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Evaluation of a new flow cytometric HPA 1a screening method
Transfusion and Apheresis Science 30 (2004) 89–92 intl.elsevierhealth.com/journals/tras
Evaluation of a new flow cytometric HPA 1a screening method A rapid and reliable tool for HPA 1a screening of blood donors and pregnant women Mette Kjær Killie a
a,*
, Jens Kjeldsen-Kragh b, Ingrid Randen b, Bjørn Skogen a, Anne Husebekk a
Department of Immunology and Transfusion Medicine, University of Northern Norway, 9038 Tromsø, Norway b Ullev al University Hospital, Oslo, Norway Received 25 October 2003; accepted 31 October 2003
Abstract We have evaluated a flow cytometric screening method for identification of HPA la negative individuals, using a commercially available monoclonal anti-CD61 antibody specific for the HPA 1a allotype, and compared the method with an ELISA based method for HPA la phenotyping and two methods for PCR genotyping. HPA 1a phenotyping by fluorochrome conjugated monoclonal anti-HPA la and analysis by flow cytometry is a rapid, reliable and inexpensive technique, suitable for screening purposes. Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction The HPA 1a epitope is located on the glycoprotein IIIa, and has two alleles (a and b) defined by a single amino acid substitution. Anti-HPA 1a antibodies often induce severe neonatal alloimmune trombocytopenic purpura (NAITP) requiring immediate transfusion with HPA 1a negative platelets to the neonate. Screening programs for HPA 1a negative pregnant women or blood donors require a simple and reliable assay. Suitable methods for screening purposes have been available since the introduc-
tion of PCR methods such as 50 nuclease assay (50 NA) or melting curve analysis [1–3], as well as different HPA 1a/b discriminating ELISA methods [4,5]. The identification of a monoclonal anti-HPA 1a specific antibody [6,7] adds a new option for HPA 1 screening. The aim of this study was to evaluate a flow cytometric assay by comparing this new method with established PCR and ELISA typing methods.
2. Materials and methods 2.1. Test samples
*
Corresponding author. Tel.: +47-776-26312; fax: +47-77626304. E-mail address: [email protected] (M.K. Killie).
Anticoagulated blood (EDTA) from pregnant women or blood donors and platelet concentrates produced from buffy-coats from four blood
1473-0502/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.transci.2003.10.004
90
M.K. Killie et al. / Transfusion and Apheresis Science 30 (2004) 89–92
donors were used for platelet phenotyping. The Committee for Medical Human Research Ethics approved the part of the study involving typing of pregnant women. 2.2. Platelet phenotyping by flow cytometry Fluorescein isothiocyanate (FITC)-labelled mouse IgG1 anti-CD61 (clone SZ21, Immunotech, Marseille, France) was used for phenotyping. Forty microlitres of the antibody (final dilution 1:125, 16 lg/ml) were added to 10 ll whole blood, platelet rich plasma or platelet concentrates and incubated for 10 min at room temperature. Five hundred microlitres of PBS containing 0.3% EDTA was added to each sample before analysis. The fluorescence intensities obtained were analysed by FacsCalibur instrument using CellQuest software system (Becton Dickinson, CA, USA). Well-characterised HPA 1a positive and negative platelets were used as controls. 2.3. Platelet genotyping HPA 1a genotyping was carried out by 50 nuclease assay and melting curve analysis as described earlier [1–3]. 2.4. Platelet phenotyping by ELISA HPA 1 phenotyping was performed with an ELISA kit according to the manufacturerÕs instructions (Platelet HPA 1a Typing Assay, DiaMed AG, Switzerland).
