Rajendra Chaudhary

Seroprevalence of Toxoplasma gondii antibodies in North Indian blood donors: Implications for transfusion transmissible toxoplasmosis

INTRODUCTION Transfusion transmitted Toxoplasma gondii (T. gondii) can result in significant clinical consequences in immunocompromised and multiply transfused patients, pregnant women and fetus etc. Anti-T. gondii seroprevalence, specifically IgM antibodies reflect the risk of transfusion transmission. METHODS Four hundred and ninety-three blood donors in a tertiary care hospital in North India were screened for IgG and IgM anti-T. gondii antibodies by enzyme linked immunosorbent assay (ELISA). RESULTS The prevalence of IgG and IgM anti-T. gondii antibodies was 51.8% and 5% respectively. The prevalence was higher in females (M=51.6%, F=89.2%) and in replacement donors (replacement donors=63.2%, voluntary donors=33.5%). CONCLUSION The donor population constitutes a significant risk of transfusion transmitted toxoplasmosis. Effective strategies are required to prevent transfusion transmitted toxoplasmosis.

Application of flow cytometry in detection of red-cell-bound IgG in Coombs-negative AIHA.

Abstract Coombs negative autoimmune hemolytic anemia (AIHA) is characterized by laboratory evidence of in vivo hemolysis along with a negative direct antiglobulin test (DAT) performed by conventional tube technique (CTT) in clinically suspected AIHA patients. The sensitive gel test (GT) and flow cytometry (FC) can effectively diagnose such patients where CTT does not detect low level of red cell autoantibodies. We investigated the use of FC in the serological evaluation of CTT DAT negative AIHA and its comparison with GT DAT. Of the 50 patients with suspected AIHA, CTT DAT was negative in 5 patients (Coombs negative AIHA). GT DAT could detect red cell autoantibodies in 4 of these 5 patients. Monospecific GT DAT showed IgG and/or C3d as the responsible autoantibody. FC was considered as reactive when MFI was > 3.6 (mean of 20 healthy negative volunteers +2SD). FC was reactive in all five Coombs negative AIHA patients. The mean MFI in five known CTT DAT positive samples taken for comparison was significantly higher compared to 5 DAT negative AIHA (18.3 ± 7.78 vs. 7.88 ± 1.35, p < 0.05). There was poor correlation between strength of GT DAT and MFI by FC. We conclude that FC is more sensitive test than the CTT and helps in the serological diagnosis of Coombs negative AIHA. However, in resource poor settings, GT DAT can be a good alternative to FC.

Quality evaluation of four hemoglobin screening methods in a blood donor setting along with their comparative cost analysis in an Indian scenario

Background: Despite the wide range of methods available for measurement of hemoglobin, no single technique has emerged as the most appropriate and ideal for a blood donation setup. Materials and Methods: A prospective study utilizing 1014 blood samples was carried out in a blood donation setting for quality evaluation of four methods of hemoglobin estimation along with cost analysis: Hematology cell analyzer (reference), HCS, CuSO4 method and HemoCue. Results: Mean value of HemoCue (mean ± SD = 14.7 ± 1.49 g/dl) was higher by 0.24 compared to reference (mean ± SD = 13.8 ± 1.52 g/dl) but not statistically significant ( P > 0.05). HemoCue proved to be the best technique (sensitivity 99.4% and specificity 84.4%) whereas HCS was most subjective with 25.2% incorrect estimations. CuSO4 proved to be good with 7.9% false results. Comparative cost analysis of each method was calculated to be 35 INR/test for HemoCue, 0.76 INR /test for HCS and 0.06-0.08 INR /test for CuSO4. Conclusion: CuSO4 method gives accurate results, if strict quality control is applied. HemoCue is too expensive to be used as a primary screening method in an economically restricted country like India.

A comparison of conventional tube test and gel technique in evaluation of direct antiglobulin test

Abstract In vivo coating of red cells by antibody and/or complement is detected using various sensitive techniques, however most hospitals even today rely on the conventional tube technique (CTT). We compared the performance of the CTT and recently introduced gel test (GT) in the evaluation of direct antiglobulin test (DAT). The CTT and GT were first compared using in-house prepared control cells. The polyspecific DATs were performed simultaneously by CTT and GT on 170 consecutive blood samples. Positive samples were further tested for monospecific IgG and C3d by both techniques. GT demonstrated stronger agglutination scores (60 vs. 43) compared to CTT using control cells. The sensitivity and specificity of the GT was 98.4 and 95.2%, respectively as compared to CTT for polyspecific DAT. Discordance between the two test systems was seen in 6/170 patients. Of these, 5 were missed by CTT while GT failed to detect in vivo coating in only 1 case. The agreement between two methods of DAT was 96.4% (κ = 0.926) using polyspecific AHG and 95.7% (κ = 0.379) with mono specific anti-IgG. We conclude that GT is a better alternative to CTT for detecting red cell bound antibodies in various clinical conditions.

