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.

Prestorage gamma irradiation induces oxidative injury to red cells.

BACKGROUND: Gamma irradiation of blood results in the formation free radicals, which interact with lipids and proteins in the membranes of red blood cells. We have investigated oxidative injury to gamma-irradiated red cells by measuring markers of oxidative injury and its correlation with red cell membrane damage. METHODS: Thirty red cell blood units were irradiated at 25 Gy using Gamma Irradiator (Nordion, Canada) and stored at 4°C for 28 days. Markers of oxidative injury such as MDA levels, methemoglobin formation and osmotic fragility and markers of membrane damage including supernatant Hb, supernatant K(+), and LDH were studied. RESULTS: There was a progressive and statistically significant increase in markers of oxidative injury such as MDA (3.76 v/s 5.01), and methemoglobin formation (1.87 v/s 3.58) in irradiated red cells compared to control non-irradiated cells. Exposure to gamma irradiation caused significant increase in markers of hemolysis such as supernatant Hb (0.087 v/s 0.363), K(+) (35.1 v/s 51.2) and LDH (366.9 v/s 587.4) over the storage period of 28 days. CONCLUSION: Gamma irradiation increases lipid peroxidation and oxidative injury to the red cells.

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.

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.

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.

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.

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.

Evaluation of efficacy of Valsalva for attenuating needle puncture pain in first time nonremunerated voluntary plateletpheresis donors: A prospective, randomized controlled trial.

BACKGROUND: Plateletpheresis is generally safe but may have adverse reactions. Adverse reactions can negatively influence donor recruitment and retention. Valsalva is a proven method of attenuating pain caused by venipuncture. AIMS: The aim was to evaluate the efficacy of the Valsalva maneuver on the attenuation of needle pain and donor anxiety. SETTINGS AND DESIGN: This prospective randomized controlled trial was conducted between November 2015 and April 2016 at the Department of Transfusion Medicine. SUBJECTS AND METHODS: One-hundred and sixty consecutive donors were grouped into control group (C) and Valsalva group (V) each of sample size 80. The Valsalva group performed a Valsalva maneuver and control did nothing before the venipuncture. Anxiety and pain were scored using a 10 cm visual analog scale (VAS). Severity was graded as VAS = 0 defines no pain and anxiety, VAS = 1-3 as mild pain and anxiety, VAS = 4-6 as moderate pain and anxiety, VAS = 7-9 as severe pain and anxiety, whereas VAS = 10 denotes extreme pain and anxiety. STATISTICAL ANALYSIS: Statistical Package for Social Sciences, version 23 was used for analysis. Independent samples t-test/Mann-Whitney U-test was used to compare between treatment and control group, whereas the Wilcoxon signed-rank test was used to test the difference between pre- and postobservations. RESULTS: In the Valsalva group, post-Valsalva anxiety levels were significantly reduced to (1 [0-2]) from their pre-Valsalva values of (2 [0-3]); (P < 0.001). Pain was significantly lower (2[1-2]) in Valsalva group compared to control (4[2-5]); (P < 0.001). CONCLUSIONS: Valsalva reduced both severity of venipuncture pain and anxiety. Valsalva can be performed by donors as it is an easy, painless, and nonpharmacological method of pain and anxiety attenuation.

Prevalence and predictors of adverse reactions in plateletpheresis donors with the perspective of donor safety in a tertiary care hospital of Northern India.

BACKGROUND: Plateletpheresis procedures are generally safe and associated with low adverse reactions. Although donor reactions and injuries are self-limited events, they may discourage donors from future platelet donations. AIM: The purpose of this study was to determine the prevalence and predictors of adverse donor reactions in plateletpheresis donors, which could serve as targets for interventions to reduce reactions. MATERIALS AND METHODS: The study included 106 platelet donors over a period of 2 years. The demographic, biometric, and clinical parameters were noted. The data were analyzed for predictors of adverse donor reactions. STATISTICAL ANALYSIS USED: The data were analyzed using independent sample t-test to correlate donor variables such as gender. To correlate other variables such as age, weight, and whole blood processed, Chi-square test was used. RESULTS: A total of 106 plateletpheresis donations were performed and 13.2% of vasovagal reactions were observed. The significant predictive factors for reactions were young female donors with low body weight in which more than 2.5 L volume of whole blood was processed and more than 250 ml of acid, citrate, and dextrose-A was infused and with single venous access procedures. CONCLUSIONS: The results of this study are encouraging and helpful in identifying donors at risk for developing adverse reactions during plateletpheresis so that proper and close observation during and after donation as well as timely intervention can prevent most of the unpleasant events of plateletpheresis donors.

