Collaborative studies to establish the first World Health Organization International Standard for detection of human antibody against human platelet antigen-3a

BACKGROUND AND OBJECTIVES: The platelet-specific antibody anti-human platelet antigen-3a (anti-HPA-3a) is involved in neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet refractoriness. However, HPA-3a antibodies are often difficult to detect, probably because the antigen is labile. This report describes the production of a freeze-dried preparation of pooled human plasma, coded 03/190, containing IgG antibodies against the HPA-3a. The material is intended for use as a minimum sensitivity reagent in glycoprotein-specific assays currently used for anti-HPA-3a detection. Laboratories can use it to assess the sensitivity of their 'in-house' assays for anti-HPA-3a and to calibrate local controls for routine use in each batch of tests. MATERIALS AND METHODS: Plasma containing anti-HPA-3a was obtained from a mother of two babies both born with severe thrombocytopenia, and following dilution it was freeze dried in glass ampoules. RESULTS: Two collaborative studies demonstrated that the candidate material contained anti-HPA-3a and human leucocyte antigen (HLA) class I antibodies, but no other HPA antibodies that might confuse the detection of the anti-HPA-3a. The minimum dilution that should give a positive result was determined to be 1 : 8 by two further international collaborative studies involving a total of 49 laboratories in 23 countries. CONCLUSION: The material also contains HLA antibodies and is suitable for use only in techniques that are glycoprotein specific (i.e. monoclonal antibody immobilization of platelet antigens and enzyme-linked immunosorbent assay) where only HPA antibodies will be detected. This standard will allow laboratories to measure their sensitivity of detection of anti-HPA-3a and will also allow those laboratories with relatively insensitive techniques to monitor their performance as they improve their methodology.     

An International Reference Reagent (minimum sensitivity) for the detection of anti-human platelet antigen 1a

Background and Objectives   The platelet-specific alloantibody anti-human platelet antigen (HPA) 1a is involved in feto-maternal alloimmune thrombocytopenia, post-transfusion purpura and platelet refractoriness. The existing minimum potency preparation for the detection of anti-HPA-1a (NIBSC code 93/710) was established by World Health Organization in 1997 and is used by laboratories to validate new assays or to calibrate 'in-house' controls. However, it has been well-used and a replacement is required. This report describes the production and comparative evaluation of a freeze-dried preparation of pooled human plasma, coded 05/106, containing anti-HPA-1a.
Materials and Methods   Plasma containing anti-HPA-1a was obtained and 2974 1-ml aliquots were prepared and freeze-dried in glass ampoules. In order to characterize the material and compare it to the existing reference material, three collaborative studies were organized, involving a total of 50 different laboratories in 23 countries.
Results   As expected only anti-HPA-1a could be detected in the plasma and no additional HPA or human leucocyte antigen antibodies were detected. When tested in titration, there was a wide variation in the sensitivity of antibody detection by different laboratories, irrespective of the technique used. However, there was no significant difference between the two materials when compared using a t -test.
Conclusions   When diluted 1 in 2, most laboratories were able to detect the presence of anti-HPA-1a in both materials and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. In October 2007, the World Health Organization Expert Committee on Biological Standardization approved the material 05/106 as an International Reference Reagent.     

Collaborative study to establish the first international standard for quantitation of anti-HPA-1a

BACKGROUND AND OBJECTIVES: This report describes the production of a freeze-dried preparation of pooled human plasma, containing immunoglobulin G (IgG) antibodies against the human platelet antigen 1a (HPA-1a). The material, coded 03/152, is proposed as an International Standard containing 100 arbitrary units of anti-HPA-1a for use in quantitative assays to determine the anti-HPA-1a activity in clinical samples. MATERIALS AND METHODS: Plasma samples containing potent anti-HPA-1a were pooled and freeze dried in 1-ml ampoules. In addition, three individual plasma samples were selected which had varying levels of anti-HPA-1a activity. The anti-HPA-1a activity of these three samples was determined by using a variety of quantitative assays with the proposed standard as a reference. RESULTS: An international collaborative study, which was part of the 2004 ISBT Platelet Immunology Workshop, involved 39 laboratories in 24 countries and showed that the anti-HPA-1a activity in three test samples could be reliably determined by using the proposed standard. CONCLUSIONS: Laboratories can use this standard to measure the anti-HPA-1a activity in patient's samples. Further studies are required to determine the relationship between anti-HPA-1a activity and clinical outcome in patients with neonatal alloimmune thrombocytopenia (NAIT).     

