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Berzuini et al report the observation that nearly half of patients with COVID-19 tested at their blood center had a positive direct antiglobulin test (DAT). However, eluates did not react with any test cells but did react with red cells from other patients with COVID-19 that were DAT negative. This suggests that COVID-19 may modulate the red cell membrane and present novel antigenic epitopes.

Introduction: The Rh antigens are encoded by the highly polymorphic RHD and the RHCE genes. Individuals with D variants may make anti-D alloantibodies, which in pregnant women cause severe or fatal hemolytic disease of the fetus and newborn. Accurate D typing using reliable routine methods must be performed. A number of unusual RHD alleles are being currently reported due in large part to the growing implementation of molecular analyses. Case Report: A sample from a Caucasian male blood donor at Northern Italy was serologically classified as RhD negative (Ccee). Instead, the sample was genotyped with all SSP-PCR kits as RhD positive, Dd and the result obtained with Partial D-TYPE (Dva or Va like or Va associated or DBS) was consistent with a suggestion of a new variant. Through a microarray platform analysis (BLOODchip Reference), the sample was genotyped as DV/DBS [RHD-CE(S)-D hybrid], associated with a predicted partial D phenotype for the RhD group, due to the lack of a hybridization signal for RHD exon 5. The PCR analysis of the exon 5 of the RHD gene gave a positive amplification result and the sequencing revealed a polymorphism in hemizygous or homozygous state in position RHD*IVS5+1t. This novel splice-site mutation alters or completely abolishes the specific sequence where the splicing of RHD gene intron 5 takes place. Conclusion: The novel RHD mutation hereby described highlights the significance of using RHD genotyping to confirm and/or clarify D antigen uncertainties in order to prevent the unnecessary immunization of patients.

Classic methods to determine human red blood cell (RBC) antigens are based on serologic testing. Thanks to increased knowledge of the molecular basis associated with many blood group antigens, it is currently possible to predict their presence or absence on the red cell membrane. Several molecular techniques have been developed to detect the most important allelic variations attributable to single nucleotide polymorphisms. The human erythrocyte antigen (HEA) BeadChip™ system manufactured by BioArray Solutions (Immucor, Warren, NJ) is one of the commercial DNA array platforms currently available to predict HEAs by DNA analysis. This technology provides a useful tool to increase the inventory of antigen-negative RBC units and prevent immunization of patients who require chronic transfusion by providing compatible RBC units based on matching by DNA testing. Immunohematology 2015;31:81–90.

The diagnosis of autoimmune hemolytic anemia (AIHA) is based on a positive direct antiglobulin test (DAT), which is performed using various methods with different sensitivities. Recently, mitogen-stimulated (MS)-DAT was suggested to be able to identify latent anti-erythrocyte autoimmunity. Traditional methods (tube, microcolumn, and solid phase) and MS-DAT were compared in 54 consecutive cases of suspected AIHA, 28 idiopathic AIHA in clinical remission, and 12 difficult-to-diagnose cases of DAT-negative AIHA, and the results (all cases) were correlated with hematologic and hemolytic parameters. DAT tube was confirmed as the gold standard to diagnose AIHA since almost all positive cases showed hemolytic anemia and positive eluates; 10 out of 26 tube-negative cases were positive on microcolumn and solid phase antiglobulin tests, and 22 out of 26 using MS-DAT, although only half of them showed clear signs of hemolysis. Mitogen stimulation increased the amount of IgG bound to red blood cells in all groups; moreover, MS-DAT was the only positive test in 10 cases of AIHA, and mitogen stimulation facilitated the identification of autoantibody specificity in culture supernatants. We conclude that a battery of tests rather than a single test is useful for the diagnosis of AIHA, including MS-DAT as an additional test for selected cases, although the results have to be cautiously interpreted based on the overall clinical context.

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). STUDY DESIGN AND METHODS: Single-nucleotide polymorphisms associated with some clinically important blood group antigens, as well as with certain rare blood antigens, were evaluated: 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 . Polymerase chain reaction (PCR)-amplified targets were detected by direct hybridization to microspheres coupled to allele-specific oligonucleotides. Cutoff values for each genotype were established with phenotyped and/or genotyped samples. RESULTS: The method was validated with a blind panel of 92 blood donor samples. The results were fully concordant with those provided by hemagglutination assays and/or sequence-specific primer (SSP)-PCR. The method was subsequently evaluated with approximately 800 blood donor and patient samples. CONCLUSION: This study presents a flexible, quick, and economical method for complete genotyping of large donor cohorts for RBC alleles.

