Platelet Crossmatching Research Articles

A rare case of hemolytic disease-associated thrombocytopenia mimicking alloimmune thrombocytopenia: Salvaging a precious baby

Abstract: Thrombocytopenia associated with hemolytic disease of fetus and newborn (HDFN) is usually mild-to-moderate severity. Neonatal alloimmune thrombocytopenia (NAIT) is one of the leading causes of severe thrombocytopenia in neonates. We describe a rare case of very severe thrombocytopenia (5 g/L) with bleeding manifestations associated with HDFN mimicking NAIT. The baby was a preterm female, born to a 34-year-old multiparous mother with a bad obstetric history and multiple alloantibodies (anti-D, anti-C, anti-E, and anti-Jka). She was managed with phototherapy, exchange transfusion with phenotype-matched packed red blood cells, random donor platelets and intravenous immunoglobulin as platelet crossmatching could not provide compatible platelets. NAIT was ruled out with human platelet antigen (HPA) genotyping of the baby, the mother, and the father. It was concluded that the very severe thrombocytopenia might be due to HDFN after ruling out the other causes. HDFN can rarely cause very severe thrombocytopenia. However, when a newborn presents with severe/very severe thrombocytopenia, NAIT is to be ruled out with HPA genotyping, even in resource-limited settings, to plan out subsequent pregnancies.

Establish a Graded Method to Avoid HLA Class I Antibodies Corresponding Antigen and Combining HLAMatchmaker Application in Improving the CCI Value after Platelet Transfusion for Patients with IPTR

To establish a graded method to avoid mean fluorescence intensity (MFI) threshold of HLA Class I antibodies corresponding antigen, and the HLAMatchmaker program has been used to select the minimum mismatch value of donor-patient epitopes. Evaluate the application value of combining both methods in selecting HLA compatible platelets (PTL) for patients with immune platelet transfusion failure (IPTR) in improving platelet the corrected count increment (CCI). A total 7 807 PLT cross-matching compatible were performed by the solid-phase red cell adherence (SPRCA) method for 51 IPTR patients. The Luminex single antigen flow cytometry was used to detect HLA Class I antibodies in patients, and detected the MFI value for different specificity antigens of HLA Class I antibodies, was graded into strong positive group (MFI>4 000, level 1), medium positive group (1 000< MFI≤4 000, 2), weak positive group (500< MFI≤1 000, 3), and one negative control group (MFI≤500). The results of 7 807 SPRCA their negative/positive reaction wells were enrolled and statistically analyzed in different grades and the four groups, the statistical differences between the four groups were compared. Multiple applications for the select HLA Class I compatible donor events were made for patients in two cases, and HLAMatchmaker program was used to calculate the number of HLA Class I epitopes mismatches between the donors and patients. The donor with the minimum number of epitopes mismatches was selected, while avoiding the corresponding antigens of HLA Class I antibodies in levels 1 and 2, the provision of HLA compatible platelets for IPTR. After the transfusions, the CCI value of the platelet transfusion efficacy evaluation index was calculated, and the clinical evaluation of the transfusion effect was obtained through statistical analysis. There were statistically significant differences in the positive results of SPRCA immunoassay among the strong positive group, medium positive group, and weak positive group of 51 IPTR patients with different specific of HLA -I class antibodies and corresponding antigens(all P <0.001). The positive results showed a range from high to low, with strong positive group>medium positive group>weak positive group. There were a statistical difference among between the strongly positive or moderately positive groups and the negative control group(P <0.001). There was no statistical difference between the weakly positive group and the negative control group(P >0.05). The strong positive group was set as the corresponding specific HLA Class I site corresponding antigen grade 1 avoidance threshold, the medium positive group as the grade 2 avoidance thresholds, and the weak positive group as the grade 3 avoidance threshold. In the case of donor platelet shortage, it is not necessary to avoid the weak positive group. Avoiding the strategy of donor antigens and HLAMatchmaker program scores ≤7 corresponding to HLA Class I antibodies of levels 1 and 2, with CCI values>4.5×109/L within 24 hours, can obtain effective clinical platelet transfusion conclusions. When selecting HLA Class I compatible donors for IPTR patients, the grading avoids HLA Class I antibodies corresponding to donor antigens, and the donor selection strategy with the minimum scores of HLAMatchmaker program is comprehensively selected. The negative result confirmed by platelet cross-matching experiments has certain practical application value for improving platelet count in IPTR patients.

