Major histocompatibility complex (MHC) antigens polymorphism and alloimmunization study in thalassemia patients with febrile non-hemolytic transfusion reaction (FNHTR).

OBJECTIVES: HLA alloimmunization is one of the most troublesome consequence of regular transfusion which is itself a mainstay measure to provide longevity to the thalassemia patients. Febrile non-hemolytic transfusion reaction (FNHTR) is one of the most common complication which might be related to the HLA alloimmunization. Here, we studied the HLA antigenic system and alloimmunization rate in the Iranian ß-thalassemia patients who suffered from FNHTR compare to the ß-thalassemia patients without FNHTR. MATERIALS & METHODS: Total of 60 ß-thalassemia patients with FNHTR (case group) and 20 ß-thalassemia patients without FNHTR (control group) randomly have been selected and enrolled in the study. All were tested for HLA-A and -B loci by PCR-SSP method and also for the presence of anti-lymphocyte antibodies by LIFT method. Comparisons between two groups were performed by Pearson's χ2 test. RESULTS: Totally, a significant predominance was noted for two HLA alleles, HLA-A*24 (P = 0.029) and B*55 (P = 0.034) which have higher prevalence in control group. Although no significant association was found between the presence of anti-leukocyte antibodies and the development of FNHTR, the HLA-A*32 (P = 0.047) allele was considered as possible genetic markers in the susceptibility to the development of anti-leukocyte antibodies. CONCLUSION: Here some evidences about the possible role of HLA polymorphism in susceptibility to FNHTR are provided. Those results indicated that HLA-A*24 and HLA-B*55 might play protective role on inducing FNHTR in ß-thalassemia patients. Further studies which investigate the allele level of HLA-I alongside with specific reactivity of HLA-I antibodies might reveal more deep data about these phenomena.

Fatal haemolytic transfusion reaction due to anti-Ena and identification of a novel GYPA c.295delG variant in a Thai family.

BACKGROUND AND OBJECTIVES: High-frequency antigen Ena (MNS 28) is expressed on glycophorin A (GPA). En(a-) individuals can form anti-Ena when exposed to GPA. A Thai patient formed an antibody that reacted against all reagent red blood cells (RBCs). The patient received incompatible blood resulting in a fatal haemolytic transfusion reaction (HTR). This study aimed to characterize the antibody detected in the patient and investigate the cause of HTR. MATERIALS AND METHODS: Blood samples from the patient and three of his family members were investigated. Massively parallel sequencing (MPS) and DNA-microarray were used for genotyping. Standard haemagglutination techniques were used for phenotyping and antibody investigations. RESULTS: DNA sequencing showed the patient was homozygous for GYPA*M c.295delG (p.Val99Ter) predicting En(a-). Three family members were heterozygous for GYPA c.295delG. MPS and DNA-microarray predicted the patient was N- discordant with the N+ RBC phenotype. The patient's plasma was positive with enzyme/chemical-treated reagent RBCs but failed to react with En(a-) and Mk Mk RBCs. CONCLUSION: The GYPA c.295delG variant prevented GPA expression on RBCs resulting in En(a-) phenotype. The N+ phenotype result was probably due to the anti-N typing reagent detecting 'N' (MNS30) on GPB. The patient's alloantibody has anti-Ena specificity.

Relationship between allergic sensitisation-associated single-nucleotide polymorphisms and allergic transfusion reactions and febrile non-haemolytic transfusion reactions in paediatric cases.

BACKGROUND: Allergic transfusion reactions (ATR) and febrile non-haemolytic transfusion reactions (FNHTR) are common transfusion-related adverse reactions; however, their pathogenesis remains unclear and it is difficult to predict their occurrence. Single-nucleotide polymorphisms (SNP) are related to the onset of various diseases and therapy-related adverse events; therefore, identification of SNP related to transfusion-related adverse reactions may help to elucidate the underlying mechanism and predict the onset of these reactions. MATERIALS AND METHODS: We retrospectively analysed the association between the onset of ATR or FNHTR and 22 allergic sensitisation-related SNP in 219 children (aged ≤20 years) who had haematological and oncological diseases and who had received transfusions of platelets and/or red blood cell concentrates. RESULTS: Among the 219 children, 105 had developed an ATR and/or FNHTR at least once. The patients who developed ATR frequently had a risk allele in rs6473223, while the patients who developed FNHTR frequently had a risk allele in rs10893845. Furthermore, patients who developed ATR accompanied by febrile symptoms also frequently had a risk allele in rs10893845, similar to patients who developed FNHTR. DISCUSSION: The results suggested that allergic sensitisation is associated with the onset of ATR and/or FNHTR in some patients. Although further prospective evaluation is necessary, analysis of these SNP might help to provide safer transfusion therapy by predicting patients at higher risk of transfusion-related adverse reactions and further clarifying the pathogenic mechanism underlying such reactions.