The value of alloantibody detection in predicting response to HLA-matched platelet transfusions

Alloantibody tests demonstrate immunological causes of insufficient increments in random platelet transfusions. The value of a positive or negative test result in predicting the outcome of human leucocyte antigen (HLA)-matched transfusions in patients refractory to leucodepleted random platelet transfusions has not been assessed. We retrospectively evaluated the outcome of the first HLA-matched platelet transfusion in 72 patients with haematological diseases in two ways: first, the strategy according to which the patient was selected for HLA-matched platelet transfusions was analysed. The strategies were: (i) results of alloantibody tests were not available, (ii) a positive alloantibody test, (iii) a negative alloantibody test. Secondly, the outcome of the first HLA-matched transfusion was investigated relative to the results of alloantibody tests, irrespective of the decision strategy. No significant association was found between the decision strategy and the outcome of the first HLA-matched platelet transfusion. Positive alloantibody tests, however, predicted a better outcome of the first HLA-matched platelet transfusion (P = 0.04 and P = 0.03 after 1 and 16 h respectively). In patients refractory to random platelet transfusions, positive alloantibody tests predicted a better outcome of HLA-matched platelet transfusions. Patients with negative alloantibody tests, however, may benefit from HLA-matched platelet transfusions.     

Interlaboratory variation in the detection of clinically significant alloantibodies against human platelet alloantigens

This report describes the results of eight workshop exercises which were designed to test the proficiency of laboratories in the detection of antibodies to human platelet antigens (HPA). Detection of the most clinically significant alloantibody, anti-HPA-1a, is adequate. However, despite improvements in consistency of test results between laboratories over the last 3 years, there is still a high probability that clinically significant antibodies against other HPA alloantigens will not be detected.     

Advances in alloimmune thrombocytopenia: perspectives on current concepts of human platelet antigens,antibody detection strategies,and genotyping

Alloimmunisation to platelets leads to the production of antibodies against platelet antigens and consequently to thrombocytopenia. Numerous molecules located on the platelet surface are antigenic and induce immune-mediated platelet destruction with symptoms that can be serious. Human platelet antigens (HPA) cause thrombocytopenias, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet transfusion refractoriness. Thirty-four HPA are classified into 28 systems. Assays to identify HPA and anti-HPA antibodies are critically important for preventing and treating thrombocytopenia caused by anti-HPA antibodies. Significant progress in furthering our understanding of HPA has been made in the last decade: new HPA have been discovered, antibody-detection methods have improved, and new genotyping methods have been developed. We review these advances and discuss issues that remain to be resolved as well as future prospects for preventing and treating immune thrombocytopenia.     

A double antibody radioassay for the detection of platelet associated IgG

A double antibody radioimmunoassay has been developed for the detection of platelet associated IgG (PAIgG). The assay employs conventional radioassay technology and, as such, readily may be adopted by laboratories familiar with radioassay techniques. The assay is sensitive to 0.6 ng IgG and can be undertaken on 10⁶-10⁷ platelets, or fewer in the case of thrombocytopenia with raised PAIgG. The assay can be completed within 2 working days. Normal PAIgG levels in EDTA blood were 0.8 to 5.7 μg IgG/10⁹ platelets. However, normal levels depended upon the anticoagulant used for blood collection and were on average five-fold higher for blood taken into EDTA than for blood taken into ACD; normal range 0.1–1.2 μg/IgG 10⁹ platelets. PAIgG was assayed on blood taken simultaneously into ACD and EDTA from 15 patients with chronic idiopathic thrombocytopenia (ITP). PAIgG was raised in 14 out of 15 EDTA samples, but in only 11 of 15 ACD samples. EDTA was therefore regarded as the anticoagulant of choice. A total of 20 of 23 (87%) patients with chronic ITP had PAIgG values above the normal range. The PAIgG in chronic ITP was weakly related to platelet count, weakly and inversely related to platelet volume, but was independent of serum IgG concentration.     