3. Results and discussion 3.1. Optimal concentration of FITC-SZ21 In order to determine the optimal concentration of FITC conjugated SZ21, the antibody preparation, which is delivered at a concentration of 2 mg/ ml, was tested in different dilutions against HPA 1aa platelets, and platelets of 1ab and 1bb phenotypes. The fluorescence intensities (FI) were recorded, and the fluorescence ratio (FR) between the FI values for 1aa or 1ab platelets over the FI
for 1bb platelets were calculated (Fig. 1). At the highest concentration of the antibody, the fluorescence ratio was about 1, indicating that unspecific binding to HPA 1a negative platelets occur. By diluting the antibody 50–125 fold, the FR for HPA 1aa and ab platelets increased to about 35 and 15, respectively. Further dilution of the antibody to 1:1000 gave a successive decline in fluorescence ratio. Thus, by using the FITC-conjugated anti-HPA 1a antibody at dilution 1:125, HPA 1aa and 1ab platelets could efficiently be distinguished from HPA 1bb platelets. A PE-conjugated antibody was also tested, but gave low fluorescence ratio (mean 3.8, range 1.3– 7.0) and was not suitable to distinguish between HPA 1a positive and negative samples. The reason for this is not known, but it is conceivable that the large size of the PE molecule may cause steric hindrance for binding of the antibody. 3.2. Phenotyping of fresh platelets by flow cytometry Thirty-four HPA 1a positive samples (HPA 1aa and 1ab) were phenotyped by flow cytometry technique, using the SZ21 antibody at dilution 1/ 125. The mean of the fluorescence intensity (FL1, geometric) for those samples was 158 (1 SD ± 63). All HPA 1a negative samples had FL1 mean values less than 10. 3.3. Phenotyping of stored platelets by flow cytometry In some cases it is desirable to perform typing of platelets that have been stored for several days. Therefore, the flow cytometry phenotyping assay was tested with platelets that were isolated from whole blood that had been stored for 3, 5, 8 and 10 days, respectively. In samples kept for more than 7 days, the peak in the histogram mimicking unstained platelets tended to increase (data not shown). This was probably due to the accumulation of dying cells in the gated region. Therefore we may conclude that samples more than 7 days old are not suited for HPA 1a phenotyping by flow cytometry.
M.K. Killie et al. / Transfusion and Apheresis Science 30 (2004) 89–92
91
40 HPA 1aa/HPA 1bb HPA 1ab/HPA 1bb
35
30
Ratio
25
20
15
10
5
0 1
10
50
125
200
400
800
1000
Titer
Fig. 1. A diagram illustrating optimal dilution of monoclonal anti-HPA 1a (SZ21). An optimal dilution of antibody was 1/50–1/125. X-axis: dilution of antibody. Y-axis: ratio between HPA 1a positive and negative samples.
3.4. Phenotyping of pooled platelet concentrates by flow cytometry In our search for HPA 1a negative donors we performed phenotyping of platelet concentrates produced from pools of four buffy-coats. A histogram showing a large HPA 1a positive population and a smaller HPA 1a negative population indicated that one of the four donors in the pool had the HPA 1a negative phenotype. Each donor from a pool containing HPA 1a negative platelets were tested separately the next time they donated blood. Donors that had both phenotype and genotype HPA 1bb were defined as HPA 1bb donors.
genotyped HPA 1ab. The reason for this HPA 1 phenotype/genotype discrepancy, has not been identified. Discrepancy in genotype and phenotype is not a unique finding and has been published earlier by others; an ARG93GLN substitution, disrupting the HPA 1a epitope, was recently reported [8]. An alternative explanation could be a genetic defect in the HPA 1a allele, causing a lack of expression of the actual protein. Individuals lacking the HPA 1a phenotype, although possessing a defect HPA 1a allele, may be immunised, and should be regarded as HPA 1bb. This finding points out the importance of phenotyping as target for HPA 1a typing. To prevent immunisation by a cell surface antigen, phenotyping is the only method of real interest.
3.5. Comparison of phenotyping with genotyping Testing of 45,960 samples by flow cytometry using the monoclonal SZ21 antibody gave 1121 HPA 1a negative samples, which corresponds well with the expected frequency of HPA 1bb in Caucasians (2.4%). By typing the 1121 samples with PCR technique, 1112 were genotyped HPA 1bb. Nine samples with the phenotype HPA 1bb, were
3.6. Comparison of cost and time investment between the different test systems Cost accounting and time investment for the described screening methods were performed. Phenotyping by flow cytometry was the least expensive, provided that a flow cytometer is already available. Both phenotyping by flow
92
M.K. Killie et al. / Transfusion and Apheresis Science 30 (2004) 89–92 700000 ELISA 5´NA Flow Light cycler
600000
Euro
500000 400000 300000 200000 100000 0 1
1000
2000
4000
8000
16000
32000
Number of samples analysed
Fig. 2. Comparison of time cost (a) of three different HPA 1a typing methods. The graph (x-axis) starts with the investments of necessary equipment, hence 93.094 a for a flow cytometer and 1.500 a for an Elisa washer and reader. Operating expenses were set to 25 a/h for all methods. Time investment for phenotyping by Elisa or flow cytometry was calculated to be 4 h/200 samples. An automatic sample preparation unit will reduce the time investment, but add to instrument investment.
