Molecular basis of RhD-negative phenotype in North Indian blood donor population

Background & objectives: RHD gene typing is highly complex due to homology with RHCE genes. Molecular polymorphism of the RHCE and RHD genes have been characterized among various populations, but no studies have been undertaken among Indians. This study was undertaken to assess the genetic basis of RHD-negative phenotype in Indian blood donor population. Methods: Sample from a total of 200 phenotypically RhD-negative blood donors were analyzed for presence of RHD gene using polymerase chain reaction (PCR). RHD genotyping was done using three primer sets designed for exons 4 and 10 and one set for identification of pseudo (RHD?) gene between introns (int) 3 and 4. Amplified PCR products were analyzed by gel-electrophoresis (XY Loper, Uvitech, Cambridge) and confirmed by nucleotide sequencing (ABI 3730 xl 96 capillary system). Results: No PCR product was found in 195/200 (97.5%) of study samples indicating homozygous gene deletion. Of the 5/200 (2.5%) showing RHD gene polymorphisms, 4/200 (2%) were positive for presence of exon 10 only (RHD-CE-D hybrid). RHD? gene was not detected in any of the samples tested. One sample showed presence of all three tested regions and was negative for RHD? gene. Interpretation & conclusions: RHD gene deletion was found to be the most common cause of an RHD-negative phenotype while RHD? gene was, reported to be present in up to 39 per cent of various ethnic populations, but was not detected. RHD-CE-D hybrid gene (found in 2.5% individuals) is important for predicting the requirement of Rh prophylaxis during the antenatal period.

Cytokine generation in stored platelet concentrate: comparison of two methods of preparation.

BACKGROUND & OBJECTIVES: Contaminating white blood cells (WBCs) in stored platelet concentrates (PC) are the main source of pro-inflammatory cytokines including interleukin-6 (IL-6), interleukin- 8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha) that are implicated in transfusion reactions. We compared the levels of these cytokines in stored platelet preparations prepared by two methods. Effect of pre-storage leucofiltration on these cytokine levels was also studied. METHODS: Twelve units of pooled PCs were prepared by platelet rich plasma (PRP) method and buffy-coat (BC) method each and stored for 5 days. IL-6, IL-8 and TNF-alpha levels were measured in platelet supernatants on day 0, 1, 3 and 5 of the storage using commercially available immunoassays. Pre-storage leucofiltration was done in 4-pooled units of PRP-PC and cytokine levels compared with unfiltered PCs. RESULTS: Median IL-6 levels increased from day 0 to day 5 in both PRP-PC and BC-PC. In PRP-PC, IL-8 increased from <3 pg/ml on day 0 to 817 pg/ml on day 5, while in BC-PC the corresponding levels were 10 and 346.5 pg/ml, respectively. No significant increase in levels of TNF-alpha was observed in BC-PC during storage period, while levels increased significantly in PRP-PC on day 1 only. There was no significant change in the levels of all three cytokines in leucofiltered PCs over 5 days of storage. INTERPRETATION & CONCLUSION: Findings of our study showed that method of preparation and WBC content are the critical factors in determining the cytokine levels in stored PCs.

Low prevalence of IgA deficiency in north Indian population.

BACKGROUND AND OBJECTIVES: IgA deficient patients are at risk of severe anaphylactic reaction on being transfused blood and blood products, and its prevalence varies in different parts of the world. No data are available from India.We did a blood donor survey to look for prevalence of IgA deficiency in north India. METHODS: A sensitive enzyme linked immunosorbent assay developed in-house was used to detect IgA deficiency in a total of 3818 blood donors. Complete IgA deficiency was defined as value less than 5 mg/dl whereas partial IgA deficiency was defined as value between 5-30 mg/dl. RESULTS: Of the 3818 blood donors screened, 3640 (95.3%) were males with a mean age of 31.2 yr. No donor was found to have complete IgA deficiency; however, 257 (6.7%) had partial IgA deficiency. INTERPRETATION AND CONCLUSION: Our study shows that IgA deficiency is rare in north India.

Evaluation of platelet storage lesions in platelet concentrates stored for seven days.

BACKGROUND & OBJECTIVES: Platelet storage lesions (PSL) are detrimental to the post transfusion functional capacity of platelets. As EDTA enhances the storage-induced changes, changes in platelet indices with and without EDTA incubation are promising new tests to monitor the PSL. The present study was undertaken to monitor the PSL in 40 units of pooled platelet concentrates harvested by platelet rich plasma (PRP) method and stored for seven days in second generation platelet storage containers using conventional in vitro tests and platelet indices. METHODS: Morphological changes in platelets were monitored by automated haematological cell counter for platelet count and mean platelet volume (MPV). Samples were incubated with K2EDTA for 1 h and platelet indices were repeated on the EDTA incubated samples. Difference between pre-and post-EDTA incubation of platelet count (dPLT) and MPV (dMPV) were calculated. Metabolic parameters such as pH, pO2, pCO2 and ATP were measured. RESULTS: There was no significant change in the indices without EDTA during storage, however, after EDTA incubation, significant changes were noted in dPLT and dMPV. The mean dPLT on day 0 was 75.15 x 10(3)/microl decreasing to 44.4 x 10(3)/microl on day 7, while dMPV from 0.76 fl on day 0 increased to 1.34 fl on day 7 (P < 0.05). Metabolic parameters showed a significant decrease in pH and pCO2 concurrent with increasing pO2 during storage (P < 0.05). Average ATP level on day 0 was 21.09 micromol/dl falling to 10.59 micromol/dl on day 7. INTERPRETATION & CONCLUSION: The results indicate that storage induced lesions take place even in second generation platelet storage containers under recommended conditions of storage. Platelet indices especially after EDTA incubation are useful in monitoring PSL. However, how much these changes contribute to poor post transfusion survival and haemostatic function of platelets need to be investigated.