Development of quantitative monoclonal antibody-specific immobilization of platelet antigens assay for antibodies against human platelet antigen-1a, 3a,and 5b

Human platelet antigens (HPAs) are platelet-specific alloantigens that cause alloimmunization. Alloantibodies induced via pregnancy and transfusion may result in various clinical syndromes. The monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay is the gold standard for the detection and identification of HPA antibodies. The present study aimed to develop standardized quantitative MAIPA assays for antibodies against HPA-1a, 3a, and 5b. MAIPA protocol was applied on serial dilutions of standard or reference reagents containing antibodies against HPA-1a, 3a, and 5b. Optimal experimental parameters were determined, and standard curves were constructed with a linear regression. The sensitivity, specificity, and reproducibility of the assays were also evaluated. The optimum reagents and conditions for the MAIPA assays were decided through repeated testing and comparison. The intra-assay coefficients of variation (CVs) are 1.3~3.6%, 2.7~5.2%, and 4.7~5.3%; the inter-assay CVs are 4.0~5.6%, 4.4~5.9%, and 3.2~6.3%, respectively, for HPA-1a, HPA-3a, and HPA-5b antibodies. In specificity tests, some monoclonal antibodies and sera with other antibodies were tested and no obvious positive result was observed. The standardized quantitative MAIPA assays for antibodies against HPA-1a, 3a, and 5b have good sensitivity, reproducibility, and specificity. They have important application value in clinical diagnosis of alloimmunization disease caused by HPA antibodies and safe transfusion of platelets.     

Polymorphism of the human platelet antigen-5 system is a risk factor for occlusive vascular complications in patients with sickle cell anemia

BACKGROUND: Polymorphisms of platelet membrane glycoproteins such as human platelet antigen (HPA)-1b, HPA-2b, the -5T/C Kozak sequence and C807T have been described as risk factors for vascular disease. Vaso-occlusion episodes are a common feature of sickle cell anaemia (SCA), leading to complications such as stroke, acute chest syndrome, avascular head femur necrosis and priapism. Complex interactions are involved in vaso-occlusion, and activated platelets may play an important role. These data raised the question of whether platelet polymorphisms could be implicated in occlusive vascular complications (OVC) of SCA. MATERIALS AND METHODS: In this study, 97 patients with SCA were analysed in two groups: 34 patients presenting with OVC (SCA-VC) and 63 without these complications (SCA-N). The distribution of the HPA-1, -2 and -5 systems, as well as C807T dimorphism and -5T/C Kozak sequence alleles, was evaluated using DNA-based methods. RESULTS: Patients of the SCA-VC group showed a higher frequency of the HPA-5b allele (0.324) compared with those of the SCA-N group (0.111) (chi2 = 13.19, P = 0.0002). None of the other polymorphisms, isolated or associated as haplotypes, demonstrated any correlation with the development of OVC in these patients. CONCLUSIONS: The findings of this study suggest that the HPA-5b allele is a genetic risk factor for the development of OVC in patients with SCA. This allele could be explored as a target for the development of new therapeutic approaches.     

