Sudipta Sekhar Das

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.

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.

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.

Managing uncontrolled postsplenectomy reactive thrombocytosis in idiopathic thrombocytopenic purpura: Role of thrombocytapheresis

Reactive thrombocytosis occurs in response to infection, trauma, or surgery. Splenectomy alone accounts for 19% of all possible causes of extreme thrombocytosis. We performed thrombocytapheresis in a young lady with chronic idiopathic thrombocytopenic purpura (ITP) who developed postsplenectomy reactive thrombocytosis. Her post splenectomy platelet count was 227 × 10(6)/ml which elevated to 1623 × 10(6)/ml on the 7th postoperative day. A single thrombocytapheresis procedure reduced her platelet to 403 × 10(6)/ml. She was discharged on the 10th postoperative day and then maintained a count of 204-238 × 10(6)/ml with aspirin. Thrombocytapheresis reduces the platelet count rapidly in thrombocytosis and prevents patients from having thrombotic events. However, such procedures should be performed very meticulously to ensure patient safety.

Utility of adsorption techniques in serological evaluation of warm autoimmune haemolytic anaemia.

BACKGROUND Various adsorption techniques are available to remove serum autoantibodies and subsequently detect the underlying alloantibody in previously transfused patients with autoimmune haemolytic anaemia. We planned to establish a suitable adsorption technique in our transfusion service which can remove all autoantibodies and detect underlying alloantibodies rapidly, cheaply and effectively. STUDY DESIGN AND METHODS We evaluated 71 direct antiglobulin test-reactive patients with warm AIHA over a period of 20 months. Twenty-three of these 71 patients who had a previous history of blood transfusion or pregnancy and were confirmed carriers of autoantibodies (indirect antiglobulin test-reactive) were considered for the adsorption study. Depending on the adequacy of samples, history of blood transfusion and severity of anaemia either autoadsorption or alloadsorption or both using polyethylene glycol (PEG) or low ionic strength saline (LISS)-papain were performed. RESULTS Underlying alloantibodies were detected in 7 of the 23 patients (30.4%) and all these were specific to Rhesus antigens. The mean number of alloadsorptions for complete autoantibody removal using PEG was 1.43 which was significantly lower than the 3.9 using the LISS-papain method (p<0.05). The mean time required by PEG alloadsorption and LISS-papain alloadsorption for autoantibody removal was 93.6 minutes and 177.7 minutes, respectively (p<0.05). Discordant results were not observed in any case and identical alloantibodies were detected by both the techniques. CONCLUSION We found that the PEG method is a rapid, cheap and effective way to remove autoantibodies and detect underlying alloantibodies.

In-house preparation of lectin panel and detection of Tn polyagglutination.

Polyagglutination is a condition in which red cells are agglutinated by ABO-compatible adult human sera, but not by cord blood sera and may be acquired or inherited. Lectins are invaluable reagents in the investigation of red cells polyagglutination. We prepared in-house lectin panel and confirmed Tn polyagglutination in a pregnant lady. The lady was anemic and refused blood transfusion elsewhere due to serological discrepancy. We found ABO discrepancy and an incompatible minor cross-match in the initial investigation and suspected polyagglutination. Confirmation of polyagglutination was done using adult and cord sera. We then used the in-house lectin panels to detect the type of polyagglutination. The agglutination pattern with the various lectins was suggestive of Tn polyagglutination, which was further supported by the enzyme study. Most blood banks in India lack commercial lectin panels because of cost and procurement difficulty. Lectins play an important role in the diagnosis and differentiation of polyagglutination and immunohematological management of patient. The important and basic lectins can be prepared in-house using specific raw seeds following standardized protocol.

Significance of Quantitation of Autoantibodies in the Eluate of Sensitized Red Cells in Warm Autoimmune Hemolytic Anemia

Background Concentrated autoantibodies obtained by elution can be subjected for total serological characterization including their quantitation. The present study was performed to correlate the quantity of immunoglobulin (Ig) G (IgG) autoantibodies in eluate with direct antiglobulin test (DAT) reactivity and markers of in vivo hemolysis in warm autoimmune hemolytic anemia (WAIHA). Methods Detailed autoantibody characterization was performed on 34 samples with regards to antibody class, IgG subclass, DAT dilution and quantification study on eluate. Cold acid elution was performed on the DAT positive red cells and quantitation of IgG in eluates was done using ELISA. Number of IgG molecules eluted per RBC was calculated using Avogadro’s number. Results We observed a significant correlation between the concentration of IgG in eluate and DAT strength (r = 0.53, P = 0.014), hemoglobin (Hb)(r = −0.41, P = 0.04), serum bilirubin (r = 0.67, P = 0.000) and serum lactate dehydrogenase (LDH) (r = 0.50, P = 0.02). The number of IgG molecules/RBC also correlated significantly with the DAT strength (r = 0.69, P = 0.001) as well as DAT dilutions (r = 0.53, P = 0.004). Conclusions Quantification of autoantibodies in eluate in WAIHA helps to assess the severity of the disease and plan appropriate management.