Study of Clinical, Hematological and Molecular Characteristics of Patients of Thalassemia and Hemoglobinopathies in Tertiary Care Hospital.

To study clinical, hematological and molecular characteristics of patients of thalassemia and hemoglobinopathies and to correlate the molecular characteristics with clinical and hematological presentations. Material: This observational cross sectional study included 100 patients of age >12 years of all genders with chronic haemolytic anemia and history of multiple blood transfusion. Blood and radiological investigations were done. Clinical, hematological and molecular characteristics were studied. Observation and Clinical: Pallor was present in all cases and icterus in 32% cases. Total 48% of the patients had hepatomegaly and 98% had splenomegaly. Among genotypes, 15% cases had α-thalassemia, 62% had ß thalassemia + 뫧 thalassemia, 7% had HbS hemoglobinopathy, and 16% had HbE hemoglobinopathy. Hematological: Hemoglobin showed significant association with molecular genotypes of thalassemia with lowest being present in ß-thalassemia + 뫧 thalassemia and HbE.MCV showed significant association with molecular genotypes, with HbE having the lowest MCV of 65.5 fl. LDH levels showed a significant association with molecular genotype with highest being in HbS hemoglobinopathy. Molecular Characteristics: Common mutations in compound α-thalassemia were 3.7, 4.2 and 20.5 deletion. As for ß-thalassemia and 뫧 thalassemia, 47 cases had heterozygous type and 15 cases had homozygous types. In ß-thalassemia, the homozygous type showed IVS1- 5(G→C),CD 41/42(→CTT) and IVSII-654(G→T) while heterozygous type showed CD16(→G), CD 41/42(→CTT), IVS1-5(G→C), and IVSII-654(G→T) . In 뫧 thalassemia, the heterozygous type showed 뫧 inversion mutation.In HbS hemoglobinopathy, heterozygous type showed Codon 6(A→T) and compound heterozygous type showed IVS1- 5(G→C) and Codon 6(A→T). In HbE hemoglobinopathy,the homozygous type showed CD26(G→A) and compound heterozygous type showed IVS1-5(G→C) and IVSII 654(G→T). Conclusion: The common thalassemia genotypes observed in our study were α-thalassemia (15%), ß thalassemia + 뫧 thalassemia, (62%) HbS hemoglobinopathy (7%), and HbE hemoglobinopathy (16%). The patients presented with pallor, icterus, hepatomegaly, and splenomegaly which were comparable among all molecular genotypes of thalassemia and hemoglobinopathies. α-thalassemia had compound α-thalassemia with common mutations being 3.7, 4.2 and 20.5 deletion. As for ß-thalassemia and 뫧 thalassemia, 47 cases had heterozygous type and 15 cases had homozygous types. In 뫧 thalassemia, the heterozygous type showed 뫧 inversion mutation in 5 cases. MCH, Retic count, ferritin stores, and peripheral blood smear were similar in all molecular genotypes. Hemoglobin, MCV and LDH showed a significant association with molecular genotypes. Microcytic hypochromic anaemia was commonest among all.The findings of the present study show that the genotypes of thalassemia are characterized by diversity as well as significant genetic heterogeneities.

Prevalence of arrhythmia in COVID-19 patients with mild/moderate and severe illness: a prospective cohort study.

OBJECTIVES: The type of arrhythmias, and their prevalence in mild/moderate and severe COVID-19 patients admitted to the hospital are unknown from a prospective cohort study. METHODS: We did continuous electrocardiograms along with multiple ECGs in 305 consecutive hospitalized COVID-19 patients. RESULTS: The incidence of arrhythmias was 6.8% (21/305) in the target population. The incidence of arrhythmias was 9.2% (17/185) in patients with severe COVID-19 illness and 3.3% (4/120) in patients with mild/moderate COVID-19 illness with no significant difference (p = 0.063). All the arrhythmias were new-onset arrhythmias in this study. 95% (20/21) of these arrhythmias were atrial arrhythmia with 71.42% (15/21) being atrial fibrillation and one episode of sustained polymorphic ventricular tachycardia. No episode of high-grade atrioventricular block, sustained monomorphic ventricular arrhythmia, or torsades de pointes arrhythmias were observed in this study. The patients with arrhythmias were admitted to the intensive care unit (80.9% vs. 50.7%; p: 0.007), were on a ventilator (47.6% vs. 21.4%; p: 0.006), and had high in-hospital mortality (57.1% vs. 21.1%; p: 0.0001) than patients without arrhythmias. CONCLUSION: Atrial arrhythmias were the most frequent arrhythmias in hospital-admitted COVID-19 patients with atrial fibrillation being the most common arrhythmia. TRIAL REGISTRATION: Clinical Trial Registry India (CTRI) (CTRI/2021/01/030788). (https://www.ctri.nic.in/).

