2Minimal diagnostic criteria in MDS.

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cases, diseases causing chronic cytopenia and dysplasia can be ruled out, yet only some of the minimum criteria of MDS aremet, therefore an MDS diagnosis cannot be established. In the case of ICUS, refractory cytopenia can be observed, accompaniedby mildly or unmodified morphology and normal karyotype, while patients classified as having IDUS exhibit the reverse, that is,unequivocal dysplastic morphological features without the cytopenia necessary for diagnosis of MDS [13,22,23].

SUMMARY

In summary, the diagnosis and prognostic classification of MDS seems to be the greatest challenge among all myeloid neoplasms.The uncertainty is sustained by several factors. On one hand, MDS is a rather heterogeneous group of diseases; on the otherhand, the correct evaluation of morphology—which serves as the basis for diagnosis and prognosis—is a difficult task even forexperienced examiners. Therefore in the past decades, to facilitate the more precise classification of patients with a number ofobjective studies, such as well-defined anamnestic data (e.g., number of transfusions), laboratory parameters (WBC, absoluteneutrophil count, platelet count, lactate dehydrogenase value (LDH), ferritin, β2 microglobulin, etc.), cytogenetic, flow cytometric,and molecular genetic research were int he center of interest. The most recent prognostic scoring systems reflect these efforts.In case of the Revised International Prognostic Scoring System (R-IPSS), the prognostic power of several parameters (cytogeneticalterations, degree of cytopenia, LDH, ferritin, β2 microglobulin, myelofibrosis, age, sex, FAB, WHO classifications) was testedon numerous patients (IPSS n=816, R-IPSS n=7012). The analysis of such a large sample allowed the demonstration of theprognostic effect of less frequent cytogenetic alterations, thus, instead of the three cytogenetic groups of IPSS, here five groupsfacilitate the more precise anticipation of clinical outcome. Beside cytogenetics, the percentage of blasts, the hemoglobinconcentration, platelet and absolute neutrophil count proved to be the most determining parameters. On the basis of thesefactors, patients are assigned to five risk groups, making the assessment of low-risk patients more precise [24,25] (Table 2).

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Figure 3Normal granulocyte, monocyte maturation. The arrows indicate the maturation process.

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The most up-to-date flow-cytometric scoring scales – such as the one prepared by European LeukemiaNET – also aid in thediagnosis and prognostic classification of low-risk as well as ICUS and IDUS patients. In that study, 797 patient samples (417 low-risk MDS, 380 pathologic control samples) were analyzed by flow cytometry. According to the results, merely four cytometricparameters facilitate effectively the diagnosis of low-risk patients. These are the followings: the percentage of bone marrowblasts, the percentage of progenitor B cells within CD34 positive cells, the mean fluorescence intensity of CD45 expression inlymphocytes as compared to myeloblasts, and the granulocyte to lymphocyte side scatter ratio [18].The results of these new studies contributed to the more objective and more precise diagnosis and clinical follow-up of MDSthroughout a wider institutional spectrum.

ACKNOWLEDGEMENTS

This work was supported by a TAMOP-4.2.2.B-11/1/KONV of the Medical and Health Science Center, University of Debrecen (B.K:).

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Figure 4Characteristic distinctions of antigen-expression patterns in MDS. Histograms show one of the most important antigen expression on dysplastic(red frame) and normal (yellow frame) granulocytes or monocytes.

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22. Valent P; Jäger E; Mitterbauer-Hohendanner G; Müllauer L; Schwarzinger I; Sperr WR; Thalhammer R; Wimazal F. Idiopathic bone marrowdysplasia of unknown significance (IDUS): definition, pathogenesis, follow up, and prognosis. Am J Cancer Res 2011; 1:531-541.

23. Valent P; Bain BJ; Bennett JM; Wimazal F; Sperr WR; Mufti G; Horny HP. Idiopathic cytopenia of undetermined significance (ICUS)and idiopathic dysplasia of uncertain significance (IDUS), and their distinction from low risk MDS; Leukemia Research 2012; 36:1-5.

24. Greenberg PL; Tuechler H; Schanz J; Sanz G; Garcia-Manero G; Solé F; Bennett JM; Bowen D; Fenaux P; Dreyfus F; Kantarjian H; KuendgenA; Levis A; Malcovati L; Cazzola M; Cermak J; Fonatsch C; LeBeau MM; Slovak ML; Krieger O; Luebbert M; Maciejewski J; Magalhaes SM;Miyazaki Y; Pfeilstöcker M; Sekeres M; Sperr WR; Stauder R; Tauro S; Valent P; Vallespi T; van de Loosdrecht AA; Germing U; Haase D.Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012; 120:2454-2465.

25. Schanz J; Tüchler H; Solé F; Mallo M; Luño E; Cervera J; Granada I; Hildebrandt B; Slovak ML; Ohyashiki K; Steidl C; Fonatsch C; PfeilstöckerM; Nösslinger T; Valent P; Giagounidis A; Aul C; Lübbert M; Stauder R; Krieger O; Garcia-Manero G; Faderl S; Pierce S; LeBeau MM; BennettJM; Greenberg P; Germing U; Haase D. New comprehensive cytogenetic scoring system for primary myelodysplastic syndromes andoligoblastic AML following MDS derived from an international database merge. J Clin Oncol 2012; 30:820-829.

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PREDICTION OF THERAPY RESPONSE AND PROGNOSIS IN LEUKEMIAS BY FLOW CYTOMETRIC

MDR ASSAYS

János Kappelmayer1, Zsuzsa Hevessy1, András Apjok2, Katalin Tauberné Jakab2

1Department of Laboratory Medicine, Medical and Health Science Center, University of Debrecen, Hungary, 2Solvo Biotechnology,Szeged, Hungary


János Kappelmayer, Department of Laboratory Medicine, Medical and HealthScience Center, University of Debrecen, HungaryTel: +36 52-340-006Fax: +36 52-417-631e-mail: [email protected]

Key words: flow cytometry, leukemia, drug-resistance

ABSTRACT

Multidrug resistance (MDR) is an unwanted phenomenon, that may cause therapy failure in several neoplasms includinghematological malignancies. The purpose of any type of laboratory MDR assay is to reliably identify such patients and to provideuseful data to clinicians with a relatively short turnaround time. MDR can be multicausal and several previous data identified agroup of transmembrane proteins - the ATP-binding casette (ABC) proteins - that may be involved in MDR in varioushematological malignancies. The prototype of these proteins is the P-glycoprotein (Pgp, MDR1, ABCB1) that is a seven-membranespanning transmembrane protein capable of extruding several cytotoxic drugs that are of key importance in the treatmentof hematological disorders. Similarly other ABC proteins – Multidrug resistance associated protein 1 (ABCC1) and breastcancer resistance protein (ABCG2) are both capable of pumping out cytotoxic drugs. Here, we present flow cytometric methodsto identify MDR proteins by antigen and activity assays. The advantage of flow technology is the short turnaround time and itsrelative easiness compared to nucleic acid based technologies. However, for the activity assays, it should be noted, that thesefunctional tests require live cells, thus adequate results can only be provided if the specimen transport can be completed within6 hours of sample collection. Identification of MDR proteins provides prognostic information and may modulate therapy, thussignifies a clinically useful information in the evaluation of patients with leukemias.

INTRODUCTION

Drug resistance may be intrinsic or acquired and it severely impairs the progress in cancer chemotherapy [1,2]. The importanceof this phenomenon is underlined by the fact that its presence is not limited to malignancies but may hamper the success oftherapy in several other diseases like rheumatoid arthritis and epilepsy. In MDR, resistance occurs to several chemically unrelateddrugs, lipid-soluble drugs like anthracyclines, vinca alkaloids, epipodophyllotoxins, antibiotics and the resistance can be causedby one or more of several mechanisms. A frequently observed phenomenon is when the drug is quickly extruded from the cellby transporters before any cytotoxic action can be elicited. These efflux proteins are localized in the cell membrane, howeverfurther intracellular sites were also described and these are thought to contribute to resistance by accumulating the drug inintracellular compartments and preventing it from reaching its nuclear targets [3].The best studied efflux pump is a permeability glycoprotein (P-glycoprotein, Pgp) which is a 170 kDa protein that cleaves ATP tocover the energy needed for expelling many xenobiotics. Pgp consists of two homologous halves, each consisting of sixtransmembrane segments and one ATP-binding domain. The most accepted model is based on the presence of a catalytic

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intermediate where both catalytic sites of Pgp are active and ATP is hydrolysed alternatively alternatively (Figure 1.). ATPhydrolysis at one site triggers conformational changes within the protein resulting in drug transport, while hydrolysis of a secondATP at the other site is required for resetting the original high- affinity binding conformation [4-7]. The transmembranesegments containing the drug-binding site are quite mobile so drug binding occurs through a substrate-induced fit mechanism.This mechanism explains how Pgp can accommodate a broad range of compounds [7]. Pgp was designated as MDR1 and it wasshown 25 years ago, that the expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine,doxorubicin, and vinblastine [8]. The human Pgp belongs to a large group of transport proteins, known as ATP-binding casette(ABC) superfamily, that share common structural and functional properties. To date, about 50 human ABC transporter geneshave been identified. Their protein products are classified into seven groups entitled: ABCA-ABCG.In addition to MDR proteins other membrane pumps extrude somewhat different substrates than Pgp. A 190 kDa protein calledMRP (multidrug resistance related protein) is a group of transporters that can contribute to clinical resistance. In contrast toPgp, MRP1 expression is predominant in the basolateral plasma membrane. MRP protein functions as a multispecific organicanion transporter, with oxidized glutathione, cysteinyl leukotrienes, and activated aflatoxin B1 as substrates. This protein alsotransports glucuronides, sulphate conjugates of steroid hormones, bile salts and other hydrophobic compounds in the presenceof glutathione [9-12]. MRP proteins play an important physiological role in the protection of the body against xenobioticsoccurring in the environment. MRP2 and MRP3 seem to play a role in organic conjugate transport while MRP4 and MRP5may have a nucleotide transporter function.The third most studied efflux protein is the breast cancer resistance protein (BCRP, ABCG2). Its mRNA encodes this 663 aminoacid member of the ATP-binding cassette superfamily of transporters. Enforced expression of the full-length BCRP cDNA inMCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulationand retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells [13].Furthermore its over-expression was identified as a negative prognostic marker in acute myeloid leukemia patients and it wasdescribed that the survival significantly worsened in case of BCRP over-expression concomitant with Pgp and other unfavourableprognostic markers. [14, 15].

IMPORTANCE OF DRUG RESISTANCE IN HEMATOLOGICAL MALIGNANCIES

The importance of MDR in hematological malignancies seem to be well-established, since from the historic paper of Ueda et al.in 1987 over 1400 hits can be found in Medline about MDR and leukemias. The mechanisms that may contribute to the enhancedPgp expression are the activity of its transcription factor [16], gene rearrangement [17], or hypomethylation of the mdr-1promoter region [18]. In addition, Pgp has been investigated in lymphoid malignancies and it has been demonstrated thatpatients with increased level of Pgp, either at diagnosis or upon relapse have poorer prognosis than those patients who do notexpress Pgp [19-21]. Undoubtedly however, the majority of literature deals with MDR in AML patients, where drug resistance isprimarily determined by Pgp [22-24] although other transporters were also found to have significance [25-27].

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Figure 1Function of MDR proteins. Drug-efflux proteins like Pgp expel xenobiotics already from the cell membrane, thus most of these molecules cannot enter the cell.

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Aside from de novo resistance, MDR can develop during chemotherapy as sensitive cells are killed off and genetic resistance isinduced either by the chemotherapy itself or emerges spontaneously during treatment. Although normal tissues possessconstitutive Pgp activity, acquired resistance never develops in noncancerous cells. Pgp may not only influence disease outcomeas a pump protein, but because of its involvement as an intramembranal substrate transport, it may have anti-apoptotic effectas well.In addition to the analysis of MDR pump function at the protein level, several studies have lead the way to delineate the role ofMDR-1 genetic polymorphisms in disease outcome. MDR-1 single nucleotide polymorphisms were found to be associated withan achievement of complete remission and event-free survival in AML patients, however they were not found to be associatedwith overall survival [28].

METHODS TO DETECT DRUG RESISTANCE

A few considerations are important when MDR diagnostic assays are discussed. One is that in hematological malignancies morethan one mechanism of drug resistance may be present that may impose a special diagnostic problem. The other is that mostof the data on MDR assays are derived from measurements on cell lines. However in clinical samples the efflux proteins like Pgpis present in several orders of magnitude lower copy numbers. There are three different approaches to identify MDR.(i) RNA measurement for different MDR proteins(ii) determination of protein expression by flow cytometry or immunohistochemistry(iii) functional tests that measure the transport activity of MDR proteins.

The 3-4,5 dimethylthiazol-2-yl-2,5-diphenyltetrazolium-bromide (MTT) assay or that is more simply referred to as cell survivalassays is used in some laboratories. It reports results in quantitative terms, however it is laborious and requires 4 days and thusis unsuitable for routine analysis of clinical samples. According to a consensus paper published in 1996 [29] two methods haveto be executed in order to obtain reliable results for MDR testing. The problem with RNA measurement is that mRNA level doesnot necessarily correlate with the expression of the relevant proteins and the mRNA results are often provided only insemi-quantitative terms.

MDR ANTIGEN AND ACTIVITY MEASUREMENTS

Antigenic assays are easy to execute and thus can easily fit into the general routine of a flow cytometry laboratory as all CDmarkers are detected by using directly conjugated antibodies. However, the major drawback of antigenic detection of MDR isthat the different clones of antibodies have variable sensitivity and thus the results obtained are often highly variable.Furthermore, reporting of MDR antigenic data is not straightforward as in many cases the conventional percent positivity is notvery meaningful due to the low expression rate in clinical samples. Thus, authors report the results in terms of fluorescenceunits or mean fluorescence intensities. Here, the ratio of the sample mean fluorescence channel and the isotypic controlfluorescence channel was calculated and MFI ratios exceeding values found on normal blood cells were reported as positive.Thus, relative fluorescence intensity values (RFI) are usually provided for various MDR proteins when measured by antibodies[30]. Some papers, however points to the possibility that certain antibodies detecting MDR1 have been shown to be sensitiveto conformational changes [31,32], thus an increase in antibody binding capacity may be exploited in the investigation of clinicalsamples. Functional assays definitely offer an advantage over antigen measurements since they measure the clinically relevantproperty, the transport activity of MDR proteins. The most widely used functional assay to detect MDR activity is basedon the measurement of a fluorescent substrate. These are mainly accumulation type assays where the fluorescent dye iscontinuously accumulated in the cell either due to its binding to intracellular structures like doxorubicin or due to its intracellularenzymatic modification like in case of calcein-acetoxymethyl ester. The fluorescence tracers in the majority of these assays arethe rhodamine 123 [33], calcein-AM, [34] or JC1 [35]. The tracer JC1 has proven to be useful for the simultaneous detection ofPgp activity and apoptosis in leukemic cells [36]. These functional assays are evaluated by flow cytometry and the principle ofthe measurement is to measure fluorescence of cells in the presence and absence of efflux pump inhibitors. An advantageof functional assays is, that they can be combined with surface staining of the leukemic cells, thus the cell population of interestcan be gated out [37] by labelling the sample by the appropriate antibody and the efflux activity of the desired cells populationis analysed with and without the efflux pump inhibitor (Figure 2). It should be noted that in this assay the positive population isrepresented with the lower fluorescence values– since the indicator dye is removed from the cells – while in an antigen assaythe positive cells are always displayed with higher fluorescence values as observed with any other flow cytometric antigen assay(Figure 3.).The quantitative results of the functional assay can be expressed in multidrug resistance activity factor (MAF) units by usingselective inhibitors. The difference in fluorescence is proportional to the activity of the efflux pump. In a typical flow activityassay, MDR1 and MRP1 are inhibited separately and thus total MDR activity can more appropriately be dissected. A definite

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disadvantage of the functional assays, however that they require living cells, thus prolonged storage of samples is not possible.This is probably one reason why the detection of MDR in flow cytometry laboratories is relatively rare.In our previous work the functional assay was correlated to an antigenic assay carried out by quantitative flow cytometry [38].We performed the determination of the antibody binding capacity by incubating cells with the anti-Pgp antibody MRK16 andsubsequently with a FITC labeled anti-mouse IgG. Results were expressed as ABC by using a calibration curve obtained bymeasuring the fluorescence intensity of precalibrated beads. The multidrug resistance activity factor (MAF) values weredetermined with the MDR inhibitor Verapamil (Vp). In triplicate samples by the preincubation of samples with (Vp+) or without(Vp-) the MDR blocker and subsequently loading them with calcein-AM. The calcein fluorescence was measured and the valuesMFI (Vp+) – MFI (Vp-)/MFI (Vp+) x100 were reported as the MAF value corresponding to MDR activity. This quantitativemeasurement of MDR activity is characteristic for the cumulative activity of Pgp and MRP1 as verapamil inhibits bothtransporters. When MAF was determined in the presence of the MRP1 inhibitor MK571 the MAFVp-MAFMK571 value referred tothe Pgp specific resistance.We found that in the high Pgp expressor KB-V1 cell line an extremely high MDR activity was detectable along with high numberof Pgp molecules/cell while in the low expressor KB-8-5 cell line the functional assay resulted only in a 20% decrease while thenumber of Pgp molecules decreased by over 90%. This also refers to the better sensitivity of the functional assay.