Assessment of Platelet Crossmatch Results by Solid Phase Red Cell Adherence Assay among Pediatric Hemato-oncology Patients in a Tertiary Care Oncology Center

ABSTRACT Background and Objectives: This study employed platelets cross-matched by the solid-phase red cell adherence (SPRCA) method and oc-related it with posttransfusion platelet count increment in terms of corrected count increment (CCI) and percent platelet recovery (PPR) among pediatric hemato-oncology patients. It did not study subgroups of patients nor did it have a control group. This was an observational study. The objective of the study was to assess platelet crossmatch results by the SPRCA method and find its correlation with posttransfusion platelet count increment among pediatric hemato-oncology patients in a tertiary care oncology center. Methods: This was a prospective observational study performed on pediatric patients with hematological malignancies requiring platelet transfusions during August 2019–January 2020. An assessment of the response to platelet transfusion on the participants was analyzed against the crossmatch results using SPRCA to measure the response in terms of CCI and PPR. Results: A total of 80 pediatric multiply transfused patients of hematological malignancies were transfused with ABO-identical single donor platelet unit which was subsequently tested for compatibility SPRCA assay. Among a total of 80 platelet crossmatches, 86% (69/80) were found to be compatible. Among 69 patients with compatible platelet crossmatches, 91% (63/69) had adequate CCI (&gt;7500). Among 11 patients with incompatible crossmatches, 73% (8/11) had adequate CCI, and 27% (3/11) had inadequate CCI. Statistically significant association was found between crossmatch compatibility and PPR. Conclusions: Transfusion of crossmatched platelets from the available inventory to multiply transfused patients of hematological malignancies can be an option. It is very important to assess the posttransfusion platelet responses in such patients in terms of CCI as well as PPR. The SPRCA assay is a rapid and effective method to select the compatible unit from the inventory and is feasible to implement in an oncology setup to cater the requirement of multiple platelet transfusions to patients with hematological malignancies.

Virtual platelet cross-matching as transfusion management for patients with immune platelet refractoriness.

This study describes the use of the Epvix platform for virtual cross-matching (VC) of human leucocyte antigen (HLA)-compatible platelets for patients with immune platelet refractoriness, and demonstrates effectiveness of the selected platelets. A prospective cohort of haematological patients was evaluated from 2018 to 2022. HLA-typed donor bank profile was previously uploaded to the Epvix platform. Each patient's antibody reactivity panel (PRA) was included in the platform. Then, search, selection and VC were performed, and 24-h-corrected count increment (CCI) platelet transfusion was calculated (reference ≥2500). Six patients were included (four female, two male), with mean age of 61 years. HLA antibodies were detected as the cause of immunity for all patients, whereas four patients also had non-immune causes. High percentage of alloimmunization was detected in all studied patients (mean PRA: 85.7%). Thirty different donors were able to schedule and perform platelet donations. The mean 24-h CCI count was 9882. All platelet transfusions achieved a satisfactory CCI count except for two transfusion events. Presence of non-immune causes identified in these two cases could account for the unsatisfactory CCI. Epvix is a free application hosted on the Web and uses the HLAMatchmaker algorithm to generate histocompatibility reports. This study demonstrates the efficiency of VC performed by Epvix. However, physical cross-matching will still be necessary in some instances, as the platform does not support human platelet antigen polymorphism.

Comparison of platelet antibody screen, crossmatching and HLA antibody testing in patients refractory to platelet transfusions

Patients undergoing recurrent platelet transfusions can become refractory to these transfusions. Platelet antibody screens (Immucor), platelet crossmatching assays (Immucor), and HLA antibody testing are commonly used to test these patients. The relative effectiveness of these tests has not been determined. A higher incidence of strongly positive screen results that did not predict crossmatch results was anecdotally noted. Therefore, the results of the platelet antibody screens and crossmatches were systematically compiled over a 12-year period from 2010 to 2021. Of note, the Immucor Capture-P Ready Screen (platelet antibody) had a recall in March 2013 after which the performance of the test appears to have changed. The positivity rate of the platelet antibody screen increased over the course of the study, and this was statistically significant when analyzing year as a continuous variable and when grouping years by four-year periods (2010–13,2014–17,2018–21). In contrast, platelet crossmatch reactivity decreased slightly throughout this period. During the 2018–21 period, HLA antibody testing was commonly performed and correlated well with the crossmatch testing but not with the screen. These results suggest that the drastic increase in positivity we observed in the platelet antibody screen over this period is due to increased analytic sensitivity (with possible reduced specificity) of the screen and not a change in our patient population. Based on these results, the platelet antibody screen has little clinical utility and directly performing platelet crossmatching or HLA antibody testing is recommended for patients suspected to be refractory to platelet transfusions due to alloimmune-mediated factors.

