A comparison of serological phenotyping and molecular genotyping for Kell, Kidd, and Duffy antigens in multi-transfused thalassemia patients.

BACKGROUND: In multi-transfused thalassemia patients, serological phenotyping fails to test patient's actual blood group antigen profile due to the presence of donor red blood cell (RBC) in the circulation. This limitation of serological tests can be overcome by genotype determination using the polymerase chain reaction (PCR)-based methods. The aim of this study is to compare the serological phenotyping of Kell, Kidd, and Duffy blood group systems with molecular genotyping in the normal blood donors and multi-transfused thalassaemia patients. MATERIALS AND METHODS: Blood samples from 100 normal blood donors and 50 thalassemia patients were tested using standard serological techniques and PCR-based methods for Kell (K/k), Kidd (Jka/Jkb), and Duffy (Fya/Fyb) blood group systems. The results were compared for concordance. RESULTS: Genotyping and phenotyping results were 100% concordant for normal blood donors whereas those for thalassemia patients showed 24% discordance. The frequency of alloimmunization in thalassemia patients was 8%. The results of genotyping were used to provide Kell, Kidd, and Duffy matched blood for transfusion therapy to thalassemia patients. CONCLUSION: The actual antigen profile in multitransfused thalassaemia patients can be reliably determined using genotyping. This would benefit in providing better antigen matched transfusion therapy to such patients hence reducing the rate of alloimmunization.

Implementation of internal quality control program for monitoring of enzyme-linked immunosorbent assay performance at a blood center.

BACKGROUND: Internal quality control (IQC) samples may be incorporated in enzyme-linked immunosorbent assay (ELISA) routinely for detection of errors occurring due to change in environmental conditions, test system, or operator performance. We have described methodology for preparation of IQC samples, monitoring of results using Levey-Jennings (LJ) charts and their interpretation. We have also described our experience of quality control in ELISA using IQC samples, identification of errors and corrections applied. MATERIALS AND METHODS: IQC samples for anti-HIV, hepatitis B surface antigen (HBsAg), and anti-HCV ELISA were prepared "in-house" using standard methodology. After validation of run, E-ratio of IQC sample was calculated and plotted on LJ chart. Further interpretation was done to detect the errors. LJ charts illustrating the performance of IQC samples on 180 runs for each ELISA were drawn and analyzed. RESULTS: For anti-HIV ELISA, violation of warning rule was found in 2 runs (1.11%). Only one run (0.55%) was rejected due to violation of rejection rule. For HBsAg ELISA, violation of warning rule was indicated in two runs (1.11%). Two runs (1.11%) were rejected due to violation of rejection rules. For anti-HCV ELISA, violation of warning rule was indicated in two runs (1.11%), whereas two runs were rejected due to violation of rejection rules. Comprehensive checks were performed for the evaluation of equipment calibration, handling, and storage temperature of reagents and operator's technique. A thorough investigation was undertaken according to the type of error. CONCLUSION: Inclusion of IQC with each ELISA run is valuable to check the assay performance, ensuring reliability and reproducibility of test results.

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.

A study on factors influencing the hemostatic potential of fresh frozen plasma.

BACKGROUND: Fresh frozen plasma (FFP) is administered to correct deficiencies of various coagulation factors. The level of these factors in FFP varies with donor demographics and ex-vivo processing of plasma. In this study we have compared the quality control parameters of FFP collected from donors of different genders, age groups, ABO blood groups, smoking and alcohol intake habits. MATERIALS AND METHODS: Four ABO group matched plasma units were pooled, split and further processed by four different freeze-thaw algorithms: frozen by contact shock freezer; thawed at 37°C, frozen by contact shock freezer; thawed at 45°C, frozen by mechanical freezer; thawed at 37°C, frozen by mechanical freezer; thawed at 45°C. The coagulation factor levels in plasma units were compared. RESULTS: There were no significant differences in the quality parameters with donor age, gender and alcohol intake. Factor VIII levels were significantly lower in O group FFP (P < 0.05). Smokers had significantly higher levels of fibrinogen (P < 0.05). There were no significant differences in PT, fibrinogen and factor VII levels of FFP processed through various algorithms. Plasma frozen rapidly through contact shock freezer had significantly lower aPTT and higher levels of factor V and VIII compared to mechanical freezing. There were no significant differences between PT, aPTT, fibrinogen, factor V, factor VII and factor VIII levels of FFP thawed at 37°C and 45°C. Mean thawing time was 28 minutes at 37°C and 17 minutes at 45°C. CONCLUSION: Rapid freezing is recommended for optimum preservation of coagulation factors. Thawing may be done at 45°C in cases of emergency, without compromising hemostatic potential.

