Fast and low-cost direct ELISA for high-throughput serological HPA-1a typing.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay. STUDY DESIGN AND METHODS: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500. RESULTS: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03. CONCLUSION: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.

De Novo Donor-Specific Human Leukocyte Antigen Antibody Screening in Kidney Transplant Recipients After the First Year Posttransplantation: A Medical Decision Analysis.

Screening for de novo donor-specific antibodies (dnDSA) in stable kidney transplant recipients is routine practice in some centers. Patients with DSA are at increased risk of graft loss and early intervention may improve outcomes. However, the costs and benefits of dnDSA surveillance are unknown. A medical decision analysis to examine a screening strategy was developed for kidney transplant recipients who had stable graft function and were DSA negative 1 year posttransplant. In the base case, a modest 25% reduction in graft loss in dnDSA-positive patients treated with increased immunosuppression resulted in 0.04618 quality-adjusted years (QALYs) gained. However, benefits from reduced graft loss were eliminated if there was a small increased risk of death from added therapy. The incremental cost effectiveness was marginal at approximately $120 000-250 000 per QALY, but could be more or less favorable depending on several key variables such as efficacy of treatment, screening costs, incidence rate of subclinical dnDSA, and patient survival. Screening performed the best in patients with lower mortality rates and higher baseline incidence rates of dnDSA. Further study is warranted to gather the necessary high-quality evidence to justify screening.

Significance of semiquantitative assessment of preformed donor-specific antibody using luminex single bead assay in living related liver transplantation.

AIM: To analyze the risks of preoperatively produced donor-specific antibody (DSA) in liver transplantation. METHODS: DSA was assessed using direct complement-dependent cytotoxicity (CDC) and anti-human globulin- (AHG-) CDC tests, as well as the Luminex Single Antigen assay. Among 616 patients undergoing blood type identical or compatible living donor liver transplantation (LDLT), 21 patients were positive for CDC or AHG-CDC tests, and the preserved serum from 18 patients was examined to determine targeted Class I and II antigens. The relationships between the mean fluorescence intensity (MFI) of DSA and the clinical outcomes were analyzed. RESULTS: Patients were divided into 3 groups according to the MFI of anti-Class I DSA: high (11 patients with MFI > 10,000), low (2 patients with MFI < 10,000), and negative (5 patients) MFI groups. Six of 11 patients with high Class-I DSA showed positive Class-II DSA. Hospital death occurred in 7 patients of the high MFI group. High MFI was a significant risk factor for mortality (P = 0.0155). Univariate analysis showed a significant correlation between MFI strength and C4d deposition (P = 0.0498). CONCLUSIONS: HLA Class I DSA with MFI > 10,000 had a significant negative effect on the clinical outcome of patients with preformed DSA in LDLT.

Varied distribution of RhD epitopes in the Indian population.

BACKGROUND: Inhabited by more than 4000 caste and tribal groups, India has an extremely heterogenous population. For thousands of years many tribal groups have practised endogamy and are practically genetically isolated. Traditionally, polyclonal anti-D reagent has been used for RhD typing; though monoclonal antibodies are increasingly being used. As a result, blood banks find it difficult to assign the RhD status to an increasing number of people. As monoclonal anti-D typing reagents may not detect all RhD antigen epitopes, we studied the RhD antigen epitope heterogeneity in different population groups in India. METHODS: Red cells of 5315 RhD-positive individuals belonging to different castes and tribes of India were tested with 30 different epitope-specific monoclonal anti-D antibodies. RESULTS: No single monoclonal antibody could detect all RhD-positive red cells detected by polyclonal antisera. The highest proportion of D antigen was detected by LHM 76/55 and BRAD-8 (98%) monoclonal antibodies. CONCLUSION: We need to determine the correct mix of monoclonal antibodies that will detect nearly all RhD antigens detected by polyclonal anti-D sera. Similarly, before accepting monoclonal anti-D for therapeutic use, it would be necessary to determine the appropriate ones for use in the Indian population.

Comparison of the performance of microtube column systems and solid-phase systems and the tube low-ionic-strength solution additive indirect antiglobulin test in the detection of red cell alloantibodies.