A commercially available kit is available that measures the activity of multiple transporters. Preliminary results with thisMultiDrugQuant assay kit were published and the authors were able to show a significant correlation between the expressionof the multidrug resistant proteins (P-gp and MRP1) and their functional activity in adult AML and pediatric ALL samples [39].This test was also suitable to identify drug resistance in solid tumors as collagenase disintegration preserved the MDR activityand the antigenicity of tumor cells. The extended calcein assay provided sufficient viable and functionally active tumor cells

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Figure 2Functional MDR assay in an AML sample. The cells are identified on the FS-SS dot plots (panel A) than subsequently the CD45-dim myeloidblasts are gated out (panel B). Calcein is measured on the blasts in the presence (panel C) or absence (Panel D) of an MDR inhibitor. Note thatthe fluorescence values are lower in the absence of the inhibitor.

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from surgical biopsies to determine the functional MDR activity [40].In 2011 the SOLVO-MDQ kit received a CE/IVD qualification. It provides all necessary ingredients to execute functional MDRassays for Pgp, MRP1 and BCRP by using selective inhibitors and two different fluorescence dyes and as a minimum requirementa 4-color flow cytometer is required.

CONCLUSIONS

Flow cytometry is increasingly been used in clinical laboratories and today already small benchtop cytometers can provide theadvantage of using multiple colors. We anticipate that in hematology, as many novel probes emerge functional assays maybecome more popular and one such application is likely to be the detection of MDR protein activities, that is easier with a CE-marked diagnostic kit.

ACKNOWLEDGEMENT

The author would like to thank Valéria Sziráki Kiss for technical assistance. This study was supported by the IVDMDQ08 NKTHproject.

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Figure 3Comparison of the functional an antigen assays in cell lines. In the functional MDR assays (panel A) positive cells display reducedfluorescence values since they expel the fluorescent dye. In the antigen assays (panel B) the positive cell line displays higher fluorescence dueto binding the fluorescent antibody.

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22. Karászi E, Jakab K, Homolya L, Szakács G, Holló Z, Telek B, Kiss A, Rejtô L, Nahajevszky S, Sarkadi B, Kappelmayer J.Calcein assay for multidrugresistance reliably predicts therapy response and survival rate in acute myeloid leukaemia. Br J Haematol. 2001;112(2):308-314.

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36. Chaoui D, Faussat AM, Majdak P, Tang R, Perrot JY, Pasco S, Klein C, Marie JP, Legrand O. JC-1, a sensitive probe for a simultaneous detectionof P- glycoprotein activity and apoptosis in leukemic cells. Cytometry B Clin Cytom. 2006;70(3):189-96.

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FLOW CYTOMETRIC INVESTIGATION OF CLASSICAL AND ALTERNATIVE PLATELET ACTIVATION

MARKERS

Béla Nagy Jr, Ildikó Beke Debreceni, János KappelmayerDepartment of Laboratory Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary


Béla Nagy Jr, MD, PhDDepartment of Laboratory Medicine, Medical and Health Science Center, University ofDebrecen, Debrecen, HungaryTel: +36 52 340 006Fax: +36 52 417 631e-mail: [email protected]

Keywords: P-selectin, heterotypic aggregates, microparticle, coated-platelets, flow cytometry, heparin-induced thrombocytopenia

ABSTRACT

Platelets show a substantial role in the maintenance of vascular integrity when these cells after a rapid activation adhere to thevessel wall lesion, aggregate with other platelets and leukocytes resulting in an arterial thrombosis. Analysis of in vivo plateletactivation at an early time point is crucial in the detection of developing thrombotic events. In addition, the forecast of futurecomplications as well as the evaluation of the efficacy of anti- platelet medication are also essential in a large group of patients.Changes in the levels of platelet receptors or alteration in other surface properties due to intra- and extracellular responses toa stimulus can be measurable primarily by flow cytometry with specific antibodies via the assessment of classical and alternativeplatelet activation markers. Some of these biomarkers have been already used in routine laboratory settings in many cases,while others still stand in the phase of research applications. Deficiency in platelet receptors is also accessible with this techniquefor the diagnosis of certain bleeding disorders. We here describe the most important types of platelet activation markers, andgive an overview how the levels of these markers are altered in different diseases.

INTRODUCTION

Platelets are involved in the regulation of hemostasis, as activated platelets normally adhere to the injured vessel wall.Thrombocytes form aggregates with each other, but also interact with leukocytes to avoid a substantial blood loss from thecirculation. These cellular complexes also contribute to the development of local inflammatory events. In contrast, abnormalplatelet function may result in thrombotic or bleeding complications.In arterial thrombosis, the level of platelet reactivity increases, and the expression of several platelet activation proteins (markers)can be measured on the cell surface. In addition, distinct platelet subpopulations (e.g. coated-platelets) may be also investigatedusing such experiments. Discovery of novel biomarkers is still of interest to predict emergency thrombotic states, and to monitorthe effects of anti-platelet therapy. The deficiency or lack of platelet receptors may generate a dysfunction in platelet aggregationand cause hemorrhage. Early detection of all these anomalies is demanding, and flow cytometry is a reliable laboratory methodto analyze platelet function in ex vivo clinical samples. This tool is now getting available in more and more laboratories, and acombination of two or three antibodies against platelet receptors allows a sensitive and specific analysis of platelets. However,there are several preanalytical and methodological pitfalls, which may influence the measurement and the interpretation ofthese results. In this review, the classic and alternative platelet activation markers on flow cytometry are summarized, whichhave been assessed in a large number of studies to evaluate altered platelet function.

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I. CLASSICAL PLATELET ACTIVATION MARKERS

I/1. CD62PP-selectin (CD62P) is one of the most abundant proteins in the α-granules of platelets, which is exposed on the cell surfacewithin seconds after platelet activation [1]. Flow cytometric analysis of surface-bound CD62P alone or in a combinationwith other markers (see below) has been used as the ‘gold standard’ marker for the assessment of platelet activation in ex vivopatient samples in the last 3 decades reviewed in [2].

Detection of elevated surface P-selectin was the subject of numerous clinical studies in acute coronary syndrome (ACS) [3-5], intype 1 and 2 diabetes mellitus (DM) [6-8], untreated hypertension [9], obesity with or without DM [10-11], peripheral arterydisease (PAD) [12], acute ischemic stroke [13-16], essential thrombocythemia (ET) [17], and in those clinical conditions whereplatelet activation is one part of the disease pathomechanism such as in primary Raynaud’s disease [18]. Platelet-bound P-selectin values are typically determined in percent positivity (%), however, even a small change in mean fluorescence intensity(MFI) values may demonstrate a larger alteration in surface P-selectin measured on a logarithmic scale. P-selectin analysisin non-activated samples is for ‘baseline’ CD62P positivity in the current in vivo platelet activation status, while platelet reactivitycan be evaluated with CD62P levels on stimulated platelets by using submaximal concentrations of classic agonists adenosine-diphosphate (ADP) (0.5-5 µM), collagen (1-2 µg/mL), or thrombin-receptor activating peptide (TRAP) (1-8 µM) [7,10,19,20].Significantly higher P-selectin levels were independently correlated with the body mass index (BMI) [10], the atherosclerosisindicator carotid intima-media thickness (IMT), and the inflammation marker C-reactive protein (CRP) [11]. Stellos et al. furtherinvestigated surface-bound P-selectin as a prognostic marker in myocardial infarction (MI), and they found a positive associationbetween the extent of myocardial injury measured with the levels of troponin-I plus creatine kinase-MB and CD62Ppositivity independently of age, gender, and baseline medication [5]. CD62P values were significantly increased in ST-segmentelevation MI (STEMI) patients that reflected a greater degree of occlusive thrombus formation in these patients versus otherswith non- ST-segment elevation MI (NSTEMI) or Troponin-I-positive unstable angina (UA). On the other hand, P-selectin positivityshowed a limited sensitivity (57.5%) and specificity (69%) for detection of ACS and discrimination of chest pain of differentorigins [5]. In monitoring of anti-platelet medication, CD62P had a minor sensitivity to the effects of ADP-receptor blockerclopidogrel and acetylsalicylic acid (ASA) therapy in stroke [21]. Surprisingly, opposite findings were also reported when lessCD62P positive platelets were measured in MI [20,22] and (convalescent) cerebral infarction as baseline values and in responseto agonist stimulation compared to clinical control cohorts [19,23,24]. Likewise, CD62P expression rapidly declined after theonset of acute ischemic stroke [25]. These phenomena were explained to be due to the rapid shedding of P-selectin fromcirculating platelets, and the sequestration of these activated cells into heterotypic aggregates [26,27]. In fact, the plasmaconcentration of released/shed receptors (i.e. soluble P-selectin) was measured in parallel with immunoassays as an additionalplatelet marker in these studies [11,19,20,24]. It was also suggested that platelets were exhausted and failed to respond tothrombin in vitro after a substantial cellular activation during stroke [28]. Therefore, detection of CD62P by flow cytometryseems to be a more reliable tool for monitoring platelet function at acute but not chronic stimulus of platelets.

I/2. CD40LCD40L expression was first described on activated T-cells [29], and was later shown to be liberated to the platelet surface fromα-granules, similarly to P-selectin [30]. It is now considered as an emerging platelet activation marker, and its level (CD154) wasalso increased when platelet activation was associated with endothelial dysfunction and inflammation in MI and UA [31]. Patientswith UA who needed coronary angioplasty or had recurrent angina showed even higher CD40L expression on platelets comparedwith those without such complications [32]. Moreover, significant increase in CD40L on platelets was already detected in transientischemic attack (TIA), not only complete stroke [33]. Especially in atherosclerotic ischemic stroke, CD40L positivity was enlargedcompared to that in asymptomatic carotid stenosis [14]. Consequently, upregulated CD40L level was thought to initiateischemic stroke from large artery atherosclerosis, and the concentration of this marker was correlated with worse clinicaloutcome after cerebral infarction [16,34].

I/3. CD63CD63 (granulophysin, LAMP-3) is translocated from dense-granules and lysosomes to the plasma membrane after plateletactivation [35]. CD63 expression was higher on day 1 in the stroke group versus control group, which remained significantlyelevated until day 90 [25]. Similarly, Cha et al. found significantly higher CD63 platelet positivity in patients withatherosclerotic ischemic stroke than in normal subjects; however, no significant differences were seen between atheroscleroticischemic stroke and asymptomatic carotid stenosis [14]. Additionally, increased CD63 level was predominantly detected in theacute stage of ischemic stroke compared with its convalescent stage and the control group [16,36]. In contrast, others found noelevation in CD63 positivity in either acute or convalescent stroke patients versus subjects without vascular disease [15]. Similarlyto P-selectin, CD63 had an inferior role to detect the effects of clopidogrel and ASA in stroke patients [21].Immunofluorescence analysis of CD63 by flow cytometry was a suitable method for the diagnosis of Hermansky-Pudlak syndrome

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FLOW CYTOMETRIC INVESTIGATION OF CLASSICAL ANDALTERNATIVE PLATELET ACTIVATION MARKERS

B.NAGY JR, I. BEKE DEBRECENI, J. KAPPELMAYER

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accompanied with bruise and bleeding complications, where the significantly lower number of dense-granules and lysosomesin platelets was recognized by using anti-CD63 antibody versus a normal sample [35].

I/4. GPIIb/IIIa receptor (PAC-1 binding)Fibrinogen receptors undergo a conformational change during platelet activation [37]. PAC-1 antibody was formerly developedby Shattil and his coworkers [37], and nowadays it is a commercially available monoclonal antibody, which specifically bindsto the activated form of GPIIb/IIIa receptor complex induced by shear stress upon platelet aggregation. Increasing level ofactivated GPIIb/IIIa receptors was studied from clinically stable to unstable coronary artery diseases [38]. Also, constantly elevatedPAC-1 binding at 3-month follow-up was associated with an increased incidence of recurrent stroke [36]. On the contrary, McCabeet al. did not find any difference in PAC-1 percent positivity between those with acute or convalescent cerebrovascular disease[15]. In manifest metabolic syndrome, higher expression of PAC-1 with augmented fibrinogen binding was observed comparedto subjects with vascular disease [39]. PAC-1 was also found as a sensitive parameter in following clopidogrel effect along withdecreased level of the intracellular vasodilator-stimulated phosphoprotein (VASP) [40].

II. ALTERNATIVE BIOMARKERS OF PLATELET ACTIVATION

II/1. PMPsPlatelet-derived microparticles (PMPs) has been employed as an alternative evaluation of platelet activation in recent years.These vesicles are formed during platelet ‘budding’, and thus contain several components from platelet cytoplasm and outermembrane. Consequently, PMPs were positive for CD62P and CD63 [41]. Moreover, those PMPs shed from phosphatidylserine(PS)-positive platelets were also positive for PS, and had 50- to 100-fold higher procoagulant activity than activated platelets[42]. Definition and analysis of PMPs are still a debated area of clinical flow cytometry. Due to the variable storage andpreparation of samples, isolation of PMPs as well as differences in the settings of measurement, PMP numbers fairly varied evenin the same disease causing potential inappropriate interpretations [43]. Yet, the need for standardized protocols is stilldemanding. The analysis of PMPs was first set by using fluorescent beads with standard size and amount for enumerating PMPsbelow 1 µm [44]. Beads were initially processed, and then clinical samples were measured within a standard collection time of30 seconds. The numbers of PMPs were calculated based on the event count from the bead tube collected for the same timeperiod. PMPs were gated into a restricted area by FSC and SSC parameters, and then identified by the presence of PS withAnnexin V- FITC and their CD41 positivity. In addition, CD62P expression was also measured on these vesicles [44] (Figure 1).Previous studies described elevated number of PMPs in MI, atrial fibrillation, and ischemic stroke with severe carotidatherosclerosis compared to healthy controls [23,41,45,46]. Others recently claimed that PMPs act as an independent markerof cardiovascular events in high-risk ACS patients, because atherosclerotic burden did not affect PMP number in stable anginasubjects [47]. Furthermore, in ACS patients who underwent coronary stenting had even higher PMP numbers at 15 minutesafter the intervention induced by the procedure-mediated trauma compared to those with diagnostic catheterization alone[44] (Figure 1). In terms of abnormal metabolic and inflammatory conditions, Csongrádi and her colleagues demonstrated

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Figure 1Representative dot plots of PMP analysis. PMPs were collected in patients after diagnostic catheterization without stenting (A) and clinicalsubjects after coronary stenting (B). PMPs were identified in R1 gate according to FSC and SSC parameters, and then by their Annexin V(PS) positivity (PMP number: 881 events [A] vs. 1117 events [B] in R2). During further analysis, PMPs were stained by anti-CD41-PECy5 andanti-CD62-PE antibodies to measure the activation status of PMPs (20.5% vs. 33.7%) (adapted after some minor modifications with permissionfrom [44]).

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significantly higher PMP concentrations in obese subjects and type 2 DM patients versus healthy individuals, where PMP levelswere strongly associated with carotid IMT, BMI values, and CRP concentrations [11]. In agreement with this study, PMP levelswere positively and independently correlated with carotid IMT in the convalescent phase of ischemic stroke as well [19]. Finally,PMPs could be also measured in the flow cytometric detection of heparin-induced thrombocytopenia (HIT) (see below). Insummary, although increased PMP levels were documented even in the early phase of vascular diseases, it is still questionablewhether flow cytometric analysis of PMPs is ready to be used as a biomarker for routine laboratory purposes due torelatively lower sensitivity and specificity, and the rather variable conditions of measurement [47].

