Genomewide Association Study of Severe Covid-19 with Respiratory Failure.

BACKGROUND: There is considerable variation in disease behavior among patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (Covid-19). Genomewide association analysis may allow for the identification of potential genetic factors involved in the development of Covid-19. METHODS: We conducted a genomewide association study involving 1980 patients with Covid-19 and severe disease (defined as respiratory failure) at seven hospitals in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe. After quality control and the exclusion of population outliers, 835 patients and 1255 control participants from Italy and 775 patients and 950 control participants from Spain were included in the final analysis. In total, we analyzed 8,582,968 single-nucleotide polymorphisms and conducted a meta-analysis of the two case-control panels. RESULTS: We detected cross-replicating associations with rs11385942 at locus 3p21.31 and with rs657152 at locus 9q34.2, which were significant at the genomewide level (P<5×10-8) in the meta-analysis of the two case-control panels (odds ratio, 1.77; 95% confidence interval [CI], 1.48 to 2.11; P = 1.15×10-10; and odds ratio, 1.32; 95% CI, 1.20 to 1.47; P = 4.95×10-8, respectively). At locus 3p21.31, the association signal spanned the genes SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6 and XCR1. The association signal at locus 9q34.2 coincided with the ABO blood group locus; in this cohort, a blood-group-specific analysis showed a higher risk in blood group A than in other blood groups (odds ratio, 1.45; 95% CI, 1.20 to 1.75; P = 1.48×10-4) and a protective effect in blood group O as compared with other blood groups (odds ratio, 0.65; 95% CI, 0.53 to 0.79; P = 1.06×10-5). CONCLUSIONS: We identified a 3p21.31 gene cluster as a genetic susceptibility locus in patients with Covid-19 with respiratory failure and confirmed a potential involvement of the ABO blood-group system. (Funded by Stein Erik Hagen and others.).

HEA BeadChip™ technology in immunohematology.

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.

The Lombardy Rare Donor Programme.

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.

Comparison of traditional methods and mitogen-stimulated direct antiglobulin test for detection of anti-red blood cell autoimmunity.

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.

Blood group genotyping 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) 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). 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.

Strategies for the transfusion of subjects with complex red cell immunisation: the Bank of rare blood donors of the Region of Lombardy.

INTRODUCTION: Selecting units of rare blood for transfusion to patients with complex immunisation is one of the most critical processes of a Transfusion Centre. In January 2005 the 'Rare Blood Components Bank - Reference Centre of the Region of Lombardy' w as established with the following goals: 1) identifying regional rare blood donors; 2) creating a regional registry of rare donors; 3) organising a regional bank of liquid and frozen rare blood units; 4) setting up a regional Immunohaematology Reference Laboratory (IRL) to type donors and resolve complex cases. METHODS: The key elements in establishing the Bank were periodic meetings organised by the directors and representatives of the regional Departments of Transfusion Medicine and Haematology (DTMH) and the institution of three working groups (informatics, regulations, finance). RESULTS: The regional IRL was set up, the relevant operating procedures were distributed region-wide, software features were defined and later validated upon activation, and the funds assigned were allocated to various cost items. The number and characteristics of the donors to be typed were identified and 14 regional DTMHs started to send samples. Overall, 20,714 donors were typed, for a total of 258,003 typings, and 2,880 rare donors were identified. Of these, 97% were rare donors because of combinations of antigens (2,139 negative for the S antigen and 659 negative for the s antigen) and 3% (n=82) because they were negative for high-frequency antigens. In the first 2 years of activity, the IRL carried out investigations of 140 complex cases referred from other Centres and distributed 2,024 units with rare phenotypes to 142 patients. CONCLUSIONS: The main goal achieved in the first 24 months from the start of the project was to set up a regional network able to meet the transfusion needs of patients with complex immunisation.

Immune hemolytic anemia associated with teicoplanin.

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.

Outcomes of an automated procedure for the selection of effective platelets for patients refractory to random donors based on cross-matching locally available platelet products.

In 1999, we implemented an automated platelet cross-matching (XM) programme to select compatible platelets from the local inventory for patients refractory to random donor platelets. In this study, we evaluated platelet count increments in 40 consecutive refractory patients (8.3% of 480 consecutive platelet recipients) given 569 cross-match-negative platelets between April 1999 and December 2001. XM was performed automatically with a commercially available immunoadherence assay. Pre-, 1- and 24-h post-transfusion platelet counts (mean +/- SD) for the 569 XM-negative platelet transfusions containing 302 +/- 71 x 109 platelets were 7.7 +/- 5.5, 32.0 +/- 21.0 and 16.8 +/- 15.5 x 109/l respectively. Increments were significantly higher (P < 0.05, t-test) than those observed in the same patients given 303 random platelet pools (dose = 318 +/- 52 x 109 platelets) during the month before refractoriness was detected, when pre-, 1- and 24-h post-transfusion counts were 7.0 +/- 8.6, 15.9 +/- 16.1 and 9.6 +/- 12.8 x 109/l respectively. The cost of the platelet XM disposable kit per transfusion to produce 1-h post-transfusion platelet count increments >10 x 109/l was euro 447. This programme enabled the rapid selection of effective platelets for refractory patients, from the local inventory.