Coronavirus infection and ABO blood grouping: Correlation or coincidence?

Background: Association between the ABO blood group and patient outcomes in COVID-19 patients is still unexplored. A known association may help to understand possible risks in advance to the management of such COVID-19 patients. The present study was designed to test such association if there is any, between the ABO blood group and the severity of COVID-19 patients. Methods: The present hospital-based observational study was conducted at a COVID-19 dedicated tertiary care hospital in North India over a period of six months during the first wave of the pandemic in the country. Five hundred consecutive patients, who tested positive for COVID-19 using RT-PCR on oropharyngeal/nasopharyngeal swabs, admitted to the hospital were included in the study. ABO and Rhesus (Rh) blood grouping was done on leftover hematology blood samples using gel column agglutination technology. Required clinical details of patients including age, gender, clinical symptoms, comorbidities, outcomes, etc., were obtained from the patient's case sheets. Results: The most common blood group was 'B' (42.8%) followed by 'O' (23.4%), and 'A' (22.4%) while the least common was 'AB' (11.4%). Rh positive was seen in 96.2% while 3.8% were negative. Baseline characteristics were comparable including length of hospital stay, duration of symptoms, and associated comorbid illnesses. The need for intensive care unit (ICU) admissions (P = 0.05) and intubations (P = 0.20) was similar across all four blood groups. Differences in the severity of COVID-19 disease and mortalities among the groups were non-significant. Conclusion: There was no observed association found between the ABO blood group and COVID-19 infection requiring hospitalization, ICU admission, intubation, and outcomes. However, there was a higher proportion of breathlessness and the presence of at least one comorbidity in blood group O as compared to others.

Establishment of normal reference range of serum hepcidin in Indian blood donors.

BACKGROUND: Hepcidin-25, a polypeptide hormone, plays a major role in iron metabolism and is found to be reduced during iron deficiency; therefore, testing for hepcidin can be utilized as an indicator of bioavailability of iron. Globally, reference range values for hepcidin have been established in different communities. The aim of the present study was to establish the normal reference range values of serum hepcidin in Indian blood donors to fathom the baseline and reference point of hepcidin. MATERIALS AND METHODS: A total of 90 donors fulfilling the eligibility criteria were recruited in the study consisting of 28 males and 62 females. Blood samples collected were used to execute hemoglobin (Hb), serum ferritin, and hepcidin assays. Serum hepcidin-25 isoform was detected by a commercial competitive enzyme-linked immunosorbent assay kit according to the manufacturer's instructions. Hb and ferritin were evaluated by the standard methods. RESULTS: The mean ± standard deviation (SD) of Hb level in males was 14.62 ± 1.34 g/dL and females was 13.33 ± 0.76 g/dL. The mean ± SD of ferritin level in males was 113 ± 56.12 ng/mL and females was 62.65 ± 40.8 ng/mL. Similarly, the mean ± SD of hepcidin level in male donors was 22.18 ± 12.17 ng/mL and female donors was 10.95 ± 6.06 ng/mL. The established reference range values of Hepcidin were 6.32-46.06 ng/mL for males and 3.44-24.78 for females. CONCLUSION: These findings suggest that further studies with larger groups of donors are mandatory to produce reference values of hepcidin that can be précised to the whole populace in India.

Reassessment of Critical Anti-D Antibody Titer in RhD Alloimmunized Antenatal Women.

OBJECTIVE: In the setting of RhD-alloimmunized pregnancy, laboratory variations in critical titer (CT) of anti-D antibody may result in needless referrals or a compromised fetal outcome. METHODS: RhD-alloimmunized pregnant women were included. Fetal outcome was categorized based on cord hemoglobin and interventions required. For 3 commonly used CTs of 8, 16, and 32, sensitivity and specificity as well as positive and negative predictive values were computed. RESULTS: When compared with CTs of 16 and 32, we detected 6.9% and 19.4% more cases of moderate-severe hemolytic disease of the fetus and newborn by using 8 as the CT. However, this leads to greater rate of unnecessary referral (12.1%, 10/82) than a CT of 16 (8.2%, 6/73) and 32 (4.9%, 3/61). A CT of 8 demonstrated 100% sensitivity, but 12.1% (10/82) of patients were referred needlessly. CONCLUSION: Because of its 100% sensitivity, we advocate decreasing the CT to 8. However, this may lead to unwarranted referrals.

