Comparison of the solid-phase red cell adherence assay and tube method for detection and identification of red blood cell antibodies.

OBJECTIVES: Identifying antibodies to red blood cell antigens is one of transfusion medicine's critical responsibilities. The International Society of Blood Transfusion recognizes 354 red blood cell antigens. Accurate identification of clinically significant alloantibodies is imperative for preventing hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. We compared the performance of the tube (polyethylene glycol-indirect antiglobulin test [PEG-IAT]) and solid-phase red cell adherence assay techniques. METHODS: We performed a retrospective study on all antibody screens performed between 2007 and 2021 at Stanford Transfusion Services. Initially, 631,535 antibody screens were performed using a solid-phase technique. Subsequent antibody identifications were performed using a combination of tube testing and solid-phase techniques. RESULTS: Antibody screening resulted in 28,316 (4.5%) positive samples. Antibody identification performed on both platforms identified 50 discordant samples. The anti-E antibody had the lowest sensitivity (98.99%) in the automated solid-phase technique, while anti-Jkb had the lowest sensitivity (98.78%) with the PEG-IAT method. CONCLUSIONS: To our knowledge, this is the first robust, 15-year study comparing methodologic sensitivity to detect clinically significant alloantibodies. The incidence of discordant results between PEG-IAT and the solid-phase technique was low. Among discordant samples, anti-Jka was commonly detected using the solid-phase method but not with the PEG-IAT. In contrast, anti-E was commonly detected by PEG-IAT but not by the solid-phase method.

Evaluation of Platelet Alloimmunization by Filtration Enzyme-Linked Immunosorbent Assay.

The current methods for detecting antiplatelet antibodies are mostly manual and labor-intensive. A convenient and rapid detection method is required for effectively detecting alloimmunization during platelet transfusion. In our study, to detect antiplatelet antibodies, positive and negative sera of random-donor antiplatelet antibodies were collected after completing a routine solid-phase red cell adherence test (SPRCA). Platelet concentrates from our random volunteer donors were also prepared using the ZZAP method and then used in a faster, significantly less labor-intensive process, a filtration enzyme-linked immunosorbent assay (fELISA), for detecting antibodies against platelet surface antigens. All fELISA chromogen intensities were processed using ImageJ software. By dividing the final chromogen intensity of each test serum with the background chromogen intensity of whole platelets, the reactivity ratios of fELISA can be used to differentiate positive SPRCA sera from negative sera. A sensitivity of 93.9% and a specificity of 93.3% were obtained for 50 µL of sera using fELISA. The area under the ROC curve reached 0.96 when comparing fELISA with the SPRCA test. We have successfully developed a rapid fELISA method for detecting antiplatelet antibodies.

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

Background and Aims: Platelet 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. Methods: A 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. Results: The 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. Conclusion: The 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.

Comparative evaluation of DTT treated ABO isoagglutinin titres performed by two methods with solid phase red cell adhesion (SPRCA) titres.

BACKGROUND AND AIMS: Measurement of actual concentration of IgG requires methods like treatment of serum with dithiothreitol (DTT). This study was aimed at comparing of DTT treated ABO titres performed by conventional test tube technique (CTT) and column agglutination technique (CAT) with HA/SPRCA. MATERIALS AND METHODS: This was a prospective, observational study conducted from October 2019 to March 2020. All consecutive A, B and O group donors who gave consent for participation were included. All samples were tested by CTT and CAT before and after DTT treatment (pCTT, pCAT) and with HA/SPRCA. RESULTS: A total of 300 donors were included; 100 each from A, B and O blood group donors. Group O titres were higher than group A/B titres. Group O titres were highest when performed by pCAT, followed by pCTT and lowest by HA/SPRCA. Group A/B titres were highest when performed by HA/SPRCA, followed by pCAT and pCTT for anti-A and highest when performed by pCAT, followed by HA/SPRCA and lowest by pCTT for anti-B. CONCLUSION: Results obtained by pCAT were closer to results obtained by pCTT, whereas those obtained by HA/SPRCA were variable. SPRCA offers the advantage of automation, no inter-observer variation and less time consumption because IgM interference is not observed with SPRCA, thus providing an alternative to pCTT. However, these methods cannot be used interchangeably and to discern the most suitable method, a clinical impact of these results needs to be studied.

Application of lyophilised human platelets for antibody detection in solid phase red cell adherence assay.

Antibodies against human platelets cause a variety of thrombocytopenic disorders, which lead to potentially fatal haemorrhage. Therefore, their prompt detection is mandatory for successful patient treatment. Solid phase red cell adherence (SPRCA) assay allows for platelet antibody detection widely. However, preparation of fresh platelets with HLA-I and human platelet antigens (HPA)1-5,15 genotyped as target cells is inconvenient and fresh platelets have a short shelf life. In this study, the lyophilised human platelets for antibody detection in SPRCA were prepared. Firstly, platelets were resuspended in lyophilisation buffer and freeze-dried. Then the characteristics of lyophilised platelet were analysed. Rehydrated platelets were recovered with a mean rate of 80.91% ± 2.87%, and still retained spherical morphology. Indirect flow cytometry showed that glycoproteins IIb/IIIa, Ia/IIa, Ib/IX, IV, CD109, and HLA class I were present on the surface of the lyophilised platelets at a comparable level to that of fresh platelets. The consistent results obtained with WHO reference reagents containing anti-HPA-1a, anti-HPA-3a, and anti-HPA-5b, as well as clinical samples from the same donors containing anti-HLA antibodies when reacting with lyophilised versus fresh platelets confirmed good antigenicity preservation of platelets after freeze-drying. Further investigation showed that the lyophilised platelets could be stored at 2-8 °C for up to 14 months and the reconstituted suspension was stable for 48 h. Therefore, lyophilised platelets can be a convenient alternative to fresh platelets to use for anti-platelet antibody detection in SPRCA tests.

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.

AIMS: To assess platelet crossmatch result by SPRCA and find its correlation with post-transfusion platelet count increment among adult hemato-oncology patients. METHODS: A 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. RESULTS: Out 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. CONCLUSIONS: Transfusion 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.

Facts and Fallacies of Kidd Antibodies: Experience in a Tertiary Care Hospital in North India.

We have analyzed the method used in our laboratory to detect the most elusive, clinically significant alloantibody: the Kidd alloantibodies and find the most convenient procedure. A retrospective analysis of the method used in our laboratory for determining Kidd alloantibodies from January 2013 to May 2015 was conducted. The details of the event that sensitized the patient for red cell antibody formation and procedure used to detect the alloantibody were retrieved from the departmental records. Of 405 red cell antibody identification cases, 24 (5.9 %) had Kidd antibody (anti-Jka in 12: 50 % cases; anti-Jkb in 4: 16.7 % cases; multiple antibodies in 8: 32 % cases). Thirteen of 24 patients (54.2 %) had autocontrol positive of which 6 cases needed adsorption procedures whereas antibody/ies could be identified without adsorption procedure in the remaining 7 cases. All the 7 cases had autocontrol of 1+ strength. Of the 11 patients (45.8 %) with autocontrol negative, the antibody was identified using solid phase in 7 cases whereas tube panels were also used in the remaining 4 cases. Kidd alloantibodies though deceptive can be identified by sensitive techniques like the solid phase and simple but laborious techniques using the tube cell panels. Depending upon the reaction strength of the autocontrol, the routine autoadsorption process may be skipped and tube cell enzyme treated cells or solid phase techniques be used to get the results.