Journal of Virological Methods, 35 (1991) 217-226 0 1991 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/91/$03.50 217 VIRMET 01252 Storage and preservation of whole blood samples for use in detection of human immunodeficiency virus type-l by the polymerase chain reaction Steve Kaye, Clive Loveday and Richard S. Tedder Division of Virology, University College and Mi{dlesex School of Medicine, London, U.K. (Accepted 16 July 1991) Summary Methods used in the diagnosis of human immunodeficiency virus type-l (HIV-l) infection by the polymerase chain reaction (PCR) usually require the separation of lymphocytes from a whole-blood sample within 24 hours of patient sampling. A method is described in which blood samples are mixed with a cryopreservative (‘Glycigel’), stored frozen, and DNA suitable for use in an HIV PCR recovered. Samples can be stored at -20°C for up to 3 months and still give positive results with all samples from infected patients; storage at -80°C for at least 3 months shows no loss of titre. The method shows no loss of sensitivity compared to previously described sample preparation methods. Deglycerolised Glycigel supernatants were found to be suitable for conven- tional anti-HIV-l serological studies and loss of sensitivity only represented the dilution effect due to sample preparation. Application of the method as a means of storing samples frozen at the point of sampling and transporting them to a central laboratory for processing is demonstrated using samples taken from HIV-l-infected mothers and their babies. HIV PCR; Sample preparation; DNA extraction Intreduction The polymerase chain reaction (PCR) technique has proved to be a useful tool for the diagnosis of human immunodeficiency virus type-l (HIV-l) Correspondence fo: Steve Kaye, Division of Virology, University College and Middlesex School of Medicine, 67 Ridinghouse Street, London WI, U.K.
Transcript
Journal of Virological Methods, 35 (1991) 217-226 0 1991 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/91/$03.50
217
VIRMET 01252
Storage and preservation of whole blood samples for use in detection of human immunodeficiency virus
type-l by the polymerase chain reaction
Steve Kaye, Clive Loveday and Richard S. Tedder Division of Virology, University College and Mi{dlesex School of Medicine, London, U.K.
(Accepted 16 July 1991)
Summary
Methods used in the diagnosis of human immunodeficiency virus type-l (HIV-l) infection by the polymerase chain reaction (PCR) usually require the separation of lymphocytes from a whole-blood sample within 24 hours of patient sampling. A method is described in which blood samples are mixed with a cryopreservative (‘Glycigel’), stored frozen, and DNA suitable for use in an HIV PCR recovered. Samples can be stored at -20°C for up to 3 months and still give positive results with all samples from infected patients; storage at -80°C for at least 3 months shows no loss of titre. The method shows no loss of sensitivity compared to previously described sample preparation methods. Deglycerolised Glycigel supernatants were found to be suitable for conven- tional anti-HIV-l serological studies and loss of sensitivity only represented the dilution effect due to sample preparation. Application of the method as a means of storing samples frozen at the point of sampling and transporting them to a central laboratory for processing is demonstrated using samples taken from HIV-l-infected mothers and their babies.
HIV PCR; Sample preparation; DNA extraction
Intreduction
The polymerase chain reaction (PCR) technique has proved to be a useful tool for the diagnosis of human immunodeficiency virus type-l (HIV-l)
Correspondence fo: Steve Kaye, Division of Virology, University College and Middlesex School of Medicine, 67 Ridinghouse Street, London WI, U.K.
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infections (Kwok et al., 1987; Ou et al., 1988). The latency of the virus as an integrated proviral DNA copy in CD6positive lymphocytes and macrophages (Dalgleish et al., 1984) makes DNA extracted from peripheral blood mononuclear cells (PBMCs) a convenient sample for use in the PCR. Previous investigators have used cryopreserved PBMCs separated from fresh blood samples within 24’ hours of sampling.
We present here a method of storage and preservation of whole blood samples in a glycerol/gelatin based medium (‘Glycigel’; Huggins, 1973) such that samples may be stored frozen after sampling and DNA suitable for use in PCR extracted up to 3 months later. The method allows samples to be stored simply at the point of sampling and transported to a central laboratory where they can be batch processed. This obviates the need for PBMC preparation and cryopreservation in the field.
An initial evaluation of the method was made using samples collected from asymptomatic HIV-l-infected homosexual men. An application of the method in which samples taken from children born to HIV-l-infected mothers were collected, stored and transported to our laboratory for analysis is described.
