Atul Sonker

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.

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.

The external quality assessment scheme: Five years experience as a participating laboratory

Background and Aim: Quality assurance in blood banking includes active participation in the external quality program. Such a program offers valuable benefits to patient care, their safety, and an overall quality of laboratory practices. In the year 2002, we participated in the External Quality Assessment Scheme (EQAS) under the World Health Organization (WHO), Bureau of Laboratory Quality Standards, Thailand. Materials and Methods: In the current study we evaluated our EQAS test result of the past five years, from 2003 to 2007. Test results of all blood samples such as ABO grouping, D typing, antibody screening, antibody identification, and transfusion transmitted infection (TTI) testing were analyzed and documented. Results: Discordant results in one or more instances were observed with antibody identification, weak D testing, and tests for anti-HIV1/2 and HBsAg. Twice we failed to detect the ‘anti-Mia’ antibody in the issued sample and that could be attributed to the absence of the corresponding antigen in the used cell panel. HBsAg was missed due to its critically low titer in the serum and the comparatively low sensitivity of our Enzyme-Linked Immunosorbent Assay (ELISA) test kit. Conclusion: All these failures in the last five years have helped us to significantly improve our transfusion service in terms of performance evaluation, patient care and safety issues, and the overall quality of laboratory practices. We therefore recommend all laboratories and hospitals to participate in the EQAS program, which will definitely help them to improve from what they learn.

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.

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

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.

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.

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.