Prevalence of the Direct Antiglobulin Test and Its Clinical Impact on Multiply Transfused Thalassemia Patients: A Prospective Study Conducted at a Tertiary Care Center in Northern India.

OBJECTIVE: This study was conducted to estimate prevalence of direct antiglobulin test (DAT) positivity and its impact on transfusion support in patients with thalassemia. METHODS: The DAT testing was performed for patients with ß-thalassemia who received transfusion from November 2021 to March 2022. Elution was done for DAT-positive samples. RESULTS: Of 180 patients, 21 (11.6%) were DAT positive. Immunoglobulin G (IgG) was present in 4 (19%) and IgG+C3d was present in 8 (38%). Only complement was present in 9 (42.8%) patients. The IgG-reactive DATs were associated with pan-reactive eluate. Patients who were DAT-positive had significantly higher levels of serum bilirubin, ferritin, and IgG than those who were DAT-negative. CONCLUSION: Autoantibody formation in multiply transfused thalassemia patients is common and merits equal attention as alloimmunization. It is particularly important as DAT-positive red blood cells may undergo clinically significant hemolysis, which may increase the transfusion requirements with associated sequalae such as increased serum ferritin and splenomegaly.

Red cell alloimmunization and associated risk factors in multiply transfused thalassemia patients: A prospective cohort study conducted at a tertiary care center in Northern India.

BACKGROUND: One of the complications of chronic transfusions in thalassemia is the development of red cell alloimmunization. AIMS: The aim of the study was to determine the frequency, specificity of red cell alloantibodies, and factors influencing alloimmunization in multiply transfused thalassemia patients. MATERIALS AND METHODS: The study was carried out prospectively on beta-thalassemia patients over 10 months. Plasma samples were used for antibody screening and identification using the column agglutination technique. Patients' clinical, laboratory, and transfusion details were obtained from hospital information system and patient files. STATISTICAL ANALYSIS: Continuous variables were reported as median and quartile, whereas categorical variables were provided as numbers and proportions. P < 0.05 was considered statistically significant. RESULTS: Out of 255 patients, 17 (6.6%) patients developed alloantibodies. Alloimmunized patients had significantly higher median ages at their first transfusions (1 year vs. 0.5 years; P = 0.042) than nonalloimmunized patients. Alloimmunized patients had significantly higher conjugated bilirubin (P = 0.016) and serum ferritin (P = 0.007). The majority of alloantibodies had specificity toward K antigen, followed by E, C, D, JKa, and JKb antigens. Alloimmunized patients received more units per year than nonalloimmunized patients (median, 30 vs. 24 units; P < 0.001). The average transfusion interval time between two successive transfusions showed a significant difference (P < 0.001). CONCLUSIONS: The prevalence of alloimmunization in thalassemia patients in North India is relatively low. Since most of the alloantibodies belong to Rh and Kell blood group system, extended phenotype-matched blood for Rh and Kell will be helpful in further preventing or decreasing the development of alloantibodies in multiply transfused thalassemia patients.

Effect of donor parameters and cell separators on yield of apheresis platelet and their impact on corrected count increment in aplastic anemia patients.

BACKGROUND: The new cell separators make it simple to collect single donor platelets (SDP), although the platelet yield may vary depending on the cell separator used and donor-related clinical and laboratory variables. AIMS: This study aims to study the factors affecting SDP yield and corrected count increment (CCI). MATERIALS AND METHODS: This retrospective study was carried out at a tertiary care facility in northern India, over 4 years (May 2017-April 2020), data were retrieved and analyzed. STATISTICAL ANALYSIS: Categorical variables were presented as proportions, while continuous variables were presented as mean with standard deviation, P < 0.05 was considered significant. RESULTS: We found a positive correlation between predonation platelet count and yield (r = 0.243, P = 0.000). No such significant correlation was found with Hb concentration (r = 0.025, P = 0.720), age (r = 0.016, P = 0.820), sex (r = -0.038, P = 0.584), and weight (r = -0.025, P = 0.714). Maximum platelet yield and minimum time were seen with Trima. Only 39.3% (33/84) meet the 24 h CCI. The majority of patients did not meet the desired CCI could be due to the patients' clinical condition. On logistic regression, we found a significant association of 24 h CCI with product yield (odds ratio [OR] = 0.168, P = 0.015) and posttransfusion platelet count (OR = 0.454, P < 0.05). CONCLUSION: The only donor-related factor that influences yield is predonation platelet count, whereas 24 h CCI may depend on the clinical status of the patient and yield.