Stability studies on samples stored at - 20°C and - 80°C have been carried out.
Materials and Methods
Patients
Two-ml aliquots of heparinised blood from 19 consecutive asymptomatic homosexual men (CDC disease group II or III) sent to the laboratory for other routine investigations were mixed with Glycigel and stored at -20°C or -80°C. Aliquots of the same specimens were stored frozen without Glycigel, and were also separated on Ficoll before storage. Similar specimens were collected and stored from HIV-l-infected mothers (n = 10) and their newborn babies for up to 6 months. Throughout this work samples from seronegative, PCR-negative patients were processed in parallel to control for cross- contamination.
Storage and preservation of samples
Patient blood was mixed thoroughly with an equal volume of melted Glycigel(l0 mM NaCl, 0.5 mM EDTA, 39% glycerol (v/v), 1.5% gelatin (w/v), 0.1% sodium azide (w/v)), and stored frozen at -20°C or -80°C. Samples were frozen. slowly in an insulated box and thawed quickly in a waterbath at 37’C
PBMCs were separated from duplicate blood samples at the time of patient sampling by Ficoll centrifugation, and stored frozen at -80°C in cell-freezing medium (RPM1 containing 30% FCS, 10% DMSO).
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DNA extraction from Glycigel-preserved samples - method A
The method of Higuchi (1989) was used to extract DNA from a cell nucleus pellet. Briefiy, 1 ml of Glycigel-prese~ed sample was thawed, spun for 1 min at 14 000 rpm (15 800 x g) in an Eppendorf centrifuge and the supernatant removed for serological studies. The cell pellet was then washed 3 times with lysis buffer (0.32 M sucrose, 10 mM Tris-HCl, pH 7.5, 5 mM MgC&, 1% Triton X-100 (v/v)) and DNA extracted from the resulting cell nucleus pellet in 100 1.11 of extraction buffer-l (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgClz, 0.01% gelatin {w/v), 0.45% NP40 (v/v), 0.45% Tween 20 (v/v), 60 pg/ ml proteinase K) for 2 h at 6O”C, followed by 10 min at 95°C to inactivate the proteinase K. This extract was used directly in the PCR. Heparinised samples stored without Glycigel were extracted by this method for comparison. DNA was also extracted by this method from lymphocytes separated by Ficoll centrifugation at the time of sampling.
DNA extraction from GlycigeE-preserved samples - method B
4 ml of the Glycigel-preserved sample was thawed, centrifuged at 400 x g for 10 min and the supernatant removed for serological studies. The cells were washed once with 10 ml normal saline containing 50% dextrose, twice with 10 ml normal saline containing 10% dextrose, and once with phosphate-buffered saline. The cells were then fractionated by Ficoll centrifugation and DNA was extracted from the PBMCs in 0.5 ml extraction buffer-2 (10 mM Tris HCl, pH 8.3, 10 mM EDTA, 10 mM NaCl, 0.5% SDS (w/v), 100 fig/ml proteinase K) for 2 h at 60°C. The extracted DNA was purified by extracting twice with phenol~~hlorofo~, and ethanol pr~ipitation. The DNA was finally redis- solved in 100 ~1 water.
DNA from PBMCs separated by Ficoll centrifugation at the time of initial sampling was also extracted by the same method.
PCR
A double. PCR (Simmonds et al., 1990) using nested primers in the gag and pol genes was used for all samples (Table 1). Thermal cycling for the first round consisted of 1 cycle at 94°C 4 min, 35 cycles at 94°C 1 min, 60°C (PO/) or 56°C (gag), 1 min, 72”C, 1 min, and 1 cycle at 72”C, 7 min. The second round consisted of 25 cycles at 94”C, 1 min, 5O”C, 1 min, 72°C 1 min, and 1 cycle at 72”C, 7 min. The reactions were carried out in a 50-~1 reaction mix containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgClz, O:Ol% gelatin (w/v), 1 unit Taq polymerase (‘Amplitaq’, Perkin Elmer Cetus), 200 ,uM each dNTP, 0.1 PM each primer.
The samples were 25 ,ul of DNA extracted from Glycigel samples processed by method A, 2.5 ~1 of DNA extracted from Ficoll separated PBMCs by method A, or 1 pg of DNA extracted from Ficoll-separated PBMCs and purified by method B.