Procalcitonin for Detecting Culture-Positive Sepsis in Neonates: A Prospective, Multicenter Study.

INTRODUCTION: It is unclear if serum procalcitonin (PCT) estimated at sepsis suspicion can help detect culture-positive sepsis in neonates. We evaluated the diagnostic performance of PCT in culture-positive sepsis in neonates. METHODS: This was a prospective study (February 2016 to September 2020) conducted in four level-3 units in India. We enrolled neonates suspected of sepsis in the first 28 days of life. Neonates with birth weight <750 g, asphyxia, shock, and major malformations were excluded. Blood for PCT assay was drawn along with the blood culture at the time of suspicion of sepsis and before antibiotic initiation. The investigators labeled the neonates as having culture-positive sepsis or "no sepsis" based on the culture reports and clinical course. PCT assay was performed by electrochemiluminescence immunoassay, and the clinicians were masked to the PCT levels while assigning the label of sepsis. Primary outcomes were the sensitivity, specificity, and likelihood ratios to identify culture-positive sepsis. RESULTS: The mean birth weight (SD) and median gestation (IQR) were 2,113 (727) g and 36 (32-38) weeks, respectively. Of the 1,204 neonates with eligible cultures, 155 (12.9%) had culture-positive sepsis. Most (79.4%) were culture-positive within 72 h of birth. The sensitivity, specificity, and positive and negative likelihood ratios at 2 ng/mL PCT threshold were 52.3% (95% confidence interval: 44.1-60.3), 64.5% (60.7-68.1), 1.47 (1.23-1.76), and 0.74 (0.62-0.88), respectively. Adding PCT to assessing neonates with 12.9% pretest probability of sepsis generated posttest probabilities of 18% and 10% for positive and negative test results, respectively. CONCLUSION: Serum PCT did not reliably identify culture-positive sepsis in neonates.

Expression of genes related to multiple drug resistance and apoptosis in acute leukemia: response to induction chemotherapy.

Resistance to chemotherapy is a major impediment to the successful treatment of acute leukemia (AL). Expression of genes involved in drug resistance and apoptosis may be responsible for this. This study aimed to investigate the expression of drug resistance (MDR1, MRP1, LRP, BCRP, GSTP1, DHFR) and apoptotic genes (p53, BCL-2, Survivin) in adult acute leukemias and compare them with clinical and hematological findings and response to induction chemotherapy. Eighty-five patients with AL [45 with acute myeloid leukemia (AML) and 40 with acute lymphoblastic leukemia (ALL)] were used as a study group. Real-time PCR results showed that expression level of MDR1 was significantly higher in AML whereas expression of DHFR, BCRP and Survivin was significantly higher in ALL patients. In AML, significant correlation was observed between LRP and MRP1 (r(s)=0.44, p=0.016), LRP and DHFR (r(s)=0.41, p=0.02), MDR1 and BCL-2 (r(s)=0.38, p=0.03). Expression of GSTP1 and LRP correlated with high white blood count (p=0.03 and p=0.03) and BCL-2 with high peripheral blast count (p=0.009). MDR1 expression was significantly associated with the expression of immature stem cell marker CD34 (p=0.002). In ALL, significant association was found between LRP gene and female sex (p<0.0001), LRP and B-ALL patients (p=0.04) and LRP and BCR/ABL positive patients (p=0.004). High expression of MDR1 and BCL-2 in AML and MRP1 gene in ALL was associated with response to induction chemotherapy (p=0.001, p=0.02 and p=0.007 respectively). These results showed the potential clinical relevance of MDR1, MRP1 and BCL-2 in adult patients with acute leukemia in the context of induction chemotherapy.

Disseminated intravascular coagulation with intracranial haematoma in neonatal congenital syphilis.

Disseminated intravascular coagulation (DIC) although a well known complication in neonatal sepsis is extremely rare in congenital syphilis and there are scanty reports of this entity in the literature. Intracranial bleeding following DIC in neonatal congenital syphilis is even rarer, and has been reported only once earlier. We are reporting the second case of neonatal DIC with intracranial haematoma due to congenital syphilis in a newborn. Our patient also had clinical and biochemical evidence of hepatitis which predisposes to DIC. Extensive investigations and emergent use of imaging modalities including ultrasound and CT scan led to early diagnosis and treatment in our patient, who could therefore be salvaged from an otherwise life threatening disease.

Hb Sun Prairie: A rare cause of chronic hemolysis in an Indian patient.