Prevalence of hepatitis E virus viremia and antibodies among healthy blood donors in India.

BACKGROUND: Hepatitis E virus (HEV) is transmitted primarily through contaminated water and food. Recently, HEV viremia in blood donors and transfusion-related transmission of HEV have been reported, leading to calls to screen donated blood for this virus. However, these data are from regions where genotype 3 HEV is predominant. In India, where human infections are caused only by genotype 1 HEV, the frequency of subclinical HEV viremia is unknown. METHODS: Minipools of sera prepared from three donor units each from our institution's  blood bank in Lucknow, India, were tested for HEV RNA using a sensitive amplification-based assay. A randomly selected subset was also tested for IgG anti-HEV antibodies using a commercial (Wantai) immunoassay. RESULTS: Sera from 1799 donors (median [range] age 30 [18-63] years; 1746 [97.0%] men) were collected (June-July 2016, 900; November-December 2016, 899). Of these, 17 (0.95%), 16 (0.90%), and 3 (0.17%) tested positive for HBsAg, anti-HCV, and anti-HIV antibodies, respectively. None of the donors tested positive for HEV RNA. Of 633 randomly selected donors (age 30 [18-63] years, 613 [96.8%] male) tested for IgG anti-HEV, 383 (60.5%) tested positive. Seropositivity rate increased with age, being 70/136 (52%), 177/299 (59%), 100/154 (65%), 30/34 (88%), and 6/10 (60%) in the 18-24, 25-34, 35-44, 45-54, and 55 years or older age groups, respectively. CONCLUSIONS: In healthy blood donors from northern India, HEV viremia is infrequent though anti-HEV antibody prevalence is high. This suggests that asymptomatic HEV viremia may be less frequent in areas with genotype 1 predominance than those with genotype 3 predominance.

Identification of iron status of blood donors by using low hemoglobin density and microcytic anemia factor.

BACKGROUND: Two new parameters low hemoglobin density (LHD) and microcytic anemia factor (Maf) have been used by Beckman-Coulter LH series analyzers as an easy screening tool for the early detection of iron deficiency. The main objective of this study was to assess if LHD and Maf could be used for assessment of iron status in blood donors and also to establish a cut-off for these two parameters at which a tentative iron deficiency could be reported conclusively. MATERIALS AND METHODS: LHD% and Maf could be calculated by knowing mean cell hemoglobin (Hb) concentration, Hb, and mean cellular volume and we used SPSS in calculating LHD and Maf from these parameters. RESULTS: : Significant differences were detected in LHD% and Maf values when iron deficient and iron-depleted donors were compared with control donors, while these were insignificant for iron reduced donors. LHD and Maf were able to differentiate between iron deficient and iron-depleted donors from normal donors. A cutoff of 9.18% for LHD% was able to differentiate iron deficient and depleted state from normal iron states with a sensitivity and specificity of 91.9% and 71% respectively. Similarly, a cutoff of 10.16 and10.71 for Maf was able to differentiate between iron-deficient and iron-depleted donors from normal donors, respectively. CONCLUSION: : LHD% and Maf in the screening of blood donors raise the possibility of early detection of iron deficiency, without the need of extra cost and blood sampling.

Techniques used for the screening of hemoglobin levels in blood donors: current insights and future directions.

Blood donor hemoglobin (Hb) estimation is an important donation test that is performed prior to blood donation. It serves the dual purpose of protecting the donors' health against anemia and ensuring good quality of blood components, which has an implication on recipients' health. Diverse cutoff criteria have been defined world over depending on population characteristics; however, no testing methodology and sample requirement have been specified for Hb screening. Besides the technique, there are several physiological and methodological factors that affect accuracy and reliability of Hb estimation. These include the anatomical source of blood sample, posture of the donor, timing of sample and several other biological factors. Qualitative copper sulfate gravimetric method has been the archaic time-tested method that is still used in resource-constrained settings. Portable hemoglobinometers are modern quantitative devices that have been further modified to reagent-free cuvettes. Furthermore, noninvasive spectrophotometry was introduced, mitigating pain to blood donor and eliminating risk of infection. Notwithstanding a tremendous evolution in terms of ease of operation, accuracy, mobility, rapidity and cost, a component of inherent variability persists, which may partly be attributed to pre-analytical variables. Hence, blood centers should pay due attention to validation of test methodology, competency of operating staff and regular proficiency testing of the outputs. In this article, we have reviewed various regulatory guidelines, described the variables that affect the measurements and compared the validated technologies for Hb screening of blood donors along with enumeration of their merits and limitations.

