CPDA-1 Stored Blood Induced Effect on Hematological and Biochemical Parameter up to 28 Days

Introduction: When blood is stored outside the body, some hematological and biochemical changes take place resulting in reduced red blood cells survival which is an important drawback when transfused into the circulation of a recipient. Objective: The stability of hematological parameters like RBC count, WBC count, differential count, platelet count, MCV, MCH, MCHC and biochemical parameters like S. Sodium, S. Potassium, S. Chloride and albumin during extended storage at 4°C for up to 28 days was evaluated. Materials and Methods: The present research was conducted in L.N. Medical College and J.K. Hospital, Bhopal, in collaboration with blood bank department of our institute. 450 mL of blood was drawn from 30 healthy volunteer donors into citrate phosphate dextrose adenine (CPDA-1) anticoagulant (63 mL). The blood was kept for 28 days and samples were evaluated on days 1, 7, 14, 21 and 28. Results: Among the hematological parameters, there was a constant decline in WBC and platelet counts from day 0 to 28. RBC count, Hb, MCV, HCT showed increasing values; MCH was almost constant, while MCHC decreased. PDW increased while PCT increased till 4th day and then decreased. Neutrophils, Eosinophils, Monocytes decreased, Basophils remained constant while lymphocytes increased. Among the biochemical parameters, values of S. Sodium decreased, S. Chloride decreased till 3rd day, increased on 4th day and then again decreased on 5th day. S. Potassium and albumin showed increasing values. Conclusion: Extended storage of blood in blood banks leads to changes in biochemical and hematological parameters of stored blood. RBC stored for a period of time at 4°C loses viability. Some may undergo spontaneous hemolysis while in storage; others lose the ability to survive in the recipient’s circulation following transfusion. The structural and biochemical changes that RBCs go through during storage are likely to contribute to adverse transfusion effects

Comparison of microwave decalcification with conventional decalcification method by using different decalcifying agents

Background: In routine histopathology, decalcification of teeth is an essential and important step during tissue processing. The present study was attempted to decalcify teeth using microwave method and to compare it with conventional decalcification method. The aim of the study was to compare microwave decalcification with conventional decalcification method with respect to the speed of decalcification, preservation of tissue structure, and efficacy of staining.Methods: In our study, the total sample size used for both routine and microwave decalcification was 30 premolar teeth. The three solutions were diluting nitric acid (5%), formic acid (5%), and EDTA (14%).Results: The results in the present study confirmed the fact that the microwave method using nitric acid was indeed the fastest decalcifying method needing just about 4 days for premolars, compared with routine decalcification. The results also showed that the overall structural details and good staining characteristics were better in teeth decalcified by 5% nitric acid in comparison to EDTA and formic acid in both the methods used. But nitric acid showed good staining details in microwave method in comparison to conventional method.Conclusions: 5% nitric acid by microwave method proved to be the best decalcifying agent as it was fast and gave good structural details and staining characteristics.

Scalable noninvasive amplicon-based precision sequencing (SNAPseq) for genetic diagnosis and screening of β-thalassemia and sickle cell disease using a next-generation sequencing platform

β-hemoglobinopathies such as β-thalassemia (BT) and Sickle cell disease (SCD) are inherited monogenic blood disorders with significant global burden. Hence, early and affordable diagnosis can alleviate morbidity and reduce mortality given the lack of effective cure. Currently, Sanger sequencing is considered to be the gold standard genetic test for BT and SCD, but it has a very low throughput requiring multiple amplicons and more sequencing reactions to cover the entire HBB gene. To address this, we have demonstrated an extraction-free single amplicon-based approach for screening the entire β-globin gene with clinical samples using Scalable noninvasive amplicon-based precision sequencing (SNAPseq) assay catalyzing with next-generation sequencing (NGS). We optimized the assay using noninvasive buccal swab samples and simple finger prick blood for direct amplification with crude lysates. SNAPseq demonstrates high sensitivity and specificity, having a 100% agreement with Sanger sequencing. Furthermore, to facilitate seamless reporting, we have created a much simpler automated pipeline with comprehensive resources for pathogenic mutations in BT and SCD through data integration after systematic classification of variants according to ACMG and AMP guidelines. To the best of our knowledge, this is the first report of the NGS-based high throughput SNAPseq approach for the detection of both BT and SCD in a single assay with high sensitivity in an automated pipeline

A Clinico-Haematological study of hemoglobin E disease and trait

Introduction: HbE (Haemoglobin E) is one of the most important and common haemoglobinopathy. Its definitive diagnosis can be made on capillary electrophoresis. Our study aimed to analyze the clinico-haematological profile of patients having haemoglobin E disease and trait, including findings on capillary electrophoresis and iron profile. Materials and Methods: The samples were taken from patients who were referred to the haematology section of Department of Pathology, Silchar Medical College from January 2013 to December 2013. These patients were suspected of having haemoglobinopathy based on Peripheral Blood Film and Complete Blood Count. Study of complete haematological, eletrophoretic (by Capillary electrophoresis) and iron profile of the patients was done. Result: In our study, abnormal haemoglobins were detected in 61 out of the 100 cases examined, out of which HbE was detected in 45 cases. These patients presented with an asymptomatic to symptomatic phenotype, a decrease in mean corpuscular volume , microcytosis and target cells , a normal iron profile and increased HbE as well as HbA2 (Haemoglobin A2) levels on Capillary electrophoresis. Conclusion: Haemoglobin E constitutes an important haemoglobinopathy in lower Assam. An important finding was raised HbA2 (usually <6% on) capillary electrophoresis due to the β-thalassemic nature of HbE mutation. It needs to be differentiated from double heterozygous HbE-β thalassemia cases, as they also have elevated HbA2 levels (usually >6%) along with raised HbF levels. Therefore a proper diagnosis is essential so that preventive measures could be undertaken to reduce the burden of this haemoglobinopathy

