Retrospective study on the distribution of hemoglobinopathies in Karnataka-A laboratory experience.

AIMS: The importance of screening for hemoglobinopathies is well-documented in India. However, information on the distribution of hemoglobinopathies in Karnataka is lacking. The present study focuses on determining the spectrum of hemoglobinopathies for various districts of Karnataka. MATERIALS AND METHODS AND RESULTS: A retrospective analysis of samples registered for hemoglobinopathies for a period of 5 years (2017-2021) was carried out. A total of 17066 records registered only from the Karnataka region, were anonymized and retrieved. The data included gender, age, district, and results of the tests. The results were based on complete blood count, peripheral smear, and capillary electrophoresis (CE) pattern. The data were revalidated by pathologists, and the unambiguous data were analyzed for the study. One-fourth of the records (25%) showed abnormal hematological parameters. The number of female records (66%) was twice that of males and both genders showed higher distribution of thalassemia, followed by variants and double heterozygotes (DH). Several cases of thalassemia major were identified below the age of 17 years. The majority of thalassemia cases were ß thal and 93% of them were ß thal trait. Among the variants, HbS was more prevalent than HbE. Among the districts, Hassan had a 35.2% thal, Mysuru had a 7.2% variant, and Chitradurga had a 5.5% DH. Thalassemia, variants, and DH were distributed across several districts of Karnataka to various levels. CONCLUSION: The comprehensive retrospective analysis of the spectrum of hemoglobinopathies in various districts of Karnataka serves as evidence to carry out a prospective study on population screening where the incidence of thalassemia and structural variants is high.

A Qualitative Method to Detect Paraproteins from Serum Using Ultra Performance Liquid Chromatography Electrospray Triple Quadrupole Mass Spectrometry.

BACKGROUND: Mass spectrometry-based techniques are increasingly reported in the literature for identifying paraproteins due to their improved specificity and sensitivity. The present study demonstrates the capability of ultra performance liquid chromatography (UPLC) electrospray ionization triple quadrupole mass spectrometry for the qualitative analysis of paraproteins. METHODS: Paraproteins from patient serum (n = 40) were immunopurified using agarose beads coated with camelid antibodies that are specific for various subtypes of immunoglobulins (Igs; G, A, M, and light chains κ, λ). The extracted Igs are reduced to separate light chains from heavy chains in solution. The reduced sample was subjected to UPLC and mass measured using electrospray ionization-mass spectrometry. The mass spectral peaks at specific retention times were deconvoluted after clean-up to obtain the mass of light chains. The interpretation of liquid chromatography peaks and LC-MS data was validated by comparing them with immunofixation electrophoresis (IFE) results. RESULTS: The interpretation from the chromatographic pattern had a 92.5% (37/40) agreement when compared with mass information. The correlation of mass spectrometry data to IFE was 90% (36/40). The high mass of light chains (>25 kDa) was suggestive of glycosylation. Patient sera positive for IgGκ on IFE (n = 15) were analyzed for the interference of tAbs. The mass of Daratumumab observed in a sample was confirmed by the treating physician. A biclonal of same isotype (IgGκ) was identified. CONCLUSIONS: The feasibility of using liquid chromatography triple quadrupole mass spectrometry for the identification of the subtype of paraproteins has been demonstrated. The method's applicability to screen for interference from tAbs and identification of biclonals of the same isotype has been highlighted.

Multicentric evaluation of a novel point of care electrochemical ELISA platform for SARS-CoV-2 specific IgG and IgM antibody assay.

New diagnostics technologies for the efficient detection and quantification of SARS-CoV-2 antibodies are very crucial to manage the COVID-19 pandemic, especially in the context of emerging vaccination paradigms. Herein, we report on a novel point-of-care Electrochemical ELISA platform with disposable screen printed electrodes functionalized with SARS-CoV-2 Spike Glycoprotein S1, to enable fast and accurate quantitative estimation of total antibody concentration (IgG and IgM) in clinical samples. The quantification is performed with a comparison of electrochemical redox current against the current produced by the spiked monoclonal antibodies with known concentration. The assay is validated through multicentric evaluation against 3 different FDA authorized Laboratory standard techniques, using both EDTA whole blood and serum samples. We demonstrate that the proposed assay has excellent sensitivity and specificity, making it a suitable candidate for epidemiological surveys and quantification of antibodies in COVID-19 vaccination programs.

Glycation of albumin and its implication in Diabetes: A comprehensive analysis using mass spectrometry.