II/2. Heterotypic aggregatesPlatelet-leukocyte aggregates are generated in the blood stream when activated leukocytes and platelets rapidly producecellular complexes with each other via exposed receptors, notably with CD62P through an interaction with P-selectin GlycoproteinLigand-1 (PSGL-1) [48]. These heterotypic aggregates accumulate in the site of thrombus formation facilitating the developmentof variable vascular infarctions. Thus, therapeutic interference of these interactions may be a potential target of anti-plateletmedication reviewed in [49]. The half-life of circulating interactions (platelet-monocytes) is much longer (about 30 minutes)compared to P-selectin expression [26]. Thus, the analysis of platelet-leukocyte aggregates may be a more consistent indicatorof platelet activation than measuring the amount of P-selectin positive single platelets.Patients with UA showed a significant increase in the level of neutrophil-platelet aggregates compared with patients with stableangina [50]. In ACS, not only the total level of platelet-monocyte complexes was augmented, but such tissue factor (TF)-positive population as well in contrast to stable angina or controls [51]. Significantly elevated levels of platelet-monocyteaggregates were published in the acute stage of cerebral infarction compared to control groups [15,16,33] that showed a goodpredictive value in early outcome and long-term prognosis after stroke in a recent study [34]. Yet, there were some contradictorydata on the presence of platelet-leukocyte interactions in atrial fibrillation showing decreased levels versus control healthyindividuals [20]. In terms of metabolic diseases, neutrophil-platelet aggregates were higher in type 1 DM patients withnephropathy compared to DM patients with normal renal function as well as non-diabetic persons [7]. There was a significantdifference in the percentage of monocyte-platelet aggregates but not platelet-neutrophil or platelet-lymphocyte interactionsbetween the diabetic especially with proliferative retinopathy and nephropathy and control groups [52]. Similarly to these data,enhanced leukocyte-platelet adhesion was correlated to platelet hyperreactivity among DM patients especially those withmicroangiopathy [53]. In chronic myeloproliferative diseases, the increased level of platelet-monocyte aggregates may alsocontribute to the vascular complications [54].

II/3. FXIIICoagulation factor XIII (FXIII) is a protransglutaminase that is essential for maintaining hemostasis as a key regulator of fibrinolysis,and accelerates the fibrin cross-linking process [55]. FXIII is targeted and concentrated at the site where platelet-rich thrombiare formed. FXIII binds to activated platelets (Figure 2), and this interaction occurs via GPIIb/IIIa and αVβ3 receptors [56,57],and the surface-bound form was suggested to cross-link secreted α-granule proteins when coated-platelets are generated [58].In a clinical study [59] in patients with PAD, platelet-associated FXIII was found significantly higher than in healthy controls, andthe detection of FXIII on platelets was proposed as an alternative marker of platelet activation [59,60].

II/4. PhosphatidylserinePhosphatidylserine (PS), a negatively charged lipid in the inner-leaflet of phospholipid membranes, is exposed to cell surfaceupon platelet activation to propagate coagulation events. Via cleaving FX and prothrombin into their active form, PS facilitatesthe assembly and activation of tenase and prothrombinase complexes. As a result, fibrin fibers are formed in the early phase ofclot formation reviewed in [61]. PS exposure can be detected by the binding of Annexin V to platelets, which requires extracellularCa2+, so it should be supported during such experiments [62]. Interestingly, this marker did not become a conventional plateletactivation marker for ex vivo clinical samples, but was an available tool for studying in vitro procoagulant platelet responses,and identifying PMPs by flow cytometry.Apart from these processes, PS expression also occurs during platelet apoptosis via caspase and calpain activation, whenplatelets undergo a cellular death pathway resulting in their clearance from the circulation by scavenger cells [63,64]. Theseevents could be also induced in vitro by the classic platelet agonist, thrombin [65]. Aging, and stored platelets after several dayswere also positive for PS [66,67]. Overall, analysis of other biomarkers is necessary with PS to distinguish platelet activation andapoptosis-mediated changes from each other.

III. DEFICIENCY IN PLATELET GLYCOPROTEINS

Inherited platelet disorders are characterized by abnormalities of platelet function and production causing mucocutaneousbleeding symptoms with distinct intensity reviewed in [68]. When platelets show defects with an absence or malfunction of

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receptor(s) in adhesion receptors (GPIb/V/IX complex; Bernard-Soulier syndrome [BS]), or aggregation receptors (GPIIb/GPIIIacomplex; Glanzmann-thrombasthenia [GT]), platelets fail to bind to the main ligands von Willebrand factor (vWF) and fibrinogen,respectively. BS was characterized with lacking ristocetin-induced aggregation, prolonged bleeding time, large platelets andthrombocytopenia resulting in epistaxis, gingival and cutaneous bleeding in an adult female patient [69]. GT platelets showedimpaired aggregation to natural agonists (ADP, collagen, arachidonic acid) causing mucosal bleeding or epistaxis in a young malesubject [70]. In both diseases, flow cytometric analysis of surface glycoprotein expression was essential for the finaldiagnosis when surface properties of patient platelets were compared to those from a healthy age-matched sample. In the typeII GT patient, platelets were identified by anti- CD42a (GPIX) with no difference between patient and control in this term, whileGPIIb receptors (CD41) were hardly detectable (Figure 3A). In addition, GPIIIa receptors were also absent by using anti-CD61antibody (data not shown) [70]. In case of the BS person, GPIX by anti-CD42a showed a significantly lower expression, and GPIbreceptors with anti-CD42b antibodies demonstrated a null level versus those of a healthy individual. Platelets were identifiedby their CD41 positivity [69] (Figure 3B).

IV. Reticulated plateletsPercent of reticulated/immature platelets was suggested as a useful marker of augmented production or turnover of plateletsin subjects with increased platelet activation long ago [71]. These platelets have large size with higher density compared tonormal platelets. They also demonstrate an enhanced reactivity as they secrete more granule contents upon activation thansmaller platelets [72]. Elevated level of reticulated platelets was measured in increased thrombopoiesis such as in ET, or whena compensatory mechanism occurs due to a large platelet loss (e.g. immune thrombocytopenic purpura) [73]. Flowcytometric analysis of reticulated platelets was formerly set using thiazole orange staining to detect their mRNA contentand a platelet-specific (e.g. anti-GPIb) antibody for platelet gating [71]. ACS patients had significantly higher level of reticulatedplatelets versus healthy individuals [74]. Moreover, reticulated platelet percent was increased in both early and late phase ofischemic stroke/TIA after adjustment for age [75]. In terms of monitoring of anti-platelet drugs, larger immature platelet fractionwas observed in aspirin treatment in those after stent thrombosis showing an increased platelet turnover [76]; however, it wasnot confirmed in subjects with stroke [75].

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Figure 2Representative dots plot series on TRAP- activated (20 µM) platelets (R1) in a normal whole blood sample analyzed by flow cytometry inthree-color labeling experiments with anti-FXIIIA-FITC, anti- CD62-PE and anti-CD42a-PerCP antibodies. FXIII-A showed a co-expression withCD62P (23%). CD62% was 94% due to full platelet activation.

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V. HEPARIN-INDUCED THROMBOCYTOPENIA (HIT)HIT is one of the most common immune-mediated reactions caused by platelet activating IgG antibodies, which usually bind toheparin/PF4 complexes after heparin administration. Heparin/PF4/IgG complexes may induce platelet aggregation with increasedthrombin generation resulting in a prothrombotic state [77]. Three types of HIT can be distinguished according to the onset ofthrombocytopenia. Typically, thrombocytopenia begins between 4 and 15 days after the start of heparin therapy. Sometimes HITdevelops within the first 24 hours of heparin administration (rapid- onset), or several days after the discontinuation of heparin(delayed-onset) [77]. Functional test for HIT laboratory diagnosis is available on flow cytometers as well [78,79]. Oláh and co-workersrecently analyzed a patient sample from a rapid-onset HIT with the following methodology [80]. Normal platelets were incubatedwith the serum of a HIT patient and the therapeutic concentration of heparin (0.3 IU/mL). Annexin V binding on the surface ofplatelets and microparticle release were measured, and platelets were identified by CD41 positivity. For instance, in a negative control,PRP were incubated with heparin alone, while Ca-ionophore-stimulated sample (10 µM) was used as a positive control. Then, PRPwith the patient plasma was studied, and finally PRP with plasma plus heparin (0.3 IU/ml). Due to the presence of HIT, a significantlyincreased Annexin V positivity could be measured compared to samples with heparin or plasma alone (Figure 4).

VI. COATED-PLATELETS

Coated-platelets are produced by a simultaneous activation of collagen and α-thrombin, and represent a subpopulation ofactivated platelets with high PS exposure and a substantial prothombinase activity [58]. In addition, coated-platelets arecharacterized by the retention of several α-granule-derived coagulation factors e.g. factor V, vWF, thrombospondin, andfibrinogen on their surface [58], which are covalently bound together via serotonin creating a potentially procoagulantsurface matrix [81]. Elevated levels of coated-platelets were measured in patients with TIA and ischemic stroke compared tohealthy subjects [82,83]. In contrast, significantly lower levels of coated- platelets were also shown in spontaneous cerebralbleeding, severe hemophilia A, and asymptomatic ET versus healthy cohorts [17,84,85]. Dale and his coworkers previously seta standardized methodology [58]. Accordingly, subsequent immunostaining and platelet activation were assessed in gel-filteredplatelets by biotinylated-fibrinogen and anti-CD41 antibody with convulxin and α-thrombin. Coated-platelets were then indirectlylabeled with streptavidin-PE to detect enhanced fibrinogen binding compared to the rest of platelets. Detection of P-selectinpercent positivity was simultaneously performed (Figure 5).

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Figure 3Representative dot plots of a complex flow cytometry analysis of platelet glycoproteins in a GT (A) and a BS (B) patient. Platelets were identifiedby anti-CD42a (GPIX), and GPIIb receptors (CD41; 1.5%) were hardly detectable in GT (A). In contrast, in a BS individual, markedly less GPIX(CD42a) receptors could be detected (67%), and there was no GPIb receptors (2%; CD42b) compared to a healthy person (CD42a: 99%;CD42b: 91%). Results from these patients were depicted with “P”, while data of healthy controls were marked with “C”. These dot plotswere adapted with permission after some minor modifications from [69,70].

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CONCLUSIONS

Investigation of platelet biomarkers has not been only an approach to study platelet reactivity in variable diseases, but alsoprovided new insights for a better understanding of the complexity of platelet physiology. For instance, modulating the activityof intracellular proteins (e.g. protein phosphatases) via activation signaling with potential anti-platelet drugs could be easilytested through platelet biomarkers by flow cytometry [86]. Novel aspects can be also studied, i.e. two distinct subpopulationsof procoagulant platelets have been recently described after high concentrations of thrombin or collagen- related peptide basedon the quantification of PS positivity, PAC-1 binding, and intracellular Ca2+ concentration [87,88]. The relative function of theseplatelet subpopulations needs further analysis.

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Figure 4Representative dot plots of HIT investigation by flow cytometry. Normal platelets were incubated with the serum of a HIT patient and heparin(0.3 IU/mL).Platelets were identified by their CD41 positivity. In a negative control, PRP were incubated with heparin alone (2%; A), and Ca-ionophore-stimulated sample (99%; 10 µM) was used as a positive control (B). PRP with the patient plasma was studied (12%; C), and PRP with plasmaplus heparin (0.3 IU/ml) (25%; D). Due to the presence of HIT, a significantly increased Annexin V positivity could be measured compared tosamples with heparin or plasma alone.

Figure 5Representative dot plots of coated-platelet measurement in a normal sample on flow cytometer. Platelets were gated (P1) based on FSC-SSCparameters, and then these events were further analyzed in P2 gate where only CD41-positive cells were counted (92%). Finally, coated-platelets were separated from the rest of platelets according to their increased fibrinogen binding detected by biotinylated- fibrinogen andstreptavidin-PE (38%; P3).

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In summary, platelet activation markers generally show good sensitivity and specificity even in the detection of lower degree ofchange in platelet reactivity, and except for PMP analysis, these biomarkers provide a good reproducibility as well.

ACKNOWLEDGEMENTS

This work was supported by a Mecenatura grant (Mec-10/2011) of the Medical and Health Science Center, University of Debrecen(B.N.Jr).

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37. Shattil SJ, Hoxie JA, Cunningham M, Brass LF. Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation.J Biol Chem 1985; 260:11107-11114.

38. Tantry US, Bliden KP, Suarez TA, Kreutz RP, Dichiara J, Gurbel PA. Hypercoagulability, platelet function, inflammation and coronaryartery disease acuity: results of the Thrombotic RIsk Progression (TRIP) study. Platelets 2010;21:360-367.

39. Serebruany VL, Malinin A, Ong S, Atar D. Patients with metabolic syndrome exhibit higher platelet activity than those with conventionalrisk factors for vascular disease. J Thromb Thrombolysis 2008; 25:207-213.

40. Schwarz UR, Geiger J, Walter U, Eigenthaler M. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment ofactivating and inhibitory signal transduction pathways in human platelets--definition and detection of ticlopidine/clopidogrel effects.Thromb Haemost 1999; 82:1145-1152.

41. van der Zee PM, Bíró É, Ko Y, de Winter RJ, Hack CE, Sturk A, Nieuwland R. P- selectin- and CD63-exposing platelet microparticles reflectplatelet activation in peripheral arterial disease and myocardial infarction. Clin Chem 2006; 52:657-664.

42. Sinauridze EI, Kireev DA, Popenko NY, Pichugin AV, Panteleev MA, Krymskaya OV, Ataullakhanov FI. Platelet microparticle membranes have50- to 100-fold higher specific procoagulant activity than activated platelets. Thromb Haemost 2007; 97:425-434.

43. Shah MD, Bergeron AL, Dong JF, López JA. Flow cytometric measurement of microparticles: pitfalls and protocol modifications. Platelets2008; 19:365-372.

44. Nagy B Jr, Szük T, Debreceni IB, Kappelmayer J. Platelet-derived microparticle levels are significantly elevated in patients treated by electivestenting compared to subjects with diagnostic catheterization alone. Platelets 2010, 21:147-151.

45. Katopodis JN, Kolodny L, Jy W, Horstman LL, De Marchena EJ, Tao JG, Haynes DH, Ahn YS. Platelet microparticles and calcium homeostasisin acute coronary ischemias. Am J Hematol 1997; 54:95-101.

46. Choudhury A, Chung I, Blann AD, Lip GY. Elevated platelet microparticle levels in nonvalvular atrial fibrillation: relationship to p-selectinand antithrombotic therapy. Chest 2007; 131:809-815.

47. Biasucci LM, Porto I, Di Vito L, De Maria GL, Leone AM, Tinelli G, Tritarelli A, Di Rocco G, Snider F, Capogrossi MC, Crea F. Differences inmicroparticle release in patients with acute coronary syndrome and stable angina. Circ J 2012;76:2174-2182.

48. Li N, Hu H, Lindqvist M, Wikström-Jonsson E, Goodall AH, Hjemdahl P. Platelet-leukocyte cross talk in whole blood. ArteriosclerThromb Vasc Biol 2000; 20:2702-2708.

49. Nagy B Jr, Miszti-Blasius K, Kerényi A, Clemetson KJ, Kappelmayer J. Potential therapeutic targeting of platelet-mediated cellular interactionsin atherosclerosis and inflammation. Curr Med Chem 2012; 19:518-531.

50. Ott I, Neumann FJ, Gawaz M, Schmitt M, Schömig A. Increased neutrophil- platelet adhesion in patients with unstable angina. Circulation1996; 94:1239-1246.

51. Brambilla M, Camera M, Colnago D, Marenzi G, De Metrio M, Giesen PL, Balduini A, Veglia F, Gertow K, Biglioli P, Tremoli E. Tissue factorin patients with acute coronary syndromes: expression in platelets, leukocytes, and platelet- leukocyte aggregates. Arterioscler ThrombVasc Biol 2008; 28:947-953.

52. Káplár M, Kappelmayer J, Veszprémi A, Szabó K, Udvardy M. The possible association of in vivo leukocyte-platelet heterophilic aggregateformation and the development of diabetic angiopathy. Platelets 2001; 12:419-422.

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53. Hu H, Li N, Yngen M, Ostenson CG, Wallén NH, Hjemdahl P. Enhanced leukocyte-platelet cross-talk in Type 1 diabetes mellitus:relationship to microangiopathy. J Thromb Haemost 2004; 2:58-64.

54. Káplár M, Kappelmayer J, Kiss A, Szabó K, Udvardy M. Increased leukocyte- platelet adhesion in chronic myeloproliferative disorders withhigh platelet counts. Platelets 2000; 11:183-184.

55. Muszbek L, Bereczky Z, Bagoly Z, Komáromi I, Katona É. Factor XIII: a coagulation factor with multiple plasmatic and cellular functions.Physiol Rev 2011; 91:931-972.