Evaluation of fresh frozen plasma usage at a tertiary care hospital in North India

Background:  Concerns regarding transfusion‐transmitted infections and non‐availability of blood components in developing nations, make it crucial to optimize fresh frozen plasma (FFP) transfusions and reduce wastage.

Platelet transfusions in clinical practice at a multidisciplinary hospital in North India

Specialty wise utilization pattern of platelet concentrates (PLT) over a period of 2 months was evaluated prospectively for appropriateness. Overall 4.87 random donor platelets (RDP) (total 1672) units were issued per request. A total of 1101 RDP (66%) were transfused prophylactically against 221 requests (64.4%) while, 571 RDP were transfused for therapeutic (requests=122, 35.6%) reasons. Twenty-three percent of prophylactic requests and 15% of the therapeutic requisitions were not justified. Most common reason for unjustified prophylactic transfusion was unavailability of pre-transfusion platelet count. Concurrent screening of request forms to ensure optimized PLT usage may further decrease platelet misuse at our center.

Pre- and post- donation haematological values in healthy donors undergoing plateletpheresis with five different systems.

BACKGROUND Although automated cell separators have undergone a lot of technical refinements, attention has been focused more on the quality of platelet concentrates than on donor safety. We planned this prospective study to observe the effects of automated plateletpheresis on normal haematological values of healthy donors and to determine whether the haematological alterations had any clinical consequences. STUDY DESIGN AND METHODS The study was conducted on 457 healthy, first-time plateletpheresis donors over a period of 26 months. The plateletpheresis procedures were performed using five different cell separators and various pre- and post-donation haematological values such as haemoglobin concentration (Hb), haematocrit (Hct), platelet and white blood cell (WBC) counts, mean platelet volume and platelet distribution width were measured in all donors. RESULTS We observed that the Hb, Hct, platelet and WBC counts decreased significantly in the donors (p<0.01) after each procedure, without there being significant changes in mean platelet volume or platelet distribution width. The decreases in Hb and Hct were significantly greater with the CS 3000 and Amicus machines, while the decreases in platelet and WBC counts were significantly greater with the CS 3000 and Fresenius separators. CONCLUSION Although a significant drop in complete blood count was observed in all donors, none manifested features of thrombocytopenia or anaemia. Nevertheless, more prospective studies on this aspect are required in order to establish guidelines for donor safety in apheresis and also to help in assessing donor suitability, especially given the present trend of double product apheresis collections.

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.

Transfusion-associated immunomodulation: Quantitative changes in cytokines as a measure of immune responsiveness after one time blood transfusion in neurosurgery patients

Very few studies in humans have investigated the laboratory evidences suggestive of transfusion-associated immunologic changes. In this prospective study, we examined the effects of perioperative blood transfusion on immune response, by measuring various cytokines production, namely, interferon-gamma (IFN-γ), interleukin-10 (IL-10), and Fas Ligand (FasL). A total of 40 patients undergoing neurosurgery were randomly allocated into four groups: (a) no transfusion, (b) allogeneic non-leukofiltered transfusion, (c) prestorage leukofiltered transfusion, (d) autologous transfusion. Samples were collected before operation (day 0) and postoperative days (post-op) 1, 7, and 14. IFN-γ and IL-10 production capacity was measured in supernatant after whole blood culture and serum FasL levels in patients’ sera using commercially available ELISA kits. Change in ratios (cytokine value after PHA stimulation/control value) of IFN-γ and IL-10 and percentage change from baseline for serum FasL levels across different transfusion groups during the sampling period were calculated. There was an increase in IL-10 production in patients receiving allogeneic non-leukofiltered transfusion on days 1 and 7 (mean ratio 2.22 (± 2.16), 4.12 (± 1.71), 4.46 (± 1.97) on days 0, 1, and 7, respectively). Similarly there was a significant (P<0.05) decrease in IFN-γ production in patients who received allogeneic non-leukofiltered red cell transfusion on post-op days 1, 7, and 14 (mean ratio 6.88 (± 4.56), 2.53 (± 0.95), 3.04 (± 1.38) and 2.58 (± 1.48) on day 0, 1, 7, and 14, respectively). Serum FasL production was increased across all patients till 7th day except for ‘no transfusion’ group and this increase was most significant in the non-leukofiltered group. We conclude that one time transfusion leads to quantitative changes in levels of these cytokines largely through interplay of Th2/Th1 pathways in allogeneic nonleukofiltered blood transfusion; however, soluble mediators like FasL which are also present in autologous and leukofiltered blood products may contribute toward minor immunologic effect in these settings.

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.