Comparison of HLA antibody identification methods for the selection of platelet products for HLA‐mediated platelet refractory patients

In an ineffective transfusion context, solid‐phase immunoassays using the Luminex platform for the detection and characterization of HLA antibodies are currently used to select HLA‐compatible platelet products. A new HLA antibody identification method, the HISTO SPOT® HLA AB test (BAG Health care GmbH, Lich, Germany), based on the detection of antibodies directed against a recombinant single antigen (SA) by colored spots detected by HISTO MATCH HLA AB module software, runs fully automated on the MR.SPOT®. The aim of this study was to compare the ability of the HISTO SPOT HLA AB and C1qScreen™ (C1q SAB) assays with that of the Labscreen single antigen class I (OL SAB) assay to detect anti‐HLA class I antibodies in 56 serum samples from 54 platelet refractory acute myeloid leukemia patients who received HLA mismatch platelet concentrates at a single oncohematology center. In total, 1414 class I specificities, 433 HLA‐A and 981 HLA‐B, were detected by the OL SAB test. The mean fluorescence intensity (MFI) was >5000 for 874 antigens and <5000 for 655 antigens. The HISTO SPOT® HLA AB and C1q SAB tests identified 85% and 79% of OL SA‐detected antigens with an MFI >5000, respectively, but did not identify 34% and 44% of OL SAB‐detected antigens, highlighting the lower sensitivity of these techniques. Interestingly, the donor‐specific antibodies (DSAs) identified by the HISTO SPOT® HLA AB and C1q SAB assays reacted against HLA mismatch platelet concentrates with the same specificity (86%) and positive predictive (77%) value as in the OL SAB test when the MFI threshold was >2000 for DSA detection. Although the HISTO SPOT® HLA AB test is less sensitive than the OL SAB test, this test could be used for the selection of HLA‐compatible platelet products.

In an ineffective transfusion context, solid-phase immunoassays using the Luminex platform for the detection and characterization of HLA antibodies are currently used to select HLA-compatible platelet products.A new HLA antibody identification method, the HISTO SPOT ® HLA AB test (BAG Health care GmbH, Lich, Germany), based on the detection of antibodies directed against a recombinant single antigen (SA) by colored spots detected by HISTO MATCH HLA AB module software, runs fully automated on the MR.SPOT ® .
The aim of this study was to compare the ability of the HISTO SPOT HLA AB and C1qScreen™ (C1q SAB) assays with that of the Labscreen single antigen class I (OL SAB) assay to detect anti-HLA class I antibodies in 56 serum samples from 54 platelet refractory acute myeloid leukemia patients who received HLA mismatch platelet concentrates at a single oncohematology center.In total, 1414 class I specificities, 433 HLA-A and 981 HLA-B, were detected by the OL SAB test.The mean fluorescence intensity (MFI) was >5000 for 874 antigens and <5000 for 655 antigens.The HISTO SPOT ® HLA AB and C1q SAB tests identified 85% and 79% of OL SA-detected antigens with an MFI >5000, respectively, but did not identify 34% and 44% of OL SAB-detected antigens, highlighting the lower sensitivity of these techniques.Interestingly, the donor-specific antibodies (DSAs) identified by the HISTO SPOT ® HLA AB and C1q SAB assays reacted against HLA mismatch platelet concentrates with the same specificity (86%) and positive predictive (77%) value as in the OL SAB test when the MFI threshold was >2000 for DSA detection.Although the HISTO SPOT ® HLA AB test is less sensitive than the OL SAB test, this test could be used for the selection of HLA-compatible platelet products.