PCR products were analysed by gel electrophoresis on ethidium-bromide- stained 2% agarose gels.
Serological studies
Studies were performed on paired sera and Glycigel supernatants from 9 consecutive homosexual men, stored at -20°C for 28 days. Glycigel supernatants were deglycerolised by loading 2.5 ml of each supernatant onto a disposable Sephadex G25 column (Pharmacia, type PDlO) equilibrated with 25 ml phosphate-buffered saline (PBS), and eluting with 3.5 ml PBS. Assuming the supernatant is a 1:4 dilution of the original plasma, the further processing resulted in a 1:5.6-final dilution. All samples were tested, undiluted, in a commercially available particle agglutination assay (Serodia-HIV, Fujirebio), by Western blotting (DuPont) and in an in-house anti-reverse transcriptase (anti-RT) assay (Loveday et al., 1989); and at final dilutions of 1: 10, 1: 100 and 1:lOOO in a competitive enzyme-linked immunosorbent assay (EIA: Well- cozyme HIV Recombinant, Wellcome Diagnostics).
Results
Comparison of Glycigel and directly frozen samples
Blood samples from 9 patients were stored at -20°C for 28 days frozen directly or preserved in Glycigel. DNA was extracted by method A. The yield of DNA was estimated by ethidium bromide staining and comparison to known standards according to the method of Maniatis et al. (1982) (Table 2).
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The mean yield from the Glycigel preserved samples was 2.9 x higher. PCR using nested primers in the pol gene gave a positive signal in 9 out of 9
samples preserved in Glycigel and 7 out of 9 samples frozen directly.
Comparison of DNA extraction methods on Glycigel-preserved and fresh samples
Glycigel-preserved samples from 11 asymptomatic HIV-1-seropositive homosexual men were stored at -20°C for 7 days. DNA was extracted by the two methods described. DNA was also extracted by the same methods from PBMCs separated by Ficoll centrifugation at the time of sampling.
DNA yields are shown in Table 3; the number of samples giving a positive PCR signal is also shown in Table 3.
A crude titre was estimated for 4 of these samples by carrying out a PCR at sample dilutions of neat, 1: 10, 1: 100 and 1: 1000. A geometric mean titre was calculated (Table 4).
Stability studies
Samples were stored at -20°C and -80°C and DNA was extracted by method A after 3 and 6 months. The numbers of samples giving a positive PCR signal are shown in Table 5. A crude titre was estimated by performing PCR on samples diluted neat, l:lO, 1:lOO and 1:lOOO.
TABLE 2
Yields of and detection of HIV-I in DNA from 9 Cilycigel extracted by method A
preserved and directly frozen samples
Samples
Glycigel preserved Frozen directly
Range Mean yield (pg DNA/ml blood) @g DNA/ml blood)
2&10.0 6.3 I .o-5.0 2.2
PCR (pos./total)
919 719
TABLE 3
Yields of and detection of HIV-I in DNA from I I patients extracted by methods A and B from Glycigel-preserved and Ficoll-separated samples
Samples
Glycigel preserved
Ficoll separated
Extraction Range Mean yield PCR method (pg DNA/ml blood) @g DNA/ml blood) (pos./total)
A 2.8-35.4 pg/ml 14.8 pg/ml II/II
B 0.1-11.7 pg/ml I .5 pg/ml 2/l I
A 0.6-2.3 pg/ml I .3 pg/ml II/II
B 0.6-5.4 pg/ml 3.4 pg/ml II/II
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TABLE 4
Approximate HIV-I PCR titres for DNA from 4 patients extracted by method A from Glycigel- preserved or Ficoll-separated samples
Investigation of babies born to HIV-l-infected mothers
Specimens collected into Glycigel at a hospital in Italy, stored at - 80°C for between 1 week and 3 months, and transported frozen to our laboratory, were tested by PCR using gag and pal primers. ‘Results are shown in Table 7.
Serological results
In all commercially available antibody assays tested and the in-house anti- RT assay all serum and deglycerolised supernatants were positive with loss of sensitivity in the latter that corresponded to the dilution associated with Glycigel sample preparation (Table 8, Fig. 1). In the competitive EIA where lo- fold dilutions of samples were tested all specimens remained positive at a dilution of 1:lOO (actual dilution of Glycigel supernatants was 1:560), and all Glycigel supernatants were negative at a dilution of 1:lOOO (actual dilution was 1:5600).