A case report on non-D HDFN: Highlighting the role of antibody screening in RhD positive antenatal women.

The widespread use of anti-D immunoglobulin has resulted in a relative increase in the importance of non-D alloimmunization as a cause of hemolytic disease of the fetus and newborn (HDFN). Non-D alloantibodies that are capable of causing severe HDFN include anti-K, anti-E, and anti-c. Anti-c is clinically the most important Rh system antibody after anti-D. Here, we report three cases of neonates presenting with anemia and hyperbilirubinemia with strongly positive direct antiglobulin test who required phototherapy and neonatal exchange transfusion due to non-D antibody in RhD positive antenatal women. Anti-c was common in all the three cases while two cases have one additional non-D antibody. Due to faulty practices, antenatal antibody screening was not done for any case considering the mother's RhD positive status. Hence, antenatal antibody screening should be performed routinely, in all RhD positive pregnant women to reduce the delay in diagnosis and the management of HDFN occurring due to non-D antibodies.

Pattern of Conventional Coagulation and Thromboelastographic Parameters in Patients with COVID-19 Diseases and Association of COVID-Associated Coagulopathy with Mortality in Intensive Care Unit.

Background: Knowledge of underlying pathophysiology of coagulopathy is evolving and the pattern of coagulation parameters in coronavirus disease 2019 (COVID-19)-associated diseases is still not very clear. Aims: In the present study, we aimed to find out the pattern and distribution of conventional coagulation parameters and thromboelastographic (TEG) parameters in COVID-19-associated coagulopathy (CAC) in survivors and nonsurvivors at 28 days. Setting and Design: The present prospective observational study was conducted at a tertiary care COVID-19 intensive care unit (ICU) facility from March 21, 2020, to July 15, 2021. Materials and Methods: Admission clinical and laboratory data (conventional coagulation, inflammatory and TEG parameters, and disease severity parameters) of 64 COVID-19 patients admitted to the ICU were collected. Patients were divided into two groups, i.e., survivors and nonsurvivors. Statistical Analysis: Data were compared between two groups, i.e., survivors versus no survivors on 28 days using Student's t-test/Mann-Whitney U-test or Chi-square test/Fisher's exact test. Results: Admission mean plasma fibrinogen levels (474.82 ± 167.41 mg.dL-1) and D-dimer were elevated (1.78 [0.66, 3.62] mg.mL-1) in the COVID-19 ICU patients. Overall, COVID-19 patients had mean lower normal platelet count (150 ± 50 × 103 cells.mm-3), with marginally elevated prothrombin time (16.25 ± 3.76 s) and activated partial thromboplastin time (38.22 ± 16.72 s). A 65.6% (42/64) TEG profile analysis showed a normal coagulation profile, and the rest 21.9% (14/64) and 12.5% (8/64) had hypercoagulable and hypocoagulable states, respectively. Plasma D-dimer level was markedly elevated in nonsurvivors compared to survivors (P < 0.05), while no other conventional coagulation parameters and TEG profile demonstrated statistically significant between the two groups. Conclusion: Markedly elevated plasma D-dimer level was observed in nonsurvivors of COVID-19 ICU patients. A large portion of COVID-19 ICU patients had a normal TEG profile. Conventional coagulation parameters and TEG profile were similar between survivors and nonsurvivors.

Molecular basis of RhD-negative phenotype in North Indian blood donor population.