Establishment of a cell line panel for the detection of antibodies against human platelet antigen 4b

Antibodies against human platelet antigens (HPAs) play important roles in thrombocytopenia. In Japan, HPA-4b antibody is frequently responsible for HPA-related neonatal alloimmune thrombocytopenia. A highly sensitive assay using platelets has been developed for the detection of antibodies against HPAs. However, it is difficult to obtain the platelets expressing specific HPAs required for the assay. Therefore, an alternative method not requiring platelets would be helpful to detect antibodies against HPAs. Glycoprotein IIIa (GPIIIa) cDNA encoding HPA-4b was individually co-transduced with that of wild-type GPIIb in K562 cells, which is a non-adherent cell line, using a retroviral vector. The expression of transgene products in cultured cells were observed for over 6 months. Next, to evaluating the sensitivity and specificity of this cell line panel, we performed monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay with a serum previously identified by another method. All HPA-4b antibodies in serum samples were positive, and all serum samples, including normal serum and serum containing HLA antibodies were negative. No difference was observed in the specificity and sensitivity between our method and conventional MAIPA using platelets. The present results indicate that this established cell line panel permits highly sensitive detection of specific antibodies against HPA-4b.     

Inter-laboratory comparison of different rapid methods for the detection of bacterial contamination in platelet concentrates

Background Bacterial contamination of platelet concentrates still represents a major risk in transfusion medicine, and a variety of screening methods have been available to improve the safety of PCs. In the present study, the analytical quality of three different rapid screening methods (BactiFlow flow cytometry, Pan Genera Detection Assay, 23S rRNA RT‐PCR) was evaluated in an inter‐laboratory comparison in three different German blood services. Methods Samples were inoculated with different bacteria [Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli (two strains), Klebsiella pneumoniae (two strains), Enterobacter aerogenes (one strain), Serratia marcescens (one strain)] at different counts (4·5 × 10³–4·5 × 10⁸ CFU/ml) alternating with negative samples in one transfusion facility. Samples were blinded with a random order for each screening method, shipped to partners and analysed immediately after receipt with different rapid screening methods. Results The inter‐laboratory comparison revealed that the BactiFlow assay and 23S rRNA RT‐PCR‐screening detected all samples correctly (positive: 12/12, negative: 8/8). The Pan Genera Detection Assay test detected only four of the positive samples. Four of the non‐detected positive samples were below the assay’s detection limit. Another four inoculated samples with comparatively high bacteria counts were detected false negative (E. coli (two strains): 9·87 × 10⁵ and 2·10 × 10⁷ CFU/ml, respectively, K. pneumoniae: 4·79 × 10⁶ CFU/ml, S. aureus: 6·03 × 10⁵ CFU/ml). All rapid screening methods revealed no false‐positive results. Conclusions Both BactiFlow and 23S rRNA RT‐PCR demonstrated a high sensitivity to detecting bacterial contamination in PCs. The Pan Genera Detection Assay had some shortcomings regarding sensitivity, especially for the detection of Gram‐negative strains.     

Incidence of transfusion-induced platelet-reactive antibodies evaluated by specific assays for the detection of human leucocyte antigen and human platelet antigen antibodies

BACKGROUND AND OBJECTIVES: The aim of this work was to study the incidence of transfusion-induced platelet-reactive antibodies in a selective patient population and evaluate different methodologies for platelet antibody screening (PAS). MATERIALS AND METHODS: The patients were retrospectively selected and divided into three separate groups: haematological malignancies (Group 1: n = 33); cardiac and orthopaedic patients (Group 2: n = 31) and a control group (Group 3: n = 23) selected with the same diagnoses of Group 2. PRE- and POST-transfusion samples were tested for PAS by the following tests: PIFT (platelet immunofluorescence test), MAIPA (monoclonal antibody immobilization of platelet antigen), Flow PRA(R) and LCT (lymphocytotoxicity test). RESULTS: There was not a 100% concordance among the methodologies used. PIFT, MAIPA and Flow PRA presented very similar results whereas that of LCT differed from the other methods. A high rate of positive results (32%) was found in the PRE samples followed by an increase of almost 50% after blood transfusion (POST samples: 42.5% of positivity), but there was a statistical difference (P < 0.05) between the PRE and POST transfusion sample only for the Flow PRA(R) technique tested on Group 2. Human leucocyte antigen (HLA) class I antibodies were present on 97.4% of POST positive samples, 5.4% presented anti-human platelet antigen (HPA)-1b antibodies and 8.1% presented a mix of pan-reactive antibodies against glycoprotein IIbIIIa, IaIIa and IbIX. CONCLUSIONS: Blood transfusion did not increase the rate of alloimmunization in our haematological patients (Group 1); however, the patients were already admitted with a high rate of alloimmunization (12%). Group 2 patients are being immunized and the impact of this procedure remains to be studied as these patients may eventually undergo further hospitalization and receive more blood transfusion.     