cytometry and PCR analysis require investment in expensive instruments. The ELISA test is a simple assay suitable for large-scale testing (3) with less expensive equipment required. According to our calculations, ELISA ought to be the best method for laboratories that accomplish testing of less than 4000 samples/year, if an investment of instrument is necessary. However, if screening of more than 40,000 samples are performed during the instruments operating time, investment in flow cytometer is justified and phenotyping by flow cytometry supplemented by genotyping could be methods of choice (Fig. 2). References [1] Kjaer MK, Jaegtvik S, Husebekk A, Skogen B. Human platelet antigen 1 (HPA 1) genotyping with 50 nuclease assay and sequence specific primers reveals a single nucleotide deletion in intron 2 of the HPA 1a allele of platelet glycoprotein IIIa. Br J Haematol 2002;117:405–8. [2] Randen I, Sørensen K, Killie MK, Kjeldsen-Kragh J. Rapid and reliable genotyping of the human platelet alloantigens
[3]
[4]
[5]
[6]
[7]
[8]
HPA-1, -2, -3, -4, -5 a/b and Gov a/b by melting curve analysis. Transfusion 2003;43:445–50. Nauck MS, Gierens H, Nauck MA, M€arz W, Wieland H. Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCyclere. Br J Haematol 1999;105:803–10. Garner SF, Smethurst PA, Merieux Y, Aeby C, Smith G, Armour KL, et al. A rapid one-stage whole-blood HPA1a phenotyping assay using a recombinant monoclonal IgG1 anti-HPA-1a. Br J Haematol 2000;108:440– 7. Bessos H, Mirza S, McGill A, Williamson LM, Hadfield R, Murphy WG. A whole blood assay for platelet HPA (PLA1) phenotyping applicable to large-scale screening. Br J Haematol 1996;92:221–5. Weiss EJ, Goldschmidt-Clermont PJ, Grigoryev D, Jin Y, Kockler TS, Bray PF. A monoclonal antibody (SZ21) specific for platelet GPIIIa distinguishes PlA1 from PlA2 . Tissue Antigens 1995;46:374–81. Schwippert-Houtermans B, Strapatsakis S, Roesen P, Tschoepe D. Evaluation of antibody-based genotype classification of the platelet Fibrinogen receptor (GPIIb/IIIa). Cytometry 2001;46:238–42. Watkins NA, Schaffner-Reckinger E, Allan DL, Howkins GJ, Brons NH, Smith GA. HPA-1a phenotype-genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet beta 3 integrin that disrupts the HPA1a epitope. Blood 2002;99:1833–9.
Evaluation of a new flow cytometric HPA 1a screening method A rapid and reliable tool for HPA 1a screening of blood donors and pregnant women Mette Kjær Killie a
a,*
, Jens Kjeldsen-Kragh b, Ingrid Randen b, Bjørn Skogen a, Anne Husebekk a
Department of Immunology and Transfusion Medicine, University of Northern Norway, 9038 Tromsø, Norway b Ullev al University Hospital, Oslo, Norway Received 25 October 2003; accepted 31 October 2003
Abstract We have evaluated a flow cytometric screening method for identification of HPA la negative individuals, using a commercially available monoclonal anti-CD61 antibody specific for the HPA 1a allotype, and compared the method with an ELISA based method for HPA la phenotyping and two methods for PCR genotyping. HPA 1a phenotyping by fluorochrome conjugated monoclonal anti-HPA la and analysis by flow cytometry is a rapid, reliable and inexpensive technique, suitable for screening purposes. Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction The HPA 1a epitope is located on the glycoprotein IIIa, and has two alleles (a and b) defined by a single amino acid substitution. Anti-HPA 1a antibodies often induce severe neonatal alloimmune trombocytopenic purpura (NAITP) requiring immediate transfusion with HPA 1a negative platelets to the neonate. Screening programs for HPA 1a negative pregnant women or blood donors require a simple and reliable assay. Suitable methods for screening purposes have been available since the introduc-
tion of PCR methods such as 50 nuclease assay (50 NA) or melting curve analysis [1–3], as well as different HPA 1a/b discriminating ELISA methods [4,5]. The identification of a monoclonal anti-HPA 1a specific antibody [6,7] adds a new option for HPA 1 screening. The aim of this study was to evaluate a flow cytometric assay by comparing this new method with established PCR and ELISA typing methods.
2. Materials and methods 2.1. Test samples
*
Corresponding author. Tel.: +47-776-26312; fax: +47-77626304. E-mail address: [email protected] (M.K. Killie).