To compare the performance of seven currently available test systems in the detection of erythrocyte alloantibodies (ab), we tested in parallel 446 sera samples containing red cell ab [368 sera samples with ab that are assumed to be clinically significant (cs-ab) and 78 sera samples with ab that are assumed to be of minor clinical significance (ms-ab)] using the tube spin low-ionic-strength solution (addition method) indirect antiglobulin test (tube LISS-IAT), three microtube column agglutination techniques (DiaMed-ID, Ortho BioVue and Bio-Rad Scangel), one affinity adherence test system (CLB/Mast CellBind Screen) and two solid-phase tests [Biotest Solidscreen II and Immucor Capture-R Ready-Screen (4)]. To address the specificity of the three test systems under routine conditions, results of 4566 patient samples obtained using the tube LISS-IAT, results of 5205 patient samples obtained using the Scangel and results of 3560 samples obtained using the Capture-R were evaluated. The DiaMed-ID detected 344 cs-ab and 43 ms-ab, BioVue 333 cs-ab and 48 ms-ab, Scangel 348 cs-ab and 62 ms-ab, CellBind Screen 346 cs-ab and 47 ms-ab, Solidscreen 330 cs-ab and 38 ms-ab, Capture-R 358 cs-ab and 45 ms-ab and LISS-IAT 159 cs-ab and 12 ms-ab. In routine practice, erythrocyte cs-ab could be identified in 61 (67.8%) of 90 reactive sera (specificity: 98.6%) in the tube LISS-IAT, in 169 (58.7%) of 288 (94.4%) in Bio-Rad Scangel and in 101 (51.0%) of 198 reactive sera (94.3%) in Capture-R. We conclude that the sensitivity of the microcolumn, affinity adherence and solid-phase test systems in the detection of cs-ab was similar and was markedly superior to that of the conventional tube LISS-IAT. All high-sensitive test systems produced higher rates of false positives and ms-ab compared to the tube test. An individual cost-benefit analysis, considering the recent knowledge about the clinical significance of weak-reactive cs-ab, should be performed in every institution to decide whether and if so which high-sensitive screening system should be applied.

Inhibitor economics.

The majority of direct costs associated with caring for patients with hemophilia are attributed to replacement therapy with clotting factor concentrates (CFC). For patients who develop high-titer inhibitors, CFC are ineffective and bypassing therapy is used to achieve hemostasis, thus changing the direct costs associated with treatment. As bypassing agents are less predictably effective than CFC (often necessitating more frequent dosing) and are more costly on a per-unit basis, treatment costs for patients with inhibitors are usually much higher than those for patients without inhibitors. In addition, the immune tolerance induction protocols used to eradicate inhibitors are costly due to the frequent dosing of CFC over prolonged periods. It is estimated that the cost of hemostatic therapy for patients with inhibitors can be 2.5 times higher than the cost for patients without inhibitors. However, some studies have reported an outlier effect caused by a small percentage of inhibitor patients who require a disproportionate amount of treatment. These outliers magnify overall treatment costs, making cost assessments for hemostatic therapy less predictable in patients with inhibitors than in those without inhibitors.

External quality assessment scheme in red blood cell serology: a 5-year experience in Thailand.

From 2000 to 2004, 36, 58, 72, 78, and 86 laboratories participated in an external quality assessment scheme (EQAS) organized by the Department of Transfusion Medicine, Faculty of Medicine Siriraj Hospital. Each year the staff was requested to perform ABO grouping, D typing, antibody screening, antibody identification, and DATs on eight blood samples. Each participant received information on the correct test results and a coded summary. Regarding ABO grouping, the error rate ranged from 0.3 to 1.3 percent, mostly due to human errors. Error rates in D typing ranged from 0.7 to 5.7 percent, the most problematic being weak D phenotype interpretation. Although every sample was negative by the DAT, error rates due to false positive test results were determined to be 0.4 to 2.1 percent. Antibody screening errors were also found; however, errors steadily decreased from 4.2 percent in 2000 to 0.3 percent in 2004. Only 69.4 to 87.2 percent of laboratories performed antibody identification; however, correct results increased from 78.4 to 91.0 percent. In conclusion, an EQAS in RBC serology should be used to compare results from different laboratories and to identify those laboratories that need improvement in testing procedures.

Flow cytometric assessment of HLA alloantibodies.

Antibodies against HLA molecules are formed in response to exposure to foreign HLA molecules, which can occur as a result of blood transfusion, pregnancy, or transplant. Blood components, particularly those containing cellular elements, are the most common cause of HLA antibodies. This unit describes technical aspects of the flow cytometric crossmatch (FCXM), flow cytometric microparticle assays, and cell-based flow cytometric screening assays. The collective goal for these assays is to clearly identify the presence of HLA antibody, determine the titer of antibody, and elucidate the specificities (i.e., HLA antigens) to which they will react. Knowledge of this information is critical for organ allocation and accurate assessment of the immunological risk for a patient at the time of transplantation. In addition, the identification of HLA antibodies in blood components may be useful in planning appropriate transfusion support strategies for selected patients.