Haemolytic anaemia is a commonly encountered condition in clinical haematology practise. Dissecting the aetiology of haemolytic anaemia is of paramount importance for appropriate management. We describe a 29-years-old lady of Indian origin, who presented with fatigue and recurrent jaundice for 2 years. Examination revealed pallor, mild icterus, and splenomegaly. Blood tests showed anaemia, reticulocytosis, indirecthyperbilirubinemia, and high serum lactate dehydrogenase, consistent with haemolytic anaemia. Peripheral smear showed severely microcytic hypochromic red cells and polychromasia. Heinz bodies and inclusion bodies were seen with supravital staining. Haemoglobin high pressure liquid chromatography showed low HbA2 and normal HbF. Work-up for iron deficiency was negative. Polymerase chain reaction of the genomic DNA failed to identify common deletions in the HBA genes. Sangers sequencing of HBA2 gene revealed a homozygous missense mutation NM_000517.6: c.391G > C (p.Ala131Pro) leading to a highly unstable hemoglobin, Hb Sun Prairie. Mother was heterozygous for the same mutation, and father was unavailable for genetic testing. We highlight the role of sangers sequencing in unravelling the underlying aetiology of haemolytic anaemia. Pathophysiology and existing literature of Hb Sun Prairie has been discussed.

Coagulation profile in diabetes and its association with diabetic microvascular complications.

OBJECTIVES: To investigate the haemostatic parameters and to assesss their relationship with microvascular complications in type 2 diabetes mellitus. MATERIALS AND METHODS: Coagulation and fibrinolysis parameters were measured in 60 type 2 diabetic patients (M:F 1:1) with (n=40) and without (n=20) diabetic microvascular complications and in 30 nondiabetic healthy subjects (M:F 1:1). RESULTS: The mean age of diabetic patients and healthy controls was 56.9 +/- 8.78 and 53.2 +/- 7.58 respectively (p = 0.05). The plasma levels of PAI-1 (22.6 +/- 6.85 vs 44.8 +/- 20.8, p = 0.00), serum fibrinogen (227.5 +/- 22.8 vs. 252.75 +/- 40.23, p = 0.002) and vWF activity (99.4 +/- 28.18 vs. 144.78 +/- 36.21, p = 0.00) were found to be increased in diabetics compared to healthy controls. Plasma PAI-1 levels (37.15 +/- 15.18 vs 48.65 +/- 22.29, p = 0.0) and vWF activity (123.19 +/- 29.63 vs. 155.57 +/- 34.61, p = 0.007) were significantly increased in diabetic patients with microvascular complications than those without microvascular complications.Amongst the diabetic patients, protein S activity (63.05 +/- 16.85 vs. 51.59 +/- 10.7, p = 0.002) was significantly lower in patients with microvascular complications than in patients without these complications. Diabetic retinopathy was associated with decreased protein S levels (63.05 +/- 16.85 vs. 48.48 +/- 8.72, p = 0.005) and vWF activity (123.19 +/- 29.63 vs. 151.85 +/- 29.74, p = 0.009). Diabetic nephropathy was associated with increased PAI-1 levels (39.55 +/- 13.20 vs. 51.69 +/- 26.53, p = 0.02) and vWF activity (134.99 +/- 32.54 vs. 157.57 +/- 37.37, p = 0.007). Diabetic neuropathy did not show any significant relationship with any of the haemostatic variables. CONCLUSION: Hypercoagulable state as indicated by decreased fibrinolysis and increased coagulability is responsible as one of the factors for the development of microvascular complications of diabetes mellitus.

Prospective evaluation of variables affecting platelet function in patients with newly diagnosed chronic myeloid leukemia.

: Platelet function in chronic myeloid leukemia (CML) could be affected by either hyperleucocytosis, clonal megakaryopoiesis, or tyrosine kinase inhibitors. However, these variables have never been prospectively evaluated. We conducted a prospective study over a period of 1.5 years in a tertiary care center of north India. Patients with CML in chronic phase, more than 18 years, and treated with imatinib were enrolled (n = 32). Age, and sex-matched controls were also included. Platelet function test was performed using two-channel Chrono-Log aggregometer 490 at four time-points: first, at diagnosis; second, after leucoreduction (total leucocyte count, <10 × 10/l) achieved with hydroxycarbamide; third, on-imatinib at BCR-ABL less than 1%; and fourth, in an independent cohort (off-imatinib) at deep molecular response (DMR) (BCR-ABL < 0.01%). Statistical analysis was performed using IBM SPSS statistics (version 22.0). Median age of patients was 42 years (15-65), and M : F ratio was 1 : 1. At diagnosis, platelet function correlated negatively with total leucocyte count, but not with platelet count. As compared with baseline, platelet aggregation with ADP (2.5 µl), and collagen (2.5 µl) improved significantly after leucoreduction (P = 0.05 and 0.009, respectively). Imatinib further caused significant impairment of aggregation with ADP (2.5 µl), collagen (2.5 µl), and collagen (1 µl) (P = 0.04, 0.008, and 0.02, respectively). Patients in DMR also demonstrated a significant impairment of platelet aggregation with all the agonists as compared with controls. While leucoreduction alone can improve the baseline platelet function derangement in CML, imatinib further impairs it. Residual CML stem cells, or effect of imatinib on normal common myeloid progenitors might account for platelet function derangement at DMR.