AIM: To understand the mechanism of glycation of albumin and effects on cysteinylation and methionine oxidation. METHODS: The in vitro glycation of HSA and BSA was studied with varying concentrations of glucose. Clinical blood samples of diabetic subjects with varying HbA1c values, were analyzed to assess in vivo glycation. All samples and their tryptic digests were analyzed using liquid chromatography/mass spectrometry. Glycation sites were mapped on to the three-dimensional structure of the HSA and BSA. RESULTS: A total thirty-one sites for glycation and eight sites of Nε-carboxymethyl-lysine (CML) modification were identified on albumin. The site selectivity of glycation was correlated with the environment of the reactive residue in the three-dimensional structure. CONCLUSIONS: The maximum percentage glycation under extreme conditions was in the range of ~55 to 88% in four weeks. Two major glycation sites K-233 and K-525 were identified, which together accounted for 40-50% of total glycation. A correlation was observed between glycation and oxidation of methionine residues in samples glycated in vitro. The role of spatially proximate residues in facilitating the glycation process is evident. The tri- and tetra-glycated isoforms of albumin can serve as biomarkers for the severe uncontrolled diabetic state.

Chronic granulomatous disease presenting with small bone osteomyelitis in a young child: A case report.

Chronic granulomatous disease (CGD) is a life threatening inherited disorder with varied clinical presentations often characterized by recurrent bacterial and fungal infections along with widespread granulomatous tissue response. The disease results from phagocytic defects characterized by deficiencies in oxidative burst of neutrophils. Nitroblue tetrazolium reduction test (NBT) and Dihydrorhodamine (DHR) with PMA stimulation by flow cytometry are quick, simple, sensitive and specific laboratory tests that help establish early and reliable diagnosis of CGD with an overall improvement in survival and disease prognosis. We report a case of 2-year old child who presented with small bone osteomyelitis involving bilateral feet and was later diagnosed to have autosomal recessive CGD due to mutation in NCF1 gene.

Flow Cytometric Eosin-5'-Maleimide Test is a Sensitive Screen for Hereditary Spherocytosis.

Hereditary spherocytosis (HS) is a clinically heterogeneous disease characterized by mild to moderate hemolysis resulting from red cell membrane protein defects. Diagnostic tests include hemogram, reticulocyte count and blood smear evaluation, osmotic fragility, cryohemolysis, SDS-PAGE, flow cytometry using eosin-5'-maleimide (EMA) and genetic studies. We evaluated the flow cytometric EMA-binding test and compared it with osmotic fragility in 51 consecutive cases of suspected HS aged between 10 days and 62 years. In addition, 4 cases suspected on blood smears underwent EMA testing alone. The 16 EMA-positive cases who were determined to have HS had overlapping hemoglobin levels and reticulocyte counts with the 35 patients with normal EMA results, highlighting the importance of the flow cytometric test in providing a definitive diagnosis. Flow cytometric EMA binding test was thus a simple and relatively faster method to confirm HS in our experience.

Pilot Study on the Performance of a New System for Image Based Analysis of Peripheral Blood Smears on Normal Samples.

Image analysis based automated systems aiming to automate the manual microscopic review of peripheral blood smears have gained popularity in recent times. In this paper, we evaluate a new blood smear analysis system based on artificial intelligence, Shonit™ by SigTuple Technologies Private Limited. One hundred normal samples with no flags from an automated haematology analyser were taken. Peripheral blood smear slides were prepared using the autostainer integrated with an automated haematology analyser and stained using May-Grunwald-Giemsa stain. These slides were analysed with Shonit™. The metrics for evaluation included (1) accuracy of white blood cell classification for the five normal white blood cell types, and (2) comparison of white blood cell differential count with the automated haematology analyser. In addition, we also explored the possibility of estimating the value of red blood cell and platelet indices via image analysis. Overall white blood cell classification specificity was greater than 97.90% and the precision was greater than 93.90% for all the five white blood cell classes. The correlation of the white blood cell differential count between the automated haematology analyser and Shonit™ was found to be within the known inter cell-counter variability. Shonit™ was found to show promise in terms of its ability to analyse peripheral blood smear images to derive quantifiable metrics useful for clinicians. Future enhancement should include the ability to analyse abnormal blood samples.

Aza-heterocyclic Receptors for Direct Electron Transfer Hemoglobin Biosensor.

Direct Electron Transfer biosensors, facilitating direct communication between the biomolecule of interest and electrode surface, are preferable compared to enzymatic and mediator based sensors. Although hemoglobin (Hb) contains four redox active iron centres, direct detection is not possible due to inaccessibility of iron centres and formation of dimers, blocking electron transfer. Through the coordination of iron with aza-heterocyclic receptors - pyridine and imidazole - we report a cost effective, highly sensitive and simple electrochemical Hb sensor using cyclic voltammetry and chronoamperometry. The receptor can be either in the form of liquid micro-droplet mixed with blood or dry chemistry embedded in paper membrane on top of screen printed carbon electrodes. We demonstrate excellent linearity and robustness against interference using clinical samples. A truly point of care technology is demonstrated by integrating disposable test strips with handheld reader, enabling finger prick to result in less than a minute.