Background & objectives: RHD gene typing is highly complex due to homology with RHCE genes. Molecular polymorphism of the RHCE and RHD genes have been characterized among various populations, but no studies have been undertaken among Indians. This study was undertaken to assess the genetic basis of RHD-negative phenotype in Indian blood donor population. Methods: Sample from a total of 200 phenotypically RhD-negative blood donors were analyzed for presence of RHD gene using polymerase chain reaction (PCR). RHD genotyping was done using three primer sets designed for exons 4 and 10 and one set for identification of pseudo (RHDΨ) gene between introns (int) 3 and 4. Amplified PCR products were analyzed by gel-electrophoresis (XY Loper, Uvitech, Cambridge) and confirmed by nucleotide sequencing (ABI 3730 xl 96 capillary system). Results: No PCR product was found in 195/200 (97.5%) of study samples indicating homozygous gene deletion. Of the 5/200 (2.5%) showing RHD gene polymorphisms, 4/200 (2%) were positive for presence of exon 10 only (RHD-CE-D hybrid). RHDΨ gene was not detected in any of the samples tested. One sample showed presence of all three tested regions and was negative for RHDΨ gene. Interpretation & conclusions: RHD gene deletion was found to be the most common cause of an RHD-negative phenotype while RHDΨ gene was, reported to be present in up to 39 per cent of various ethnic populations, but was not detected. RHD-CE-D hybrid gene (found in 2.5% individuals) is important for predicting the requirement of Rh prophylaxis during the antenatal period.

Red blood cell alloimmunization among recipients of blood transfusion in India: A systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: There is a varied prevalence of red cell alloimmunization being reported from different parts of India. This study aimed to estimate the overall prevalence of alloimmunization in India by performing a systematic review of the literature and to establish the most suitable antigen-matching strategy to reduce the red blood cell (RBC) alloimmunization rate among transfusion recipients. MATERIALS AND METHODS: A systematic search of all the original articles published in English on RBC alloimmunization among transfusion recipients from India in MEDLINE, SCOPUS, CINAHL and Google Scholar bibliographic databases was conducted. After screening the articles as per inclusion/exclusion criteria, data extraction was done independently by two sets of investigators. Meta-analysis was performed by the binary random-effects model using the restricted maximum likelihood method. RESULTS: A total of 44 studies on RBC alloimmunization, with a cumulative sample size of 309,986 patients, were grouped into hospital-based and multiply-transfused patients, which yielded a prevalence of 0.5 (95% confidence interval; 0.3-0.8) and 4.8 (95% confidence interval; 3.9-5.7) per 100 patients, respectively. As many as 1992 alloantibodies were identified among the 1846 alloimmunized patients. The most common antibody identified was anti-E (127; 31.99%), followed by anti-c (75; 18.89%) in multiply-transfused patients. CONCLUSION: The rate of alloimmunization was 0.5 per 100 patients tested for antibodies and 4.8 per 100 patients receiving transfusion. Considering E- and c-antigen-matched red cells along with ABO and RhD matching may significantly reduce the overall occurrence of alloimmunization among Indian population who are transfusion-dependent.

Plateletpheresis donor deferral pattern: A retrospective 4-year data analysis at tertiary care center in India.

BACKGROUND: Increasing demand of single donor platelet requires blood banks to expand the donor pool. A reassessment of donor deferral criteria for plateletpheresis is required to ensure that this increasing demand is met without compromising on product quality and donor safety. AIMS: (1) To list the various causes of SDP donor deferral. (2) To discuss various approaches to minimize it. MATERIALS AND METHODS: Data of plateletpheresis donor deferral were collected from records retrospectively over a period of 4 years from January 2017 to December 2020. STATISTICAL ANALYSIS: All statistical tests were performed using IBM SPSS software for Windows version 20. Categorical variables were presented as proportions, while continuous variables were presented as mean with standard deviation, mean calculated P < 0.05 was considered statistically significant. RESULTS: Out of the 7478 donors screened for plateletpheresis procedure, 3232 (43.2%) were deferred among which 3089 (42.5%) were male and 142 (63.1%) were female donors. Majority (96.5%) of deferral were temporary. These included low platelet count (47.4%) followed by poor venous access (22.4%) and low hemoglobin (Hb) (7.2%). Among the donors deferred for low Hb, 24.7% (58 out of 234) had Hb between 12 and 12.4 g%. Similarly, among donor deferred for low platelet count, 12% (184 out of 1532) had platelet count between 140 and 149 × 103/µl. CONCLUSION: There is potential for increasing the number of eligible plateletpheresis donors if the present donor selection criteria were relaxed to a minimum Hb of 12 g/dl and minimum platelet count of 140 × 103/µl.

Application of flow cytometry in transfusion medicine: The Sanjay Gandhi Post Graduate Institute of Medical Sciences, India experience.