Anti-Fya-mediated delayed hemolytic transfusion reaction following emergency-release red blood cell transfusion: possible involvement of HLA-DRB1*04:03 in the Japanese population.

A 43-year-old Japanese male, who had undergone open liver surgery for tumor resection, presented with decreased hemoglobin levels on Day 13 post-emergency-release transfusion of 16 units of Fy(a +) red blood cells. As the anemia was accompanied by increased lactate dehydrogenase, indirect bilirubin, and reticulocytes, as well as decreased haptoglobin, it was attributed to hemolysis. In the diagnostic workup for hemolytic reaction, the direct antiglobulin test result for IgG was positive and the antibody dissociated from the patient's peripheral red blood cells was identified as anti-Fya (titer, 4). The hemolytic reaction was transient (approximately 10 days), of moderate severity, and did not result in any obvious organ damage. However, a single compatible red blood cell transfusion of 2 units was required on Day 17 after the causative transfusion. Notably, HLA typing revealed that the patient carried the HLA-DRB1*04:03 allele, which has been implicated in immunogenicity and induction of anti-Fya response in Caucasian populations. In summary, this is the first documented case of definitive anti-Fya-mediated delayed hemolytic transfusion reaction associated with HLA-DRB1*04:03 in the Japanese population.

Development of DO*A and DO*B Allele Detections to Predict Transfusion-Induced Alloimmunization Risks in Thai Populations.

BACKGROUND: Two antithetical antigens, Doa and Dob of the Dombrock (DO) blood group system are implicated in acute to delayed hemolytic transfusion reactions among patients with anti-Doa or anti-Dob. Given the unavailability of specific antiserum, a polymerase chain reaction with sequence-specific primer (PCR-SSP) was developed to identify DO*A and DO*B alleles. This study aimed to determine DO*A and DO*B allele frequencies and to predict transfusion-induced alloimmunization risks in three Thai blood donor populations. METHODS: DNA samples obtained from 1,300, 300, and 400 blood donors from central, northern, and southern Thailand, respectively, were genotyped for the DO*A and DO*B allele detections using developed PCR-SSP. The results were confirmed by DNA sequencing. RESULTS: The validated genotyping results by PCR-SSP were in concordance with DNA sequencing. The DO*B/ DO*B was the most common genotype (77.0, 76.0, and 71.0%), followed by DO*A/DO*B (21.0, 22.7, and 25.2%) and DO*A/DO*A (2.0, 1.3, and 3.8%) among central, northern and southern Thais, respectively. The alleles found among central Thais showed significant differences from those found among southern Thais but not from those of northern Thais. The risk of anti-Doa production was higher than anti-Dob production among Thais. Concerning regional groups, the risk of Doa alloimmunization among southern Thais (0.2059) was higher than those among central (0.1771) and northern Thais (0.1824). CONCLUSIONS: This was the first study to distinguish DO*A and DO*B genotypes in Thai populations using in-house PCR-SSP. This would be useful to predict alloimmunization risks that might result from transfusion-induced reactions of undetermined red cell antigens among blood donors and in reagent red cells.

Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice.