Anticoagulated blood (EDTA) from pregnant women or blood donors and platelet concentrates produced from buffy-coats from four blood
1473-0502/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.transci.2003.10.004
90
M.K. Killie et al. / Transfusion and Apheresis Science 30 (2004) 89–92
donors were used for platelet phenotyping. The Committee for Medical Human Research Ethics approved the part of the study involving typing of pregnant women. 2.2. Platelet phenotyping by flow cytometry Fluorescein isothiocyanate (FITC)-labelled mouse IgG1 anti-CD61 (clone SZ21, Immunotech, Marseille, France) was used for phenotyping. Forty microlitres of the antibody (final dilution 1:125, 16 lg/ml) were added to 10 ll whole blood, platelet rich plasma or platelet concentrates and incubated for 10 min at room temperature. Five hundred microlitres of PBS containing 0.3% EDTA was added to each sample before analysis. The fluorescence intensities obtained were analysed by FacsCalibur instrument using CellQuest software system (Becton Dickinson, CA, USA). Well-characterised HPA 1a positive and negative platelets were used as controls. 2.3. Platelet genotyping HPA 1a genotyping was carried out by 50 nuclease assay and melting curve analysis as described earlier [1–3]. 2.4. Platelet phenotyping by ELISA HPA 1 phenotyping was performed with an ELISA kit according to the manufacturerÕs instructions (Platelet HPA 1a Typing Assay, DiaMed AG, Switzerland).
3. Results and discussion 3.1. Optimal concentration of FITC-SZ21 In order to determine the optimal concentration of FITC conjugated SZ21, the antibody preparation, which is delivered at a concentration of 2 mg/ ml, was tested in different dilutions against HPA 1aa platelets, and platelets of 1ab and 1bb phenotypes. The fluorescence intensities (FI) were recorded, and the fluorescence ratio (FR) between the FI values for 1aa or 1ab platelets over the FI
for 1bb platelets were calculated (Fig. 1). At the highest concentration of the antibody, the fluorescence ratio was about 1, indicating that unspecific binding to HPA 1a negative platelets occur. By diluting the antibody 50–125 fold, the FR for HPA 1aa and ab platelets increased to about 35 and 15, respectively. Further dilution of the antibody to 1:1000 gave a successive decline in fluorescence ratio. Thus, by using the FITC-conjugated anti-HPA 1a antibody at dilution 1:125, HPA 1aa and 1ab platelets could efficiently be distinguished from HPA 1bb platelets. A PE-conjugated antibody was also tested, but gave low fluorescence ratio (mean 3.8, range 1.3– 7.0) and was not suitable to distinguish between HPA 1a positive and negative samples. The reason for this is not known, but it is conceivable that the large size of the PE molecule may cause steric hindrance for binding of the antibody. 3.2. Phenotyping of fresh platelets by flow cytometry Thirty-four HPA 1a positive samples (HPA 1aa and 1ab) were phenotyped by flow cytometry technique, using the SZ21 antibody at dilution 1/ 125. The mean of the fluorescence intensity (FL1, geometric) for those samples was 158 (1 SD ± 63). All HPA 1a negative samples had FL1 mean values less than 10. 3.3. Phenotyping of stored platelets by flow cytometry In some cases it is desirable to perform typing of platelets that have been stored for several days. Therefore, the flow cytometry phenotyping assay was tested with platelets that were isolated from whole blood that had been stored for 3, 5, 8 and 10 days, respectively. In samples kept for more than 7 days, the peak in the histogram mimicking unstained platelets tended to increase (data not shown). This was probably due to the accumulation of dying cells in the gated region. Therefore we may conclude that samples more than 7 days old are not suited for HPA 1a phenotyping by flow cytometry.
M.K. Killie et al. / Transfusion and Apheresis Science 30 (2004) 89–92
91
40 HPA 1aa/HPA 1bb HPA 1ab/HPA 1bb
35
30
Ratio
25
20
15
10
5
0 1
10
50
125
200
400
800
1000
Titer
Fig. 1. A diagram illustrating optimal dilution of monoclonal anti-HPA 1a (SZ21). An optimal dilution of antibody was 1/50–1/125. X-axis: dilution of antibody. Y-axis: ratio between HPA 1a positive and negative samples.
