Anju Dubey

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 β1 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.

A case of severe foetal anaemia due to anti-M isoimmunisation salvaged by intrauterine transfusions.

Haemolytic disease of the foetus and newborn (HDFN) is caused by transplacental passage of maternal IgG antibodies directed against an antigen of paternal origin present on the foetal red blood cells. IgG antibodies coat foetal antigens and cause decreased red blood cell survival leading to fetal anaemia. The antibodies causing severe forms of HDFN are anti-D, anti-c and anti-K1. However, more than 50 different red-cell antigens have been reported to be associated with HDFN and no prophylactic immune globulins are available to prevent the formation of these antibodies1. Anti-RhD was once the major aetiology of HDFN, but with routine antenatal administration of anti-D to all vulnerable women, maternal allo-immunisation to other red cell antigens as a cause of HDFN is becoming a greater concern. Anti-M is typically a naturally occurring immunoglobulin M (IgM) antibody, optimally reactive at 4 °C and considered clinically insignificant. This saline agglutinin was first identified by Wolff and Johnsson in 19332. Anti-M rarely causes agglutination of red cells at 37 °C or in the antiglobulin phase of testing. Rarely, the alloanti-M can be of IgG type or a combination of both IgG and IgM. These have a potential to cause haemolytic transfusion reactions and HDFN but have been very rarely implicated as the cause of severe HDFN requiring intrauterine transfusion (IUT). The MN determinants are carried on the transmembrane protein glycophorin A, are fully developed on foetal red cells, and can be detected as early as 9 weeks’ gestation3. This case report describes a woman with a history of six intrauterine deaths, all between 11 to 32 weeks of gestation. Her seventh affected pregnancy was salvaged by intrauterine administration of packed red blood cell transfusions at 29 weeks of gestation, after she was found to be allo-immunised against M antigen.

Spectrum of anti-M: a report of three unusual cases.

Following the discovery of the ABO blood group system, the MNS system, discovered in 1927, was the second blood group system to be recognised. The anti-M antibody was first discovered by Wolf and Johnson in 19331. This is a relatively common, naturally occurring, clinically insignificant antibody, usually of IgM type, which is a cold reactive saline agglutinin; sometimes, however, the anti-M antibody can be reactive at 37 °C or at antiglobulin phase. Some examples of anti-M are pH dependent reacting best at pH 6.5 and mostly inactive at pH 7.5. The antibody does not agglutinate papain- or ficin-treated red cells and is known to show a dosage effect. Rarely, the alloanti-M can be of IgG type or a combination of both IgM and IgG. Anti-M antibodies have a potential to cause haemolytic disease of the newborn and haemolytic transfusion reactions (HTR)2. Naturally occurring anti-M antibodies are more commonly found in children than in adults. Here we report three separate cases of clinically significant anti- M antibodies with different Ig types and presentations.

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.

Sudden increased incidence of transfusion reactions reported from a ward: root cause analysis

The transfusion medicine department of a teaching hospital in north India was inundated with reports of transfusion reactions with red blood cells (RBCs) seen in patients admitted to the hematology ward (seven adverse transfusion reactions in a <2-week period). Buffy coat–removed RBCs were routinely being transfused to these patients without frequent reactions previously. The transfusions had to be stopped immediately after initial 10 to 20 mL of blood infusion in most cases. Patients presented with chills, rigors, fever, dyspnea, and wheezing but no red-colored urine. The RBC units with the attached blood administration (BA) set and patients’ blood samples were collected immediately after the reactions as per departmental standard operating procedure. No ABO and Rh blood group discrepancy or incompatible crossmatch with preand posttransfusion samples from the patients could be demonstrated using gel cards (DiaMed-ID, Cressier, Switzerland) and standard tube methods. Immunohematologic workup including direct and indirect anti-human globulin tests by gel cards was also negative. The posttransfusion blood samples from recipients showed no hemolysis. There was no evidence of any hemolysis in the bag, no clots, and no evidence of any compromise in integrity of the

A report of a rare case of autoimmune haemolytic anaemia in a patient with Hodgkin's disease in whom routine serology was negative.