56. Nagy B Jr, Simon Z, Bagoly Z, Muszbek L, Kappelmayer J. Binding of plasma factor XIII to thrombin-receptor activated human platelets.Thromb Haemost 2009; 102:83-89.

57. Magwenzi SG, Ajjan RA, Standeven KF, Parapia LA, Naseem KM. Factor XIII supports platelet activation and enhances thrombus formationby matrix proteins under flow conditions. J Thromb Haemost 2011; 9:820-833.

58. Dale GL, Friese P, Batár P, Hamilton SF, Reed GL, Jackson KW, Clemetson KJ, Alberio L. Stimulated platelets use serotonin to enhance theirretention of procoagulant proteins on the cell surface. Nature 2002; 415:175-179.

59. Devine DV, Andestad G, Nugent D, Carter CJ. Platelet-associated factor XIII as a marker of platelet activation in patients with peripheralvascular disease. Arterioscler Thromb Vasc Biol 1993; 13:857-862.

60. Devine DV, Bishop PD. Platelet-associated factor XIII in platelet activation, adhesion, and clot stabilization. Semin Thromb Haemost1996; 22:409-413.

61. Heemskerk JW, Bevers EM, Lindhout T. Platelet activation and blood coagulation. Thromb Haemost 2002; 88:186-193.62. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine

expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995;184:39-51.63. Maugeri N, Rovere-Querini P, Evangelista V, Covino C, Capobianco A, Bertilaccio MT, Piccoli A, Totani L, Cianflone D, Maseri A,

Manfredi AA. Neutrophils phagocytose activated platelets in vivo: a phosphatidylserine, P- selectin, and {beta}2 integrin-dependentcell clearance program. Blood 2009;113:5254-5265.

64. Jackson SP, Schoenwaelder SM. Procoagulant platelets: are they necrotic? Blood 2010; 116:2011-2018.65. Leytin V, Allen DJ, Mykhaylov S, Lyubimov E, Freedman J. Thrombin-triggered platelet apoptosis. J Thromb Haemost 2006; 4:2656-2663.66. Mason KD, Carpinelli MR, Fletcher JI, Collinge JE, Hilton AA, Ellis S, Kelly PN, Ekert PG, Metcalf D, Roberts AW, Huang DC, Kile BT.

Programmed anuclear cell death delimits platelet life span. Cell 2007; 128:1173-1186.67. Albanyan AM, Harrison P, Murphy MF. Markers of platelet activation and apoptosis during storage of apheresis- and buffy coat-

derived platelet concentrates for 7 days. Transfusion 2009; 49:108-117.68. Nurden AT, Freson K, Seligsohn U. Inherited platelet disorders. Haemophilia 2012; 18 Suppl 4:154-160.69. Schlammadinger A, Tóth J, Nagy B Jr, Fazakas F, Hársfalvi J, Kappelmayer J, Muszbek L, Radványi G, Boda Z. Bernard-Soulier szindróma: a

herediter thrombocytopéniák ritka oka. Hemat Transzf 2007; 40:40-46.70. Kerényi A, Szegedi I, Sarudi S, Kappelmayer J, Kiss C, Muszbek L. Glanzmann thrombasthenia II. típusa. Esetleírás. Klin Kisérl Lab Med 1999;

25:162-168.71. Ault KA, Rinder HM, Mitchell J, Carmody MB, Vary CP, Hillman RS. The significance of platelets with increased RNA content (reticulated

platelets). A measure of the rate of thrombopoiesis. Am J Clin Pathol 1992; 98:637-646.72. Matic GB, Chapman ES, Zaiss M, Rothe G, Schmitz G. Whole blood analysis of reticulated platelets: improvements of detection and assay

stability. Cytometry 1998; 34:229-234.73. Harrison P, Robinson MS, Mackie IJ, Machin SJ. Reticulated platelets. Platelets 1997; 8:379-383.74. Lakkis N, Dokainish H, Abuzahra M, Tsyboulev V, Jorgensen J, De Leon AP, Saleem A. Reticulated platelets in acute coronary syndrome: a

marker of platelet activity. J Am Coll Cardiol 2004; 44:2091-2093.75. McCabe DJ, Harrison P, Sidhu PS, Brown MM, Machin SJ. Circulating reticulated platelets in the early and late phases after

ischaemic stroke and transient ischaemic attack. Br J Haematol 2004; 126:861-869.76. Würtz M, Grove EL, Wulff LN, Kaltoft AK, Tilsted HH, Jensen LO, Hvas AM, Kristensen SD. Patients with previous definite stent thrombosis

have a reduced antiplatelet effect of aspirin and a larger fraction of immature platelets. JACC Cardiovasc Interv 2010; 3:828-835.77. Haas S, Walenga JM, Jeske WP, Fareed J. Heparin-induced thrombocytopenia: the role of platelet activation and therapeutic implications.

Semin Thromb Hemost 1999; 25 Suppl 1:67-75.78. Lee DH, Warkentin TE, Denomme GA, Hayward CP, Kelton JG. A diagnostic test for heparin-induced thrombocytopenia: detection of platelet

microparticles using flow cytometry. Br J Haematol 1996; 95:724-731.79. Tomer A, Masalunga C, Abshire TC. Determination of heparin-induced thrombocytopenia: a rapid flow cytometric assay for direct

demonstration of antibody-mediated platelet activation. Am J Hematol 1999; 61:53-61.80. Oláh Z, Kerényi A, Kappelmayer J, Schlammadinger A, Rázsó K, Boda Z. Rapid- onset heparin-induced thrombocytopenia without previous

heparin exposure. Platelets 2012; 23:495-498.81. Szász R, Dale GL. Thrombospondin and fibrinogen bind serotonin-derivatized proteins on COAT-platelets. Blood 2002; 100:2827-2831.82. Prodan CI, Joseph PM, Vincent AS, Dale GL. Coated-platelets in ischemic stroke: Differences between lacunar and cortical stroke. J Thromb

Haemost 2008; 6:609-614.83. Prodan CI, Vincent AS, Dale GL. Coated-platelet levels are elevated in patients with transient ischemic attack. Transl Res 2011; 158:71-75.84. Prodan CI, Vincent As, Dale GL. Coated platelet levels correlate with bleed volume in patients with spontaneous intracerebral

hemorrhage. Stroke 2010;41:1301-1303.85. Saxena K, Pethe K, Dale GL. Coated-platelet levels may explain some variability in clinical phenotypes observed with severe hemophilia. J

Thromb Haemost 2010;8:1140-1142.86. Simon Z, Kiss A, Erdödi F, Setiadi H, Debreceni IB, Nagy B Jr, Kappelmayer J.Protein phosphatase inhibitor calyculin-A modulates activation

markers in TRAP- stimulated human platelets. Platelets 2010; 21:555-562.

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87. Topalov NN, Yakimenko AO, Canault M, Artemenko EO, Zakharova NV, Abaeva AA, Loosveld M, Ataullakhanov FI, Nurden AT, AlessiMC, Panteleev MA. Two types of procoagulant platelets are formed upon physiological activation and are controlled by integrinα(IIb)β(3). Arterioscler Thromb Vasc Biol 2012; 32:2475-2483.

88. Heemskerk JW, Mattheij NJ, Cosemans JM. Platelet-based coagulation: different populations, different functions. J Thromb Haemost2012; DOI:10.1111/jth.12045.

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MEASUREMENT OF SOLUBLE BIOMARKERS BY FLOW CYTOMETRY

Péter Antal-Szalmás, Béla Nagy Jr, Ildikó Beke Debreceni, János KappelmayerDepartment of Laboratory Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary


Péter Antal-SzalmásDepartment of Laboratory Medicine, Medical and Health Science Center, University of Debrecen, HungaryTel: +36 52-340-006Fax: +36 52-417-631e-mail: [email protected]

Key words: bead technology, soluble markers, flow cytometry

ABSTRACT

Microparticle based flow cytometric assays for determination of the level of soluble biomarkers are widely used in severalresearch applications and in some diagnostic setups. The major advantages of these multiplex systems are that they can measurea large number of analytes (up to 500) at the same time reducing assay time, costs and sample volume. Most of these assaysare based on antigen-antibody interactions and work as traditional immunoassays, but nucleic acid alterations – by using specialhybridization probes –, enzyme- substrate or receptor-ligand interactions can be also studied with them. The applied beads arenowadays provided by the manufacturers, but cheaper biological microbeads can be prepared by any user. One part of thesystems can be used on any research or clinical cytometers, but some companies provide dedicated analyzers for their multiplexbead arrays. Due to the high standardization of the bead production and the preparation of the assay components the analyticalproperties of these assays are quite reliable with a wide range of available applications. Cytokines, intracellular fusion proteins,activated/phosphorylated components of different signaling pathways, transcription factors and nuclear receptors can beidentified and quantitated. The assays may serve the diagnostics of autoimmune disorders, different viral and bacterial infections,as well as genetic alterations such as single nucleotide polymorphisms, small deletions/insertions or even nucleotide tripletexpansions can be also identified. The most important principles, technical details and applications of these systems arediscussed in this short review.

INTRODUCTION

Changes in the concentration of different proteins in human serum or plasma may indicate the presence of several normal orpathological processes and show the progression of different disorders. The measurement of total serum protein and itssubfractions or quantitation of individual proteins have been applied in routine laboratory diagnostics for several decades. Thefirst assays determined the total protein content of serum using mainly protein-specific dyes and spectrophotometry, while thesubfractions were analyzed by electrophoresis. Later, serum proteins were studied based on their enzymatic activity as thesemolecules were easily measured by the conversion of their substrate to a colored product measured by photometry. A majorstep was the introduction of antibody-antigen based immunoassays that could considerably enhance the number of testedindividual proteins. Several different types of immunoassays were then developed for measuring the light scatter alterationcaused by the immunocomplexes formed due to the antibody-antigen interactions (turbidimetry, nephelometry). Furthermore,a variety of methods was introduced that used antigens or antibodies labeled with radioactive, enzyme, fluorescence orluminescence components in competitive or sandwich immunoassays. Several of these tests were applied on automatedimmunoanalyzers enhancing the efficacy and the precision of these assays. In spite of all advantages the major drawback ofthese methods is the measurement of only one single analyte at one time that increases the time period and sample volume

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MEASUREMENT OF SOLUBLE BIOMARKERSBY FLOW CYTOMETRY

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required for analysis. Multiplex immunoassays can solve these problems. One possibility is the development of antibody-basedprotein chips – like the RANDOX QuantiPlasma 69 system – where antibodies are coated to small carriers (chips) and can measurelarge number of proteins – 69 plasma proteins in that case – simultaneously in a “sandwhich” or competitive way. The otheroption is the application of multicolor flow cytometric bead arrays.The introduction of microparticles into flow cytometry opened a brand new field for determination of the level of solublebiomarkers in different human body fluids. The method is very robust, and the standardized production of the microbeadsprovides a very reproducible and accurate technique. The introduction of multicolor beads further enhanced its applicability,since each type (color) of microparticles supports an individual test, and thus a large number of assays can be run at the sametime. Besides high-throughput, the flexibility of the systems is also excellent as different vendors offer high freedom for thecustomers in choosing the proper bead mixes for the proper clinical/research solutions. Another advantage of the technique isthe versatility of the system, since several biomolecules and markers can be tested on the same platform from proteins to nucleicacids and from enzyme-substrate interactions to receptor-ligand binding [1-4].

BASIC PRINCIPLES OF THE MICROPARTICLE BASED FLOW CYTOMETRIC ASSAYS

Protein determination - immunoassaysThe most widely used application of the system is based on antigen-antibody interaction and works as most of the classicalimmunoassays. The solid base is provided by the fluorescently labeled microparticles, and in the “two-site” or “sandwich” typeof this assay a capture antibody is coated on them. This antibody recognizes the serum protein of interest and the detection ofthe captured protein is managed by a fluorescently labeled second antibody (Figure 1A). The second type of the immunoassaysbased on the competition of different assay components (competitive immunoassays). One option is when a capture antibodyis coated on the beads and a fluorescently labeled antigen is competing with the appropriate “cold” antigens of the testedsample. The higher the amount of the protein in the serum the lower the signal we can detect. In case of another type thetested antigen is immobilized on the surface of the beads, and their soluble analogues in the tested sample compete for bindingto a fluorescently labeled antigen-specific antibody (Figure 1B). The microparticles make also possible the detection ofautoantibodies when autoantigens are present on the surface of the beads. The bound autoantibodies are identified by asecondary anti-human immunoglobulin specific antibody labeled by a second/third fluorescent dye. Similarly, microbe-specificantibodies can be identified supporting the rapid diagnostics of different bacterial/viral/fungal infections (Figure 1C) [1-4].

Nucleic acid detection – hybridizationAnother possible application of the system is the detection of certain nucleic acid sequences based on the hybridization tooligonucleotide probes coated on the microparticles. Such an assay makes possible for instance the identification of singlenucleotide polymorphisms (SNPs) or point mutations in the tested sample. The target region of the genomic DNA is amplifiedin a specific PCR reaction using fluorescently labeled primers and then the single stranded PCR products are hybridized to theprobes present on the beads. In this case two types of beads capture the labeled DNA; one carries an oligonucleotide containingthe wild type nucleotide of the SNP, while the other bead carries the mutant one. The positivity/negativity and the fluorescentintensity of the two beads measured will define the proper genotype. The system can also support gene-expression studies.The RNA extracted from e.g. two differently treated cell populations can be transcribed into cDNA, and one of the cDNA samplescan be simultaneously labeled fluorescently. The beads of the multiplex system contain special probes for special genes, andthe competition between the two cDNA samples for binding to these probes will provide information about the relativeexpression of these genes in the two differently treated samples (Figure 1D) [4-5].

Enzyme-substrate and receptor-ligand assaysA more research and development orientated application of the system is the search for proper substrates or ligands for certainenzymes or receptors. These assays can work simply using fluorescently labeled test ligands and receptor coated particles, or acompetitive assay is also applicable, when known ligand(s) of the receptor labeled fluorescently compete with the new testmolecules for binding to receptors coated onto the microparticles (Figure 1E, F) [6,7].

TYPES OF THE MICROPARTICLES

The most widely used microparticles are plastic beads that can be easily manufactured with a high throughput and a largeprecision. The latest technologies also allow the color-coding up to 500 different bead entities; furthermore, the size of thebeads can be also a usable variable in creating more-and-more complex arrays. Certain companies also incorporate the possibilityfor magnetic separation of the beads in their systems [1-3].Concerning the utility of these beads one part of the assays produced by different companies are predefined, because well-defined capture antibodies are coated on the surface of the microparticles. Thus, these beads are suitable for measuring onlyone type of an analyte. Another option is the purchase of “multifunctional” beads coated by free carboxyl or amino groups

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suitable for covalent adhesion of certain proteins according to the design of the user. Avidin/streptavidin or goat anti-mouseimmunoglobulin label can also support the labeling of the beads by user defined molecules and antibodies [2].Historically, other easily accessible bioparticles were also applied in similar microparticle assays. More than a decade ago asimple flow cytometric test was developed by our group that could measure the serum soluble CD14 (sCD14) concentrationin an easy way. The membrane bound CD14 of isolated human monocytes competed with sCD14 in the serum of the testedpatients for the binding to a fluorescently labeled anti-CD14 monoclonal antibody. The fluorescence intensity of the monocyteswas measured by flow cytometry and the lower the fluorescence we observed the higher the sCD14 concentration was in theserum. A serial dilution of a serum sample with known concentration of sCD14 served as calibrator for the assay (Figures 1Band 2) [8].The application of normal human cells as target microparticle itself might also supply the detection of different autoantibodies(such as anti-neutrophil cytoplasmic antibodies). Moreover, certain microbes can help the identification of specific antimicrobialantibodies thought to be important in the diagnostics of special immune-mediated disorders (e.g. in colitis ulcerosa or Crohn’sdisease). A typical example is the evaluation anti-Saccharomyces cerevisiae antibodies (ASCA) using bakers’ yeast suspension asa substrate particle of the detection. In these cases the gates are set around the target bioparticles based on their scatterproperties and the fluorescence of the secondary anti-human immunoglobulin antibody is measured (Figure 1C) [2].A rather novel approach is to create low cost and easily reproducible bioparticles (mainly bacteria and fungi) coated byavidin/streptavidin for capture of biotin-labeled antibodies, antigens or even special nucleotide probes. The fluorescent labelingof these microbes is easy handled and even multiplex labeling is achievable that makes this type of particles a real alternativeespecially in research applications [4].