Three Cases Highlighting Three Potential Pitfalls in the Platelet Refractory Workup

Abstract Introduction/Objective Platelet refractory (PR) patients do not achieve expected post-transfusion platelet counts. We investigate such patients with a post-transfusion platelet count, indirect Platelet Antibody Screen (ind-PAS), Class I HLA Antibody (HLA-Ab) testing, and platelet crossmatch (PXM). The following three cases highlight potential pitfalls of laboratory investigations for PR patients. Methods/Case Report Case #1 (57-year-old male with AML) revealed discrepant predicted and observed donor compatibility. Antibody testing demonstrated no platelet glycoprotein antibodies and HLA-ab to B13, corresponding to 4% calculated PRA (CPRA), indicating 96% predicted donor compatibility. However, PXM revealed that the patient was compatible with 11/14 (79%) screened donors. Communication with the reference laboratory revealed that two PXM incompatible units were ABO incompatible. Case #2 (27-year-old female with aplastic anemia) is a highly sensitized patient (99% CPRA). PXM revealed compatibility with 1/14 screened donors; however, she failed to respond to the compatible donor. To investigate this poor response, patient’s plasma was tested against HLA incompatible donors. Neat serum demonstrated compatibility with all donors, whereas 1:8 dilution resulted in positive crossmatches with half the donors. The false negative crossmatches are likely explained by prozone in the setting of high HLA-ab burden. Case #3 (79-year-old male with AML) demonstrated a discrepancy between the ind-PAS (PakPlus, Immucor), which was negative for HLA-ab, and HLA specificity testing (LABScreen, OneLambda), which revealed Class I antibodies, corresponding to 38% CPRA. Per the ind-PAS package insert, the sensitivity of the assay was 85%-91% compared to selected HLA-ab tests; other sources have demonstrated lower sensitivity of 68%. Results (if a Case Study enter NA) NA. Conclusion The three cases highlight the importance of investigating incongruent results. Cases #1 and #2 demonstrate pitfalls in the PXM: ABO incompatibility can result in positive PXM and false-negative PXM can occur in the setting of high HLA-ab burden. Case #3 reveals the importance of knowing a test’s limitation, especially sensitivity. Centers that only perform ind-PAS may miss HLA-ab detection.

Evaluation of the effectiveness of platelet crossmatching by the solid-phase red cell adherence assayin adult patients of a tertiary care hospital in Thailand: A retrospective study.

Background and AimsPlatelet transfusion refractoriness is well aware to be associated with poor clinical outcomes. Patients with the alloantibody causing refractoriness required cross‐matched compatible products to improve the platelet number. This study aims to evaluate the effectiveness and availability of platelet crossmatching provided by the solid‐phase red cell adherence (SPRCA) technique in the context of a tertiary university hospital.MethodsA retrospective chart review was performed of the records of 214 patients with platelet refractoriness in Siriraj Hospital, a tertiary university hospital in Thailand, between January 1, 2017, and December 31, 2020.ResultsThe SPRCA technique successfully provided cross‐matched compatible platelets to 114 patients (69.7%). Platelet crossmatching significantly improved the platelet counts, as shown by the increased 1‐ and 24‐h corrected‐count increments (p< 0.0001). No acute transfusion reactions were observed in these patients. Of the 114 patients who received cross‐matched platelets, 82 patients (71.9%) survived at 30‐day posttransfusion; whereas, 16 patients (14.0%) died within 7‐day posttransfusion.ConclusionThe SPRCA method can provide a high availability rate of cross‐matched platelets, which is effective at stopping and preventing clinical bleeding conditions. This method is appropriate to apply for platelet crossmatching in the context of a hospital blood bank.

Evaluation of Human Leucocyte Antigen Mediated Platelet Transfusion Refractoriness and Platelet Crossmatching Assay in Patients with Hematologic Disorders.