Autoimmune haemolytic anaemia (AIHA) is an acquired clinical condition which is characterised by the production of auto-antibodies that bind to the surface of circulating erythrocytes, leading to haemolysis and decreased survival of the red blood cells. The estimated yearly incidence of AIHA is 1–3 cases per 100,000 persons in the general population1. The direct antiglobulin test (DAT) is considered to be a cornerstone in establishing the diagnosis of AIHA, since there is uptake of autoantibodies and/or complement components onto the affected red blood cells. In suspected cases of AIHA, a positive DAT is predictive in 83% of patients, but not all cases of AIHA are DAT-positive2. Between 5% to 10% of all cases of AIHA are DAT-negative3. Three causes for this situation have been identified: (i) red blood cell bound IgG molecules, below the threshold of detection of the DAT, (ii) low-affinity IgG autoantibodies that are washed off the red cells during the washing phase for the test, and (iii) red cell bound IgA and rare warm IgM autoantibodies that are not detectable by the routine anti-human globulin reagent3. Given these different possibilities, a negative DAT must be weighed in the light of clinical suspicion. If there is clinical evidence of haemolysis, a thorough investigation with more specialised testing is needed. We report here a case of DAT-negative AIHA in a patient with Hodgkin’s lymphoma which was detected in our hospital blood bank laboratory.

A case of passenger lymphocyte syndrome following minor ABO incompatible renal transplantation

Immune hemolysis is one of the adverse effects that can occur following solid organ transplantation. Understanding the clinical settings and the various causes is necessary for prompt diagnosis and appropriate management. One such condition is passenger lymphocyte syndrome (PLS). This case report describes the case of a 27-year-old male renal allograft recipient of the B-positive blood group who received a kidney from an O-positive donor. Postoperatively, the patient showed declining hemoglobin (Hb) level and was transfused with B-group packed RBCs (PRBCs), following which there was steep fall in Hb level. A request for PRBCs was sent to the blood bank and this time cross-match with B-group PRBCs showed incompatibility. The patient′s RBCs were found to be strongly DAT (direct anti-globulin test) positive and the eluate showed the presence of anti-B with a titer of 32. Thus, diagnosis of probable PLS was made. The patient was managed with methylprednisolone, plasmapheresis and O-group PRBCs. Gradually his condition improved and was discharged in stable condition.

RhD blocking phenomenon implicated in an immunohaematological diagnostic dilemma in a case of RhD-haemolytic disease of the foetus

Of all red blood cell antigens, RhD is second only to the ABO antigens in importance in blood transfusion. The Rh antigens are fully expressed at the time of birth unlike the weak expression of ABO antigens in neonates1. Immunisation to D antigen can occur in reaction to less than 0.1 mL of foetal blood, resulting in anti-D alloantibody in the maternal circulation2. Haemolytic disease of the foetus and newborn (HDFN) occurs when there is a destruction of foetal/neonatal red cells by IgG antibodies produced by the mother. When Anti-D is the cause of HDFN, the severe anaemia can cause foetal hydrops, tissue hypoxia and even foetal death in utero3. Sensitised pregnant women require close monitoring for early detection of foetal anaemia and to decide whether and, if so, when intrauterine transfusion is required. As a key part of any pre-transfusion testing, the ABO and RhD groups of recipient samples are determined. It has been reported that if a neonate’s red cells are heavily saturated with IgG antibodies, RhD typing with anti-D reagents may give either false negative or false positive results3. We report here the case of blocked RhD in a cord blood sample in a suspected case of RhD-HDFN which was detected in our hospital blood bank laboratory.

Evaluation of transfusion-related complications along with estimation of inhibitors in patients with hemophilia: A pilot study from a single center.

Background: Apart from inhibitor development in patients with hemophilia (PWH) the old problems of blood borne viral infections and red cell alloimmunization still persist in PWH from developing countries. This study was planned to detect the presence of inhibitors in our PWH and to determine the presence of transfusion transmitted infections (TTI) markers and clinically significant red cell alloantibodies in these patients. Materials and Methods: One hundred fourteen PWH were screened for various laboratory tests. Screening for inhibitors was done by mixing study. Blood grouping, TTI testing and red cell alloantibody detection were done as per the departmental standard operating procedures. Results: Out of 114 patients evaluated 98(86%) had hemophilia A and remaining 16(14%) had hemophilia B. Five (5.1%) patients of hemophilia A were positive on inhibitor screening. On Bethesda assay, one patient was high responder (14.4 BU/ml) and rest 4 were low responders (<5 BU/ml). Overall, 19 PWH were positive for TTI markers and two had clinically significant red cell alloantibody (anti-E and anti-Jkb). Conclusion: This is probably first comprehensive study from our state on laboratory testing in PWH. The specialty of Transfusion Medicine can be a core part of hemophilia care. The overall prevalence of inhibitors in our hemophilia A patients was 5.1%, which is less as compared to majority of published studies.