K E Y W O R D S
Histospot, HLA alloantibody platelet refractoriness, Luminex

| INTRODUCTION
Currently, refractory status remains the major problem of platelet transfusions in patients suffering from hematological diseases or aplasia.Platelet transfusion efficacy can be impaired by multiple factors, and the mechanisms involved are of either immunological or nonimmunological origin (hypersplenism, fever, infection, disseminated intravascular coagulation, etc.). 1 The first etiology of platelet transfusion ineffectiveness due to immunological conflict is the presence of anti-HLA class I allo-immunization. 2 The clinical effectiveness of substitute transfusions can then gradually decrease during repeated transfusions until a refractory state is determined, for which the transfused platelets do not modify the platelet count and stop only transitory hemorrhage.
The introduction of solid-phase immunoassays using recombinant single antigens (SAs) for the detection and characterization of HLA antibodies in transfusion has resulted in a new paradigm with respect to the selection of HLA platelet products.This is because SA tests provide a much higher sensitivity than complement-dependent lymphocytotoxicity (CDC) assays and because each HLA antigen is independently detected.However, although the SA assays allow the detection of antibodies not detectable by CDC assays, the clinical significance of these antibodies is incompletely understood. 3Serum samples from the patients included in the Trial to Reduce Alloimmunization to Platelets (TRAP) study (Accession Number: HLB00700909a) were retrospectively analyzed in LUMINEX ® technique.In patients negative to the less sensitive lymphocytotoxicity assay, the MFI level did not predict transfusion ineffectiveness. 4In addition, SA testing raises technical issues that require resolution and careful consideration when interpreting antibody results. 5Indeed, anti-HLA alloantibodies were found in 63% of healthy subjects in the absence of immunizing events.7][8][9] While some studies show that these antibodies have no impact on organ transplantation, their involvement in platelet transfusion has not yet been studied.The use of more specific techniques to identify only complement-dependent antibodies in SA assays provides an alternative to restrict prohibited antigens to those most pathogenic and to increase the number of compatible donors on file. 10The value of these new tests (C1q assays or C3d assays) for predicting the pathogenic effect of antibodies, particularly in organ transplantation, is still debated. 11,12evertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients.Some publications suggest that the LUMINEX ® technique makes it possible to better predict transfusion efficacy as a function of the level of mean fluorescence intensity (MFI) associated with the antibodies identified. 13The presence of antibodies associated with an MFI >5000 makes it possible to identify patients at risk of refractory status.However, the relationship between platelet transfusion ineffectiveness and the MFI threshold has not been established, leading to a variable transfusion threshold depending on the laboratory.Additionally, thresholds can also be locus-specific.
The HISTO SPOT ® HLA AB test (BAG Health care GmbH, Lich, Germany) is a new HLA antibody identification method based on the interaction between the HLA antibodies present in the sample and the recombinant single antigen immobilized on the microarray.The micro HLAABT2-01/eng ELISA for the HISTO SPOT ® HLA AB test runs fully automated on the MR.SPOT ® processor.The antibodies binding to their target antigen are recognized by horseradish peroxidase-conjugated anti-IgG.The presence of the antigen/antibody/anti-IgG product is detected by a colored spot formed by tetramethylbenzidine (TMB). 14,15he aim of this study is to retrospectively evaluate this new method to select virtual compatible platelet transfusion in refractory platelet patients.All serum from platelet refractory samples was tested with the HISTO SPOT ® HLA AB test.The HLA identification results were compared to the results for the other SA test established in the market (LABScreen ® Single Antigen, One Lambda) and those reported in the complementdependent SA test (C1q test assays, one lambda).Then, the efficacy of platelet concentrate was evaluated by the corrected count increment at 24 h (CCI_24h) as a function of the HLA specificities detected by the three SA tests.

| Study design and patient characteristics
Fifty-six serum samples from November 2016 to December 2019 were collected for anti-HLA antibody screening and identification from 54 refractory platelet acute myeloid leukemia (AML) patients who received HLA mismatch platelet concentrates in an oncohematology single center, Institut Paoli-calmettes, Marseille (Figure 1).All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.
Patient-identifying information was anonymized, and institutional review board approval was obtained from the Ethical Committee (approval number IPC 2022-039). 16atient characteristics are described in Table 1.
Serum samples from all patients were prospectively tested with Labscreen single antigen class I (OL SAB) and retrospectively simultaneously tested with both the C1q SAB and HISTO SPOT ® HLA AB assays.All serum samples were thawed for 5 min at 4500 rpm to separate the serum from the whole blood, pretreated with EDTA (0.1 mM), aliquoted (100 μL/tube), and stored at À40 C.

| HLA genotyping
Recipients were genotyped for low-resolution HLA-A and HLA-B by LABType SSO (Thermo Fisher, One Lambda, Canoga park, CA) according to the manufacturer's specifications, and the retrieved output was analyzed for allele identification using HLA Fusion v 1.2.1.Software (One Lambda, Canoga park, CA).