Snake in the grass: A case report of transfusion reactions due to contaminated donor arm disinfectant.

Bacterial contamination of blood components remains an on-going challenge. In the majority of cases, organisms contaminating the blood components are a part of normal skin flora. Here, we report a case of bacterial contamination of blood units through contaminated donor arm disinfectant. There was a series of reactions due to random donor platelet (RDP) transfusion. The patients had features of septic transfusion reactions. On root cause analysis, spirit swabs used for disinfection of donors' arm were identified as the culprit and presence of Clostridium difficile was established. All the blood components prepared on the dates of implicated RDP units were removed from the stock and we replaced the existing 70% alcohol disinfectant with chlorhexidine-alcohol-based antiseptic rub. Further, no such transfusion reactions were reported. Implementation of good donor arm disinfection technique in addition to the use of blood bags with diversion pouch is proposed to be best preventive strategy for resource-poor settings.

Enhancement of Antibody Titre and Development of Additional Red Cell Alloantibodies Following Intrauterine Transfusion.

Intrauterine blood transfusion is the mainstay of managing foetuses with severe anemia. It may however result in fetomaternal hemorrhage, which in cases of Rh isoimmunisation may increase the severity of the disease by enhancing the maternal immunological response to fetal antigens. This study was conducted to determine the frequency, specificity and origin of additional red cell antibodies which developed after IUT. The change in the titre of allo anti-D following IUT was also determined. Antibody detection and titration was done on the blood samples of all the patients before and after intrauterine blood transfusion to check for the development of additional antibody and change in the titre of existing anti-D. Severe anemia was found in 17 (58.6 %) fetuses who received a total of 42 ultrasound-guided IUTs. Development of antibodies additional to anti-D in maternal serum was seen in 5 (29.4 %) cases. The specificity of additional alloantibodies was anti-C in four cases whereas it was anti-E in one case. Four fold or greater increase in existing allo-anti D titre was seen in 6 (35.3 %) cases after IUT. Enhancement of maternal sensitisation leading to an increase in maternal antibody titre is particularly seen after the first IUT. Matching of the donor RBCs particularly for Rh antigens might prevent the induction of additional alloantibodies against these antigens. IUT as a treatment modality should be given judiciously and only when the need is inevitable.

Importance of donor history of restless leg syndrome and pica to asses iron deficiency.

Iron deficiency is associated with neuropsychological changes such as restless leg syndrome (RLS), pica, hair loss, etc. Our objective was to assess usefulness of history of RLS and pica in relation with iron stores in blood donors. During medical examination, apart from routine questionnaires specific history of RLS and pica was elicited. Along with hemoglobin markers of iron deficiency such as s. iron, s. ferritin and mean corpuscular volume were analyzed. Out of 400 blood donors 41 had h/o pica/RLS/pagophagia. Positive and negative predictive value of above history is 73.17% and 80.5% respectively. We recommend the use of a screening question for pica and/or RLS in blood donor questionnaire.

Platelet growth factors from allogeneic platelet-rich plasma for clinical improvement in split-thickness skin graft.

BACKGROUND AND OBJECTIVES: Platelets are a source of numerous growth factors which facilitate repair and healing. Thus platelet rich plasma has been increasingly used as a treatment modality in the field of reconstructive surgeries for wound healing. This preliminary study was carried out to explore whether platelet growth factors from platelet rich plasma could be used for enhancement of split thickness skin graft survival. MATERIALS AND METHODS: Twenty patients (13 males and 7 females) requiring split thickness skin graft for various clinical reasons were enrolled in the study. Platelet rich plasma was collected by apheresis and frozen at -80° C. It was thawed at room temperature immediately before its intended application. PRP was applied only on one half of the wound, while another half served as control. Patient was followed for 6 weeks. The effect was assessed at first dressing in terms of graft uptake and subsequently as time taken for complete healing. RESULTS: There was 100% uptake of the graft in the area where platelet rich plasma was applied. In the control area, there was complete graft loss in 4 cases, partial loss in 7 cases and complete uptake in 9 cases. CONCLUSION: This study demonstrated promising results on application of PRP to split thickness skin grafts. Further randomized studies with greater sample size may be undertaken to establish platelet rich plasma as a validated treatment modality.