BACKGROUND: Several drugs can cause immune hemolytic anemia. Here a patient who developed hemolytic anemia after treatment with teicoplanin is described. CASE REPORT: Owing to a two-vessel disease, a 68-year-old white man underwent coronary artery bypass grafting. He was readmitted for superficial sternal wound infection and sternal instability. Rewiring was required and worsening anemia characterized the course after the reoperation. Drugs used in the second admission were gentamycin, teicoplanin, paracetamol, and codeine. They were considered as a possible cause of drug-induced hemolytic anemia. RESULTS: The DAT was positive for complement and IgG. Autoanti-e was identified in the patient's undiluted serum sample. The eluate was reactive with all RBCs tested only after adding teicoplanin; when diluted 1:4, anti-e specificity was observed in the presence of teicoplanin. CONCLUSION: To our knowledge, this is the first report of immune hemolytic anemia owing to teicoplanin.

BACKGROUND: In 2005, the government of Lombardy, an Italian region with an ethnically varied population of approximately 9.8 million inhabitants including 250,000 blood donors, founded the Lombardy Rare Donor Programme, a regional network of 15 blood transfusion departments coordinated by the Immunohaematology Reference Laboratory of the Ca' Granda Ospedale Maggiore Policlinico in Milan. During 2005 to 2012, Lombardy funded LORD-P with 14.1 million euros. MATERIALS AND METHODS: During 2005-2012 the Lombardy Rare Donor Programme members developed a registry of blood donors and a bank of red blood cell units with either rare blood group phenotypes or IgA deficiency. To do this, the Immunohaematology Reference Laboratory performed extensive serological and molecular red blood cell typing in 59,738 group O or A, Rh CCDee, ccdee, ccDEE, ccDee, K- or k- donors aged 18-55 with a record of two or more blood donations, including both Caucasians and ethnic minorities. In parallel, the Immunohaematology Reference Laboratory implemented a 24/7 service of consultation, testing and distribution of rare units for anticipated or emergent transfusion needs in patients developing complex red blood cell alloimmunisation and lacking local compatible red blood cell or showing IgA deficiency. RESULTS: Red blood cell typing identified 8,747, 538 and 33 donors rare for a combination of common antigens, negative for high-frequency antigens and with a rare Rh phenotype, respectively. In June 2012, the Lombardy Rare Donor Programme frozen inventory included 1,157 red blood cell units. From March 2010 to June 2012 one IgA-deficient donor was detected among 1,941 screened donors and IgA deficiency was confirmed in four previously identified donors. From 2005 to June 2012, the Immunohaematology Reference Laboratory provided 281 complex red blood cell alloimmunisation consultations and distributed 8,008 Lombardy Rare Donor Programme red blood cell units within and outside the region, which were transfused to 2,365 patients with no untoward effects. DISCUSSION: Lombardy Rare Donor Programme, which recently joined the ISBT Working Party on Rare Donors, contributed to increase blood transfusion safety and efficacy inside and outside Lombardy.

In 2005, the Centro Transfusionale e di Immunomatologia, Dipartimento di Medicina Rigenerativa, an Immunohematology Reference Laboratory in Milan, Italy, instituted a rare donor program to address the transfusion needs of patients with complex immunization to red cell antigens with a rare phenotype. From June 2005 to December 2008, the laboratory used a high-productivity system (Galileo, Immucor, Norcross, GA) for mass-scale antigen screening with profile 1 and 2 antigens for select donors, where 48,715 blood donors were typed with the identification of 6,634 rare blood donors. In April 2009, the laboratory adopted the BeadChip™ platform (BioArray Solutions, Ltd., Warren, NJ) for large-scale DNA typing. The decision to implement was to expand the panel of red blood cell and platelet antigens using the human erythrocyte antigen (HEA) and human platelet antigen (HPA) BeadChip™ formats. As recommended by international guidelines, a validation plan was used to evaluate the sensitivity and specificity of the method. The results of our testing are described in this chapter.