ObjectivesPlatelet refractoriness complicates the platelet transfusion, which is essential for managing thrombocytopenia in patients with hematological disorders. It is associated with adverse clinical outcomes and increased health care costs. We conducted a prospective study to determine the effectiveness of cross-matched compatible platelets in a group of patients refractory to platelets from random donors and to evaluate human leukocyte antigen (HLA)-mediated refractoriness.MethodsThis prospective study was conducted on 40 patients with different hematological disorders requiring platelet transfusions who were refractory to random platelets and presented to the hematology unit of Alexandria's main university hospitals between May 2020 and March 2021. They received 60 ABO-compatible platelet transfusions, either leuco-reduced or random donor platelets, stored for no more than 72 hours. A solid-phase red cell adherence technique (SPRCA) was used for platelet crossmatching. The corrected count increment (CCI) was used to monitor the effectiveness of each platelet transfusion with a cut-off value of 5 × 103/µL at 1 hour and 2.5 × 103/µL at 24 hours. Anti-HLA antibodies were assessed using the enzyme-linked immunosorbent assay technique.ResultsOut of 60 cross matches, 47 (78.3%) were compatible, and 13 (21.7%) were incompatible. Among 47 compatible results, 30 (63.8%) showed adequate CCI and 17 (36.2%) showed inadequate CCI at 1-hour post-transfusion. Among the incompatible results, 3 (23.1%) had adequate CCI and 10 (76.9%) had inadequate CCI. Significant improvements were found in the mean CCI when comparing cross-matched compatible platelets and incompatible platelets at 1hour or 24 hours (p=0.009 and p < 0.001, respectively). From the 40 studied patients, HLA alloimmunization was present in 18 patients (45.0%) and absent in the remaining 22 patients (55.0%). In the absence of HLA alloimmunization, patients showed significantly better responses at 1 hour and 24 hours (p =0.001 and p =0.015, respectively). There was better sensitivity of platelet crossmatching with random donor platelet concentrates than single donor platelet concentrates.ConclusionsPlatelet crossmatching using SPRCA and HLA screening are effective and rapid tools for better management of patients' refractory to platelet transfusions.

Methodological Study on the Establishment of HLA/HPA Gene Bank of Platelet Donors and Its Clinical Application.

The current study aimed at establishing a large-scale platelet donor database with known HPA and HLA genotypes in Ningbo, to provide matched platelets for the clinic and prevent the occurrence of ineffective immune platelet transfusion refractoriness (PTR). The platelet banks of clinical donor wtih HLA-A, HLA-B, HPA1, HPA2, HPA3, HPA4, HPA5, HPA6, HPA10, HPA15 and HPA21 genotyps were established. Meanwile, the platelet gene matching was performed on the donor. It was found that there were phenotype polymorphisms in 1000 donnors with HPA1, HPA2, HPA3, HPA4, HPA5, HPA6, HPA10, HPA15 and HPA21 genotyps, and allel polymorphism distribution in donnors with HPA1, HPA2, HPA3, HPA4, HPA5, HPA6, HPA15, HPA21 genotyps. The frequency of HPA10 was a gene, and not b gene, showing a single linear distribution. The HPA 2, HPA3, HPA15 system were the most polymorphic with three phenotypes: aa, ab, bb. In the HLA-A allele, the highest frequency is A*11:01 (24.25%). There were 13 alleles that were greater than 1%, such as A*24:02, A*02:01, A*33:03, and the accumulated frequency reached 96.20%. In the HLA-B allele, the highest gene frequency was B*40:01 (13.40%). There were 24 alleles that were greater than 1%, such as B*46: 01, B*58: 01, B*15: 01, and the accumulated frequency reached 91.60%. Platelet antibody cross matching was performed on 100 blood samples from patients with thrombocytopenia after multiple platelet transfusions. The number of consistent samples was 46 (46%). Twenty patients were transfused with platelet cross matching. Among them, 18 patients had obvious improvement in clinical symptoms and good hemostatic effect after transfusion, which was judged to be effective. Platelet donor HPA and HLA-A, B antigen genotyping database provided patients with individual appropriate platelets, and provided the effectiveness of immune platelet infusion ensuring effective platelet transfusion.

Prozone rates in the solid-phase platelet crossmatch assay and correlation with class I HLA antibody levels.