SINGLE VERSUS MULTIPLEX SYSTEMS

The prototype of these systems was developed using only single beads that did not differ much from single ELISA-s in terms oftheir throughput and efficacy. A large breakthrough was the introduction of multiple beads with different sizes and then withdifferent labeling color. In the recent systems the microparticles have a certain color that identifies the assay (and the proteindetected by the beads) and in the case of multiplex systems each type of bead (recognizing different soluble markers) has a

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Figure 1Basic principles of the microparticle based flow cytometric assays. Green circles: fluorescently labeled microparticles; red circles: fluorophoreused for detection; Ag: antigen; Ab: antibody; GaHu-Ig: Goat anti-human immunoglobulin specific antibody.

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separate color. The detection antibody is labeled by another fluorophore that helps the detection and quantitation of thecaptured protein. In multiplex systems the color of the beads – measured in one or two fluorescence channels of the flowcytometer – identifies the measured protein, while the fluorescence intensity, provided by the dye of the detection antibodyon a separate fluorescence channel, clarifies the amount of protein bound to a certain bead. In each assay of the multiplexsystem where we gate on a single bead population the fluorophore of the detection antibody can be the same in the case of alldetected proteins. The amount of protein in the tested sample can be accurately quantitated using standard samples containingknown amounts of the tested protein. Obviously, in case of multiplex systems one standard curve is required for each measuredanalyte [1-3].One possible limitation of the multiplex system is the different dynamic range of tested analytes. In the case of a certainexperimental setup such as in the stimulation of isolated cells the amount of one analyte released from cells can be very high,that require the dilution of the sample, while the others have to be measured in the undiluted supernatant. As nowadays theflexibility of the assays is very high – with proper pilot experiments the over- or under- expressed analytes can be identified and– the matching tests can be freely selected. Another important issue is the optimization of the assay conditions (washingbuffer, pH, ionic strength) that is suitable for each antigen-antibody pair. Moreover, the multiplexing of the assays always resultsin the elevation of the background noise and the decrease in the sensitivity of the tests that has to be kept in mind. Finally, thepossible interactions of certain antibodies or the so called “matrix effect” can alter the properties of individual tests. Becauseof these effects, an assay that is running properly on its own will not be automatically reliable in a multiplex system [1,2].

DETECTION PLATFORMS

One part of the multiplex flow cytometric bead assays can be used with everyday clinical or research flow cytometers. TheBecton-Dickinson BD™ Cytometric Bead Array (CBA) system supports the majority of Coulter, DAKO, Partec and Becton-Dickinson flow cytometers. The kit includes the appropriate data for the setup of the equipment, reagents, calibrators and evenan analysis software that can evaluate the list mode data of the measurement in an Excel-base format. Another approach isoffered by the Luminex’s xMAP Technology. In that case the company developed special flow cytometers designed for multiplexbead analysis. The Luminex 100/200™ and FLEXMAP 3D® systems have similar components like any flow cytometers but theycan be used only for measurement and evaluation of the xMAP multiplex bead arrays. A very similar approach is offered by Bio-Rad’s Bio-Plex MAGPIX, Bio-Plex 200, and Bio-Plex 3D systems. In contrast to these instruments that identify the beads basedon their fluorescent labels the Copalis (Coupled Particle Light Scattering) system of Diasorin discriminates between singlebeads with different diameters and aggregated beads based on their scatter properties [1-3].

POSSIBLE APPLICATIONS

The number of possible tests available in the form of multiplex bead assay is radically increasing. Several companies provide

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Figure 2Determination of sCD14 in human serum by a simple flow cytometry based competitive immunoassay. Isolated monocytes were incubatedwith the appropriate dilution of the tested serum and a FITC-labeled anti-CD14 monoclonal antibody. The cells were washed and thefluorescence intensity of the monocytes – gated based on their scatter properties – was analyzed by a Coulter EPICS XL flow cytometer (A). Asthe sCD14 in the serum competes with the mCD14 of the monocytes for binding to the labeled anti-CD14 antibody, the higher the concentrationof sCD14 in the sample the lower the fluorescence intensity we detected on monocytes. A serial 2-fold dilution of a serum sample containingknown amount of sCD14 served as standard. The representative FL1 histograms of the standard samples and the standard curve created fromthese data is presented on Figure 2B.

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a long range of tests covering different areas of research and diagnostic use. These possible applications are listed in Figure3. One of the first areas that is still the most widely used application of the cytometric bead array systems is the measurementof different cytokines in body fluid of patients and controls or in the supernatant of differently stimulated cells. The “cytometricbead array cytokines” search in the PubMed database provides 300 hits and the first publication was prepared in 2001, describingthe simultaneous measurement of 6 cytokines in tear samples [9]. Nowadays assays are available for IL-1 to IL-18, TNFαand β, INFα, β and γ, more than 10 chemokines and soluble cytokine receptors. In addition, complex kits are available forTh1/Th2/Th17 (IL-2, IL-4, IL-6, IL-10, IFNγ, TNFα, IL-17α) or inflammatory (IL-6, IL-10, IL-12, IFNγ, TNFα, MCP-1) cytokines [1-3].Figure 4 illustrates one of our experiments aiming the multiplex determination of cytokines in LPS stimulated whole blood andplatelet rich plasma (PRP).The detection of intracellular proteins in cell lysates is also a valuable technique that can serve diagnostic and research purposes,too. The recently developed assay for measuring the bcr/abl fusion protein in the lysate of white blood cells is a unique methodfor the rapid diagnosis of chronic myeloid or acut lymphoblastic leukaemia. The assay is based on fluorescent beads coated withmonoclonal antibodies that can recognize the bcr part of the bcr/abl fusion proteins independently of the type of the fusion(the minor or the major breakpoint cluster region of the bcr gene is involved in the translocation). The lysate of the white bloodcells is mixed with the beads and then a fluorescently-labeled anti-abl antibody detects the bound proteins [10]. In a recentwork, we evaluated the analytical properties of this assay. The intra- assay CV% of positive controls was respectable as 3.7%was in the normal and 10% was found in the pathological range. The cut-off for mean fluorescence intensity was 112 thatprovided 100% sensitivity and 100% specificity for the assay. The results of the cytometric bead assay showed 100% agreementwith the molecular biological tests used for bcr/abl transcript detection [11]. Recently, a similar assay was introduced for thedetection of the PML/RARA fusion protein [12].Another broad field within the detection of intracellular proteins is the identification of activated/phosphorylated componentsof different signaling pathways. Dozens of signaling molecules can be tested including the MAPK-family, Wnt/GSK/Akt or theJAK/STAT pathway in addition to the activation of several growth factor receptors (like EGFR, IGFR, VEGFR, c-kit, c-Met) [1,3].Several publications are also available in the international literature. Koeper and colleagues described a skin implant model totest the toxic/irritating effect of different skin sensitizers by simultaneous testing the phosphorylation of the MAP- kinases, STAT1

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Figure 3Possible applications of the flow cytometric multiplex bead assays.

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and Phospholipase C γ [13]. Dawes et al. studied the activation of the pERK, pP38, and pJNK upon TGFβ2 stimulation in lensepithelial cells during the differentiation to myofibroblasts [14], while Wong and colleagues investigated the MAPK and NF-κBactivity in IL-25 stimulated T helper lymphocytes [15]. Similarly, intracellular nuclear receptors can be also detected using themultiplex bead arrays like in the work of Schneiderhan-Marra, where 56 proteins – including oestrogen receptor – were testedin breast cancer needle biopsy samples [16].A very dynamically developing field is the use of the multiplex bead arrays for identification of valuable biomarkers in the earlydiagnostics or follow-up of malignant disorders [1]. Opstal-van Winden et al. tested simultaneously 10 biomarkers in the serumof breast cancer patients but they could not identify a panel that could help the early diagnosis of this disorder [17]. Kim andcoworkers analyzed 3 markers (CA-125, transthyretin, and apolipoprotein A1) of ovarian cancer in a multiplex system and couldshow that the combination of these markers was superior to the analytical performance of the individual ones [18]. In a veryrecent elegant study, 30 biomarkers were tested in the serum of non-small cell lung cancer patients using a multiplex bead array.Twenty-three parameters differed between the controls and the patients, and the combined application of the 5 highest-rankedbiomarkers (α1-antitrypsin, CYFRA 21-1, IGF-1, RANTES, AFP) could discriminate between controls and NSCLC patients with highaccuracy [19].

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Figure 4Multiplex detection of human cytokines in LPS-stimulated whole blood and PRP.Citrated whole blood or PRP samples were stimulated by Re-LPS (10 µg mL-1) for 1 hour at 37°C and G-CSF, IL-1ra, IL-6, TNFα, IL-4, IL-10 andIFNγ levels were determined simultaneously. Briefly, microparticles with pre-coated antigen-specific antibodies on their surface were addedto the samples and pipetted into wells on a microplate. For the analysis of the levels of microparticle-bound antigen, a biotinylatedsecondary antibody and a streptavidin-PE conjugate were applied. After microparticles were suspended in buffer, results were determined bya Luminex 100TM analyzer (Luminex, Austin, TX, USA). One laser was microparticle-specific to show which antigen level was under investigation,and another laser determined the fluorescent signal, which was directly proportional to the concentration of antigen bound. On Figure 3A-Gwe can see the standard curves of each cytokine measured. Figure 3H presents the concentration of each cytokines in the plasma ofLPS stimulated whole blood. There was a significant increase (P<0.01) in the level of two cytokines due to the Re-LPS stimulation versus thecontrol sample (IL-1ra: 4.98±0.42 pg mL-1 vs. 1.42±0.39 pg mL-1) (TNF-α: 1.0±0.12 pg mL-1 vs. 0.1±0.06 pg mL-1). The cytokine concentrationsin the PRP were below the detection levels independently of LPS stimulation.

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For the diagnostics of autoimmune disorders a relatively large number of multiplex assays are already available. The complexevaluation of antinuclear antibodies (ANA), extractable nuclear antigens (ENA) or anti-neutrophil cytoplasmic antibodies(ANCA) can be performed using these systems. Furthermore, disease specific panels are also available for celiac disease, systemiclupus erythematosus, autoimmune thyroid disorders and vasculitis (1,20,21). An import application of the technique is the HLA-typing of donors and recipients of kidney transplantation and the identification of donor-specific serum antibodies [1,22].The application of multiplex bead assays can support diagnostic microbiology, too [1]. Yu et al. developed an inhibitory multiplexbead assay to determine 26 serotypes of pathogenic Streptococcus pneumoniae strains [23], while Wagner and colleaguesdescribed an assay that can simultaneously identify antibodies specific for the outer surface protein A, C and F of Borreliaburgdorferi serving the diagnostics of Lyme disease in both humans and animals [24]. Furthermore, food poisoning caused byEscherichia coli O157:H7 can be identified easily and rapidly using this multiplex bead system [25]. Commercially available arraysare ready to use for Epstein-Barr and Herpes virus identification, and for respiratory tract viruses andmeasles/mumps/rubeola/varicella detection [1].A promising new are of this field is the combination of multiplex bead arrays with the DNA/RNA based molecular biologictechniques. The system is suitable to analyze the presence of SNPs like the IL-6 SNP distribution in different ethnic groups [26]or the 22 SNPs of the ABC transporter genes in healthy individuals [27]. It can also detect small insertions or deletions in theBRCA1 gene [5] or even the number of nucleotide triplet expansions in Huntington disease [5]. Furthermore, the cytometricarray can be used for gene expression studies [28].

DISCUSSION

The development of microbeads based flow cytometric assays for measuring soluble biomarkers started more than 10 yearsago. At that time we thought that this system would make a revolution in laboratory diagnostics as covering even severalhundreds of analytes in one run could replace the currently available laboratory techniques. The system is indeed a robust one,the number of the available assays is enormous and there are certain areas – especially in research – where this technique reallybecame a number one choice. On the other hand, the still high costs of the systems and also the evolution of laboratoryautomation and the development of the classical laboratory tests did not let the change occur. Nevertheless, these microparticlebased assays are very much useful, and will support our research and certain diagnostic activities in the future.

ACKNOWLEDGEMENTSThis work was supported by the TÁMOP-4.2.2.A-11/1/KONV-2012-0025 project. This project was co-supported with theinvolvement of the European Union and the European Social Foundation. This work was also supported by the National Officefor Research and Technology of Hungary (TECH-09-A1-2009-0113; mAB-CHIC).

References

1. Elshal MF, McCoy JP. Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods 2006; 38:317-323.

2. Hsu HY, Joos TO, Koga H. Multiplex microsphere-based flow cytometric platforms for protein analysis and their application in clinicalproteomics - from assays to results. Electrophoresis 2009; 30:4008-4019.

3. Morgan E, Varro R, Sepulveda H, Ember JA, Apgar J, Wilson J, Lowe L, Chen R, Shivraj L, Agadir A, Campos R, Ernst D, Gaur A. Cytometricbead array: a multiplexed assay platform with applications in various areas of biology. Clin Immunol 2004; 110:252-266.

4. Pataki J, Szabó M, Lantos E, Székvölgyi L, Molnár M, Hegedüs E, Bacsó Z, Kappelmayer J, Lustyik G, Szabó G. Biological microbeads for flow-cytometric immunoassays, enzyme titrations, and quantitative PCR. Cytometry A 2005; 68:45-52.

5. Imre L, Balogh I, Kappelmayer J, Szabó M, Melegh B, Wanker E, Szabó G. Detection of mutations by flow cytometric melting point analysisof PCR products. Cytometry A 2011; 79:720-726.

6. Breuer S, Sepulveda H, Chen Y, Trotter J, Torbett BE. A cleavage enzyme-cytometric bead array provides biochemical profiling of resistancemutations in HIV-1 Gag and protease. Biochemistry 2011; 50:4371-4381.

7. Curpan RF, Simons PC, Zhai D, Young SM, Carter MB, Bologa CG, Oprea TI, Satterthwait AC, Reed JC, Edwards BS, Sklar LA. High-throughputscreen for the chemical inhibitors of antiapoptotic bcl-2 family proteins by multiplex flow cytometry. Assay Drug Dev Technol 2011; 9:465-474.

8. Antal-Szalmás P, Szöllősi I, Lakos G, Kiss E, Csípő I, Sümegi A, Sipka S, van Strijp JA, van Kessel KP, Szegedi G. A novel flow cytometric assayto quantify soluble CD14 concentration in human serum. Cytometry 2001; 45:115-123.

9. Cook EB, Stahl JL, Lowe L, Chen R, Morgan E, Wilson J, Varro R, Chan A, Graziano FM, Barney NP. Simultaneous measurement of six cytokinesin a single sample of human tears using microparticle-based flow cytometry: allergics vs. non-allergics. J Immunol Methods 2001; 254:109-118.

10. Weerkamp F, Dekking E, Ng YY, van der Velden VH, Wai H, Böttcher S, Brüggemann M, van der Sluijs AJ, Koning A, Boeckx N, VanPoecke N, Lucio P, Mendonça A, Sedek L, Szczepański T, Kalina T, Kovac M, Hoogeveen PG, Flores- Montero J, Orfao A, Macintyre E, Lhermitte

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L, Chen R, Brouwer-De Cock KA, van der Linden A, Noordijk AL, Comans-Bitter WM, Staal FJ, van Dongen JJ; EuroFlow Consortium. Flowcytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients. Leukemia 2009; 23:1106-1117.

11. Hevessy Z, Hudák R, Kiss-Sziráki V, Antal-Szalmás P, Udvardy M, Rejtő L, Szerafin L, Kappelmayer J. Laboratory evaluation of a flowcytometric BCR-ABL immunobead assay. Clin Chem Lab Med 2011; 50:689-692.

12. Dekking EH, van der Velden VH, Varro R, Wai H, Böttcher S, Kneba M, Sonneveld E, Koning A, Boeckx N, Van Poecke N, Lucio P, MendonçaA, Sedek L, Szczepański T, Kalina T, Kanderová V, Hoogeveen P, Flores-Montero J, Chillón MC, Orfao A, Almeida J, Evans P, Cullen M, NoordijkAL, Vermeulen PM, de Man MT, Dixon EP, Comans- Bitter WM, van Dongen JJ; EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708). Flowcytometric immunobead assay for fast and easy detection of PML-RARA fusion proteins for the diagnosis of acute promyelocytic leukemia.Leukemia 2012; 26:1976-1985.

