Loretta Crespiatico

Blood group genotyping for Jka/Jkb, Fya/Fyb, S/s, K/k, Kpa/Kpb, Jsa/Jsb, Coa/Cob, and Lua/Lub with microarray beads

BACKGROUND: Traditionally, blood group typing has been performed with serologic techniques, the classical method being the hemagglutination test. Serotyping, however, may present important limitations such as scarce availability of rare antisera, typing of recently transfused patients, and those with a positive direct antiglobulin test. Consequently, serologic tests are being complemented with molecular methods. The aim of this study was to develop a low‐cost, high‐throughput method for large‐scale genotyping of red blood cells (RBCs).

A retrospective evaluation of HLA‐A, B and ‐DRB1 matching in liver transplantation

Abstract Studies on the influence of histocompatibility in liver transplantation have not produced clear‐cut results. We retrospectively studied the influence of HLA‐A, B and ‐DRB1 matching on the survival of 517 liver‐transplanted patients using univariate analysis. The following parameters were also considered in relation to transplant outcome: donor and recipient age, original disease, transplant center, and pre‐transplant blood transfusions. Twenty‐four‐month graft survival according to the number of HLA‐A, B, DRB1 mismatches (MM) was 70.9% (n= 28) for zero to two MM, 76.6% (n= 248) for three to four MM, and 73.1% (n= 241) for five to six MM (P= 0.7). We obtained similar results when considering HLA‐A, B MM alone. Survival rates according to HLA‐DRB1 MM were 71.7% (n= 36) for zero MM, 73.7% (n= 236) for one MM, and 76.4% (n= 245) for two MM (P= 0.6). The same analyses, performed on cirrhotic patients alone, gave identical results. In conclusion, this study suggests, on a large series of patients, that HLA compatibility has no influence on liver transplant survival. On the contrary, an influence on transplant outcome was found for donor age, transplant center, and original disease.

Flow-cytometric approach to the prompt laboratory diagnosis of TRALI: a case report

Abstract:  Objectives: Transfusion‐related acute lung injury (TRALI) is a rare but serious complication which can occur after transfusion of blood components. In this report we describe our flow‐cytometry approach to the laboratory diagnosis of a case of TRALI in a recipient of fresh frozen plasma containing human leukocyte antigen (HLA) class II antibodies. Methods: The post‐transfusion reaction work‐up included the direct and indirect Granulocyte Immunofluorescence Test (GIFT) on the recipients neutrophils collected before and after the reaction and on the serum from the recipient and from all implicated donors; flow‐cytometry bead‐based screening and identification assay for HLA class I and II antibodies in donor sera and flow cytometry cross‐matching on T and B patients lymphocytes. Finally, we investigated the reactivity of one donor serum, containing HLA class II antibodies, with the patients neutrophils activated in vitro to induce expression of HLA class II. Results: We found an increased level of IgG bound on patients granulocytes collected after TRALI, in the absence of detectable granulocyte and HLA class I antibodies in the five implicated donors. One of them showed HLA‐DR 1 and ‐DR 51 antibodies, which determined a positive cross‐match with patients B lymphocytes and in vitro activated granulocytes. Both HLA class II antigens were present in the recipient and absent in the donor. Conclusions: In some pathological conditions, HLA class II antibodies can react with activated granulocytes expressing HLA‐DR antigens, and activate TRALI reaction. HLA class II antibodies screening and flow cytometry cross‐matching techniques should be added to the current diagnostic algorithm of TRALI.

A Microsphere-Based Suspension Array for Blood Group Molecular Typing: An Update

Background: In a previous publication we described a method for Jk^a/Jk^b, Fy^a/Fy^b, S/s, K/k, Kp^a/Kp^b, Js^a/Js^b, Co^a/ Co^b, and Lu^a/Lu^b genotyping based on a microsphere suspension array. Here, an improved version of the assay is presented. Methods: Two multiplex polymerase chain reactions (PCR) were developed: one for amplification of samples routinely tested and the other for those systems that are tested less frequently. Each biotinylated PCR product is hybridized in a single multiplex assay. A total of 2,020 samples were analyzed, and the genotypes were compared to the blood group phenotypes. Results: There have been no discrepancies with the serology results other than null and/or weak phenotypes. Conclusion: In its present form, the method presented here has the capacity to genotype hundreds of a samples in few hours with a high concordance rate with serology.

Isoniazid in patient plasma may cause a false-positive result on the complement-dependent cytotoxicity test

Correct definition of clinically relevant anti-HLA antibodies is important for transplant organ allocation and outcome. We describe a candidate for kidney transplantation who was treated with isoniazid because of active tuberculosis. The patients serum gave a positive antibody result on screening with the complement-dependent cytotoxicity (CDC) test but a negative result on screening with a bead-based assay (Luminex). The clinical history indicated no immunologic stimuli. Subsequent testing on fresh serum samples confirmed the discrepancy between CDC and Luminex results. An autologous cross-match test gave negative results, and the antibodies were sensitive to dithiothreitol treatment. We postulated that nonspecific binding of drug-antibody complexes to panel lymphocytes in the CDC test may have caused the observed lympholysis. This case, although isolated, emphasizes the importance of the combined use of CDC and solid phase assays. The CDC results alone would have led to the erroneous conclusion that the patient was highly sensitized.