Solid-phase platelet crossmatch (PXM) testing is used to help manage patients with platelet transfusion-refractoriness. Recently, we published the first report of false-negative PXM results from prozone effect that was mitigated using sample dilution. This study aimed to describe the prevalence of PXM prozone effect and the levels of class I HLA antibodies (HLA-Abs) associated with positive PXM results and with false-negative PXM results from prozone effect. A cross-sectional study of patients undergoing PXM testing from July 2019 through December 2020 was performed. All PXM tests were run simultaneously using undiluted and 1:4 diluted patient plasma. Prozone effect was defined as a negative PXM result using undiluted patient plasma but a positive PXM result using 1:4 diluted patient plasma. Among 59 patients, 830 individual ABO-compatible PXM results yielded an overall positivity rate of 25.8% (214/830) and a false-negative rate from prozone effect of 4.7% (10/214). Among the 28 patients with class I HLA-Ab testing and no other anti-platelet antibodies, maximum HLA-Ab mean fluorescence intensity (MFI) was significantly associated with a positive PXM result (p < .0001; AUC approx. 0.9) and categorized into negative (<3700), indeterminate (3700-10300), and positive (>10300) maximum HLA-Ab MFI zones. Maximum HLA-Ab MFI, however, was not associated with prozone effect (p=.17; AUC approx. 0.6). While there is a strong predictive association between class I HLA-Ab levels and positive PXM results, PXM prozone effect is a common occurrence not associated with class I HLA-Ab levels, so additional testing with diluted samples should be considered.

Correlation of platelet crossmatch results by Solid Phase Red Cell Adherence Assay (SPRCA) with post-transfusion platelet count increment in adult hemato-oncology patients of a tertiary care oncology centre in India

AimsTo assess platelet crossmatch result by SPRCA and find its correlation with post-transfusion platelet count increment among adult hemato-oncology patients. MethodsA prospective observational pilot study of 50 adult hematologic malignancy patients previously transfused, but not already known to be transfusion-refractory and without any nonimmune causes for inadequate response to platelet transfusion were included after obtaining informed consent. They were transfused one unit of ABO identical single donor platelet. Ten minutes to 1 -h post-transfusion CCI was calculated.CCI ≥ 7500 was considered as adequate response. Post-transfusion crossmatching by SPRCA was performed by using preserved platelet samples from donor units with the serum of the respective patient. Statistical analysis of the correlation between platelet crossmatch results and CCI was done. ResultsOut of 50 crossmatches, 78% (39/50) showed compatible and 22% (11/50) showed incompatible results. Among 39 compatible results, 87.2% (34/39) showed adequate CCI and 12.8% (5/39) showed inadequate CCI. Among 11 incompatible results, 18.2% had adequate CCI and 81.8% had inadequate CCI. The difference between the response in terms of CCI to compatible and incompatible crossmatches was found to be statistically significant (p < 0.05). Other variables like age, sex, number of previous transfusions and underlying clinical condition of the patient were not found to have any effect on the compatibility of crossmatch. ConclusionsTransfusion of crossmatched platelets to non-refractory, multiply transfused hematological malignancy patients without serious illness might provide a small benefit over transfusing randomly selected platelets, though these data must be confirmed with a larger sample size.

Efficacy of cross-match compatible platelets in multi transfused haemato-oncology patients refractory to platelet transfusion

BackgroundPlatelet refractoriness, which leads to platelet transfusion failure resulting in significant morbidity and long hospital stay, is routinely not investigated. AimsTo determine the efficacy of cross-match compatible platelets in multi-transfused alloimmunized hemato-oncological patients refractory to platelet transfusion. Materials and method149 ABO compatible single donor apheresis platelet transfusions given to 38 alloimmunized refractory patients. Corrected Count Increment (CCI) <5000 (1 h) was taken to define refractoriness. Solid-phase red cell adherence assay was used to determine the alloimmunization status and platelet cross-matching. Post Transfusion Platelet Increment, CCI and the Percentage Platelet Recovery were used to monitor the effectiveness of platelet transfusion. ANOVA test followed by Post hoc test Tukey HSD used to compare group means and classified into three groups depending upon the cross-matching and compatibility status. Categorical data was analysed for various outcomes using Pearson's chi square test or Fischer exact test. ResultPatients showed statistically significant recovery in terms of PPI, CCI and PPR at 1 h post SDAP transfusions when they received cross-matched compatible platelets. The one-hour CCI was significantly higher for cross-match-compatible platelets (19173 ± 2692) than for incompatible (5888 ± 1526) and for uncross-matched (8140 ± 1480). Forty four (97.8%) of 45 cross-matched compatible platelet transfusion episodes showed a satisfactory response in terms of PPI and CCI values as compared to 50 % and 53.9% in uncross-matched group respectively (p < 0.0001). ConclusionPlatelet cross-matching is an effective intervention in the management of multi-transfused alloimmunized Haemato-oncological patients, refractory to platelet transfusion.