Immunoadsorption may provide a cost-effective approach to management of patients with inhibitors to FVIII.

BACKGROUND: Immunoadsorption of plasma with Staphylococcal protein A removes immunoglobulins and immune complexes; hence, it should effectively remove inhibitors to FVIII in acquired or congenital hemophilia. The procedure may be cost effective, given the expense of therapies used to treat patients with inhibitors, particularly in an acute setting. STUDY DESIGN AND METHODS: Three patients with inhibitors to FVIII were treated with the Excorim Immunosorba system (two columns used in tandem). Costs for immunoadsorption and for other therapeutic products administered to the patients were calculated. RESULTS: Two patients had acquired hemophilia and severe bleeding associated with low levels of circulating FVIII and high levels of inhibitors to FVIII. They failed to achieve a satisfactory response to management with immunosuppression, pFVIII, recombinant FVIIa or IVIG but responded rapidly, with long-term benefit, to immunoadsorption therapy. The third patient had congenital hemophilia and immunoadsorption was effective in reducing his inhibitor level, allowing him to undergo immune tolerance therapy. Costs of treatment before immunoadsorption were markedly higher than those associated with the immunoadsorption procedures (i.e., >Can 350,000 dollars and >Can 1,000,000 dollars vs. < 20,000 dollars). CONCLUSION: Immunoadsorption appears to be an effective and cost-effective alternative in the management of patients with inhibitors to FVIII.

Report on the Fourth International Granulocyte Immunology Workshop: progress toward quality assessment.

BACKGROUND: A formal quality assurance (QA) scheme has been established to facilitate proficiency testing for granulocyte antibodies and antigens. STUDY DESIGN AND METHODS: Fifteen laboratories participated in the Fourth International Granulocyte Immunology Workshop. The main objective of the workshop was to establish a formal QA scheme for granulocyte serology and molecular typing methods. A secondary objective was to determine the relative sensitivities of the granulocyte immunofluorescence test, granulocyte agglutination test, and MoAb immobilization assays using defined antisera and protocols. RESULTS: Laboratories scored between 16.7 and 100 percent (mean, 57.5%) of the maximum available in the serologic part of this QA exercise. There were particular problems in detecting granulocyte-specific human neutrophil antigen-1 (HNA-1a) IgM antibodies and HNA-2a antibodies in the presence of HNA-1b antibodies. The granulocyte immunofluorescence test was more sensitive than the granulocyte agglutination test in titration studies, but the latter method more readily identified the presence of HNA-3a antibodies. HNA genotyping was generally well performed, with nine laboratories obtaining 100-percent correct results for HNA-1a, HNA-1b, and HNA-1c. CONCLUSIONS: There is a need to standardize the detection of granulocyte-specific antibodies. Laboratories with good performance tended to use two methods for detecting granulocyte-specific antibodies and an HNA-typed panel of granulocytes. The use of a method for elucidating mixtures of granulocyte- and lymphocyte-reactive antibodies (e.g., MoAb immobilization assay) and the use of methods for detecting both cytotoxic and noncytotoxic HLA class I antibodies were also associated with a higher than average performance.

[Evaluation of the polyethyleneglycol antiglobulin test in the detection and identification of erythrocyte antibodies]. / Valoración de la prueba de antiglobulina con polietilenglicol en la detección e identificación de anticuerpos eritrocitarios.