Complement impacts innate and adaptive immunity. Using a model in which the human KEL glycoprotein is expressed on murine red blood cells (RBCs), we have shown that polyclonal immunoprophylaxis (KELIg) prevents alloimmunization to transfused RBCs when a recipient is in their baseline state of heath but with immunoprophylaxis failure occurring in the presence of a viral-like stimulus. As complement can be detected on antibody coated KEL RBCs following transfusion, we hypothesized that recipient complement synergizes with viral-like inflammation to reduce immunoprophylaxis efficacy. Indeed, we found recipient C3 and C1q were critical to immunoprophylaxis failure in the setting of a viral-like stimulus, with no anti-KEL IgG alloantibodies generated in C3-/- or C1q-/- mice following KELIg treatment and KEL RBC transfusion. Differences in RBC uptake were noted in mice lacking C3, with lower consumption by splenic and peripheral blood inflammatory monocytes. Finally, no alloantibodies were detected in the setting of a viral-like stimulus following KELIg treatment and KEL RBC transfusion in mice lacking complement receptors (CR1/2-/-), narrowing key cells for immunoprophylaxis failure to those expressing these complement receptors. In-vitro studies showed complement fixed opsonized RBCs were significantly less likely to bind to B-cells from CR1/2-/- than wild type mice, potentially implicating lowered B-cell activation threshold in the presence of complement as being responsible for these findings. We thus propose a two-hit model for inflammation-induced immunoprophylaxis failure, where the first "hit" is recipient inflammation and the second "hit" is complement production/sensing. These results may have translational relevance to antigen-antibody interactions in humans.

Blood group genotyping in alloimmunized multi-transfused thalassemia patients from Iran.

OBJECTIVES: Serological methods may not be reliable for RBC antigen typing, especially in multi-transfused patients. The blood group systems provoking the most severe transfusion reactions are mainly Rh, Kell, Kidd, and Duffy. We intended to determine the genotype of these blood group system antigens among Iranian alloimmunized thalassemia patients using molecular methods and compare the results with serological phenotyping. METHODS: Two hundred patients participated in this study. Blood group phenotype and genotype were determined using the serological method and PCR-SSP, respectively. The genotypes of patients with incompatibility between phenotype and genotype were re-evaluated by RFLP-PCR and confirmed by DNA sequencing. RESULTS: Discrepancies between phenotype and genotype results were found in 132 alleles and 83 (41.5%) patients; however, there was complete accordance between the three genotyping methods. Most discrepancies were detected in Rh and Duffy systems with 47 and 45 cases, respectively, and the main discrepancy was in the FY*B/FY*B allele when serologically showed Fy(a+b+). All 39 undetermined phenotypes, due to mixed-field reactions, were resolved by molecular genotyping. CONCLUSION: Molecular genotyping is more reliable compared with the serological method, especially in multi-transfused patients. Therefore, the addition of blood group genotyping to serological assays can lead to an antigen-matched transfusion in these patients.

NLRP3 inflammasome of renal tubular epithelial cells induces kidney injury in acute hemolytic transfusion reactions.

BACKGROUND: Blood transfusion, a common basic supporting therapy, can lead to acute hemolytic transfusion reaction (AHTR). AHTR poses a great risk to patients through kidney function damage in a short time. Previous reports found that heme from destroyed red blood cells impaired kidney function, and NLR family pyrin domain containing 3 (NLRP3) inflammasome was augmented in case of kidney injury. However, the detailed mechanism regarding whether NLRP3 inflammasome is involved in kidney function injury in AHTR is not fully understood yet. METHODS: Hemolysis models were established by vein injection with human blood plasma or mouse heme from destroyed red blood cells. The injured renal tubular epithelial cells (RTECs) were evaluated by tubular damage markers staining in hemolysis models and in primary RTECs in vitro. The activation of NLRP3 inflammasome in RTECs by hemes was investigated by Western blot, ELISA, scanning electron microscopy, immunofluorescent staining, flow cytometry, and hemolysis models. NLRP3 gene knockout mice were employed to confirm these observations in vitro and in vivo. The binding between a novel inhibitor (66PR) and NLRP3 was affirmed by molecule docking and co-immunoprecipitation. The rescue of 66PR on kidney function impairment was explored in murine hemolysis models. RESULTS: We found that heme could activate NLRP3 inflammasome in RTECs to induce kidney function injury. NLRP3 gene knockout could prevent the damage of RTECs caused by hemes and recover kidney function in AHTR. Moreover, NLRP3 inflammasome chemical inhibitor, 66PR, could bind to NLRP3 protein and inhibit inflammasome activation in RTECs, which consequently relieved the injury of RTECs caused by hemes, and alleviated kidney function damage in the AHTR model. CONCLUSIONS: Hemes could activate NLRP3 inflammasome in RTECs, and a novel NLRP3 inflammasome inhibitor named 66PR relieved kidney function damage in AHTR. Our findings provided a new possible strategy to treat kidney function failure in AHTR.

SCAR: The high-prevalence antigen 013.008 in the Scianna blood group system.

