Virginie Ferrera

HLA-DRB1 alleles and Jka immunization

BACKGROUND: In transfusion medicine, anti‐Jka has been implicated in hemolytic transfusion reactions. Development of anti‐Jka after transfusion does not always occur after Jk(a–) patients receive at least 1 unit of Jk(a+) blood unit. This study was designed to identify HLA‐DRB1 alleles associated with predisposition to Jka immunization after blood transfusion or pregnancy.

Analysis of ABO discrepancies occurring in 35 French hospitals.

BACKGROUND:  The risk of immunohemolytic reaction owing to ABO‐mismatched mistransfusion is 100 to 1000 times higher than the risk of viral infection. Like analysis of incident reports, evaluation of near‐miss events can provide useful insight into hazardous situations for mis‐matched blood transfusion. The aim of this prospective study was to assess the incidence and root causes of all ABO discrepancies, detected by a central hematology laboratory, in blood samples referred from 35 district hospitals.

HLA-DRB1 polymorphism is associated with Kell immunisation

K immunisation is observed in some polytransfused patients and pregnant women but does not occur in all cases of K incompatibility. This study analysed the role of genetic background in this selective response to K antigen by investigating HLA‐DRB1 alleles associated with K immunisation in a southern European population. HLA‐DRB1 genotyping was performed by polymerase chain reaction sequence‐specific oligonucleotide/sequence‐specific primer procedures in 54 K immunised patients and 200 healthy controls. The frequency of HLA‐DRB1*11 was significantly higher in K immunised patients than healthy controls: 31 of 54 (57%) vs. 56 of 200 (28%) (Pc < 0·001). In the remaining K immunised HLA‐DRB1*11‐negative patients, the frequency of HLA‐DRB1*13 was increased: 14 of 23 (61%) vs. 49 of 144 in healthy controls (34%) (P < 0·02). The combined frequency of the two HLA‐DRB1 alleles (HLA‐DRB1*11 and HLA‐DRB1*13) was 83% in K immunised patients when compared with 52% in healthy controls (Pc < 0·001). K and k differ by a single amino acid T193 (M). The DRB1*11 and DRB1*13 alleles share a HLA‐DRB1 gene sequence containing S in position 13, D in 70 and A in 74, and coding for the P4 pocket within the HLA‐DR binding groove. This feature of the HLA‐DRB1 gene could be involved in the K peptide presentation through a polymorphism ligand specific for the T193 (M) of K. In conclusion, this study demonstrated a high frequency of HLA‐DRB1*11 or HLA‐DRB1*13 alleles in K immunised patients, which could be due to specific K peptide presentation by HLA‐DR molecules.

Positive association of DRB1*04 and DRB1*15 alleles with Fya immunization in a Southern European population

BACKGROUND: Anti‐Fya has been implicated in hemolytic transfusion reactions. However, not all Fy(a−) patients develop anti‐Fya after transfusion with 1 unit of blood [Fy(a+)]. This study was designed to identify HLA‐DRB1 alleles associated with a predisposition to Fya immunization after blood transfusion.

Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles

BACKGROUND: Molecular RHD blood group typing is very efficient for managing donors and patients carrying any of the various molecular types of weak D and DEL. The purpose of the work was to develop a multiplex polymerase chain reaction (PCR) SNaPshot assay for simultaneous detection of weak D and DEL alleles that are prevalent in Europeans, Africans, and Asians.

Characterization of novel RHD alleles: relationship between phenotype, genotype, and trimeric architecture

BACKGROUND: RH1 is one of the most clinically important blood group antigens in the field of transfusion and prevention of fetomaternal incompatibilities. New variant RHD alleles are regularly identified and their characterization is essential to ensuring patient safety.

Erythrocyte‐magnetized technology: an original and innovative method for blood group serology

BACKGROUND: Erythrocyte‐magnetized technology (EMT) is a new fully automated method for ABO‐RH‐K phenotyping and antibody detection. The magnetization of red cells avoids centrifugation and washing phases. This report describes the results of an evaluation of this new technology on its specific automated system.

Adsorption of autoantibodies in the presence of LISS to detect alloantibodies underlying warm autoantibodies.

BACKGROUND : The safe transfusion of patients with warm autoimmune hemolytic anemia requires an efficient and time‐saving assay to detect alloantibodies underlying autoantibodies. Methods used include RBCs treated with ZZAP reagent, proteolytic enzyme, or untreated RBCs in the presence of PEG. We propose a method using LISS, which presents some advantages over previous methods.

How we evaluate panagglutinating sera

P anagglutinating sera is one of the most challenging dilemmas of the antibody identification process. It occurs when patient sera react with all red blood cells (RBCs) tested, that is, with both screening and identification panel cells used in first approach. In this situation systematic workup is necessary to reduce the risk of error and optimize sample use. Two main problems must be resolved. The first is to determine whether panagglutination is due to an autoor alloantibody against a high-frequency antigen (HFA) or to multiple antibodies recognizing antigens other than HFA. The second problem is to detect the possible concomitant presence of clinically significant alloantibodies masked by panagglutination. The purpose of this article is to describe an algorithm developed to resolve these panagglutination problems at our laboratory that performs around 8000 RBC antibody identification tests each year for routine and reference purposes in the pretransfusional and obstetrical setting. Of a total of 52,000 antibody identifications performed over the past 7 years, the technique described here was used to investigate 3124 cases of panagglutination including 3002 associated with positive autocontrol tests and 122 with negative autocontrol tests (Table 1). To illustrate the problem-solving efficacy of this approach, we will present the results achieved at each step. Many of these results, particularly those involving HFA, were subsequently confirmed by a reference laboratory.