The application of flow cytometry (FC) is diverse and this powerful tool in used in multiple disciplines such as molecular biology, immunology, cancer biology, virology, and infectious disease screening. FC analyzes a single cell or a particle very rapidly as they flow past single or multiple lasers while suspended in buffered solution. FC has a great impact in the field of transfusion medicine (TM) due to its ability to analyze individual cell population and cell epitopes by sensitive, reproducible, and objective methodologies. The main uses of FC in TM are detection of fetomaternal hemorrhage, diagnosis of paroxysmal nocturnal hemoglobinuria, quantification of D antigen, detection of platelet antibody, quality control of blood components, for example, residual leukocyte counts and evaluation of CD34-positive hematopoietic progenitor cells in stem cell grafts. In recent years, FC has been implemented as an alternative method for the detection and characterization of red cell autoantibodies in autoimmune hemolytic anemia. Many workers considered FC as a very good complement when aberrant expression of various erythrocyte antigens needs to be elucidated. It has been extensively used in the resolution of ABO discrepancies and chimerism study. FC has also been used successfully in various platelet immunological studies. In the recent past, FC has been used in several studies to assess the platelet storage lesions and elucidate granulocyte/monocyte integrity and immunology. FC analysis of CD34+ stem cells is now the method of choice to determine the dosage of the collected progenitor cells. The technique is vastly used to evaluate residual leukocytes in leukodepleted blood components. We conclude that flow cytometers are becoming smaller, cheaper, and more user-friendly and are available in many routine laboratories. FC represents a highly innovative technique for many common diagnostic and scientific fields in TM. Finally, it is the tool of choice to develop and optimize new cellular and immunotherapeutic trials.

Prognostic Significance and Prevalence of IgG Subtypes in Rh Haemolytic Disease of Fetus and Newborn.

Many authors have reported poor prognostic value of anti-D antibody titer in the setting of Hemolytic Disease of Fetus and Newborn (HDFN). According to literature, HDFN cases with IgG1 and IgG3 have more severity compared to IgG2 and IgG4.Therefore, we planned this study to evaluate the prevalence and prognostic value of IgG subtypes in the setting of Rh HDFN. This was a retrospective study performed at a tertiary care center in north India from October 2015 to November 2017. Women with anti-D antibody were included in the study and categorized on the basis of presence of specific IgG subtype. "DAT IgG1/IgG3 ID" card (BIO-RAD) was used for determining the subclass of IgG. Various clinical, laboratory & interventional parameters were used to categorize fetal outcome in severe and non-severe cases. Perinatal outcome was then compared between women with different IgG subclass profile. Subclass distribution among 80 alloimmunized women was 26.2% for IgG1, 15% for IgG3, 46.2% for IgG1 + IgG3 and the rest had neither IgG1 nor IgG3. Severity of HDFN was significantly higher when IgG1 &/or IgG3 were present alone or in combination, compared to cases with absence of IgG1 or IgG3 (p value < 0.05). Risk of severe HDFN was significantly higher in the presence of IgG1 &/or IgG3 and the severity was highest when both IgG1 and IgG3 were present. We recommend that IgG subclass determination should be included in a multi-parameter protocol for more accurate prediction HDFN severity to ensure timely referral and intervention.

Roles of Salmonella typhi and Salmonella paratyphi in Gallbladder Cancer Development.

BACKGROUND: Typhoid (Salmonella typhi and paratyphi) carriers and gall bladder cancer (GBC) are endemic in northern India. Results of previous studies about association of typhoid carriers with GBC are inconsistent. We studied antibodies against Salmonella typhi and paratyphi in serum samples of patients with GBC. METHODS: We performed modified Widal test for antibodies against Salmonella typhi (Vi and O) and Salmonella paratyphi (AO and BO) antigens in patients with GBC (n=100), xanthogranulomatous cholecystitis (XGC, n=24), chronic cholecystitis (CC, n=200) and healthy controls (HC, n=200). RESULTS: Serum antibodies against Salmonella were more frequently positive in GBC (22%) and XGC (29%), particularly in males in age ≥50 years (GBC: 47% and XGC: 50%) vs. HC (0) (p <0.01). Vi antibody was more common in GBC (13%, OR:9.8) and XGC (8%, OR:5.9) than HC (2%). O antibody was more common in GBC (8%, OR: 8.6) and XGC (8%, OR: 9.0) than HC (1%). O antibody was also more common in males with GBC (12%) than CC (1%) and HC (1%) (P=0.02 and p <0.001, respectively). AO (6%) and BO (4%) antibodies were detected in GBC, particularly in males, than HC (0), (p <0.01). Salmonella antibodies were more frequent in GBC with GS than those without GS (50% vs. 20%, OR=3.94, P=0.01). CONCLUSIONS: Salmonella carrier state was more common in GBC and XGC, particularly in elderly males than HC. The Vi antibody was more common in GBC and XGC than HC. Salmonella infection was more common in GBC with GS than those without GS.