13. Koeper LM, Schulz A, Ahr HJ, Vohr HW. In vitro differentiation of skin sensitizers by cell signaling pathways. Toxicology 2007; 242:144-152.14. Dawes LJ, Sleeman MA, Anderson IK, Reddan JR, Wormstone IM. TGFbeta/Smad4- dependent and -independent regulation of human lens

epithelial cells. Invest Ophthalmol Vis Sci 2009; 50:5318-5327.15. Wong CK, Li PW, Lam CW. Intracellular JNK, p38 MAPK and NF-kappaB regulate IL-25 induced release of cytokines and chemokines from

costimulated T helper lymphocytes. Immunol Lett 2007; 112:82-91.16. Schneiderhan-Marra N, Sauer G, Kazmaier C, Hsu HY, Koretz K, Deissler H, Joos TO. Multiplexed immunoassays for the analysis of breast

cancer biopsies. Anal Bioanal Chem 2010; 397:3329-3338.17. Opstal-van Winden AW, Rodenburg W, Pennings JL, van Oostrom CT, Beijnen JH, Peeters PH, van Gils CH, de Vries A. A bead-based

multiplexed immunoassay to evaluate breast cancer biomarkers for early detection in pre-diagnostic serum. Int J Mol Sci 2012; 13:13587-13604.

18. Kim YW, Bae SM, Lim H, Kim YJ, Ahn WS. Development of multiplexed bead- based immunoassays for the detection of early stage ovariancancer using a combination of serum biomarkers. PLoS One 2012; 7(9):e44960.

19. Lee HJ, Kim YT, Park PJ, Shin YS, Kang KN, Kim Y, Kim CW. A novel detection method of non-small cell lung cancer using multiplexed bead-based serum biomarker profiling. J Thorac Cardiovasc Surg 2012; 143:421-427.

20. González-Buitrago JM. Multiplexed testing in the autoimmunity laboratory. Clin Chem Lab Med 2006; 44:1169-1174.21. Maecker HT, Nolan GP, Fathman CG. New technologies for autoimmune disease monitoring. Curr Opin Endocrinol Diabetes Obes 2010;

17:322-328.22. Ziemann M, Schönemann C, Bern C, Lachmann N, Nitschke M, Fricke L, Görg S. Prognostic value and cost-effectiveness of different

screening strategies for HLA antibodies prior to kidney transplantation. Clin Transplant 2012; 26:644-656.23. Jigui Yu, Jisheng Lin, Kyung-Hyo Kim, William H. Benjamin, Jr., and Moon H. Nahm. Development of an Automated and Multiplexed

Serotyping Assay for Streptococcus pneumoniae Clin Vaccine Immunol 2011; 18:1900–1907.24. Wagner B, Freer H, Rollins A, Erb HN. A fluorescent bead-based multiplex assay for the simultaneous detection of antibodies to B.

burgdorferi outer surface proteins in canine serum. Vet Immunol Immunopathol 2011; 140:190-198.25. Kelly M. Leach, Joyce M. Stroot, and Daniel V. Lim Same-Day Detection of Escherichia coli O157:H7 from Spinach by Using

Electrochemiluminescent and Cytometric Bead Array Biosensors Appl Enviroment Microbiol 2010; 76:8044–8052.26. Ivanova M, Ruiqing J, Kawai S, Matsushita M, Ochiai N, Maruya E, Saji H. IL-6 SNP diversity among four ethnic groups as revealed by bead-

based liquid array profiling. Int J Immunogenet 2011; 38:17-20.27. Koo SH, Ong TC, Chong KT, Lee CG, Chew FT, Lee EJ. Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex

suspension array. Biol Proced Online 2007; 9:27-42.28. Wedemeyer N, Göhde W, Pötter T. Flow cytometric analysis of reverse transcription- PCR products: quantification of p21(WAF1/CIP1)

and proliferating cell nuclear antigen mRNA. Clin Chem 2000; 46:1057-1064.

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CALCIUM INFLUX CHARACTERISTICS DURING T LYMPHOCYTE ACTIVATION MEASURED WITH

FLOW CYTOMETRY

Enikő Biró1, Barna Vásárhelyi2,3, Gergely Toldi1

1First Department of Pediatrics, Semmelweis University, Budapest H‐1083, Hungary2Department of Laboratory Medicine, Semmelweis University, Budapest H‐1083, Hungary3Research Group of Pediatrics and Nephrology, Hungarian Academy of Sciences, Budapest H‐1083, Hungary


Enikő BiróFirst Department of Pediatrics, Semmelweis University, Bókay u. 53, Budapest H‐1083, HungaryTel: +36 30 997 6217Fax: +36 1 3138212e‐mail: [email protected]

Key words: Ca2+ influx, flow cytometry, autoimmune disease, T lymphocyte activation

ABSTRACT

T lymphocytes are of paramount importance in many intercellular reactions, such as the regulation of the inflammatory responseand immune reactivity. Until the recent past, single‐cell techniques were used for the investigation of calcium influx during Tlymphocyte activation. Therefore, over the recent years we have created a novel approach that allows simultaneous recordingof calcium influx in several lymphocyte subsets using flow cytometry. Our research group developed a robust algorithm (FacsKin)for the evaluation of the acquired data that fits functions to median values of the fluorescent marker of interest and calculatesrelevant parameters describing each function.Over the recent years, we have investigated calcium influx characteristics applying this method in a number of autoimmunedisorders and under different physiological conditions (such as the neonatal period and pregnancy). In this review, we aim togive a brief summary of our findings and of the common characteristics of calcium influx in the investigated disorders, namely:multiple sclerosis (MS), rheumatoid arthritis (RA), type 1 diabetes mellitus (T1DM), ankylosing spondylitis (AS), and preeclampsia(PE). Based on our results, a number of dominant features were identified that were present in most of the investigatedautoimmune diseases.

INTRODUCTION

T lymphocytes are of paramount importance in many intercellular reactions, such as the regulation of the inflammatory responseand immune reactivity. Upon the engagement of the T cell receptor (TCR), a number of signal transduction pathways culminatein the transient elevation of the cytoplasmic calcium concentration ([Ca2+]cyt). First, Ca2+ is released from intracellular storesthat is followed by further Ca2+ entry from the extracellular space through the store‐operated calcium release activated calcium(CRAC) channels. In the course of lymphocyte activation, K+ channels maintain the driving force for sustained Ca2+ influx asthey grant the efflux of K+ from the cytoplasm, thus conserving an electrochemical potential gradient between the intra‐ andextracellular spaces. There are two major types of K+ channels in T cells: the voltage‐gated Kv1.3 and the Ca2+‐activated IKCa1channels. The relation between the Ca2+ currents through CRAC channels and the efflux of K+ makes the proliferation andactivation of lymphocytes sensitive to pharmacological modulation of Kv1.3 and IKCa1 channels, and provides an opportunityfor targeted intervention. Specific inhibition of these channels results in a diminished Ca2+ influx in lymphocytes and a lowerlevel of lymphocyte activation.

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Previous data suggest that selective modulation of lymphocyte activation through specific inhibition of K+ channels may be apossible therapeutic approach for the treatment of autoimmune disease [1‐6]. Beeton et al. showed that terminally differentiatedeffector memory T (TEM) cells play a pivotal role in the pathogenesis of autoimmunity [1]. For instance, Wulff et al. suggest thatdisease causing TEM cells are able to home to inflamed tissues in the CNS and exhibit immediate effector function in autoimmunedisease. They described that the characteristic K+ channel phenotype of TEM cells in MS is Kv1.3high IKCa1low, contrasting naïveand central memory T (TCM) cells, which exhibit a Kv1.3low IKCa1high channel phenotype [3]. Therefore the therapeuticrelevance of specific Kv1.3 channel inhibitors is of outstanding interest, as they may offer the possibility for selective modulationof pathogenic TEM cells, while naïve and TCM cells (needed for physiological immune responses) would escape the inhibitionthrough upregulation of IKCa1 channel expression. Beeton et al. demonstrated that the symptoms of experimental autoimmuneencephalitis, a murine model of MS, significantly improved after treatment with selective Kv1.3 inhibitors [1, 4]. Besides naiveand memory cells, helper (CD4) and effector (CD8) T lymphocytes modulate the autoimmune response in different ways. CD4cells influence the immune system by producing cytokines, while CD8 cells are capable of causing immediate cell destruction.The two main arms of CD4 lymphocytes are Th1 and Th2 cells. Th1 cells mainly produce pro‐inflammatory cytokines, thussustaining autoimmune reactions. However, Th2 cells reduce the inflammatory response by producing anti‐inflammatory cytokines.Until the recent past, single‐cell techniques were used for the investigation of Ca2+ influx during lymphocyte activation. Therehas been no high‐throughput method available to study the kinetics of lymphocyte activation in more subsets simultaneously.Single‐cell techniques are restricted by not being capable of characterizing this process in complex cellular systems, thereforeignoring the interaction between the different lymphocyte subsets that may modulate the course of their activation. Therefore,over the recent years we have created a novel approach that allows simultaneous recording of Ca2+ influx in several lymphocytesubsets. Our research group developed a robust algorithm (FacsKin) that fits functions to median values of the data of interestand calculates relevant parameters describing each function. By selecting the best fitting function, this approach provides anopportunity for the mathematical analysis and statistical comparison of kinetic flow cytometry measurements of distinct samples.For example, in case of Ca2+ influx measurements, the software fits a double‐logistic function on each recording. This function isused to describe measurements that have an increasing and a decreasing intensity as time passes. The software also calculatesparameter values describing each function, such as the Maximum value, the Time to reach maximum value or the Area Underthe Curve (AUC). These parameters represent different characteristics of lymphocyte Ca2+ influx kinetics (Figure 1).Details of the method of measurements were described earlier [7, 8]. Briefly, peripheral blood mononuclear cells (PBMCs) wereseparated from freshly drawn peripheral venous blood of the investigated subjects. Cell surface markers were applied to separatethe lymphocyte subsets of interest. Cells were loaded with Ca2+ sensitive dyes and activated with aspecific stimuli. Cellfluorescence data were measured and recorded for 10 minutes in a kinetic manner on flow cytometer. Our studies were adheredto the tenets of the most recent revision of the Declaration of Helsinki.Over the recent years, we have investigated Ca2+ influx characteristics in a number of autoimmune disorders and under differentphysiological conditions. In this review, we aim to give a brief summary of our findings and of the common characteristics ofCa2+ influx in autoimmune disease.

MULTIPLE SCLEROSIS

Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system. The autoimmune reaction primarilydestroys the myelin sheath covering the nerve cells. T lymphocytes play a key role in the pathogenesis of MS. They regulate theongoing inflammatory process of the central nervous system (CNS) which leads to the damage of the myelin sheath and axons.However, only a small part of T lymphocytes are myelin‐specific autoreactive cells. Besides the demyelinating action of thesecells of the CNS, the activation of peripheral lymphocytes also contributes to the pathogenesis and disease progression [9]. Inour investigations we measured samples of healthy individuals and MS patients with no immunomodulatory therapy, as well asMS patients treated with interferon beta (IFN‐b), currently regarded as the most effective therapy of MS.In our study we discovered increased sensitivity of CD8 cells to Kv1.3 channel inhibition in MS. Therefore, from the CD4‐CD8point of view, we demonstrated specific immunomodulation that may be beneficial in the therapy of MS via the selectivesuppression of CD8 effector lymphocytes over CD4 helper cells. However, this specificity is not present within the CD4 subset,since our results suggest that Th1 and Th2 cells are similarly suppressed upon the inhibition of Kv1.3 channels. Since the cytokinebalance is of utmost importance in the regulation of the autoimmune reaction, the inhibition of the Th2 subset would probablyresult in a setback of therapeutic efforts in MS. Our findings are relevant in the light of observations regarding the contributionof TEM cells to the development of MS, as described above. Although the Kv1.3high IKCa1low pattern is found in both CD4+and CD8+ TEM cells, we can assume that the majority of TEM cells are CD8+, since TEM cells express immediate effector function.This provides an explanation for the increased sensitivity of CD8 cells to Kv1.3 channel inhibition in MS in our study.We have also demonstrated important differences in Ca2+ influx kinetics and lymphocyte K+ channel function in MS patientswithout IFN‐b compared with healthy individuals. The selective blocker of the lymphocytes Kv1.3 channel might be a promisingdrug in combination therapy, supplementing the presently used IFN‐b treatment. Our results indicate that IFN‐b therapy causes

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compensatory changes only in the Th1 subset in MS regarding Ca2+ influx kinetics and the function of K+ channels. However,the increased function of the Th2 subset, and therefore the production of anti‐inflammatory cytokines is less affected. Thismight contribute to a more effective treatment of the autoimmune process in this disorder [10].

RHEUMATOID ARTHRITIS

The short‐term activation of peripheral blood and synovial fluid T lymphocytes, especially that of autoreactive T cells plays acrucial role in initiating and maintaining the chronic inflammation in the joints of patients suffering from rheumatoid arthritis(RA). These cells regulate the inflammatory process resulting in the destruction of the articular cartilage and also play a role inextra‐articular damage. We investigated T lymphocyte Ca2+ influx kinetics following activation in peripheral blood of recentlydiagnosed RA patients compared to healthy individuals.Our results indicate that margatoxin (MGTX), a specific blocker of the Kv1.3 channel acts differentially on Ca2+ influx kinetics inmajor peripheral blood lymphocyte subsets of RA patients:

Th2 and CD8 cells are inhibited more dominantly than Th1 and CD4 cells. Kv1.3 channel expression in RA patients and healthysubjects Based on our results, the immunomodulatory effect of Kv1.3 channel inhibition is predominantly seen in cytotoxic (CD8)T cells in RA. However, this effect does not seem to be as specific as reported before by Beeton and colleagues in case of TEMcells [13], since anti‐inflammatory Th2 cells are also affected to a noteworthy extent. This subset has an important role incounterbalancing the peripheral lymphocytes might be the differential distribution of disease‐associated autoreactive T cells inRA patients on local and systemic levels. In the synovial fluid (locally), autoreactive TEM cells, expressing high numbers of Kv1.3channels are abundantly present. However, this Kv1.3 pattern was not detected in peripheral blood T cells, because autoreactiveTEM cells are infrequent in the circulation. Peripheral blood T cells were predominantly found to be naive and TCM cells [13]. A

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Figure 1A: Dot‐plot of a kinetic flow cytometry recording, where each dot represents one cell of the measured sample. Horizontal axis: Time (inseconds), Vertical axis: Fluorescent intensity of Ca2+ sensitive dyes (relative parameter value). Green line: median value of the fluorescentparameter of interest.B: FacsKin software fits a double‐logistic function (red line) on each recording. Horizontal axis: Time (in seconds), Vertical axis: Fluorescentintensity of Ca2+ sensitive dyes (relative parameter value). The calculated parameters: Max: the peak value of Ca2+ influx, tmax: time to reachMax value, AUC: Area Under the Curve.

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reason for limited specificity of Kv1.3 inhibition in peripheral lymphocytes might be the differential distribution of disease‐associated autoreactive T cells in RA patients in the synovial fluid and the circulation [11].

TYPE 1 DIABETES

Type 1 diabetes mellitus (T1DM) is an autoimmune disease affecting the pancreas by destroying the insulin‐producing beta cells.Due to the lack of insulin glucose levels increase in blood and in urine. Without insulin treatment T1DM is a fatal disease.In lymphocytes of healthy subjects both Kv1.3 and IKCa1 channels contribute to the maintenance of Ca2+ influx upon activation.On the contrary, the sensitivity of T1DM lymphocytes to the inhibition of Kv1.3 channels is increased. Our data indicate that byspecific inhibition of Kv1.3 channels, lymphocyte activation can be modulated in T1DM. It has been hypothesized that beneficialeffects of Kv1.3 channel inhibition by MGTX are due to the dominance of Kv1.3 channels on activated TEM cells [12, 13]. It wasalso presumed that MGTX would inhibit the activation of CD8 lymphocytes responsible for cytotoxic destruction of pancreaticbeta cells. Nevertheless, our findings obtained with a functional method (i.e. kinetic flow cytometry measurements) provideclear evidence for Kv1.3 channels to have an important role in each lymphocyte subset in T1DM, including Th2 lymphocytesacting as counterbalancing factors in the development of T1DM through the production of antiinflammatory cytokines [14].Therefore, administration of Kv1.3 channel inhibitors would not have an exclusive effect on cells responsible for the autoimmuneresponse in T1DM, but may have an impact on the activation characteristics of immune cells in general, leading to unpredictablealterations. For this reason, further studies are needed to describe the effects of the application of specific Kv1.3 inhibition andthe extent of beneficial consequences in T1DM [8].