| HLA antibody screening and the identification protocol
Fifty-six serum samples obtained from 54 patients were routinely assessed using Luminex single-antigen flow beads to determine antibody specificity using Labscreen single antigen class I (One Lambda, Thermo Fisher Scientific, Canoga Park, California, USA) assay according to the recommendations of the manufacturer.Briefly, 20 μL of test serum was incubated with 5 μL of LABScreen beads.The beads were washed three times with 1Â wash buffer, and 100 μL goat anti-human IgG conjugated with R-phycoerythrin (PE) and diluted 1:100 in 1Â wash buffer was added.After a 30-min incubation step at room temperature in the dark, 1Â PBS buffer was added.Samples were analyzed using a LABScan.100 (Luminex ® 100, Austin, TX, USA) system.The data were analyzed by using HLA Fusion.4.0 Software (One Lambda, West Hills, CA, USA).The mean fluorescence intensity (MFI) was calculated using the baseline formula proposed by Fusion 4.0 software.All beads with a normalized MFI threshold greater than 1000 were considered positive.
The panel-reactive antibody (PRA) percentages were calculated using the calculated panel-reactive antibody (CPRA) algorithm, established by the United Network for Organ Sharing (UNOS) and Eurotransplant allocation system.A CPRA value (%CPRA) was automatically determined as the percentage of individuals with unacceptable antigens.

| C1q SAB detection
All HLA antibodies were tested for the ability to bind C1q.The binding level was determined by the C1qScreen™ assay per the manufacturer's instructions (One Lambda, part of Thermo Fisher Scientific, Canoga Park, California, USA).The fluorescence intensity was measured using a Luminex-based LABScan™ 100 flow analyzer.The C1q binding antibody specificity and levels were analyzed and determined using Fusion 4.0 software.A C1q MFI threshold greater than 1000 was considered positive.

| HISTO SPOT ® HLA AB test
The HISTO SPOT ® HLA AB Screen/ID assay is a miniaturized and multiplexed adaptation of the conventional solidphase enzyme-linked immunosorbent assay (ELISA).Each recombinant or native HLA antigen was deposited on the bottom of a plate well and represents an independent spot.Serum samples were first diluted to a 1:20 ratio using a sample buffer (5 μL of the serum and 95 μL of SAMBUF) within a 96-well microtiter plate.The subsequent assay procedures were automated and executed using the MR.SPOT ® processor.
At the end of the run, the MR.SPOT ® processor captured images of the resulting colored dots at the bottom of each test well.These images were then imported into the HISTO MATCH interpretation software for analysis.
The image analysis program determined the intensity of each spot in the array, and the mean color intensity (MCI) was calculated by subtracting the background signal from the measured spot intensity.Based on the spot intensity and the variability of the background, the software determined a cut off value for each test.If the normalized signal was within ±2% of the cut off, the results had to be reviewed and edited by the user.

| Evaluation of platelet transfusion ineffectiveness
The pretransfusion platelet count was measured 1 to 9 h before transfusion, and the posttransfusion platelet count was determined 15 to 20 h after transfusion.Corrected count increments (CCI_24h) were calculated as follows: (posttransfusion platelet countpretransfusion platelet count in 10 9 /L) Â (body surface area in m 2 )/(platelet dose transfused Â 10 11 ).According to HAS guidelines, a successful transfusion was defined as a CCI_24h > 7, and a transfusion was ineffective if the CCI_24h was < 7 despite the dose of platelets transfused being adapted to the patient's weight and the patient being ABO compatible.

| Statistical analyses
Data from the three methods, LSA, LSA C1q and Histospot, were collected.The PRA percentage was calculated.Antigen specificities from Histospot and LSAC1q results were analyzed according to the different cutoff values (500, 2000, 5000, and 10,000) from the MFI of SA from OL SAB.The percentages of antigen specificities concordant between the different assays at each cutoff value were then calculated.A few HLA class I antigens were excluded from the analysis because the respective antigens were not present on the HISTO SPOT ® chips used at the time.If there were two antigens on the chip for one serological specificity (e.g., A*02:01 and A*02:03 for A2) and only one of the antigens was positive, the serological specificity was classified as positive.ROC curves were plotted to determine the specificity and sensitivity of predicting the MFI threshold between SA OL and HistoSPOT and between SA OL and LSA C1q.
The sensitivity and specificity for each technique were determined.A true positive was a sample for which a donor-specific antibody (DSA) was identified in the context of an ineffective transfusion (CCI_24H < 7).A true negative was a sample for which no DSA was identified in the context of an effective transfusion (CCI_24H > 7).A false positive was a sample for which a DSA was identified in the context of an effective transfusion (CCI_24H > 7).A false negative was a sample for which no DSA was identified in the context of an ineffective transfusion (CCI_24H < 7).The sensitivity (TEST+ CCI < 7)/(TEST+ CCI < 7 + TESTÀ CCI < 7) was measured as the proportion of true positives that were correctly identified, and the specificity (TESTÀ CCI > 7)/ (TESTÀ CCI > 7 + TEST+ CCI > 7) was measured as the proportion of true negatives that were correctly identified.The positive predictive value (TEST+ CCI < 7)/ (TEST+ CCI < 7 + TEST+ CCI > 7) and negative predictive value (TESTÀ CCI > 7)/(TESTÀ CCI > 7 + TESTÀ CCI < 7) (PPV and NPV, respectively) were the proportions of positive and negative results in diagnostic tests that were associated with effective transfusion true positive and true negative results, respectively.All analyses were conducted by using XLStat software.