An International Reference Reagent for the detection of anti‐human neutrophil antigen‐1a

Background and Objectives Testing for neutrophil antibodies has become more common as awareness of transfusion‐related acute lung injury (TRALI) has increased. However, unlike other areas of blood cell antibody testing, there are no certified reference reagents available with which laboratories can determine the sensitivity of detection of their assays. This report describes the production and evaluation of a freeze‐dried preparation of human plasma, code 09/284, containing anti‐human neutrophil antigen‐1a (anti‐HNA‐1a) for use as a minimum sensitivity reagent. Materials and Methods One‐millilitre of aliquots of plasma containing anti‐HNA‐1a were freeze‐dried in glass ampoules. To characterize the material, 24 laboratories took part in an international collaborative study. The participants evaluated doubling dilutions of the material using their in‐house routine assays and recorded the highest dilution in which the antibody could be detected. Results When diluted 1 in 4, most laboratories were able to detect the anti‐HNA‐1a in the material, and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. Conclusions In October 2011, the WHO Expert Committee on Biological Standardization approved the material 09/284 as an International Reference Reagent for the detection of anti‐HNA‐1a.     

A microplate reader-based method to quantify NADH-cytochrome b5 reductase activity for diagnosis of recessive congenital methaemoglobinemia

Objectives: Congenital methemoglobinemia due to NADH-cytochrome b5 reductase 3 (CYB5R3) deficiencies is an autosomal recessive disorder that occurs sporadically worldwide, A sensitive, accurate, and rapid analysis of NADH-CYB5R enzyme concentrations is necessary for the diagnosis of RCM. Here we present an alternative microplate method that is based on a standard 96-well microplate format and microplate reader that simplify the quantification of NADH-CYB5R activity.

Methods: TECAN (Infinite 200 PRO series) microplate reader with Tecan’s proven Magellan? software measured the NADH-CYB5R enzyme activity in 250 normal controls and previously diagnosed 25 cases of RCM due to NADH-CYB5R deficiency in the Indian population using 96-well microplates using 200?μl of total reaction mixture and also compared with standard spectrophotometric assay. We have also studied stability of the hemolysate stored at 4 and ?20°C temperature.

Results and discussion: Enzyme activity in all 25 samples ranged from 6.09 to 10.07?IU/g Hb (mean?±?SD: 8.08?±?1.99?IU/g Hb) where as normal control ranged (n?=?250) between 13.42 and 21.58?IU/g Hb) (mean?±?SD: 17.5?±?4.08?IU/g of Hb). Data obtained from the microplate reader were compared with standard spectrophotometer method and found 100% concordance using both methods. Microplate method allows differentiating between normal, deficient and intermediate enzyme activity. It was observed that samples had significant loss of activity when stored at 4°C and retained stable activity at ?20°C for 1 week time.

Conclusion: Our new method, incorporating a whole process of enzyme assay into a microplate format is readily applicable and allows rapid monitoring of enzyme assay. It is readily applicable to quantitative assay on pediatric sample as well as large number of samples for population screening.     