PURPOSE: The polyethylene glycol antiglobulin test has been found to enhance the reactivity of most alloantibodies. MATERIAL AND METHODS: To investigate the utility of polyethylene glycol antiglobulin test for detection and identification of red blood cell antibodies, a comparison study of polyethylene glycol (PEG), a low-ionic-strength additive solution (LISS) and bovine serum albumin (BSA) was conducted. The sera of 47 different patients with positive antibody screening test by the LISS method, were tested in parallel with reagent antibody-detection cells using PEG, LISS and BSA. RESULTS: In the sera of 47 patients, 57 antibodies were detected. We identified 39 antibodies by the three methods. Twelve antibodies reacted by the BSA method and the LISS method but did not react with the PEG method (8 anti-I, 1 anti-P1, 1 anti-Lea(a), and two antibodies missed by the PEG method because they did not react with anti-IgG: 1 anti-M and 1 anti-K). Three antibodies reacted only with the LISS method (3 anti-I). Four clinically significant antibodies were detected only by the PEG method (2 anti-Jka, 1 anti-Jkb, 1 anti-c). The serum from a patient with delayed hemolytic transfusion reaction and no antibody detectable by the LISS and the BSA methods was tested by the PEG method. We were able to detect an anti-Jka by PEG in the pretransfusion sample. In 24 (60%) of 40 samples with clinically significant antibodies, PEG antiglobulin reactions were stronger (total score 221) than LISS antiglobulin reactions (total score 170) and BSA antiglobulin reaction (total score 184); in 14 (35%) of 40 samples, they were identical, and in 2 (5%) agglutination in the PEG method was weaker. CONCLUSION: In our experience, the polyethylene glycol antiglobulin test is more sensitive than LISS and BSA in detecting clinically significant antibodies and is an acceptable technique for routine compatibility test.

Assessment of severity of haemolytic disease of newborn by monocyte monolayer assay.

The present study was undertaken to estimate the predictive value of antibody titration, antibody quantitation and monocyte monolayer assay (MMA) in assessment of severity of haemolytic disease of the newborn (HDN). Serum samples from 45 alloimmunized mothers, with anti-D(23), anti-c(10), anti-K(6), anti-E(5) and anti-e(1) were taken for the study. The results obtained were compared and the efficiency of each technique in predicting the severity of HDN was assessed. Antibody quantitation and MMA (phagocytic index) correlated well with severity of HDN in mothers with anti-D antibodies. Antibody quantitation (anti-D) had a positive predictive value of 54.5 per cent and negative predictive value of 85.7 per cent while MMA had a positive predictive value of 75 per cent and a negative predictive value of 100 per cent. These findings suggest MMA to be a good negative predictor of HDN but not a good positive predictor of haemolytic disease of the newborn.

Detection of anti-Fya, anti-S and anti-Jka in relation to the genotypes of the panel red cells: report of a U.K. NEQAS survey. U.K. National External Quality Assessment Scheme.

Three antibody-containing samples (anti-Fya, anti-S and anti-Jka), each at two dilutions, were distributed to U.K. hospitals and transfusion centres together with an antibody screening panel comprising red cells homozygous for the corresponding antigens. Participants in the study subjected the samples to their routine antibody screening procedures using both their own antibody screening panels and the screening panel provided. A within-group comparison of those participants using their own screening panels having a heterozygous expression of the antigens, with the same participants when using the screening panel provided, showed for five of the samples a greater detection rate in routine antibody screening procedures when using the panel provided, having homozygous expression of the corresponding antigens. The sixth sample, the most potent, was detected equally using both panels. The difference in overall detection rate is statistically significant (chi-square test, 2P < 0.001). The study shows that the use of red cells presumptively homozygous for Fya, S and Jka improved the detection of the corresponding antibodies.

Assessment of clinical significance of anti-Ge in an untransfused man.

A 19-year-old, untransfused Melanesian man from Papua New Guinea was admitted to the hospital for repair of an atrial septal defect. His serum contained an alloantibody that reacted strongly on the indirect antiglobulin test and was identified as anti-Ge. Gerbich-negative blood was transfused following urgent surgery. A 51Cr red cell survival study performed 2 weeks after surgery yielded zero survival of Gerbich-positive cells after 24 hours. A monocyte-driven, antibody-dependent, cell-mediated cytotoxicity assay performed on both pretransfusion and posttransfusion serum samples and on concentrated serum showed less than 1 percent specific lysis of Gerbich-positive cells. This did not correlate with the indication of clinical significance predicted by the 51Cr study. Red cell adherence and phagocytosis, not evident in a monocyte monolayer assay using native serum, were demonstrable in 16 percent of monocytes by the use of concentrated serum.

A cost-effectiveness evaluation of platelet crossmatching and HLA matching in the management of alloimmunized thrombocytopenic patients.