3.4. Phenotyping of pooled platelet concentrates by flow cytometry In our search for HPA 1a negative donors we performed phenotyping of platelet concentrates produced from pools of four buffy-coats. A histogram showing a large HPA 1a positive population and a smaller HPA 1a negative population indicated that one of the four donors in the pool had the HPA 1a negative phenotype. Each donor from a pool containing HPA 1a negative platelets were tested separately the next time they donated blood. Donors that had both phenotype and genotype HPA 1bb were defined as HPA 1bb donors.
genotyped HPA 1ab. The reason for this HPA 1 phenotype/genotype discrepancy, has not been identified. Discrepancy in genotype and phenotype is not a unique finding and has been published earlier by others; an ARG93GLN substitution, disrupting the HPA 1a epitope, was recently reported [8]. An alternative explanation could be a genetic defect in the HPA 1a allele, causing a lack of expression of the actual protein. Individuals lacking the HPA 1a phenotype, although possessing a defect HPA 1a allele, may be immunised, and should be regarded as HPA 1bb. This finding points out the importance of phenotyping as target for HPA 1a typing. To prevent immunisation by a cell surface antigen, phenotyping is the only method of real interest.
3.5. Comparison of phenotyping with genotyping Testing of 45,960 samples by flow cytometry using the monoclonal SZ21 antibody gave 1121 HPA 1a negative samples, which corresponds well with the expected frequency of HPA 1bb in Caucasians (2.4%). By typing the 1121 samples with PCR technique, 1112 were genotyped HPA 1bb. Nine samples with the phenotype HPA 1bb, were
3.6. Comparison of cost and time investment between the different test systems Cost accounting and time investment for the described screening methods were performed. Phenotyping by flow cytometry was the least expensive, provided that a flow cytometer is already available. Both phenotyping by flow
92
M.K. Killie et al. / Transfusion and Apheresis Science 30 (2004) 89–92 700000 ELISA 5´NA Flow Light cycler
600000
Euro
500000 400000 300000 200000 100000 0 1
1000
2000
4000
8000
16000
32000
Number of samples analysed
Fig. 2. Comparison of time cost (a) of three different HPA 1a typing methods. The graph (x-axis) starts with the investments of necessary equipment, hence 93.094 a for a flow cytometer and 1.500 a for an Elisa washer and reader. Operating expenses were set to 25 a/h for all methods. Time investment for phenotyping by Elisa or flow cytometry was calculated to be 4 h/200 samples. An automatic sample preparation unit will reduce the time investment, but add to instrument investment.
cytometry and PCR analysis require investment in expensive instruments. The ELISA test is a simple assay suitable for large-scale testing (3) with less expensive equipment required. According to our calculations, ELISA ought to be the best method for laboratories that accomplish testing of less than 4000 samples/year, if an investment of instrument is necessary. However, if screening of more than 40,000 samples are performed during the instruments operating time, investment in flow cytometer is justified and phenotyping by flow cytometry supplemented by genotyping could be methods of choice (Fig. 2). References [1] Kjaer MK, Jaegtvik S, Husebekk A, Skogen B. Human platelet antigen 1 (HPA 1) genotyping with 50 nuclease assay and sequence specific primers reveals a single nucleotide deletion in intron 2 of the HPA 1a allele of platelet glycoprotein IIIa. Br J Haematol 2002;117:405–8. [2] Randen I, Sørensen K, Killie MK, Kjeldsen-Kragh J. Rapid and reliable genotyping of the human platelet alloantigens
[3]
[4]
[5]
[6]
[7]
[8]
HPA-1, -2, -3, -4, -5 a/b and Gov a/b by melting curve analysis. Transfusion 2003;43:445–50. Nauck MS, Gierens H, Nauck MA, M€arz W, Wieland H. Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCyclere. Br J Haematol 1999;105:803–10. Garner SF, Smethurst PA, Merieux Y, Aeby C, Smith G, Armour KL, et al. A rapid one-stage whole-blood HPA1a phenotyping assay using a recombinant monoclonal IgG1 anti-HPA-1a. Br J Haematol 2000;108:440– 7. Bessos H, Mirza S, McGill A, Williamson LM, Hadfield R, Murphy WG. A whole blood assay for platelet HPA (PLA1) phenotyping applicable to large-scale screening. Br J Haematol 1996;92:221–5. Weiss EJ, Goldschmidt-Clermont PJ, Grigoryev D, Jin Y, Kockler TS, Bray PF. A monoclonal antibody (SZ21) specific for platelet GPIIIa distinguishes PlA1 from PlA2 . Tissue Antigens 1995;46:374–81. Schwippert-Houtermans B, Strapatsakis S, Roesen P, Tschoepe D. Evaluation of antibody-based genotype classification of the platelet Fibrinogen receptor (GPIIb/IIIa). Cytometry 2001;46:238–42. Watkins NA, Schaffner-Reckinger E, Allan DL, Howkins GJ, Brons NH, Smith GA. HPA-1a phenotype-genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet beta 3 integrin that disrupts the HPA1a epitope. Blood 2002;99:1833–9.