Phenotyping and Genotyping of HNA: Prevalence, Risk of Alloimmunization, and HNA Incompatibilities in Indians.

Background: Antibodies to human neutrophil alloantigens (HNA) are involved in the pathophysiology of several clinical conditions including transfusion-related acute lung injury (TRALI), alloimmune and autoimmune neutropenia, and febrile nonhemolytic transfusion reactions leading to neutropenia. The cognate antigens are polymorphic structures expressed on several glycoproteins on the neutrophils, i.e., antigens HNA-1a, -1b, -1c, and -1d on Fc-γ-receptor IIIb; HNA-2 on CD177; HNA-3a and -3b on choline transporter-like protein 2; HNA-4a and -4b on CD11b/αM subunit of the αMß2-integrin (CD11b/CD18, Mac-1, CR3); and HNA-5a and -5b on αL-subunit (CD11a) of the αLß2 integrin (CD11a/CD18), leukocyte function associated molecule (LFA)-1. Currently, there is a lacuna of diagnostic methods for detection of HNA in India. This study aimed to determine the HNA frequencies in Indians, estimate the risk of alloimmunization, and prepare typed neutrophil panels, which can be used to detect HNA antibodies in neutropenia cases. Material and Methods: EDTA blood samples were collected from random 1,054 blood donors. HNA-2 was phenotyped on fresh EDTA samples using FITC labelled monoclonal anti-CD177 by flowcytometry. HNA-1 (FCGR3B) genotyping was carried out by DNA sequencing and PCR-RFLP. Antigens of HNA-3 (SLC44A2) and HNA-5 (ITGAL) were genotyped by PCR-RFLP using TaqαI and Bsp1286I restriction enzymes, respectively, while HNA-4 (ITGAM) was genotyped by PCR-SSP. Results: Allele frequencies of FCGR3B*01, FCGR3B*02, and FCGR3B*03 were found to be 0.433, 0.444, and 0.087, respectively. FCGR3B*01+*02+*03- was the most common genotype (33.78%). Ten individuals showed deficiency of FCGR3B individuals, while 23 showed hyperexpression, i.e., FCGR3B*01+*02+*03+. FCGR3B*04and *05 occurred with a frequency of 0.002 and 0.024. HNA-2 was found to be a high frequency antigen occurring in 98.8% population. Four percent individuals showed atypical expression of CD177 on their neutrophils. Allele frequencies of SLC44A2*01 and SLC44A2*02were 0.812 and 0.188, respectively, and that of ITGAM*01, ITGAM*02, ITGAL*01, and ITGAL*02 were 0.9546, 0.0454, 0.2372, and 0.7628, respectively. Conclusion: This is the first study in India to report the frequencies of HNA among blood donors. Typed neutrophil panels identified in the present study will enable us to investigate suspected cases of immune neutropenia in future.

Red cell antigen phenotypes in blood donors & thalassaemia patients for creation of red cell antigen-matched inventory.

Background & objectives: Patients with thalasssaemia are at a risk of alloimmunization and the presence of RBC alloantibodies further complicates transfusion therapy. Matching for the critical antigens of Rh, Kell, Kidd and Duffy blood group systems has been shown to minimize alloimmunization. The aim of the present study was to create a database of extensively typed donors for clinically significant and common blood group antigens of Rh, Kidd, Kell and Duffy systems for transfusion therapy of multitransfused thalassaemic patients. Methods: Five hundred O group regular blood donors were phenotyped for Rh, Kell, Duffy and Kidd blood group antigens using haemagglutination technique. Eighty four non-alloimmunized and 15 alloimmunized thalassaemia major patients with known antigenic profiles (determined by polymerase chain reaction with sequence-specific primers) were selected for this study. Results: By analyzing antigen profiles of 500 O group regular donors, a database of 193 donors matching perfectly for Rh, Duffy, Kell and Kidd antigens was prepared for 15 alloimmunized patients. For non-alloimmunized 84 thalassaemic patients, a database of 405 donors was created. Interpretation & conclusions: A database of 500 regular blood donors phenotyped for common antigens of Rh, Duffy, Kell and Kidd blood group systems was created, which would be useful in providing extended antigen-matched RBCs for thalassaemia patients. This will improve the quality and effectiveness of transfusion therapy.

A Study of Clinical and Serological Correlation of Positive Direct Antiglobulin Test in Blood Bank at a Tertiary Care Center.