LETTERS TO THE EDITOR: Comparison of three low-ionic-strength solutions for routine pretransfusion testing: antibody screening/identification, cross-matching, immune anti-ABO detection, and direct antiglobulin tests

The two antiglobulin tests, that is, direct antiglobulin test (DAT) and indirect antiglobulin test (IAT), are important tools in immunohematology. The DAT is used to determine if antibodies or complement system factors have bound to red blood cell (RBC) surface antigens in vivo. The IAT is performed to detect in vitro antibody-antigen reactions for RBC antibody screening/identification, immune anti-ABO detection, and cross-matching. The IAT is performed using low-ionic-strength solutions (LISS) to increase the rate of antibody association to antigen, thereby allowing a reduction in incubation time while achieving optimal agglutination. Similarly most manufacturers of microcolumn detection systems used for DAT specifically recommend suspending patient RBCs in LISS. In addition to LISS provided by each manufacturer, numerous products are available at widely varying costs ranging from 0.034 €/ml to 0.67 €/ml. Despite the widespread use of LISS few publications have studied differences between commercially available brands. The purpose of this letter is to communicate the results of an attempt to use a single LISS, that is, LISS Diagast (Diagast, Loos, France), for all tests requiring LISS regardless of the RBC panel or microcolumn used. The following products are used with the manufacturer’s recommended LISS reagent in accordance with their instructions for use: Polyspecific Ortho BioVue system (Ortho-Clinical Diagnostics [OCD], Raritan, NJ), and ID-Card antiglobulin test anti-IgG, DC-Screening II system (Diamed, Basel, Switzerland). The Ortho BioVue system and ID-Card antiglobulin anti-IgG are used for RBC antibody screening and identification, ABO antibody detection, and cross-matching. Patient DATs are performed in DC-Screening II system. The equipment recommended by the manufacturers is used: specific heat blocks or centrifuges for the Ortho BioVue system or Diamed ID. The commercial RBC panels used include 4% panel RBCs in a storage solution (for screening, BioVue Screen Papain OCD; for identification, BioVue Top OCD with 10 or 11 RBCs and reference panel with 15 RBCs, CNRGS, Paris, France) and a 0.8% LISS panel (UPR EFS, France). The 4% RBC panel suspensions or patient RBCs are diluted with the manufacturer’s recommended LISS product as described in the use instructions. LISS (Diagast) is also used to adsorb antibody in situations involving panagglutination and the adsorbed serum must be tested against a 4% RBC suspension. For screening/identification or ABO antibody detection by IAT with the Ortho BioVue system, dilution of 4% panels is required: 10 mL of the 4% suspension is mixed with 50 mL LISS (“addition” method), 40 mL of patient plasma is added, and the cards are incubated for 20 minutes at 37°C and centrifuged for 5 minutes. If dilution of 4% panels is required for anti-human globulin anti-IgG antiglobulin test ID-Cards, 10 mL of 4% RBC panel suspension is mixed with 40 mL of LISS, 25 mL of patient plasma is added, and cards are incubated for 20 minutes at 37°C followed by centrifugation for 10 minutes. If the panel is a 0.8% suspension, 50 mL of RBC suspension is placed in the two types of cards. For DAT and cross-matching, we prepared the patient’s RBCs by suspension in LISS. In this study a selection of 222 patient samples (ethylenediaminetetraacetate anticoagulated) were tested simultaneously using RBCs suspended in either LISS Diagast or the brand recommended by the manufacturers. A total of 129 samples were tested for RBC screening/identification and cross-matching. Results showed that 63 samples had no RBC antibody and 66 contained the RBC antibody previously identified. In addition cell antibody screening/identification was performed on selected plasma with low concentration of anti-RH1, RH3, RH4, KEL1, FY1, JK1, and MNS3 (titer 4 determined by IAT filtration). For detection/identification of immune antibodies against ABO antigens, 45 plasma samples from 18 newborns (detection of passive maternal antibodies) and 27 adults were tested. Positive reactions were obtained in 32 cases. The number, specificity, and titer of RBC antibodies are listed in Table 1. The DAT was performed on 48 patients’ samples. All samples were tested within 72 hours of collection. DAT was negative in 19 cases and positive in 29 cases (17 anti-IgG, six anti-C3d, six anti-IgG + C3d). Results showed that LISS Diagast and BLISS Ortho were equivalent for RBC screening/identification, crossmatching, and immune anti-ABO detection and that LISS Diagast and ID-Diluent2 were equivalent for RBC screening/identification, cross-matching, and DAT. The reactivity strength was equal for all IAT comparisons except for the cases with slight differences ( 1+) detailed in Table 1. No difference of reactivity strength was observed for the cross-matching data. For the DAT the reactivity strength was equal except in two cases of DAT C3d positive (0.5+ and 1+ decreases with LISS Diagast compared to ID-Diluent2). Findings also showed that substitution of LISS Diagast for dilution of 4% panel cells for use in the Diamed cards did not affect sensitivity. To our knowledge this is the first report comparing different LISS for routine pretransfusion testing as antibody screening/identification, cross-matching, immune anti-ABO detection, and DAT. Data demonstrate