Title: Effect of multiple maternal red cell alloantibodies on the occurrence and severity of Hemolytic Disease of the Fetus and Newborn.

INTRODUCTION: Antenatal antibody screening in India is focused on the detection of anti-D in RhD-negative mothers. HDFN outcome can also be affected by the presence of antibodies other than anti-D. We planned this study to find the impact of 'anti-D in combination with additional antibodies' on the development and severity of HDFN compared with 'anti-D alone'. METHODS: This is a retrospective study performed at a referral center in northern India from October 2015 to March 2018. Antibody screening was performed on women with complicated obstetric history. Women with anti-D antibody were included in the study and categorized on the basis of presence of additional antibody (anti-D alone or in combination with other antibody). Various clinical, laboratory & interventional parameters were used to define HDFN and severe HDFN. Perinatal outcome was then compared between the two groups. RESULTS: A total of 176 women with anti-D antibody were included in the study. Of these, 136 cases (77.3%) had anti-D alone while at least one additional antibody was present in 40 (22.7 %) cases. Most common additional antibodies were anti-C, anti-E and anti-c. After excluding 46 women for various reasons, 130 women were left for final analysis. Approximately 57% and 78% of cases were affected by severe HDFN amongst women with anti-D alone and in combination, respectively. Relative risk of developing severe HDFN was 1.7 times higher in women with additional antibody. CONCLUSIONS: Patients with combination antibodies were found to have more severe HDFN compared to the ones with anti-D alone.

Prospective Cohort Study to Assess the Effect of Storage Duration, Leuko-Filtration, and Gamma Irradiation on Cell-Free DNA in Red Cell Components.

INTRODUCTION: Damage to a cell and the loss of integrity of its cell membrane leads to the release of endogenous immunogenic molecules, which together are classified as "damage-associated molecular patterns" (DAMPs). Cell-free DNA (cf-DNA) released from nucleosomes may serve as a proco-agulant cofactor and may be an important mediator of immunomodulatory and proinflammatory effects associated with blood transfusion. OBJECTIVES: To assess the levels of cf-DNA in supernatants of stored red cell components and the effect of leukoreduction and gamma irradiation on the release of cf-DNA during storage. METHODS: This is a prospective cohort study on 99 stored red cell components, randomly divided into three groups - buffy coat (BC)-depleted, leuko-filtered (LP), and irradiated (IR) packed red blood cells. Red cell supernatants were drawn over a period of 21 days at three different time points (day 0, 7, and 21) from the study units. cf-DNA extraction was done and quantified by a bench top fluorometer. Change in cf-DNA content, rate of change (µg/day), and percent change were estimated and compared across different groups. RESULTS: cf-DNA content increased (p = 0.000) with storage duration in the BC (median = 238.66 µg, interquartile range [IQR] = 168.42 on day 21 vs. median = 9.44 µg, IQR = 5.23 on day 0) and IR groups (p = 0.000) (median = 245.55 µg, IQR = 253.88 on day 21 vs. median = 7.07 µg, IQR = 13.58 on day 0), while there was a decreasing trend (p = 0.032) in the LP group (median = 4.55 µg, IQR = 10.73 on day 21 vs. median = 8.66 µg, IQR = 6.56 on day 0). The median rate of change in cf-DNA content (11.13 µg/day) and percent change in cf-DNA content (median = 4,106.16%) was highest in the IR group. CONCLUSIONS: Stored red cell components contain significant amount of cf-DNA. Release of cf-DNA is further aggravated by irradiation while leukoreduction leads to a decrease in cf-DNA content.