BACKGROUND: The Scianna (SC) blood group system comprises seven antigens. They reside on the erythroblast membrane-associated glycoprotein (ERMAP). The ERMAP and RHCE genes are juxtaposed to each other on chromosome 1. We report a novel SC antigen. STUDY DESIGN AND METHODS: Blood samples came from a patient and his two sisters in Saudi Arabia. To investigate the antibody specificity we used the column agglutination technique and soluble recombinant ERMAP protein. The significance of anti-SCAR was evaluated by the transfusion history and a monocyte monolayer assay. We determined the genomic sequence of ERMAP and RHCE genes. RESULTS: The patient's serum showed an antibody of titer 8 against a high-prevalence antigen. The soluble recombinant ERMAP protein inhibited the antibody. The propositus genotyped homozygous for an ERMAP:c.424C>G variant, for which his sisters were heterozygous. The c.424C>G variant occurred in the SC*01 allele in one haplotype with the RHCE*03 (RHCE*cE) allele. No signs of hemolysis occurred following an incompatible blood transfusion. The monocyte monolayer assay was negative. CONCLUSIONS: We characterized a high-prevalence antigen, with the proposed name "SCAR," which is the eighth antigen of the Scianna blood group system (proposed designation 013.008). Individuals homozygous for ERMAP:p.(Gln142Glu) protein variant can produce anti-SCAR. Although we did not observe any sign of hemolysis at this time, the anti-SCAR prompted a change of the treatment regimen. A review of the known reports indicated that all SC alloantibodies of sufficient titer should be considered capable of causing hemolysis.

FcγR2B B2.4 haplotype predicts increased risk of red blood cell alloimmunization in sickle cell disease patients.

BACKGROUND: Red blood cell (RBC) alloimmunization is an important transfusion complication which is prevalent among sickle cell disease (SCD) patients. Autoimmune diseases are a known risk factor for RBC alloimmunization, suggesting that autoimmunity and post-transfusion alloantibody development occur through similar physiopathological pathways. Polymorphisms in the FcγR2B gene have already been associated with several autoimmune disorders and hypothetically could be associated with RBC alloimmunization. Our goal was to evaluate if important polymorphisms of FcγR2B have an impact on the risk of RBC alloimmunization among SCD patients. STUDY DESIGN AND METHODS: This was a case-control study in which alloimmunized and non-alloimmunized SCD patients were compared in terms of the genotype frequency of the FcγR2B polymorphisms -386G/C, -120 T/A, and 695C/T, genotyped through direct Sanger sequencing. RESULTS: A total of 237 patients met the eligibility criteria, 120 cases (alloimmunized) and 117 controls (non-alloimmunized). RBC alloimmunization was associated with female sex (p < 0.001), lifetime number of RBC units transfused (p = 0.002) and 120 T/A FcγR2B genotype (p = 0.031). The FcγR2B promoter region haplotype 2B.4 (386C120A) was positively associated with RBC alloimunization (p = 0.045). The logistic regression (LR) model identified female sex (OR 10.03, CI 95% 5.16-19.49; p < 0.001) and FcγR2B 2B.4 haplotype (OR 4.55, CI95% 1.1118.65; p = 0.035) as independent predictors of RBC alloimmunization in SCD patients. CONCLUSION: SCD patients with the FcγR2B 2B.4 haplotype had over a fourfold higher risk for RBC alloimmunization. This highlights the role played by FcγR2B on RBC alloimmunization and may be helpful in identifying the immune responders.

Platelet concentrate and type II IL-1 receptor are risk factors for allergic transfusion reactions in children.

PURPOSE: Allergic transfusion reactions (ATRs) are immunological reactions after transfusion. Interleukin-1 (IL-1) is a critical regulator for human diseases. We performed this study to investigate the association of type II IL-1 decoy receptor (IL1R2) expression with ATRs in children. METHODS: Children received blood transfusions between January and December 2019 were included. The age, sex, number and type of blood transfusion, allergic history, and medical history were collected and statistically analyzed. The blood samples were collected from children with and without ATRs for detecting the relative expression IL1R2 mRNA. Logistics regression analysis was performed to identify the risk factors for ATRs in children. The area under the receiver operating characteristic (ROC) curve (AUC) was used to evaluate the predictive performance of risk factors. RESULTS: Totally, 28,840 transfusions in 20,230 children, with 236 ATRs (0.82%) in 117 patients (0.58%) were included. ATRs were common in children at the hematology-oncology department, in children received higher number of blood transfusions, and older children. Platelet concentrate induced a higher incidence of ATRs (3.31%) than red cell concentrate (0.22%, p < 0.0001). After the transfusion, IL1R2 mRNA level was higher in the blood samples in children with ATRs than those without ATRs (p < 0.0001). Logistics regression analysis indicated that platelet concentrate (95% CI 3.555, 293.782) and IL1R2 expression (95% CI 1.171 × 102, 1.494 × 104) were independent risk factors for ATRs in children. IL1R2 expression had high performance in predicting ATRs (AUC = 0.998, 100% sensitivity and 98.85% specificity). CONCLUSION: High IL1R2 expression level in children who received blood transfusions may predict the morbidity of ATR.