Comparative analysis of various clinical specimens in detection of SARS-CoV-2 using rRT-PCR in new and follow up cases of COVID-19 infection: Quest for the best choice.

BackgroundAn appropriate specimen is of paramount importance in Real Time reverse transcription-polymerase chain reaction (rRT-PCR) based diagnosis of novel coronavirus (nCoV) disease (COVID-19). Thus, it's pertinent to evaluate various diversified clinical specimens' diagnostic utility in both diagnosis and follow-up of COVID-19.MethodsA total of 924 initial specimens from 130 COVID-19 symptomatic cases before initiation of treatment and 665 follow up specimens from 15 randomly selected cases comprising of equal number of nasopharyngeal swab (NPS), oropharyngeal swab (OPS), combined NPS and OPS (Combined swab), sputum, plasma, serum and urine were evaluated by rRT-PCR.ResultsDemographic analysis showed males (86) twice more affected by COVID-19 than females (44) (p = 0.00001). Combined swabs showed a positivity rate of 100% followed by NPS (91.5%), OPS (72.3%), sputum (63%), while nCoV was found undetected in urine, plasma and serum specimens. The lowest cycle threshold (Ct) values of targeted genes E, ORF1b and RdRP are 10.56, 10.14 and 12.26 respectively and their lowest average Ct values were found in combined swab which indicates high viral load in combined swab among all other specimen types. Analysis of 665 follow-up multi-varied specimens also showed combined swab as the last specimen among all specimen types to become negative, after an average 6.6 (range 4-10) days post-treatment, having lowest (15.48) and average (29.96) Ct values of ORF1b respectively indicating posterior nasopharyngeal tract as primary nCoV afflicted site with high viral load.ConclusionThe combined swab may be recommended as a more appropriate specimen for both diagnosis and monitoring of COVID-19 treatment by rRT-PCR for assessing virus clearance to help physicians in taking evidence-based decision before discharging patients. Implementing combined swabs globally will definitely help in management and control of the pandemic, as it is the need of the hour

A Simple, Cost-Effective, and Extraction-Free Molecular Diagnostic Test for Sickle Cell Disease Using a Noninvasive Buccal Swab Specimen for a Limited-Resource Setting

Sickle cell disease (SCD) is the most prevalent life-threatening blood monogenic disorder. Currently, there is no cure available, apart from bone marrow transplantation. Early and efficient diagnosis of SCD is key to disease management, which would make considerable strides in alleviating morbidity and reducing mortality. However, the cost and complexity of diagnostic procedures, such as the Sanger sequencing method, impede the early detection of SCD in a resource-limited setting. To address this, the current study demonstrates a simple and efficient proof-of-concept assay for the detection of patients and carriers using extraction-free non-invasive buccal swab samples by isothermal DNA Amplification coupled Restrictase-mediated cleavage (iDAR). This study is a first of its kind reporting the use of buccal swab specimens for iDA in molecular diagnosis of a genetic disease, all the while being cost effective and time saving, with the total assay time of around 150 min at a cost of USD 5. Further, iDAR demonstrates 91.5% sensitivity and 100% specificity for detecting all three alleles: SS, AS, and AA, having a 100% concordance with Sanger sequencing. The applicability of the iDAR assay is further demonstrated with its adaptation to a one-pot reaction format, which simplifies the assay system. Overall, iDAR is a simple, cost-effective, precise, and non-invasive assay for SCD screening, with the potential for use in a limited resource setting

Development of pathophysiologically relevant models of sickle cell disease and β-thalassemia for therapeutic studies

Abstract Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications

An Epidemiological Analysis of SARS-CoV-2 Genomic Sequences from Different Regions of India

The number of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) cases is increasing in India. This study looks upon the geographic distribution of the virus clades and variants circulating in different parts of India between January and August 2020. The NPS/OPS from representative positive cases from different states and union territories in India were collected every month through the VRDLs in the country and analyzed using next-generation sequencing. Epidemiological analysis of the 689 SARS-CoV-2 clinical samples revealed GH and GR to be the predominant clades circulating in different states in India. The northern part of India largely reported the ‘GH’ clade, whereas the southern part reported the ‘GR’, with a few exceptions. These sequences also revealed the presence of single independent mutations—E484Q and N440K—from Maharashtra (first observed in March 2020) and Southern Indian States (first observed in May 2020), respectively. Furthermore, this study indicates that the SARS-CoV-2 variant (VOC, VUI, variant of high consequence and double mutant) was not observed during the early phase of virus transmission (January–August). This increased number of variations observed within a short timeframe across the globe suggests virus evolution, which can be a step towards enhanced host adaptation