Frequency of Platelet Crossmatch Positivity and Predictive Value for Poor Platelet Increment Among Paediatric Oncohaematology Patients in India.

Immune platelet destruction is a significant cause for platelet refractoriness. The platelet crossmatch-a solid phase red cell adherence assay utilizes donor platelets and patient serum to assess compatibility and appears to be a feasible option in resource constrained settings. This study was done to evaluate the frequency of platelet crossmatch positivity among Paediatric Oncohaematology patients and also to assess whether a positive crossmatch is predictive of unsuccessful platelet transfusions in this group of patients. Paediatric Oncohaematology patients who received platelet transfusions between March 2013 and September 2013 were included in the study. The pre-transfusion patient sample and a segment from the transfused donor unit were used for performing the platelet crossmatch. A blood sample was collected one hour after the transfusion to assess post-transfusion platelet count. Corrected count increment (CCI) was calculated using the standard formula. CCI ≤ 7500/µL/m2/1011 was considered evidence of an unsuccessful transfusion. Seventy-three platelet crossmatches were performed for 69 patients, of which 30 patient samples (41%) showed crossmatch positivity. 25 (89.2%) of 28 unsuccessful transfusions showed crossmatch positivity, and 40 (88.9%) of 45 successful transfusions showed negative crossmatches (p = 0.03). Crossmatch positivity among transfusion dependent Paediatric Oncohaematology patients was as high as 42%, when ABO matched platelet units were allocated without further testing. Our results indicate that this test may be a reliable tool to select compatible platelet units and an effective intervention in the management of patients at risk of immune platelet refractoriness.

A case report of neonatal alloimmune thrombocytopenia: The utility of platelet crossmatch studies in the diagnosis and management

Alloimmune thrombocytopenia is the most common cause of severe thrombocytopenia and intracranial bleed in the perinatal period, resulting from placental transfer of maternal platelet antibodies directed against the paternal-derived fetal platelet antigens. It is estimated to occur in 1 in 2500 births but continues to be an underdiagnosed and underreported condition in our country. Detection of human platelet antigen-specific antibody in the maternal serum and platelet genotyping are the gold standards in the diagnosis. This case report highlights the utility of platelet crossmatch studies in the diagnosis and management of neonatal alloimmune thrombocytopenia.

Neonatal alloimmune thrombocytopenia due to a new alloantigen Bl(a) defined by an Asp458Gly substitution in GPIIIa.

Neonatal alloimmune thrombocytopenia (NAIT) commonly arises due to antibodies against a small number of well-defined human platelet antigens (HPAs). A minority of NAIT cases occur due to maternal immunization against low-frequency polymorphisms in platelet glycoprotein that result in new immunogenic epitopes. Antibodies to these novel epitopes can be detected by the incubation of maternal serum with paternal platelets and is usually performed after initial investigation using HPA-typed panel platelets has failed to provide evidence of NAIT. The propositus and the parents from a case of suspected neonatal alloimmune thrombocytopenia (NAIT) were investigated using serologic and molecular techniques to detect and identify relevant platelet-specific antibodies and for HPA typing. Calculations of molecular dynamics were undertaken to explore potential variations in the molecular structure. Maternal antibodies were detected that were reactive only in crossmatch with paternal platelets using the platelet immunofluorescence test (PIFT) and a GPIIb/IIIa monoclonal antibody immobilization of platelet antigen (MAIPA) assay. In the propositus and father, a novel mutation c.1373 A > G was found in exon 10 of ITGB3 resulting in the substitution of an aspartic acid for a glycine (p.Asp458Gly). Recombinant GPIIIa glycoprotein mutated to contain the novel mutation and expressed in HEK293 cells with GPIIb was also specifically recognized by maternal antibodies. Calculations of molecular dynamics identified that the mutation was in a structurally constrained site. This case describes a low-frequency platelet antigen (Asp458Gly) that defines a further alloantigenic target in NAIT. The case emphasizes the role of the platelet crossmatch as the single most useful tool to establish evidence of immunization of low-frequency platelet glycoprotein polymorphisms. A crossmatch should always be performed where there is strong clinical evidence of NAIT but initial laboratory investigations are not confirmatory.