Utilising red cell antigen genotyping and serological phenotyping in sickle cell disease patients to risk-stratify patients for alloimmunisation risk.

BACKGROUND: Alloimmunisation and haemolytic transfusion reactions (HTRs) can occur in patients with sickle cell disease (SCD) despite providing phenotype-matched red blood cell (RBC) transfusions. Variant RBC antigen gene alleles/polymorphisms can lead to discrepancies in serological phenotyping. We evaluated differences between RBC antigen genotyping and phenotyping methods and retrospectively assessed if partial antigen expression may lead to increased risk of alloimmunisation and HTRs in SCD patients at a tertiary centre in Canada. METHODS: RBC antigen phenotyping and genotyping were performed by a reference laboratory on consenting SCD patients. Patient demographic, clinical and transfusion-related data were obtained from a local transfusion registry and chart review after research ethics board approval. RESULTS: A total of 106 SCD patients were enrolled, and 91% (n = 96) showed additional clinically relevant genotyping information when compared to serological phenotyping alone. FY*02N.01 (FY*B GATA-1) (n = 95; 90%) and RH variant alleles (n = 52, 49%; majority accompanied by FY*02N.01) were common, the latter with putative partial antigen expression in 25 patients. Variability in genotype-phenotype antigen prediction occurred mostly in the Rh system, notably with the e antigen (kappa: 0.17). Fifteen (14.2%) patients had a history of alloimmunisation, with five having HTR documented; no differences in clinical outcomes were found in patients with partial antigen expression. Genotype/extended-phenotype matching strategies may have prevented alloimmunisation events. CONCLUSION: We show a high frequency of variant alleles/polymorphisms in the SCD population, where genotyping may complement serological phenotyping. Genotyping SCD patients before transfusion may prevent alloimmunisation and HTRs, and knowledge of the FY*02N.01 variant allele increases feasibility of finding compatible blood.

Association between HLA-DRB1*01 and HLA-DRB1*15 with alloimmunisation in transfusion-dependent patients with thalassaemia.

BACKGROUND: Alloantibody production is one of the most challenging complications in transfusion-dependent thalassaemia patients. Haemolytic anaemia, an increase in blood consumption, difficulty in haematopoietic stem cell transplantation and reduced quality of life are consequences of alloimmunisation. The most predisposed antigens (Ags) for alloantibody development are Rh and Kell blood group Ags. OBJECTIVE: The aim of the present study is to evaluate any correlation between HLA-DRB1 alleles and Rh and Kell alloantibodies. MATERIALS AND METHODS: Fifty-two non-responders (control) and 54 responders (case) were enrolled in this study. Alloantibody detection was performed using the tube method. Genotyping of HLA-DRB1*01 and HLA-DRB1*15 was conducted by single-specific primer-polymerase chain reaction. RESULTS: In the responder group, 77.8% were hyper-responders (more than one alloantibody), and only 22.2% were mono-responders. Most detected alloantibodies were Anti-K (94.4%), followed by Anti-E (64.8%), Anti-C (29.6%) and Anti-D (25.9%). There was a significant difference in HLA-DRB1*15 between responder and non-responder groups, 73.7% vs 26.3%, respectively. (P = .029, OR = 3.290; 95%CI). Our results showed that HLA-DRB1*15 was more frequent in hyper-responders than mono-responders (92.9% vs 7.1%) (P = .007). The greatest HLA-DRB1*15 was seen in Anti-K (P = .014, odds ratio [OR = 3.784]; 95% confidence interval [CI]) and Anti-E (P = .011, OR = 3.609; 95%CI) alloantibodies. There is no association between HLA-DRB1*01 and alloimmunisation. CONCLUSION: Our findings showed that there is a significant correlation between HLA-DRB1*15 and Anti-K and Anti-E alloantibodies. These findings can be useful in detecting susceptible thalassaemic patients and improving transfusion management.