ANKYLOSING SPONDYLITIS

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease, the best characterized of the diseases belonging to theconcept of spondylarthritides. The pathogenesis of AS is still unclear, but it is considered to be a systemic autoimmune disease[15]. This is supported by the number of alterations found in lymphocyte subgroups in peripheral blood. Specifically, increasednumbers of circulating Th2 helper lymphocytes [16] were reported in AS. Along with the alterations observed in cell prevalenceone can assume that T cell activation properties may also be altered in AS. We investigated the short‐term T cell activationcharacteristics in AS before and during infliximab (IFX) therapy [17].CD4 and CD8 cells presented with a delayed increase in cytoplasmic Ca2+ levels after activation in AS compared to controls.With IFX, therapy the delay in CD4+ cytoplasmic Ca2+ levels did not normalize in AS. For CD8+ cells, cytoplasmic andmitochondrial Ca2+ kinetics during activation normalized by week 6 on IFX (but not on week 2).Of note, the increase of cytoplasmic Ca2+ levels in CD4 and CD8cells from AS is delayed compared to controls. The delayedCa2+ response of AS fits well into the observations done in in vitro tests with T cells exposed to TNF‐α. Church et al. reportedthat TNF‐α suppressed the Ca2+ peak after PHA stimulation; they have suggested that either signalling pathways upstream ofCa2+ mobilisation or the Ca2+ signalling itself were impaired by prolonged TNF‐α exposure [18]. In AS, only one study has beenperformed with a more robust analytical approach: Lee et al. did not observe a significant difference in intracellular Ca2+ betweenAS patients and normal controls in activated peripheral blood mononuclear cells [19]. The inconsistency between their and ourresults is probably due to different methodology and cell types investigated [17].

HEALTHY PREGNANCY AND PREECLAMPSIA

In healthy pregnancy (HP), the maternal immune system needs to acquire tolerance to protect the developing fetus from harmfulimmunological reactions. If this tolerance is impaired, a general maternal immune response may arise, resulting in systemicinflammation. This has been suggested to be a major factor in the pathogenesis of preeclampsia (PE) [20, 21]. This disorder ischaracterized by hypertension, proteinuria, edema and endothelial dysfunction usually evolving in the third trimester ofpregnancy. We compared the activation‐elicited Ca2+ influx in major peripheral T lymphocyte subsets of HP and PE women tothat in non pregnant women and tested the alteration of Ca2+ influx upon treatment with specific inhibitors of the Kv1.3 andIKCa1 K+ channels.Our findings suggest that the Ca2+ influx kinetics in activated T lymphocytes markedly differs in HP compared to non pregnantwomen: the decreased activation of the Th1 subset may partly be responsible for the well‐established Th2/Th1 skewness in HP[22, 24]. Indeed, the maintained activation properties of Th1 lymphocytes in patients with PE may contribute to the lack of Th2dominance associated with normal pregnancy. Similarly, CD8 cells in PE do not acquire suppressed activation kinetics either.[23] Thus, the decrease in their cytotoxic activity characteristic for HP is not present in PE. It is of particular interest that Ca2+influx of Th2 lymphocytes in HP was insensitive to K+ channel inhibition, while Ca2+ influx decreased significantly in non pregnantupon treatment with specific channel blockers. Of note, Th2 lymphocytes in PE presented with non pregnant‐like characteristicsand were sensitive to K+ channel inhibition as well. While it is unclear whether the resistance or sensitivity of Th2 lymphocytes

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to K+ channel inhibition is reflected in

Th2 function, it is tempting to speculate that this may be an element contributing to the Th2 shift present in HP, but absent inPE. This hypothesis may be supported by reports suggesting that the shape of Ca2+ influx influenced by K+ channel functionsmay determine the cytokine production profile of helper T lymphocytes. Interestingly, other differences were also observedbetween HP and PE. While Ca2+ influx in CD8 and Th1 lymphocytes was resistant to K+ channel inhibition in PE, that of HPlymphocytes was sensitive. Again, while it is unclear whether the resistance of Th1 lymphocytes to K+ channel inhibition isreflected in their function, the insensitivity of the Th1 subset in PE may be linked to the Th1 skewness. Comparing the inhibitoryproperties of lymphocyte K+ channels in non pregnant women and PE, we found that – similarly to the Ca2+ influx kinetics –they are comparable in all investigated subsets, except for the CD8 lymphocytes. Our results indicate marked differences of Ca2+influx kinetics and sensitivity to lymphocyte K+ channel inhibition in major lymphocyte subsets between non pregnant, HP andPE lymphocytes. It is of interest that these properties in PE are more comparable to non pregnant than to HP.These results suggest that there is a characteristic pattern for Ca2+ influx and its sensitivity to K+ channel inhibition in HP thatis missing in PE. This raises the notion that lymphocyte Ca2+ handling upon activation may have a role in the characteristicimmune status of healthy pregnancy [24].

NEONATES

Decreased functionality of neonatal T cells is a widely recognized experimental and clinical phenomenon. Reduced functioningis well characterized by a lower level of cytokine production compared with adult T cells [25, 26]. Several factors might beresponsible for the decreased cytokine expression compared with that of adult lymphocytes. Among many others, propositionsinclude naivity of neonatal lymphocytes. The majority of these cells is naive (CD45RA) lymphocytes in contrast to adults, whereeffector (CD45RO) cells dominate [26].Another possible factor might be the impairment of mechanisms regulating short‐term activation of lymphocytes comparedwith adults. Kv1.3 and IKCa1 lymphocyte K+ channels are essential components of these mechanisms. We hypothesized thatshort‐term T lymphocyte activation properties are different in neonates compared with adults. The aim of our study was tocharacterize the Ca2+ influx kinetics upon activation in major T lymphocyte subsets in the neonate and its sensitivity to thespecific inhibition of Kv1.3 and IKCa1 lymphocyte K+ channels, important regulators of Ca2+ influx.

Ca2+ influx following PHA activation of T lymphocytes markedly differs in the neonate from tha in the adult. Upon treatment oflymphocytes with selective inhibitors of the Kv1.3 and IKCa1 channels (MGTX and TRAM, respectively), Ca2+ influx reductionwas not demonstrated in neonatal lymphocytes only in CD8 subsets. The finding that neonatal lymphocytes are less sensitiveto the specific inhibition of K+ channels compared with adults may be due to altered functionality or a lower expression of thesechannels. Therefore, we measured the expression of Kv1.3 K+ channels on the investigated lymphocytes. Instead of lower values,we found increased expression of Kv1.3 channels on neonatal CD4, CD8 and Th2 lymphocytes compared with adults. Thus, theoption that the decreased sensitivity of lymphocytes to K+ channel inhibition is due to lower channel expression should beexcluded. Based on the lower sensitivity of lymphocyte Ca2+ influx to inhibition at higher channel expression values, it isreasonable to postulate that neonatal Kv1.3 channels are functionally altered. Signs of altered sensitivity to K+ channel inhibitionwere present in all major lymphocyte subsets investigated (i.e. Th1, Th2, CD4 and CD8 cells). The only subset in which the short‐term activation was inhibited significantly by specific blockers of both Kv1.3 and IKCa1 channels was CD8 lymphocytes. However,even in this case, the level of inhibition did not reach the extent described in adults in spite of high Kv1.3 expression values. Thissuggests that our observations are generally characteristic of all lymphocyte subpopulations studied. Furthermore, an interestingobservation is that the decreased Ca2+ influx found in the CD8 subset in neonates after MGTX and TRAM treatment is coupledwith a massive elevation in the expression of Kv1.3 channels by this subset. However, no correlation was detected betweenCa2+ influx parameters and channel expression data; thus, a causative relation between the two findings is unlikely.Our results may partly explain why neonatal lymphocytes are less responsive to activating stimuli and, hence, exert a lowerintensity of immune response. We demonstrated that short‐term activation and associated intracellular Ca2+ influx kinetics arelower in neonatal lymphocytes compared with adults. This phenomenon is associated with the altered function of lymphocyteK+ channels. Our results improve the understanding of the mechanisms that prevent neonatal T lymphocytes from adequateactivation upon activating stimuli. They show that the functional impairment of lymphocyte K+ channels may be of importancein those mechanisms [27].

SUMMARY

Based on our results, a number of dominant features were identified that were present in most of the investigated autoimmunediseases. First, the time when the peak of Ca2+ influx was reached decreased in autoimmune patients compared to healthy

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individuals, indicating that these cells are in a state of sustained reactivity due to the ongoing autoimmune reaction.Earlier studies were limited to the investigation of K+ channels in naive and memory lymphocytes. As an extension of thesefindings to other significant T lymphocyte subsets, in Th1 cells in patients suffering from an autoimmune disease we detecteda different pattern of sensitivity to the inhibition of lymphocyte K+ channels compared to healthy individuals: a greater decreaseof Ca2+ influx upon the inhibition of the Kv1.3 channel than that of the IKCa1 channel was observed. On the contrary, in healthyindividuals the IKCa1 channels had a more effective inhibition profile compared to the Kv1.3 channels. This finding is of specialinterest, since Th1 cells are regarded as key players in the mediation of pro‐infalmmatory responses.However, the selectivity of the investigated inhibitors (MGTX and TRAM) was limited in our experiments, as they did not onlyaffect a single subset, as previously suggested. Although in earlier observations the inhibition of Kv1.3 channels specificallyblocked the function of TEM cells, our investigations extending to other significant T lymphocyte subsets demonstrated that theinhibitory effect is present not only in disease‐associated CD8 and Th1 cells, but also in the anti‐inflammatory Th2 subset. Theinduced decrease in their function could lead to unexpected potential side‐effects in vivo and also in a setback of therapy, sinceTh2 cells are responsible for anti‐inflammatory responses.The advantage of our newly developed method compared with the single‐cell techniques is that it enables characterizing theprogress of lymhocyte activation in complex cellular systems, since the interaction between the different cell subsets is alsotaken into account. Substances produced during lymphocyte activation (e.g. cytokines, chemokines) modulate the function ofother cell subsets. Thus cell activation does not only affect one cell type but indirectly takes an effect on other cells in the sample.Accordingly, different lymphocyte subsets might be regarded as a network, where cells are in connection with the onessurrounding them. Furthermore, not only K+ channel blockers, but other molecules and drugs might be screened. Investigatingother lymphocyte subsets of interest is also possible with our method depending on the type of surface markers used fordiscriminating the cell types. Furthermore, our method may play a key role in several other fields of basic and clinical researchwhere the characterization of different intracellular kinetic processes are needed that can be identified with fluorescent reagents(e.g. the generation of reactive oxygen radicals, alterations of membrane potential, etc). Further details of our algorithm and itsapplication can be found at www.facskin.com.

ACKNOWLEDGEMENT

The preparation of this manuscript was supported by grant OTKA 101661.

References:

1. Beeton C, Wulff H, Standifer NE, Azam P, Mullen KM, Pennington MW, Kolski‐Andreaco A, Wei E, Grino A, Counts DR, Wang PH, LeeHealeyCJ, Andrews BS, Sankaranarayanan A, Homerick D, Roeck WW, Tehranzadeh J, Stanhope KL, Zimin P, Havel PJ, Griffey S, Knaus HG, NepomGT, Gutman GA, Calabresi PA, Chandy KG. Kv1.3 channels are a therapeutic target for T cellmediated autoimmune diseases. Proc Natl AcadSci USA 2006; 103:17414‐17419.

2. Chandy KG, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD. K+ channels as targets for specific immunomodulation. TrendsPharmacol Sci 2004; 25:280‐289.

3. Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, Beeton C, Chandy KG. The voltage‐ gated Kv1.3 K+ channel in effector memory T cellsas new target for MS. J Clin Invest 2003; 111:1703‐1713.

4. Rangaraju S, Chi V, Pennington MW, Chandy KG. Kv1.3 potassium channels as a therapeutic target in multiple sclerosis. Expert OpinTher Targets 2009; 13:909‐924.

5. Rus H, Pardo CA, Hu L, Darrah E, Cudrici C, Niculescu T, Niculescu F, Mullen KM, Allie R, Guo L, Wulff H, Beeton C, Judge SI, Kerr DA, KnausHG, Chandy KG, Calabresi PA. The voltage‐gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosisbrain. Proc Natl Acad Sci USA 2005; 102:11094‐11099.

6. Varga Z, Csepany T, Papp F, Fabian A, Gogolak P, Toth A, Panyi G. Potassium channel expression in human CD4+ regulatory and naïve T cellsfrom healthy subjects and multiple sclerosis patients. Immunol Lett 2009; 124:95‐101.

7. Kaposi AS, Veress G, Vásárhelyi B, Macardle P, Bailey S, Tulassay T, Treszl A. Cytometry‐acquired calcium‐flux data analysis in activatedlymphocytes. Cytometry A 2008; 73:246‐253.

8. Toldi G, Vásárhelyi B, Kaposi A, Mészáros G, Pánczél P, Hosszufalusi N, Tulassay T, Treszl A. Lymphocyte activation in type 1 diabetes mellitus:the increased significance of Kv1.3 potassium channels. Immunol Lett 2010; 133:35‐41.

9. Martino G, Furlan R, Brambilla E, Bergami A, Ruffini F, Gironi M, Poliani PL, Grimaldi LM, Comi G. Cytokines and immunity in multiplesclerosis: the dual signal hypothesis. J Neuroimmunol. 2000; 109:3‐9.

10. Toldi G, Folyovich A, Simon Z, Zsiga K, Kaposi A, Mészáros G, Tulassay T, Vásárhelyi B. Lymphocyte calcium influx kinetics in multiple sclerosistreated without or with interferon β. J Neuroimmunol 2011; 237:80‐86.

11. Toldi G, Bajnok A, Dobi D, Kaposi A, Kovács L, Vásárhelyi B, Balog A. The effects of Kv1.3 and IKCa1 potassium channel inhibition on calciuminflux of human peripheral T lymphocytes in rheumatoid arthritis. Immunobiology 2012 May 23 [Epub ahead of print]

12. Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, Beeton C, Chandy KG. The voltage‐gated Kv1.3 K+ channel in effector memory T cellsas new target for MS. J Clin Invest 2003; 111:1703‐1713.

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13. Chandy KG, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD. K+ channels as targets for specific immunomodulation. TrendsPharmacol Sci 2004; 25:280‐289.

14. Yoon JW, Jun HS. Cellular and molecular pathogenic mechanisms of insulin‐dependent diabetes mellitus. Ann NY Acad Sci 2001; 928:200‐211.

15. Colbert RA, Delay ML, Klenk EI, Layh‐Schmitt G. From HLA‐B27 to spondyloarthritis: a journey through the ER. Immunol Rev 2010; 233:181‐202.

16. Yang PT, Kasai H, Zhao LJ, Xiao WG, Tanabe F, Ito M. Increased CCR4 expression on circulating CD4+ T cells in ankylosing spondylitis,rheumatoid arthritis and systemic lupus erythematosus. Clin Exp Immunol 2004; 138:342‐347.

17. Szalay B, Mészáros G, Cseh Á, Ács L, Deák M, Kovács L, Vásárhelyi B, Balog A. Adaptive immunity in ankylosing spondylitis: phenotype andfunctional alterations of T‐cells before and during infliximab therapy. Clin Dev Immunol 2012; 2012:808724.

18. Church LD, Goodall JE, Rider DA, Bacon PA, Young SP. Persistent TNF‐α exposure impairs store operated calcium influx in CD4+ Tlymphocytes. FEBS Letters 2005;579:1539‐1544.

19. Lee HT, Chen WS, Chen MH, Tsai CY, Chou CT. The expression of proinflammatory cytokines and intracellular minerals in patients withankylosing spondylitis. Formosan J Rheumatol 2009; 23:40‐46.

20. Saito S, Shiozaki A, Nakashima A, Sakai M, Sasaki Y. The role of the immune system in preeclampsia. Mol Aspects Med 2007; 28:192‐209.21. Redman CW, Sargent IL. Immunology of pre‐eclampsia. Am J Reprod Immunol 2010;63:534‐543.22. Darmochwal‐Kolarz D, Leszczynska‐Gorzelak B, Rolinski J, Oleszczuk J. T helper 1‐ and T helper 2‐type cytokine imbalance in pregnant

women with pre‐eclampsia. Eur J Obstet Gynecol Reprod Biol 1999; 86:165‐170.23. Darmochwal‐Kolarz D, Saito S, Rolinski J, Tabarkiewicz J, Kolarz B, Leszczynska‐ Gorzelak B, Oleszczuk J. Activated T lymphocytes in

preeclampsia. Am J Reprod Immunol 2007; 58:39‐45.24. Toldi G, Stenczer B, Treszl A, Kollár S, Molvarec A, Tulassay T, Rigó J, Vásárhelyi B. Lymphocyte calcium influx characteristics and their

modulation by Kv1.3 and IKCa1 channel inhibitors in healthy pregnancy and preeclampsia. Am J Reprod Immunol 2011;65:154‐63.25. Cohen SB, Perez‐Cruz I, Fallen P, Gluckman E, Madrigal JA. Analysis of the cytokine production by cord and adult blood. Hum Immunol

1999; 60:331‐336.26. García Vela JA, Delgado I, Bornstein, Alvarez B, Auray MC, Martin I, Oña F, Gilsanz F. Comparative intracellular cytokine production by in

vitro stimulated T lymphocytes from human umbilical cord blood (HUCB) and adult peripheral blood (APB). Anal Cell Pathol 2000; 20:93‐98.