Objectives Detection of red cell bound immunoglobulins and/or complement by direct antiglobulin test (DAT) is a crucial serological assay in the diagnosis of autoimmune hemolytic anemia (AIHA). However, DAT may be positive in a variety of clinical conditions with or without hemolysis. We aimed at evaluating the clinical and serological correlation of positive DAT by categorizing the clinical conditions associated with positive DAT, estimating the presence of in vivo hemolysis in case of positive DAT with polyspecific and monospecific antisera and correlating the strength of positive DAT with the presence of hemolysis. Materials and Methods The prospective observational study was performed on 200 samples that were positive for DAT with polyspecific antiglobulin reagent as the baseline investigation. These samples were further tested with anti-immunoglobulin G and anti-C3 monospecific DAT reagents to evaluate the type of protein responsible for positive DAT. The antiglobulin tests were performed by tube technique. DAT positivity was graded (1+ to 4 + ) in each patient. Autocontrol test was included. The patients with positive polyspecific DAT were categorized into different clinical conditions. The presence or absence of in vivo hemolysis was evaluated in all clinical categories and also for each grade of positivity with polyspecific and monospecific antiglobulin reagents. Statistical Analysis Binomial logistic regression and Mann-Whitney U test were applied to between the group analyses. For categorical variables, Fisher's exact test and relative risk were used. The qualitative data were expressed in numbers and percentages. Results The highest number of patients (75/200, 37.5%) belonged to the autoimmune diseases group. Tuberculosis and hepatitis C were the main infectious diseases associated with positive DAT. Out of 200 DAT-positive patients, 98 (49%) had in vivo hemolysis and 102 (51%) did not have hemolysis. AIHA (22) and systemic lupus erythematosus (18) were the commonest clinical conditions associated with in vivo hemolysis. All the 11 samples that showed positivity with only anti-C3 reagent did not show any hemolysis. There was statistically significant increase in the incidence of in vivo hemolysis with increasing grades of DAT positivity with all the three antihuman globulin reagents. Conclusion There are different disease conditions which show positive DAT with or without hemolysis. So, it is important to clinically and serologically correlate positive DAT results.

Comparison Between Conventional and Automated Techniques for Blood Grouping and Crossmatching: Experience from a Tertiary Care Centre.

CONTEXT: The routine immunohematological tests can be performed by automated as well as manual techniques. These techniques have advantages and disadvantages inherent to them. AIMS: The present study aims to compare the results of manual and automated techniques for blood grouping and crossmatching so as to validate the automated system effectively. MATERIALS AND METHODS: A total of 1000 samples were subjected to blood grouping by the conventional tube technique (CTT) and the automated microplate LYRA system on Techno TwinStation. A total of 269 samples (multitransfused patients and multigravida females) were compared for 927 crossmatches by the CTT in indirect antiglobulin phase against the column agglutination technique (CAT) performed on Techno TwinStation. RESULTS: For blood grouping, the study showed a concordance in results for 942/1000 samples (94.2%), discordance for 4/1000 (0.4%) samples and uninterpretable result for 54/1000 samples (5.4%). On resolution, the uninterpretable results reduced to 49/1000 samples (4.9%) with 951/1000 samples (95.1%) showing concordant results. For crossmatching, the automated CAT showed concordant results in 887/927 (95.6%) and discordant results in 3/927 (0.32%) crossmatches as compared to the CTT. Total 37/927 (3.9%) crossmatches were not interpretable by the automated technique. CONCLUSIONS: The automated system shows a high concordance of results with CTT and hence can be brought into routine use. However, the high proportion of uninterpretable results emphasizes on the fact that proper training and standardization are needed prior to its use.

Thrombocytopenia in leptospirosis and role of platelet transfusion.

AIM: The study was designed to find out the incidence of thrombocytopenia in leptospirosis and to correlate thrombocytopenia with other parameters like renal failure, hepatic failure and bleeding manifestation like adult respiratory distress syndrome and to assess the role of platelet transfusion. MATERIALS AND METHODS: 50 cases of leptospirosis during the month of July and August 2005 were retrospectively analyzed. Criteria for selection were Lepto Tek Dri - dot test positive cases of the clinically suspected cases of Leptospirosis. Degree of thrombocytopenia was categorized as severe, moderate and mild. Presence of thrombocytopenia was clinically correlated with parameters like renal dysfunction, hepatic dysfunction and hemorrhagic manifestations (mainly ARDS). Role of platelet transfusion was assessed with reference to presence and degree of thrombcytopenia and hemorrhagic manifestations. RESULTS: Out of total 50 patients 26 were male and 24 were females. Major bleeding manifestation in the form of ARDS was seen in 15 (30%) of patients. 28 (56%) patients had thrombocytopenia and 22 (44%) patients had normal platelet counts. Total number of patients with renal dysfunction was 24 (48%). Only four (18.18%) patients with normal platelet counts had renal dysfunction while 20 (71.42%) patients with thrombocytopenia had renal dysfunction. Only two (9.09%) patients with normal platelet counts and 48 (46.42%) patients with thrombocytopenia had hepatorenal dysfunction. Total number of patients with ARDS was 15 (30%). Of these two (13.33%) had normal platelet count while 13 (86.6%) patients were thrombocytopenic. Total 47 units of platelets were transfused to 12 patients in our study. Of these seven patients with severe thrombocytopenia required total 28 units, two patients with moderate thrombocytopenia required total seven units and patients with mild thrombocytopenia were transfused total 12 units of platelets. CONCLUSION: It is important to anticipate and recognize thrombocytopenia early in the course of leptospirosis so that appropriate steps can be taken to prevent it and to treat it with platelet transfusion when it develops.