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).Single-nucleotide polymorphisms associated with some clinically important blood group antigens, as well as with certain rare blood antigens, were evaluated: 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). Polymerase chain reaction (PCR)-amplified targets were detected by direct hybridization to microspheres coupled to allele-specific oligonucleotides. Cutoff values for each genotype were established with phenotyped and/or genotyped samples.The method was validated with a blind panel of 92 blood donor samples. The results were fully concordant with those provided by hemagglutination assays and/or sequence-specific primer (SSP)-PCR. The method was subsequently evaluated with approximately 800 blood donor and patient samples.This study presents a flexible, quick, and economical method for complete genotyping of large donor cohorts for RBC alleles.

Several drugs can cause immune hemolytic anemia. Here a patient who developed hemolytic anemia after treatment with teicoplanin is described.Owing to a two-vessel disease, a 68-year-old white man underwent coronary artery bypass grafting. He was readmitted for superficial sternal wound infection and sternal instability. Rewiring was required and worsening anemia characterized the course after the reoperation. Drugs used in the second admission were gentamycin, teicoplanin, paracetamol, and codeine. They were considered as a possible cause of drug-induced hemolytic anemia.The DAT was positive for complement and IgG. Autoanti-e was identified in the patient's undiluted serum sample. The eluate was reactive with all RBCs tested only after adding teicoplanin; when diluted 1:4, anti-e specificity was observed in the presence of teicoplanin.To our knowledge, this is the first report of immune hemolytic anemia owing to teicoplanin.

Abstract 5091 Background The diagnosis of autoimmune hemolytic anemia (AIHA) is based on a positive direct antiglobulin test (DAT), which is performed by various methods with different sensitivity. Recently mitogen stimulated (MS)-DAT was suggested able to disclose a latent anti-erythrocyte autoimmunity Aims: To compare different traditional (tube, microcolumn and solid phase) and new (mitogen stimulated, MS)-DAT methods in different diagnostic conditions: 54 consecutive cases of hemolytic anemia of suspected autoimmune origin, 28 idiopathic AIHA in clinical remission, and 12 difficult cases of DAT-negative AIHA, and 2) to correlate results with hematologic and hemolytic parameters. Methods DAT tube, microcolumn, solid phase, and eluates were performed by standard techniques. MS-DAT was performed by stimulating whole blood with PHA, PMA and PWM and antibodies were detected by competitive solid phase ELISA. Results Forty out of 54 consecutive cases of suspected AIHA were positive by one or more tests namely 14 DAT-tube, 19 microcolumn, 19 solid phase and 35 MS-DAT. Among the 14 DAT-tube positive cases, 11 were confirmed by all test, and 3 by one or more (1 microcolumn, 1 solid phase, and 1 MS-DAT), eluates were positive in 11, and the majority of patients (10/14) showed hemolytic anemia. As regards the 26 DAT-tube negative cases, 7 were positive in microcolumn and solid-phase (eluates positive in 2/8, panreactive), and 16 in MS-DAT, although in both groups anemia and hemolytic signs were less clear. Mitogen stimulation increased the amount of RBC-bound IgG in all groups, suggesting that MS-DAT could disclose a latent autoimmunity. Tube-negative/other methods positive cases included patients with B-CLL, myelodysplasia/aplasia, and thalassemia intermedia, in which autoimmune phenomena are more frequently observed than overt clinical autoimmune diseases. MS-DAT failed to detect anti-erythrocyte antibodies in half cases AIHA in clinical remission which still were tube-positive. Finally, MS-DAT was the only positive test in 10 cases of AIHA of difficult diagnosis, and mitogen stimulation allowed the identification of autoantibody specificity in culture supernatants of 2 cases which gave weak positive results in microcolumn/solid phase only. Conclusion We concluded that a battery of tests rather than a single test is recommended for the diagnosis of AIHA, depending on the specific clinical context: tube is still a good choice for first screening, microcolumn and solid-phase, which are the automated routine, should be confirmed by a positive eluate to diagnose AIHA, although positivity without a clinical equivalent may be taken into account as part of an autoimmune habitus. MS-DAT could be considered as an additional test for selected cases of difficult diagnosis. Disclosures No relevant conflicts of interest to declare.