Extracellular DNA in blood products and its potential effects on transfusion.

Blood transfusions are sometimes necessary after a high loss of blood due to injury or surgery. Some people need regular transfusions due to medical conditions such as haemophilia or cancer. Studies have suggested that extracellular DNA including mitochondrial DNA present in the extracellular milieu of transfused blood products has biological actions that are capable of activating the innate immune systems and potentially contribute to some adverse reactions in transfusion. From the present work, it becomes increasingly clear that extracellular DNA encompassed mitochondrial DNA is far from being biologically inert in blood products. It has been demonstrated to be present in eligible blood products and thus can be transfused to blood recipients. Although the presence of extracellular DNA in human plasma was initially detected in 1948, some aspects have not been fully elucidated. In this review, we summarize the potential origins, clearance mechanisms, relevant structures, and potential role of extracellular DNA in the innate immune responses and its relationship with individual adverse reactions in transfusion.

Lack of persistent microchimerism in contemporary transfused trauma patients.

BACKGROUND: Following transfusion, donor white blood cells (WBCs) can persist long-term in the recipient, a phenomenon termed transfusion-associated microchimerism (TA-MC). Prior studies suggest TA-MC is limited to transfusion following traumatic injury, and is not prevented by leukoreduction. STUDY DESIGN AND METHODS: We conducted a prospective cohort study at a major trauma center to evaluate TA-MC following injury. Index samples were collected upon arrival, prior to transfusion. Follow-up samples were collected at intervals up to one year, and beyond for those testing positive for TA-MC. TA-MC was detected by real-time quantitative allele-specific polymerase chain reaction assays at the HLA-DR locus and several polymorphic insertion deletion sites screening for non-recipient alleles. RESULTS: A total of 378 trauma patients were enrolled (324 transfused cases and 54 non-transfused controls). Mean age was 42 ± 18 years, 74% were male, and 80% were injured by blunt mechanism. Mean Injury Severity Score was 20 ± 12. Among transfused patients, the median (interquartile range) number of red cell units transfused was 6 (3,12), and median time to first transfusion was 9 (0.8,45) hours. Only one case of long-term TA-MC was confirmed in our cohort. We detected short-term TA-MC in 6.5% of transfused subjects and 5.6% on non-transfused controls. CONCLUSIONS: In contrast to earlier studies, persistent TA-MC was not observed in our cohort of trauma subjects. Short-term TA-MC was detected, but at a lower frequency than previously observed, and rates were not significantly different than what was observed in non-transfused controls. The reduction in TA-MC occurrence may be attributable to changes in leukoreduction or other blood processing methods.

Initial trends of individual donation nucleic acid testing in voluntary & replacement donors from a tertiary care centre in north India.

Background & objectives: Individual donation nucleic acid testing (ID-NAT) is considered as sensitive technology to assess blood safety from viral transfusion-transmissible infections (TTIs) in blood donors. The present study was aimed to analyze the results of ID-NAT for three years (2013-2015) with special reference to different types of donors and their age ranges in a tertiary care centre in north India. Methods: The results of ID-NAT for three years were retrospectively analyzed at our centre. A total of 168,433 donations were tested with ID-NAT, of which 10,467 were tested with Procleix® Ultrio® reagents and 157,966 were tested with Procleix®UltrioPlus® reagents, and the results were compared with those of serology to calculate the NAT yield in voluntary, replacement, first-time and repeat donors. Results: A combined NAT yield was observed as one in 1031 out of 167,069 seronegative donations with HBV yield as one in 1465, HCV yield as one in 3885 and HIV-1 as one in 167,069. Yield for co-infection (HCV and HBV) was one in 41,767. A high NAT yield was observed in replacement donors (1 in 498) as compared to voluntary donors (1 in 1320). Interpretation & conclusions: Addition of NAT to serology improved the blood safety in our centre interdicting possibility of 150 TTIs annually. It has also reemphasized the safety of voluntary over replacement donors. The results also highlight the need of proper counselling, notification and referral guidelines of NAT yield donors in our country and other countries which lack them.

Type 1 IFN signaling critically regulates influenza-induced alloimmunization to transfused KEL RBCs in a murine model.