Human leukocyte antigen antibody detection technologies in platelet transfusion refractoriness, with special emphasis on functional test

In the clinical settings of transfusion medicine, human leukocyte antigen (HLA) antibody detection and platelet cross-matching are important tests applied in the management of patients with platelet transfusion refractoriness (PTR). Presently, fluorescence beads technologies using extracted and purified HLA antigens are applied for the HLA antibody screening, and highly sensitive methods such as flow cytometry crossmatch (FCXM) and immunocomplex capture fluorescence analysis (ICFA), based on the use of live intact cells, are used for the cross-matching. Such highly sensitive methods have importantly contributed for the prevention of refractoriness caused by low titer antibodies but, on the other hand, they also detect those low titer antibodies or naturally occurring antibodies without or with unknown clinical relevance. Moreover, the use of recombinant antigens has been shown to be associated with specific issues. In addition, although the relationship between the antibody features and the clinical implications, such as the relevance of antibody subclass in platelet clearance, has been investigated in the field of transplantation, presently, few evidence exists in the literature related to blood transfusion. Here, we reviewed on the available HLA antibody detection methods, the issues of the present methods, and present a new method for the evaluation of clinically significant HLA antibodies using in vitro platelet phagocytosis assay.

P095 Antibodies to human platelet antigens: when do they cause an incompatible crossmatch

Aim Our center evaluates approximately 100 platelet refractory patients each year. Previously, we have correlated single antigen bead MFI with platelet crossmatch results in an effort to optimize our process for selection of HLA-compatible platelets. However, we continue to struggle to appreciate when antibodies to human platelet antigens (HPA) are also contributing to the platelet refractory state. In this study, our aim is to understand the relationship between HPA antibody testing (PakPlus, Immucor) and platelet crossmatch (CaptureP, Immucor). Methods One patient sample with a singular & strong specificity, HPA-1a, was identified based on PakPlus testing. This sample was also negative for HLA antibodies based on PakPlus testing. The plasma was then diluted and the PakPlus result, optical density (OD), was determined at each dilution. Ten antigen-positive platelet units were crossmatched with the battery of plasma dilutions. The strength of the platelet crossmatch was graded by one technologist, blinded to the dilution, with 4+ being strong positive to negative. Results The patient sample demonstrated a fairly linear dilution of HPA-1a, beginning with a neat result of OD 1.33. All 10 antigen-positive platelet units were positive at OD 0.42 or greater, yet negative at OD 0.04. There was platelet-specific variability between OD 0.3 and OD 0.06 (see Table 1). Conclusions These results demonstrate that HPA antibodies cause crossmatch positivity at low OD. The variability seen may be due to homozygosity versus heterozygosity of antigen expression. These initial results suggest that a PakPlus result as low as OD 0.06 may cause incompatibility in the platelet crossmatch and could be considered when selecting platelets for the platelet refractory patient. Download : Download high-res image (413KB) Download : Download full-size image

How do I … manage the platelet transfusion-refractory patient?

Platelet transfusion-refractoriness is a challenging and expensive clinical scenario seen most often in patients with hematologic malignancies. Although the majority of platelet transfusion-refractory cases are due to nonimmune causes, a significant minority are caused by alloimmunization against Class I human leukocyte antigens (HLAs) or human platelet antigens (HPAs). Such platelet transfusion-refractory patients can be effectively managed with appropriate antigen-negative products. Our institution has developed a diagnostic and management algorithm for the platelet transfusion-refractory patient with an early focus on identifying those cases caused by immune-mediated factors. Using physical platelet cross-matches to initially classify platelet transfusion-refractory patients as immune-mediated or not, cross-match-compatible inventory is then provided to immune-mediated patients, whereas subsequent HLA (with or without HPA) testing is performed. Our blood donor program performs Class I HLA typing of all repeat platelet donors to facilitate the identification of antigen-negative platelet units (virtual cross-matching) as well as the recruitment of HLA-matched donors. The platelet transfusion-refractoriness algorithm realizes an initial net cost savings once two apheresis platelets are saved from use for each newly identified, immune-mediated platelet transfusion-refractory patient. An algorithm utilizing physical platelet cross-matches, Class I HLA and HPA antibody testing, and upfront Class I HLA typing of platelet donors leads to overall resource savings and improved clinical management for platelet transfusion-refractory patients.