Successful use of the cell separator hemonetics multicomponent collection system+ for therapeutic thrombocytapheresis in a low body weight child of essential thrombocythemia.

In children, essential thrombocythemia (ET) is extremely rare with an incidence of 1/million. Since thromboembolic complications are more common than hemorrhagic manifestation, immediate thromboctyapheresis by an automated cell separator can prevent untoward consequences in the form of cerebrovascular, coronary or peripheral vascular occlusive events. Due to varied options of automated cell separators, selecting an appropriate cell separator in such acute emergency situation can be confusing for a treating physician, especially if the patient is a child of low body weight. We present here the successful use of hemonetics multicomponent collection system (MCS+) for therapeutic platelet reduction (TPR) in a 12-year-old male child of 28 kg with extreme thrombocytosis (TS) (3072 × 10(9)/l) due to ET. A total of three procedures were performed without priming of the machine with allogenic blood. We observed hemonetics MCS+, best suited for TPR even in children with low body weight.

Comparative evaluation of gel column agglutination and erythrocyte magnetized technology for red blood cell alloantibody titration.

Antibody titration is traditionally performed using a conventional test tube (CTT) method, which is subjected to interlaboratory variations because of a lack of standardization and reproducibility. The aim of this study is to compare newer methods such as get column technology (GCT) and erythrocyte magnetized technology (EMT) for antibody titration in terms of accuracy and precision. Patient serum samples that contained immunoglobin G (IgG) red blood cell (RBC) alloantibodies of a single specificity for Rh or K anitgens were identified during routine transfusion service testing and stored. Titration and scoring were performed separately by and stored. Titration and scoring were performed separately by different laboratory personnel on CTT, GCT, and EMT. Testing was performed a total of three times on each sample. Results were analyzed for accuracy and precision. A total of 50 samples were tested. Only 20 percent of samples tested with GCT shoed titers identical to CTT, whereas 48 percent of samples tested with EMT showed titers identical to CTT. Overall, the mean of th titer difference from CTT was higher using GCT (+0.31) compared with that using EMT (+0.13). Precision shown by CTT was 30 percent, EMT was 76 percent, and GCT was 92 percent on repeat testing. GCT showed higher titer values in comparison with CTT but was found to be the most precise. EMT titers were comparable to CTT, and its precision was intermediate. Further studies to validate this method are required.

Determining the Effect of Preparation and Storage: An Effort to Streamline Platelet Components as a Source of Growth Factors for Clinical Application.

BACKGROUND: In the present study, different methods for preparation of platelet-rich plasma (PRP) are investigated in order to standardize the component in terms of growth factor content. The effects of concentration technique and storage duration are also analyzed. METHODS: PRP was collected from 40 donors by plateletpheresis as well as by the buffy coat and tube method. Concentration of growth factors was performed using double freeze thaw- and CaCl2-induced degranulation techniques. Growth factor estimation was performed using ELISA. RESULTS: The levels of growth factors were highest in PRP from buffy coat, moderately lower in plasma gained by plateletpheresis and lowest in that obtained by the tube method. Mean levels of platelet-derived growth factors (PDGF) AB and BB are significantly higher when CaCl2 was used for concentrating the growth factors. The mean levels of transforming growth factor ß1 and insulin-like growth factor I were higher when applying the double freeze thaw technique. There was a substantial decline in the levels of growth factors during storage. CONCLUSION: The buffy coat method is suitable as preparation method for PRP in most settings. The double freeze thaw technique is better suited as concentration technique as it causes lysis of both platelets and white blood cells for releasing growth factors and is easier to perform. Growth factors are not stable in plasma, thus PRP should be frozen immediately after preparation.

Evaluation of various methods of point-of-care testing of haemoglobin concentration in blood donors.