BACKGROUND: Only a fraction of red blood cell (RBC) transfusion recipients form alloantibodies, and variables determining responsiveness or nonresponsiveness are poorly understood. We and others have previously shown in animal models that pretreatment with toll-like receptor agonists that mimic different types of infections impacts the magnitude or frequency of RBC alloantibody responses. We hypothesized that influenza infection, coexistent with transfusion, would impact responses to transfused RBCs in a manner dependent on Type 1(α/ß) interferon (IFN) signaling and tested this in a murine model. STUDY DESIGN AND METHODS: Wild-type mice or mice lacking the ability to respond to Type 1 IFN were infected with influenza prior to the transfusion of transgenic murine RBCs (K1) expressing the human KEL glycoprotein or the triple fusion HOD protein. Alloantibody responses were measured longitudinally after transfusion by flow cytometric crossmatch, and posttransfusion RBC recovery and survival was evaluated. RESULTS: Influenza-infected mice transfused with K1 RBCs developed robust anti-KEL alloantibodies, whereas animals transfused in the absence of infection remained nonresponders; influenza-associated RBC alloimmunization was also observed after transfusion of HOD RBCs. Recipient Type 1 IFN production was critical to the mechanism of action of influenza-induced RBC alloimmunization, with alloimmunization being significantly decreased in mice unable to sense Type 1 IFN (through antibody blockade or genetic approaches). CONCLUSION: These and other data suggest that Type 1 IFN responses to toll-like receptor agonists or infections regulate RBC alloantibody responses. Studies investigating whether such a correlation exists in humans may be informative.

Nucleic acid amplification test: Bridging the gap in blood safety & re-evaluation of blood screening for cryptic transfusion-transmitted infection among Indian donors.

Background & objectives: : Nucleic acid amplification test (NAT) in blood donor screening not only detects window period (WP) donors but also those with chronic occult infections which are negative by routine serological screening. This study was conducted to determine the time trend of NAT positivity and seroprevalence of transfusion-transmitted infections (TTIs) through a period of six years and evaluate the strength of NAT as a supplementary test in identifying the cryptic carriers in blood donor population. Methods: : A total of 1,01,411 blood donations were screened between January 2011 and December 2016 by the ELISA and individual donor (ID) NAT Procleix Ultrio Plus Assay. Additional molecular and serological assays were done on the NAT yield samples to differentiate the type of cryptic carriers. Results: : NAT yields comprised 0.05 per cent (50/101411) of the total samples tested with a yield rate of 1/2028. Hepatitis B virus (HBV) contributed to 80 per cent of the total NAT yields and the rest 20 per cent due to hepatitis C virus (HCV). Majority of HBV NAT yields (75%) were from chronic occult donors and 25 per cent were WP donors. Both HBV and HCV NAT yields had a wide range of viral count. There was no HIV NAT yield. A significant decline in the prevalence rate of TTIs through the study period of six years was observed. Interpretation & conclusions: : The cryptic infections found in blood donors increase the risk of TTIs. Blood screening by both serology and NAT can reduce this threat.

Identification of genetic biomarkers for alloimmunization in sickle cell disease.

Most sickle cell disease (SCD) patients rely on blood transfusion as their main treatment strategy. However, frequent blood transfusion poses the risk of alloimmunization. On average, 30% of SCD patients will alloimmunize while other patient groups form antibodies less frequently. Identification of genetic markers may help to predict which patients are at risk to form alloantibodies. The aim of this study was to evaluate whether genetic variations in the Toll-like receptor pathway or in genes previously associated with antibody-mediated conditions are associated with red blood cell (RBC) alloimmunization in a cohort of SCD patients. In this case-control study, cases had a documented history of alloimmunization while controls had received ≥20 RBC units without alloantibody formation. We used a customized single nucleotide polymorphism (SNP) panel to genotype 690 SNPs in 275 (130 controls, 145 cases) patients. Frequencies were compared using multiple logistic regression analysis. In our primary analysis, no SNPs were found to be significantly associated with alloimmunization after correction for multiple testing. However, in a secondary analysis with a less stringent threshold for significance we found 19 moderately associated SNPs. Among others, SNPs in TLR1/TANK and MALT1 were associated with a higher alloimmunization risk, while SNPs in STAM/IFNAR1 and STAT4 conferred a lower alloimmunization risk.