Patients
and platelet concentrate transfusion characteristics: Among the 54 patients, the CCI_24h were calculated after the transfusion of platelet concentrates with phenotypes with compatible blood groups and with doses adapted to the weight of each patient for only 15 patients (Figure 1).These patients had an average cPRA of 61% (11%-100%; median, 65% +/À 30).Eleven out of fifteen (73%) had a CPRA >50%.These patients received 135 platelet concentrate transfusions within an average of 41.9 days after testing for anti-HLA antibodies (6-109 days; median, 38 +/À 32 days).Among the 135 platelet transfusions, 71 were from whole blood buffy coat concentrates, and 64 were from random singledonor apheresis platelet units.Among the 64 transfusions from apheresis, 3 platelet products were not HLA phenotyped and 2 products were transfused but the dose was not adapted to the patient's weight.Each patient received an average of 4 apheresis products during this study (1 to 9 products; median, 4 +/À 2.56 products).

| Characteristics of commercial kits
The SA OL and HISTO SPOT ® HLA AB kits were composed of 81 and 79 class I single antigens, respectively (Table 2 Comparison of OL SAB with the C1q SAB and HISTO SPOT ® HLA AB assays: Two serum samples were negative, and 54 serum samples were positive, with CPRA ranging from 2% to 98%.Thirteen serum samples had a CPRA >50%.Forty and 32 serum samples were HLA Antibody positive in the HISTO SPOT ® HLA AB test and C1q SAB assay, respectively.In total, 1414 HLA class I specificities, 433 HLA-A and 981 HLA-B, were reported by OL SAB test for the 54 positive serum samples (Table 3).Sixty-six percent (68% for HLA-A and 66% for HLA-B) of these specificities were detected by the HISTO SPOT ® HLA AB test and 57% (51% for HLA-A and 59% for HLA-B) by the C1q SAB test.The MFI of 825 HLA antigen (58%) from the OL SAB test was >5000.All the most frequent HLA-A and HLA-B antigens in the European population were the target of at least one HLA alloantibody.
The C1q SAB assay did not recognize any specificity that was not identified by an IgG OL SAB test.ROC curve analysis showed an AUC of 0.811 (0.777-0.845 (p < 0.0001)), establishing a threshold of 7293 MFI from the OL SAB test to detect antibodies with the C1q SAB test with a sensitivity and specificity of 68% and 87%, respectively (Figure 2B).
Seventy-five percent of HLA specificities were positively concordant between the HISTO SPOT ® HLA AB test and C1q SAB assay (Table 4).Eighty-four percent of C1q-binding antibodies had an MFI > 5000.Only sixty-nine (7%) HLA specificities (15 HLA-A and 54 HLA-B) were detected by the C1q SAB test but not by the HISTO SPOT ® HLA AB test.Among them, 64% had an OL SA MFI > 5000, and no HLA-A and HLA-B specificities were preferred.No ROC curve was statistically interpretable when the MFI of the OL SAB test for detecting was compared to the HLA antigens detected by HISTO SPOT ® HLA AB test.
With an MFI threshold of >5000 for DSA detection (Table 5) by OL SAB, DSAs were detected in 5/38 ineffective transfusions (mean MFI: 7100 +/À800).All OL SABdetected DSAs were also detected by the HISTO SPOT ® HLA AB test and bound to C1q.Among the 21 effective transfusions, DSAs were detected in one transfusion by both the OL SAB and HISTOSPOT tests (MFI: 6300) but did not bind C1q.Since the MFI threshold for DSA detection was >2000, the NPV and PPV were relatively similar for the three assays.