Effective platelet support for alloimmunized refractory thrombocytopenic patients may be provided by several potential strategies, the most common being HLA-matched single-donor platelets or crossmatch-compatible, pooled random- or single-donor platelets. This study used a detailed economic analysis to compare the cost-effectiveness of several techniques for platelet crossmatching and that of HLA-matched single-donor platelets. The crossmatch methods evaluated were a microlymphocytotoxicity test (LCT), an immunofluorescence technique (PSIFT), a radioactive antiglobulin test (PRAT), and an enzyme-linked immunosorbent assay (ELISA). The analysis was based on the need to support 100 refractory patients with acute leukemia with a presumed requirement of 500 transfusions. The relative costs for a successful crossmatch were: PRAT less than LCT less than LCT + PRAT less than PSIFT less than ELISA. In the comparison of the crossmatch methods, an increase in costs was generally associated with an increase in the number of successful transfusion episodes. However, decreasing marginal gains were seen. The HLA-matched single-donor platelets were relatively cost-inefficient in comparison to the crossmatch-compatible platelets. A theoretic sequence of tests for cost-effective provision of optimal platelet support in refractory patients was evaluated. Such considerations of cost are important in the selection of an optimal program for the management of alloimmunized refractory thrombocytopenic patients.

Development of an immunoassay for the detection of minute amounts of IgG-coated erythrocytes in whole blood and its application for the assessment of Fc-mediated clearance of anti-D-coated erythrocytes in vivo.

A rapid and sensitive immunoassay for the detection of minute quantities of IgG-coated erythrocytes in whole blood was developed. Washed red blood cells were incubated in two steps with anti-human IgG antiserum followed by 125I-labelled protein A. The assay was able to detect amounts of sensitized erythrocytes as small as 0.5 ml of packed erythrocytes in a total blood volume of 5 liters and hematocrit 40%. A linear relation between increasing amounts of IgG-coated red cells in whole blood and the binding of 125I-labelled protein A was obtained. We applied the technique on the assessment of the removal of IgG anti-D-coated erythrocytes from the circulation of test individuals. T1/2 for the elimination of approximately 4 ml packed red cells sensitized with 62 micrograms of anti-D in 14 normal subjects was 20 +/- 5 min (mean +/- SEM). A splenectomized person did not clear the injected cells from the circulation during the test period of 70 min. If a standard curve was constructed the total blood volume in the test subjects could be calculated. This value correlated well (r = 0.99) with the blood volume calculated from the height and weight of the test individuals.

Assessment of anti-HLA antibodies in sera being tested for platelet reactivity by a platelet lymphocyte immunofluorescence test (PLIFT).

An indirect immunofluorescence test using a platelet mononuclear cell suspension is described. The test is a simple and sensitive method to assess the contamination by anti-HLA antibodies of sera being tested for platelet reactivity and eliminates the need to support two independent test systems such as lymphocytotoxicity and immunofluorescence testing. The test could be of value in routine platelet serology testing and platelet crossmatching.

The UK National External Quality Assessment Scheme in Blood Group Serology. ABO and D grouping, antibody screening, direct antiglobulin test and antibody identification 1984-1985.

In seven exercises of blood grouping the overall rates of major error were 0.19% and 0.25% in ABO and D grouping respectively. In ABO grouping this represents an increase in error rate over that observed in 1982-1983 but the increase was due to an unusually high error rate with one particular group A2B cell. An improvement in performance was observed in simple D grouping and was largely due to a lower incidence of false positive grouping of D-negative cells in the antiglobulin test. An improvement in performance observed in D grouping IgG-coated D-negative cells appeared to be due to a better understanding of the problem rather than to any change in serological practice. Error rates in antibody screening were somewhat lower than in 1982-1983 but this may or may not represent an improvement in performance as the test materials were not the same in the two periods. The direct antiglobulin test with IgG-coated cells was reliably performed with polyspecific and with anti-IgG reagents but an excess of false positive results was obtained with anti-C3d. Error rates in antibody identification varied from 0.6% for anti-D to 74% for anti-c + E.

Comparison between a solid-phase low-ionic-strength solution antiglobulin test and conventional low-ionic-strength antiglobulin test: assessment for the screening of antierythrocyte antibodies.

A solid-phase low-ionic strength salt antiglobulin test (LISS-SPAT) has been developed using a microplate coated with dried sera as a solid phase. Before coating, the in vitro C3d fragment generation was activated by adding heat-aggregated immunoglobulin. The LISS-SPAT was compared with low-ionic strength conventional antiglobulin test (LISS-AGT) and also with a test using polybrene or papain microplates. When detecting the IgG and IgM antierythrocyte antibodies the reaction was developed in the same way in LISS-SPAT and LISS-AGT. In routine work, the LISS-SPAT provides a fast, reliable, handy and inexpensive screening of antibodies. This method appears to be an additional method to the papain and polybrene tests in microplates.