BACKGROUND: Point-of-care testing (POCT) devices for determining pre-donation haemoglobin (Hb) concentrations mark the advent of advanced technology for blood banks. POCT devices have undergone several improvements including changes in testing methodology and size of device, befitting the needs of blood donors and blood banks in terms of safety and quality of blood components. This study was planned to evaluate the suitability of non-invasive and invasive POCT devices for blood donor Hb screening. MATERIAL AND METHODS: Pre-donation Hb in apparently healthy blood donors was measured by a non-invasive spectrophotometric based method (NBM-200, OrSense) and an invasive method utilizing reagent free cuvettes (DiaSpect) along with a device using sodium azide-coated cuvettes (HemoControl, EKF diagnostic GmbH). The performance of the devices was evaluated by comparison with the reference method, i.e. an automated cell counter (KX-21). RESULTS: Hb was measured in 485 prospective blood donors. DiaSpect hemoglobin T system was found to be the most sensitive method of POCT for Hb (sensitivity 98.1%) followed by HemoControl (sensitivity 86.8%). NBM-200 was the least sensitive method (sensitivity 71.7%). The intraclass correlation coefficient was highest for DiaSpect (0.78), followed by HemoControl (0.77) and NBM-200 (0.43). The variation of results on repeat testing was high for NBM-200 with a coefficient of variation of 4.28%, compared to 2.19% for DiaSpect. On comparing the mean testing time, DiaSpect (1.9 seconds) was found to be significantly quicker than the other two POCT devices (p<0.001). DISCUSSION: NBM-200 has the apparent advantage of eliminating pain but also a substantial possibility of causing ineligible donors to be accepted. DiaSpect was fast and accurate, with its results showing perfect agreement with those of the standard method. It is, therefore, aptly suited for screening donors in blood banks.

Evaluation of a red cell leukofilter performance and effect of buffy coat removal on filtration efficiency and post filtration storage.

Prestorage leukoreduction of red cells is effective in reducing the incidence of HLA alloimmunization and improving the quality of stored packed red blood cells (PRBC). This study was conducted to evaluate the effectiveness of Imugard III-RC 4P in removing the leukocyte from packed red cells and the storage effects thereafter. The effects of buffy coat removal on the efficiency of leukofiltration, storage parameters of leukofiltered packed red blood cells and feasibility of prestorage leukofiltration were also assessed. Sixteen units each of buffy coat-depleted (LP) and nondepleted (NLP) PRBC were taken. Every unit was divided into two equal halves, one leukofiltered and other, non-leukofiltered. Cell counts, volume, hematocrit and hemoglobin were measured before and after filtration. Levels of K(+), lactate dehydrogenase (LDH) and hemolysis were assessed in all the units weekly, post leukofiltration. Post leukofiltration, red cell and volume loss was within the specified limit in all the units. Residual leukocytes were significantly lesser in LP- PRBC compared to NLPPRBC. K(+), LDH and hemolysis were significantly elevated in NLP- PRBC. Leukofiltered PRBC showed lesser elevation of K(+), LDH and hemolysis towards the end of the storage period as compared to their unfiltered counterparts. Leukofilter is capable of performing ~4 log reduction. Buffy coat removal prior to filtration improves the efficiency of leukofilter and aids in improving the storage of red cells in terms of hemolysis.

Knowledge, attitude, and beliefs of young, college student blood donors about Human immunodeficiency virus.

INTRODUCTION: Young people, who tend to be healthy, idealistic, and motivated, are an excellent pool of potential voluntary unpaid blood donors. Recruiting and retaining young blood donors improves the long term safety and sufficiency of a country's blood supply. Knowledge, attitude, and beliefs about Human immunodeficiency virus (HIV) should play an important role in prevention of disease transmission. MATERIALS AND METHODS: This study was a questionnaire based survey, conducted to explore the levels of knowledge, attitude, and beliefs about HIV in young college student blood donors. RESULTS: The results showed that the proportion of participants with comprehensive knowledge of HIV prevention and transmission was lesser than expected. Increase in education level and male gender was found to be significantly associated with high HIV-related knowledge. The responses on the different aspects of HIV-related attitude were also varied and there is still stigma associated with Acquired Immunodeficiency Syndrome (AIDS) even in the educated groups. DISCUSSION: There was a spectrum of myths and misperceptions emphasizing the need of education that recognizes the social context of attitude towards HIV. Results from this study may contribute to the development of appropriate educational and training material for this group of donors which in turn, may assist in achieving the elusive goal of safe blood supply in future.