TABLE 8
Comparative study of matched serum and Glycigel supernatants from 9 consecutive homosexual men showed no divergent results using 4 HIV-I antibody assays
Differences in signal reflect dilution effects of Glycigel supernatant (G. sup.) preparation and with IO-fold dilutions of samples in the competitive EIA all samples remained positive at a dilution of I : 100. Units: competitive EIA-normalised absorbance (cut-off/test): negative < 1, positive > 1; particle agglutination assay (PAA): highest dilution showing agglutination, negative < 1: 16. Western blot interpreted according to manufacturer’s criteria; anti-RT EIA: units of anti-RT activity from a standard curve of a known positive control (S) serially diluted on each test plate (I unit = I : 1 000s).
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gp160. gp120.
~66. --.
p55. p51. N--
gp41. -
~24.
p17.
GS GS GS GS - +
PATIENT 1 2 3 4 CONTROLS
Fig. I. Four typical results from the 9 patients showing Western-blot patterns for both Glycigel supernatants (G) and serum (S), the former showing diminished intensity due to dilution.
Discussion
It is unlikely that current PCR methodology will replace serology as a frontline diagnostic method for the diagnosis of HIV-l infection in adults, or for the screening of blood donations. PCR is, however, useful in making an early diagnosis of neonatal infection since the presence of maternal antibodies in the neonate makes serological diagnosis difficult or impossible before 15 months of age (DeRossi et al., 1988).
This limited use of PCR in routine HIV-l diagnosis, coupled with the technical difficulties involved in carrying out the method and the resources required (Clewley, 1989) is resulting in the method being used in a few specialised centres. There is a requirement, therefore, for samples collected at sites without a PCR facility or a PBMC separation capability to be stored in a way that will allow such samples to be processed at a later date without loss of sensitivity or specificity.
Glycerol preservation methods for whole blood required for transfusion purposes have been in use for many years (Smith, 1950) and we have evaluated the Glycigel method described for preservation of small blood samples
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described by Huggins (1973) for its usefulness in preserving samples for use in HIV-l PCR.
The results show that heparinised whole blood frozen directly gives a poor yield of DNA when extracted by either of the methods presented here.
Glycigel preservation will preserve PBMCs and allow standard Ficoll separation and phenol/chloroform extraction of DNA (method B, Table 3). However, the yields of DNA were low and resulted in many false-negative PCR results. This poor result is probably due to inhibition of the PCR by haemoglobin, a known powerful PCR inhibitor, from red cell fragments which layered with the lymphocytes at the Ficoll interface. Much more successful was the extraction of DNA from a cell nucleus preparation (method A) in which case it is presumed that the Glycigel is preserving the white cell nuclei. DNA extracted by method A from Glycigel samples gives comparable DNA yields, and PCR titres to samples processed conventionally by Ficoll separation of lymphocytes from fresh blood (Tables 3 and 4). It should be noted that the higher DNA yields from Glycigel samples processed by method A result from extraction of DNA from all white cells, and not only PBMCs.
The stability data for -20°C showed about a tenfold reduction in PCR titre of over a period of 3 months, although this did not generate any false-negative results in the samples tested. However, storage at -20°C is not ideal and may potentially generate false negative results, although samples may be satisfactorily stored at this temperature if this is the only freezing facility available. Storage at -80°C showed no reduction in titre after 3 months anti this temperature is therefore to be preferred for long-term storage.
Deglycerolised supernatants from Glycigel-preserved specimens could reliably be used to perform commercially available and in-house antibody assays for HIV-l, with a loss in sensitivity reflecting the dilution effect of sample preparation, and have now been used successfully in the study of over 150 patients.
The results from specimens collected from babies at a hospital in Italy indicate that investigation of patients at a centre with no PCR or lymphocyte separation facilities, at a site remote from where the PCR is to be performed, can be successfully achieved using these methods.
Acknowledgements
We would like to thank Dr Harvey Holmes, MRC AIDS Reagents Project, for supplying Tuq polymerase used in this project, and Dr Antonio Ferrazin Ospedale S. Martino, Genoa, for collection of blood samples in Italy.
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