P159 Comparison of single antigen bead and platelet crossmatch assays

Aim Satisfactory increments may be achieved in refractory patients with platelets that are antigen negative for corresponding HLA antibody. However, no HLA antibody test or cutoff is universally used. Our small study sought to identify an appropriate single antigen bead assay (SAB) and Mean Fluorescent Intensity (MFI) cutoff to select HLA mismatched platelets in lieu of platelet crossmatch (PLT XM). Methods HLA sensitized patient sera (n = 17; CPRA 24–100%, median 93%) were tested by One Lambda™ Class I IgG-SAB and C1q-SAB and Immucor™ C3d-SAB, and PLT XM against 8 Class I typed platelets. Cumulative MFI for donor specific antibodies (DSA) in 36 patient-donor pairs were calculated. The vendor’s recommended cutoff for C3d-SAB, 500 MFI for C1q-SAB and 3000, 5000 and 10,000 MFI for IgG-SAB were used to assign an expected result to compare with the observed PLT XM result. MFI values from IgG-SAB negative, PLT XM positive (presumed false negative) pairs were replaced with MFI values from 1:10 diluted serum to correct for complement interference (Mod IgG-SAB). Results were stratified by MFI and sensitivity and specificity were calculated. Results No SAB was 100% concordant with PLT XM. Two positive PLT XMs were likely due to ABO incompatibility. Two negative PLT XMs occurred in the presence of moderately strong B44 DSA. C1q-SAB compares favorably with PLT XM. C3d-SAB has the highest specificity, but lowest sensitivity. IgG- and Mod IgG-SAB (3000 MFI) have excellent sensitivity but poor specificity. When Mod IgG-SAB cutoff is raised to 5000 MFI or 10,000 MFI, specificity increases but sensitivity decreases. Mod IgG-SAB (5000 MFI) sensitivity is comparable to C1q-SAB and specificity is improved when B44 DSA Conclusions C1q-SAB is reported to correlate with acceptable platelet increments and correlates well with PLT XM in our study. Mod IgG-SAB (5000 MFI) that ignores anti-B44 DSA

Analysis of platelet-reactive alloantibodies and evaluation of cross-match-compatible platelets for the management of patients with transfusion refractoriness.

Cross-match-compatible platelets can improve corrected count increments (CCIs) in alloimmunised patients with transfusion refractoriness. However, only a few studies mentioned that the specificities of platelet-reactive alloantibodies can predict high reactivity in cross-match assays among these patients. A total of 204 medical records of patients who were refractory to random single-donor apheresis platelets between January 2014 and December 2014 were enrolled. Platelet-reactive antibodies in patients' serum were screened by an enzyme-linked immunosorbent assay (ELISA).The platelet cross-match assays were performed by a solid-phase adherence assay. The specificities of human leukocyte antigen (HLA) class I and human platelet antigens (HPAs) alloantibodies were determined by Luminex Single Antigen and Monoclonal Antibody-specific Immobilization of Platelet Antigens (MAIPA) assays, respectively. Anti-HLA and anti-HPA alloantibodies were found in 114 of 204 (55.88%) patients, including 110 (96.49%) with anti-HLA alloantibodies only, 2 (1.75%) with anti-HPA alloantibodies (anti-GPIIb/IIIa) only and 2 (1.75%) with both anti-HLA and anti-HPA alloantibodies (anti-HPA-3a and anti-HPA-5b). The most common HLA class I alloantibody phenotypes in cross-match-incompatible patients were HLA-A23 (59.38%), -A24 (50.00%), -A02 (43.75%), -B27 (65.63%), -B40 (50.00%), -B18 (46.88%) and -B07 (43.75%). A total of 480 cross-matched platelet units were administered in 82 of 114 alloimmunised patients with a mean CCI of 7800 ± 5200, a significant improvement over random platelet units (P < 0.001). No development of additional platelet alloantibodies was observed during this platelet transfusion regiment. This study showed that transfusion of cross-match-compatible platelet units offers effective and safe management of platelet transfusion refractoriness (PTR). The finding of alloantibodies among cross-match-incompatible cases can be used as predictors for platelet donor selection.