27. Toldi G, Treszl A, Pongor V, Gyarmati B, Tulassay T, Vásárhelyi B. T‐lymphocyte calcium influx characteristics and their modulation by Kv1.3and IKCa1 channel inhibitors in the neonate. Int Immunol 2010; 22:769‐774.

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ADVANCES IN ORAL COAGULANTS

Eleanor S. PollakDepartment of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, and Children's Hospital ofPhiladelphia and the Philadelphia VA Medical Center


Eleanor S. Pollak, MD, FCAPAssociate ProfessorDepartment of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania, andChildren's Hospital of Philadelphia and the Philadelphia VA Medical Center 310B Abramson Research Center3615 Civic Center Blvd.Philadelphia, PA 19104Office: (215)590-6117; Fax: (215)590-2320e-mail: [email protected]

Key words: oral anticoagulants, warfarin, pharmacogenetics, dabigatran, rivaroxaban, apixaban

ABSTRACT

This article reviews current and future treatment practices concerning oral anticoagulants. In the second decade of the 21stmillennium clinicians can finally treat thrombotic disease with long-awaited new oral anticoagulant medications. In addition,improvements have been made in managing warfarin, the traditional but far from obsolete medication. The first part of thisreview will cover current advances with warfarin treatment. The second portion will discuss specific active coagulation factorinhibitors, the new oral anticoagulants.

ADVANCES IN ORAL COAGULANTS

WarfarinThe drug warfarin has remained the principal oral anticoagulant medication used to hinder the coagulation waterfall cascade ofproteolytic enzymes.[1] Although warfarin was patented back in the 1940s and was followed by an onslaught of correlatedscientific activity, the actual gene for the warfarin target, Vitamin K epoxide reductase (VKOR), was not identified until 2004.[2,3] This discovery of VKOR has also allowed the medical field to focus on decreasing warfarin‘s dangerous safety profile bygenerating new complementary genetic tests.[4]The warfarin preparation: Technically, the warfarin compound (C19H16O4), [C19H15NaO4 -- commonly known by the brand name:Coumadin ®], is a racemic mixture of the R- and S-enantiomers of 3-(α-acetonylbenzyl)-4-hydroxycoumarin. It is a crystallizedform of warfarin sodium, an isopropanol clathrate, which essentially lacks any of the impurities of its amorphous form. In somecountries, different coumarins with either shorter (acenocoumarol) or longer (phenprocoumon) half-lives are used in place ofwarfarin. Despite worldwide use, tremendous disadvantages still accompany warfarin. Notably, the prescribed drug dosage hasa dangerously narrow therapeutic index. Vast variability exists for warfarin dosage needs depending in part on common patientgenotypes. The frequent genotypes influencing this variability have focused on two principal genetic variant groups. Thesevariants belong to vitamin K-epoxide reductase complex (VKORC1) enzymes as well as cytochrome P450-2C9 (CYP2C9) moleculesand influence drug concentration and metabolism, respectively.[4]


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Warfarin is contraindicated when the risk hazard is greater for hemorrhage than for the benefit provided by anticoagulation.The Prothrombin Time (PT) response due to warfarin may be influenced by a multitude of endogenous and exogenous factors.These not only include therapeutically prescribed medications, but herbal compounds as well as food consumed. In the UnitedStates, at the request of the FDA (United States Food and Drug Administration) in 2006, the company Bristol-Myers Squibbinserted a “black-box warning” label for Coumadin on the risk of major or fatal bleeding.It is essential that a patient’s PT/INR (Internationally Normalized Ratio) be determined on a frequent basis. Thus, warfarin usemust be carefully watched when insufficient laboratory facilities would complicate monitoring. Care should be taken to avoid orprudently consider use with pregnancy [5], blood dyscrasias, and imminent surgery of the CNS, eye or large exposed surfaces.Additionally, unsupervised patients at risk for mishaps pose significant dangers. A non-profit website provides a validatedcalculation tool to assist with warfarin dosing decisions: http://www.warfarindosing.org.[6]

In the mid 1980s, the recognition of problems of non-uniform testing of the PT ultimately led to an international method tostandardize and calibrate warfarin-like anticoagulant compounds. This resulted in the assignment of the World HealthOrganization (WHO) thromboplastin preparation with an International Sensitivity Index (ISI) of 1.1. A comparison ratio to thisISI is measured and reported for each laboratory’s thromboplastin using the unit of the International Normalised Ratios (INR).[7]

Overall, the use of warfarin has improved for compliant, regularly monitored patients. However, many of the extant difficultieshave been greatly decreased with the introduction of new drugs.

New Oral Anticoagulant Medications In this decade new oral anticoagulants are becoming the preferred therapy for indications when warfarin had been the onlyavailable oral anticoagulant therapy for over the previous half a century. The medications, dabigatran, rivaroxaban, abixaban,betrixaban, and edoxaban are small-molecule, selective inhibitors that bind to the active site of coagulation vitamin K dependentfactors IIa or Xa. These recently developed new oral anticoagulants possess general similarities to the chemical structure ofwarfarin. Molecular weights of these new oral anticoagulants are approximately 1.5 to 2 times that of warfarin. (see Table 1).The new oral anti-coagulants are attractive to patients, many healthcare providers, and healthcare system suppliers in partbecause laboratory monitoring is not routinely required; standard fixed doses are prescribed to patients with normal weightsand renal function. This saves time and energy for those patients who would have normally been required to travel to a testingsite for frequent warfarin monitoring. The new anticoagulants also reduce other drawbacks of warfarin including multiple druginteractions and problematic pharmacogenetics. Three of the novel new oral anticoagulants, dabigatran, rivaroxaban, apixabanhave each been tested head-to-head against warfarin in large clinical trials for the indication of treatment of atrial fibrillation(AF).[8-10] Although no trial has prospectively tested these agents against each other, several meta-analyses provide addedperspective regarding the utility and benefits that may be provided by these medications.[11] A semisystematic review andmeta-analysis of 44,563 patients showed the new oral anticoagulants to be superior to warfarin in patients without heart failureregardless of gender or the presence of diabetes. However, additional benefits were not seen alongside the concurrent conditionsof heart failure nor nonparoxysmal atrial fibrillation.[12]

Table 2 provides the specifics of the targeted enzyme, the drug half-life, the bioavailability, the % renal excretion, the doses /dayof medication, possible method of testing if needed, the year of FDA approval for human use, the name of trial and safety risksof bleeding. All of these new drugs reach peak plasma levels between 1 and 4 hours after administration.

However, there remain patient characteristics that commonly influence the safety, efficacy and pharmacokinetics of the neworal anticoagulants. These include obesity, reduced hepatic, gastrointestinal and renal organ function, and contemporaneousprescriptions of interfering medications. It should be noted that factor Xa inhibitors are not devoid of problems and still do havepotential interactions with other compounds including inhibitors and inducers of cytochrome P450 and the P-glycoprotein (P-gp) transporter-mediated drug interactions. Drugs that may be contra-indicated include: NSAIDs, ASA, anti-platelet drugs, protonpump inhibitors, and inhibitors or inducers of P-gp transport or CYP3A4.[13] [14] [15] The following populations were notincluded in most of the major new oral anticoagulant trials: pediatric, pregnant, elderly, and chronically ill patients. In addition,to the cited comparisons with warfarin (see Table 2), new oral anticoagulant apixaban was also shown to be more effective thanaspirin in stroke risk reduction in the AVERROES trial. [15]

Despite the overall attraction of the new anticoagulants, other advantages must be carefully balanced. The major benefit in theuse of warfarin remains the large ratio of its efficacy to its cost and availability. Besides the difficult issue with the newanticoagulants compliance due to the lack of regular monitoring the current high cost of the medications may result in patientssaving money by not filling their prescriptions or reducing the number of pills the patient takes.The most dangerous aspect of the new anticoagulants is the lack specific antidotes to reverse the medication should there be

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a problem with bleeding. This is particularly relevant in the case of catastrophic bleeding due to excess medication. Guidanceon treatment includes quickly providing routine supportive care. Because the new anticoagulants have short durations ofeffectiveness discontinuing the anticoagulant most commonly resolves the problem of excess medication.[13] However, if necessary, activated charcoal may be an option if the ingestion is within several hours of thetreatment. As dabigatran is only 1/3 bound by albumin, hemodialysis is a possibility reversal of dabigatran, particularly in caseswhen poor renal function delays natural drug elimination.[16]. Vitamin K has no indication in the scenario of reversing action ofthe new anticoagulants. However, another major benefit of warfarin includes the effectiveness of administering Vitamin K as anantidote in the event of over-anticoagulation.Specific drug-directed neutralizing antibodies are under development for oral anticoagulants dabigatran and apixaban againstFactors IIa and Xa, respectively.[17, 18]The challenge to physicians is clear. Warfarin's use since the 1950s provides practioners with expertise not yet available when

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Table 1New Oral Anticoagulants

Anti-coagulant Drug Chemical Formula and Molecular Weight Tradename & Company Chemical Structure

Warfarin C19H16O4

308 g/mol

COUMADIN®Bristol-Myers

Squibb

Dabigatranetexilate C34H41N7O5

628 g/mol

PRADAXA®BoehringerIngelheim

Rivaroxaban C19H18ClN3O5S436 g/mol

XARELTO®Bayer/Janssen

Pharmaceutical

Apixaban C25H25N5O4

459/mol

ELIQUIS®Pfizer and

Bristol-MyersSquibb

Edoxaban C24H30ClN7O4S548 g/mol

LIXIANA®Daiichi Sankyo

Table 2Oral Anticoagulant Characteristics

Anti-coagulantDrug

TargetedEnzyme

Half-Life(hrs)

% Bio-avail-ability

RenalExcretion

Method oftesting if needed Dose/ day FDA

approvalNameof Trial

Safety risksfor major

bleeding vs.warfarin

Warfarin Vitamin Kdependent Enzymes

40 92 ProthrombinTime (PT)

1 1954* inhumans

---- ----

Dabigatran Thrombin 12 to 17 6 80 Thrombin Time(TT) or Dilute TT

2 2010 RE-LY Comparable

Rivaro-xaban Factor Xa 9 80 65 anti-Xa 1 2011 ROCKET AF Comparable

Apixaban Factor Xa 9 to 14 50 25 anti-Xa 2 2012 ARIS-TOTLE Superior

P a g e 4 8

using the newer oral anticoagulants. Cost considerations are an extra burden that new medications add to decision-making.The solution to the age-old cost/benefit conundrum and the necessary substantial familiarity with the new drugs are issues tobe solved by experience and time. The end result will be better outcomes for our patients, our guiding mission.

References

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2. Link, K.P. The discovery of dicumarol and its sequels. Circulation, 1959. 19(1): p. 97-107.3. Li, T., Chang, C. Y., Jin, D. Y., Lin, P. J., Khvorova, A., Stafford, D. W, Identification of the gene for vitamin K epoxide reductase. Nature, 2004.

427(6974): p. 541-4.4. Johnson, J. A., Gong, L., Whirl-Carrillo, M., Gage, B. F., Scott, S. A., Stein, C. M., Anderson, J. L., Kimmel, S. E., Lee, M. T., Pirmohamed, M.,

Wadelius, M., Klein, T. E., Altman, R. B., Clinical Pharmacogenetics Implementation Consortium Guidelines for CYP2C9 and VKORC1genotypes and warfarin dosing. Clin Pharmacol Ther, 2011. 90(4): p. 625-9.

5. Blickstein, D. and I. Blickstein, The risk of fetal loss associated with Warfarin anticoagulation. Int J Gynaecol Obstet, 2002. 78(3): p. 221-5.6. Gage, B.F., Eby, C., Johnson, J.A., Deych, E., Rieder, M.J., Ridker, P.M., et al., Use of pharmacogenetic and clinical factors to predict the

therapeutic dose of warfarin. Clin Pharmacol Ther., 2008. 84(3): p. 326-31.7. Thomson, J.M., Tomenson, J.A., Poller, L., The calibration of the second primary international reference preparation for thromboplastin

(thromboplastin, human, plain, coded BCT/253). Thromb Haemost. 1984. 52(3): p. 336-42.8. Connolly, S.J., Ezekowitz, M.D., Yusuf, S. et al, Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med, 2009. 361: p.

1139-51.9. Patel, M.R., Mahaffey, K.W., Garg, J., et al., Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med 2011;365:883-91. N

Engl J Med 2011;365:883-91., 2011. N Engl J Med 2011;365:883-91.: p. 883-91.10. Connolly, SJ, Eikelboom, J., Joyner, C., et al., Apixaban in patients with atrial fibrillation. N Engl J Med 2011. 363: p. 806-17.11. Mantha, S. and J. Ansell, An indirect comparison of dabigatran, rivaroxaban and apixaban for atrial fibrillation. Thromb Haemost, 2012.

108(3): p. 476-84.12. Ahmad Y, L.G., Apostolakis S., New oral anticoagulants for stroke prevention in atrial fibrillation: impact of gender, heart failure, diabetes

mellitus and paroxysmal atrial fibrillation. Expert Rev Cardiovasc Ther., 2012. 10(12): p. 1471-80.13. Kaatz, S., Kouides, P. A., Garcia, D. A., Spyropolous, A. C., Crowther, M., Douketis, J. D., Chan, A. K., James, A., Moll, S., Ortel, T. L., Van Cott,

E. M., Ansell, J. Guidance on the emergent reversal of oral thrombin and factor Xa inhibitors. Am J Hematol, 2012. 87 Suppl 1: p. S141-5.14. Cabral, K.P. and J. Ansell, Oral direct factor Xa inhibitors for stroke prevention in atrial fibrillation. Nat Rev Cardiol, 2012. 9(7): p. 385-91.15. Littrell, R. and Flaker, G. Apixaban for the prevention of stroke in atrial fibrillation. Expert Rev Cardiovasc Ther, 2012. 10(2): p. 143-9.16. van Ryn, J., Stangier, J., Haertter, S., Liesenfeld, K. H., Wienen, W., Feuring, M., Clemens, A., Dabigatran etexilate--a novel, reversible, oral

direct thrombin inhibitor: interpretation of coagulation assays and reversal of anticoagulant activity. Thromb Haemost, 2010. 103(6): p.1116-27.

17. Portola Pharmaceuticals, Portola initiates phase 2 study of PRT4445, universal antidote for factor Xa inhibitor anticoagulants [press release].2012.

18. van Ryn, J., Litzenburger, T., Gan, G., Coble, K. and Schurer, J., In vitro Chacterization, Pharmacokinetics and Reversal of supratherapeuticdoses of dabigatran-induced bleeding in rats by a specific antibody fragment antidote to dabigatran., in American Heart Association:Scientific Sessions. 2012.

19. Horton, J.D., and Bushwick, B.M., Warfarin Therapy: Evolving Strategies in Anticoagulation, Am Fam Physician., 1999 59(3):635-646.20. Potpara, T. S., Polovina, M. M., Licina, M. M., Stojanovic, R. M., Prostran, M. S., Lip, G. Y., Novel oral anticoagulants for stroke prevention

in atrial fibrillation: focus on apixaban. Adv Ther., 2012. 29(6): p. 491-507.21. Doggrell, S.A., More light at the end of the tunnel - apixaban in atrial fibrillation. Expert Opin Investig Drugs, 2012. 21(8): p. 1235-9.22. Hochtl, T. and Huber, K., New anticoagulants for the prevention of stroke in atrial fibrillation. Fundam Clin Pharmacol, 2012. 26(1): p. 47-

53.23. Pollack, C.V., Jr., New oral anticoagulants in the ED setting: a review. Am J Emerg Med, 2012.24. Carter, K. L., Streiff, M. B., Ross, P. A., Wellman, J. C., Thomas, M. L., Kraus, P. S., Shermock, K. M. Analysis of the projected utility of

dabigatran, rivaroxaban, and apixaban and their future impact on existing Hematology and Cardiology Anticoagulation Clinics at The JohnsHopkins Hospital. J Thromb Thrombolysis, 2012. 34(4): p. 437-45.

25. Esmon, C.T., What did we learn from new oral anticoagulant treatment? Thromb Res., 2012. 130(Suppl 1): p. S41-3.

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