Assessment of bedside transfusion practices at a tertiary care center: A step closer to controlling the chaos.

BACKGROUND: Blood transfusion chain can be divided into three phases: preanalytical (patient bedside), analytical (steps done at transfusion services), and postanalytical (bedside). Majority (~70%) of events due to blood transfusion have been attributed to errors in bedside blood administration practices. Survey of bedside transfusion practices (pre-analytical and post analytical phase) was done to assess awareness and compliance to guidelines regarding requisition and administration of blood components. MATERIALS AND METHODS: Interview-based questionnaire of ward staff and observational survey of actual transfusion of blood components in total 26 wards of the institute was carried out during November-December 2013. All the collected data were coded (to maintain confidentiality) and analyzed using SPSS (v 20). For analysis, wards were divided into three categories: medical, surgical, and others (including all intensive care units). RESULTS: A total of 104 (33 resident doctors and 71 nursing) staff members were interviewed and observational survey could be conducted in 25 wards during the study period. In the preanalytical phase, major issues were as follows: lack of awareness for institute guidelines (80.6% not aware), improper sampling practices (67.3%), and prescription related (56.7%). In the postanalytical phase, major issues were found to be lack of consent for blood transfusion (72%), improper warming of blood component (~80%), and problems in storage and discarding of blood units. CONCLUSION: There is need to create awareness about policies and guidelines of bed side transfusion among the ward staff. Regular audits are necessary for compliance to guidelines among clinical staff.

Removal of antibodies from red cells: Comparison of three elution methods.

BACKGROUND: Direct antiglobulin test (DAT) is the most common test done in immunohematology lab, which detects immunoglobulin and fragments of complement attached to the red blood cells. These coated red blood cells are difficult to accurately phenotype, which may be required for selection of appropriate unit of red blood cells for transfusion. AIMS: We have studied the efficacy of various elution methods in removing the antibodies coating the red cells and their impact on different blood group antigen activity. MATERIALS AND METHODS: Patient samples sent for serological evaluation of autoimmune hemolysis were included in the study. DAT and Indirect antiglobulin test (IAT) were performed using gel cards (ID system, DiaMed Switzerland). Antibody coated red cells, either by in-vivo or in-vitro sensitization, were used to assess the outcome of three elution methods. RESULTS: Out of 93 DAT positive samples already sensitized in vivo, 28 (30 %) samples became DAT negative post elution using either of three methods, while 36 (38.8%) showed reduction in strength of reaction, whereas in 29 (31.2%) there was no change in strength of reaction. Similarly, out of the 17 samples prepared by in vitro sensitization, 12 samples became completely negative after glycine-HCl/EDTA elution, 9 and 5 samples became negative after heat elution and chloroquine diphosphate elution methods, respectively. CONCLUSION: On comparative analysis glycine-HCl/EDTA elution method was better than the other two methods and can be used for eluting immunoglobulins from intact red cells.

Oxidative injury as contributory factor for red cells storage lesion during twenty eight days of storage.

BACKGROUND: Storage of red blood cells at 4 °C is associated with deleterious metabolic and biochemical changes, collectively referred to as "storage lesions". Lipid peroxidation of the red cell membrane leading to lysis contributes to these storage lesions. The aim of the present study was to investigate oxidative injury to red cells during storage for 28 days and its correlation with markers of red cell membrane damage. MATERIALS AND METHODS: Samples from 30 units of red blood cells stored at 4 °C for 28 days were withdrawn aseptically on day 0, day 14 and day 28 of storage. Markers of membrane damage including plasma haemoglobin, plasma potassium and lactate dehydrogenase (LDH) concentrations and markers of oxidative injury such as malondialdehyde (MDA) levels, haemoglobin oxidation and osmotic fragility were studied in all samples. RESULTS: Statistically significant (p<0.001) increases in the mean values of plasma haemoglobin, plasma potassium, LDH and markers of oxidative injury such as MDA and haemoglobin oxidation were observed over the storage period of 28 days. Direct correlations of MDA and haemoglobin oxidation with membrane damage, as reflected by plasma haemoglobin concentration, were observed. CONCLUSION: Oxidative injury to red blood cells during storage leads to membrane damage and lysis. The role of antioxidants in the prevention of this deleterious effect of storage warrants investigation.