Prenatal diagnosis of recessive congenital methaemoglobinaemia type II: novel mutation in the NADH-cytochrome b5 reductase gene leading to stop codon read-through

A case of type II recessive congenital methaemoglobinaemia (RCM) observed in a Lebanese subject with a novel mutation in NADH-cytochrome b5 reductase gene is described. A homozygous mutation CAC to AA identified at Thr 295 with an out-of-frame 1-bp deletion leads to a frameshift with translational read-through of the natural stop codon. The molecular mechanism is demonstrated by an in vitro translation study. The model of mutated cytochrome b5 reductase protein possessing 46 additional amino acids was obtained by homology modelling. The mutation causes an alteration of hydrophobicity in the carboxyl-terminal portion, resulting in the conformation being drastically disturbed by the presence of 46 supplementary amino acids. The identical mutation was found in the heterozygous state in the patient's parents and sister. Identification of this new mutation enabled us to perform the molecular prenatal diagnosis of type II RCM at the DNA level.     

Comparison of common platelet receptors between the chacma baboon (Papio ursinus) and human for use in pre-clinical human-targeted anti-platelet studies

Anti-platelet agents play a central part in the treatment and prevention of acute thrombotic events. Discriminating animal models are needed for the development of novel agents. The chacma baboon has been extensively used as a model to evaluate anti-platelet agents. However, limited data exist to prove the translatability of this species to humans. We aimed to determine the suitability of the chacma baboon in preclinical human targeted GPIIb/IIIa, GPIbα and P2Y12 studies. Light-transmission platelet aggregometry (LTA), whole blood impedance aggregometry, receptor number quantification and genomic DNA sequencing were performed. Baboon ADP and arachidonic acid-induced LTA aggregation results differed significantly from human values, even at increased concentrations. LTA ristocetin-induced agglutination was comparable between species, but baboon platelets needed twice the concentration of ristocetin to elicit a similar response. Citrated baboon blood had significantly less aggregation than humans when evaluated with impedance aggregometry. However, hirudinised baboon whole blood gave similar aggregation as humans at the same agonist concentrations. GPIIb, GPIIIa and GPIbα numbers were significantly more on the baboon platelets. None of the amino acids deemed vital for receptor function, ligand binding or receptor inhibition, were radically different between the species. However, a conservative change in a calcium-binding region of GPIIb may render the baboon platelets more sensitive to calcium-binding agents. The chacma baboon may be used for the evaluation of human-targeted GPIIb/IIIa-, GPIbα- and P2Y12-inhibiting agents. However, the best anticoagulant, optimal agonist concentrations, increase in receptor number and sequence differences must be considered for any future studies.     

Monoclonal antibody CL5 recognizes the amino terminal domain of human phagocyte flavocytochrome b558 large subunit, gp91phox

Human phagocyte flavocytochrome b558 (Cytb) is a heterodimeric integral membrane protein that serves as the electron transferase of the beta-nicotinamide adenine dinucleotidephosphate, reduced (NADPH)-oxidase, an enzyme complex important in the host defense function of phagocytic cells. In this study, we report the characterization of monoclonal antibody (mAb) CL5 that is specific for the large subunit, gp91phox, of the oxidase protein. This antibody recognizes gp91phox by immunoblot analysis of membrane extracts and samples of the immunopurified gp91phox/p22phox heterodimer, prepared on anti-p22phox affinity matrices. Phage display analysis confirmed this specificity, indicating that the CL5 epitope contains the region 135-DPYSVALSELGDR of gp91phox. The antibody was used to probe for the presence of gp91phox in membrane preparations from neutrophils of patients with nine genetically distinct forms of X-linked chronic granulomatous disease (CGD). The causative mutations included missense errors as well as nonsense errors that result in premature termination of gp91phox synthesis. Analysis of the CGD samples by immunoblotting indicated that CL5 recognizes only the full-length wild-type and two missense mutations, consistent with the absence of stable short gp91phox peptide expression in CGD neutrophils. Interestingly, CL5 was also shown to be cross-reactive with cytosolic and membrane-bound gelsolin, identified by purification, mass spectrometry and immunoblot analysis. CL5 probably cross-reacts with the sequence 771-DPLDRAMAEL in the C-terminus of gelsolin. We conclude that mAb CL5 is a useful probe for detection of full length and possibly truncated N-terminal fragments of gp91phox from membranes of Cytb-producing cells.