Molecular characterisation of RhD variants in North Indian blood donor population.

OBJECTIVES: A molecular analysis of serologically RhD variant samples was conducted to find the incidence of various D variants in our blood donor population. BACKGROUND: Determining a blood donor's RhD phenotype and genotype is important as transfusion of units with a weak D or partial D phenotype can result in immunisation of the recipients. METHODS: Samples with discrepant D and weak D phenotypes identified on testing with at least five different monoclonal anti-D antisera were considered serological RhD variant and subjected to molecular testing (Massarray kit, Agena Bioscience, San Diego) for variant RHD gene. RESULTS: A total of 39 samples, including 19 RhD discrepant samples and 20 weak D samples, were identified as serological RhD variant from a total of 4386 samples. Thirteen (13/39) samples carried variants leading to weak D phenotype, and eight samples had variants leading to partial D categories. Seven samples (7) could not be characterised, whereas 11 samples were identified as Rh negative (RHD*01N.01) after molecular testing. Overall incidence of D variants in the study population was 0.48%. RHD*weak D type 1(5, 0.1%) and RHD*DFR1 (5, 1%) were the most common variants identified. CONCLUSIONS: Few samples with weak reaction on serological testing were found to be partial D variant and vice versa. Donor centres should develop a protocol for genotyping of samples with aberrant results on serological testing for assessing the actual RhD status of an individual as results of serological testing may be misleading.

Prevalence of DEL phenotype in D- blood donors in India.

CONCLUSIONS: Unlike weak D and partial D, DEL represents a weakened form of D that cannot be detected by conventional serology and requires use of an adsorption-elution method for its detection; therefore, DEL+ samples might be mistyped as D-. The study was undertaken to determine the prevalence of the DEL phenotype among D- blood donors from northern India. A total of 1003 D- blood donors were tested for weak D and DEL by the indirect antiglobulin test and an adsorption-elution method, respectively. Of the total 21,135 blood donors typed for D, 20,132 (95.3%) were D+ and 1003 (4.7%) gave a negative reaction for D. Of the total 1003 D- samples, 8 (0.8%) were weak D and only 2 (0.2%) were DEL+ by adsorption-elution testing. For samples that typed as D-, the majority of individuals (91.1%) were cde/cde (rr) followed by dCe/dce (r´r) in 4.8 percent, and dCe/dCe (r´r´) in 2.2 percent. Both DEL+ samples were also C+. We conclude that the prevalence of the DEL phenotype as detected by serology in D- north Indian blood donors is 0.2 percent, although it is as high as 2.8 percent in D-C+ individuals. There is an association of DEL with C, which can be used as a cost-effective marker for screening large numbers of D- blood donors for DEL.

Comparison of UV spectrometry and fluorometry-based methods for quantification of cell-free DNA in red cell components.

BACKGROUND: Stress and shear force applied on blood components during processing and storage may induce cellular damage leading to release of cell-free DNA (cfDNA). In this study, we have compared ultraviolet (UV) spectrophotometry with UV-induced fluorescence for the quantification of cfDNA in red cell supernatant. MATERIALS AND METHODS: cfDNA was extracted from 200 µL sample of supernatants from 99 packed red blood cells using QIAamp DNA Blood Mini Kit (Qiagen, Germany). Quantification of cfDNA was done using two different methods: one based on spectrophotometry (NanoDrop 2000c, ThermoFisher Scientific, USA) and another based on fluorometry (Qubit 2.0, Life Technologies, ThermoFisher Scientific, USA). Interassay variability of both the methods was estimated using serial dilutions of standard with known DNA concentration. RESULTS: DNA quantification by both the methods was close to actual amount of known standard in dilutions with higher concentration of DNA (21.68 to 2.71 ng/µl). While at higher dilutions, quantification by NanoDrop was neither precise nor accurate. Median cfDNA concentration in the study units was found to be 1.60 ng/µl (25th-75th percentile range: 1.10-2.10) by UV spectrophotometry (NanoDrop) compared to 0.080 ng/µl (25th-75th percentile range: 0.050-0.130) by fluorometry (Qubit). CONCLUSION: Due to high interassay variability between the two methods and the better precision and accuracy of Qubit, it is recommended that fluorometry-based method be used for the quantification of cfDNA in blood components.