| DISCUSSION
To our knowledge, this study is the first to compare the results of the OL SAB test, which is used routinely in many HLA laboratories, with the results of the HISTO SPOT ® HLA AB test in the context of the management of platelet inefficiencies.The study shows that the HISTO SPOT ® HLA AB method is less sensitive than the OL SAB but is relatively similar to the C1q SAB assays.Indeed, most HLA antibodies detected by the HISTO SPOT ® HLA AB test had an MFI > 6340 in the OL SAB test.However, this "less sensitive" method did not detect "natural antibodies" 17 or HLA antibodies against denatured HLA antigens, 18 which should be considered falsepositives.Recently, comparisons of the OL SAB test and the HISTO SPOT ® HLA AB test showed that the HISTO SPOT ® HLA AB test did not detect antibodies against denatured HLA antigens that were found with the OL SAB test. 14n this study, the C1q SAB test was used because some authors showed that DSA-bound C1q appeared to be more clinically relevant than DSAs identified only by the OL SAB test for patients with refractory platelet transfusions. 10However, not surprisingly as shown by the ROC curve analysis in our study and other studies of  organ transplantation, the C1q-binding ability is linked to the antibody strength (threshold: 7000-10, 000). 19,20his relationship is not perfect because the C1q SAB assay is above all used to determine the real strength of antibodies, mainly explained by the high prevalence of IgG1, as the immune-dominant subclass IgG. 21Despite a significant correlation between the results of the C1q SAB test and the HISTO SPOT ® HLA AB results, these two tests revealed different HLA specificities in a few cases.Indeed, the HISTO SPOT ® HLA AB test detected 93% HLA antibodies with C1q reactivity, suggesting that the HISTO SPOT ® HLA AB test detected alloimmunization probably associated with cellular toxicities.Fifty percent of the non-HLA C1q antibodies detected by the HISTO SPOT ® HLA AB test had an MFI < 5000, suggesting that these antibodies did not have sufficient MFI to be detected by the C1q SAB test.Surprisingly, this study indicates that OL SA MFI cutoff values >2000 could be used to determine the optimal HLA-selected platelet apheresis regimen.This threshold provides sensitive and specific values similar to those of the HISTO SPOT ® HLA AB test and C1q test, suggesting that the C1q assay does not add value to the OL SAB test.However, it is lower than in other studies, without real explanation except to the small number of platelet transfusion efficiency studied.Indeed, strong HLA antibodies (MFI > 10,000) might be clinically relevant for a refractory patient and should be avoided when selecting HLA-compatible platelet units. 22However, below this value, the MFI at which antibodies become clinically relevant has not been clearly delineated. 13A recent study showed that HLA antibodies (MFI > 4000) could be immune-reactive against a particular platelet donor. 23he high specificity and PPV highlight that the detection of HLA antibodies is definitively associated with platelet toxicity.The low sensitivity and NPV are probably secondary to causes of platelet recirculation defects, independent of all immunization phenomena, such as splenomegaly, bleeding, infection, and ABO mismatches.These factors were very common in our cohort, making it impossible to identify any major role for them in affecting the outcomes of the transfusions, especially since the platelet count was performed at approximately 24 h and not at 1 h.Moreover, this study encountered several difficulties in retrospectively obtaining platelet count data before and after transfusions, although data were obtained from only one blood transfusion service, limiting the number of studied cases of transfusion ineffectiveness.Additionally, due to the study being based on data from a single HLA laboratory over a short time frame, different HISTO SPOT ® HLA AB batches were not compared, nor was interlaboratory variability.These limits did not allow us to fully conclude on the clinical relevance of the HLA antibodies identified by HISTO SPOT ® HLA AB in the context of transfusion.
Although less sensitive than the OL SAB test, the findings from this first retrospective study suggest that the HISTO SPOT ® HLA AB test, which is an easy method with user-friendly analysis software, could be an interesting new method for managing platelet transfusion ineffectiveness.However, this study was very limited because it was a nonrandomized, single center, and small study with only 15 patients.Larger, prospective and more carefully controlled studies will be needed to validate the positioning of the HISTO SPOT ® HLA AB test with respect to the OL SAB test in the selection of phenotyped platelet concentrates.

AUTHOR CONTRIBUTIONS
Christophe Picard and Pascal Pedini contributed to the design of the study.Christophe Picard, Jean-Baptiste Baudey, Lucas Hubert, and Pascal Pedini participated in the writing of the paper.Agnes Basire, Corinne Chabrières, Patrick Ladaique, and Jean-Michel Etienne participated in data analysis, and in particular, in the collection of transfusion efficacy data.Norbert Vey and Jacques Chiaroni were involved in critical revision of the manuscript.

T A B L E 2
Characteristics of commercial kits.
T A B L E 3 Comparison of OL SAB-positive beads versus HISTOSPOT HLA AB and C1Q SAB according to the MFI threshold.
T A B L E 5 DSA detected in HISTOSPOT HLA AB, OL SAB and C1q techniques according to the MFI cutoff and transfusion effectiveness.Analytical performance of the methods according to the threshold.
T A B L E 6