Safety and effectiveness of intravenous iron sucrose versus standard oral iron therapy in pregnant women with moderate-to-severe anaemia in India: a multicentre, open-label, phase 3, randomised, controlled trial.

BACKGROUND: Intravenous iron sucrose is a promising therapy for increasing haemoglobin concentration; however, its effect on clinical outcomes in pregnancy is not yet established. We aimed to assess the safety and clinical effectiveness of intravenous iron sucrose (intervention) versus standard oral iron (control) therapy in the treatment of women with moderate-to-severe iron deficiency anaemia in pregnancy. METHODS: We did a multicentre, open-label, phase 3, randomised, controlled trial at four government medical colleges in India. Pregnant women, aged 18 years or older, at 20-28 weeks of gestation with a haemoglobin concentration of 5-8 g/dL, or at 29-32 weeks of gestation with a haemoglobin concentration of 5-9 g/dL, were randomly assigned (1:1) to receive intravenous iron sucrose (dose was calculated using a formula based on bodyweight and haemoglobin deficit) or standard oral iron therapy (100 mg elemental iron twice daily). Logistic regression was used to compare the primary maternal composite outcome consisting of potentially life-threatening conditions during peripartum and postpartum periods (postpartum haemorrhage, the need for blood transfusion during and after delivery, puerperal sepsis, shock, prolonged hospital stay [>3 days following vaginal delivery and >7 days after lower segment caesarean section], and intensive care unit admission or referral to higher centres) adjusted for site and severity of anaemia. The primary outcome was analysed in a modified intention-to-treat population, which excluded participants who refused to participate after randomisation, those who were lost to follow-up, and those whose outcome data were missing. Safety was assessed in both modified intention-to-treat and as-treated populations. The data safety monitoring board recommended stopping the trial after the first interim analysis because of futility (conditional power 1·14% under the null effects, 3·0% under the continued effects, and 44·83% under hypothesised effects). This trial is registered with the Clinical Trial Registry of India, CTRI/2012/05/002626. FINDINGS: Between Jan 31, 2014, and July 31, 2017, 2018 women were enrolled, and 999 were randomly assigned to the intravenous iron sucrose group and 1019 to the standard therapy group. The primary maternal composite outcome was reported in 89 (9%) of 958 patients in the intravenous iron sucrose group and in 95 (10%) of 976 patients in the standard therapy group (adjusted odds ratio 0·95, 95% CI 0·70-1·29). 16 (2%) of 958 women in the intravenous iron sucrose group and 13 (1%) of 976 women in the standard therapy group had serious maternal adverse events. Serious fetal and neonatal adverse events were reported by 39 (4%) of 961 women in the intravenous iron sucrose group and 45 (5%) of 982 women in the standard therapy group. At 6 weeks post-randomisation, minor side-effects were reported by 117 (16%) of 737 women in the intravenous iron sucrose group versus 155 (21%) of 721 women in the standard therapy group. None of the serious adverse events was found to be related to the trial procedures or the interventions as per the causality assessment made by the trial investigators, ethics committees, and regulatory body. INTERPRETATION: The study was stopped due to futility. There is insufficient evidence to show the effectiveness of intravenous iron sucrose in reducing clinical outcomes compared with standard oral iron therapy in pregnant women with moderate-to-severe anaemia. FUNDING: WHO, India.

Expression of Apoptosis Related and Proliferative Proteins in Malignant Lympho-Proliferative Disorders.

BACKGROUND & OBJECTIVE: The current study aimed to perform an immunohistochemical analysis of patterns of apoptotic and cell proliferative related protein expression in different histological grades and immune phenotypes of malignant lymphomas and other lymphoproliferative disorders. METHODS: This observational study was carried on 60lymph node biopsies of lymphoproliferative disorders. The biopsies were analyzed histologically and immunohistochemically. RESULTS: A total of 60 lymph node biopsies were included in the study, of which 81.6% were of malignant lympho-proliferative lesions. The majority of the biopsies were B-cell (66%) and were grouped in the intermediate grade. Bax and BCL-2 protein expression was presented by percentage of immune positive neoplastic cells per 10fields and graded on a scale of 1 to4. A Bcl-2, Bax Protein Ratio (BBPR) was determined for each case by dividing the estimated Bcl-2 protein (percentage of Bcl-2 positive cells x Bcl-2 staining intensity) by the estimated Bax protein (percentage of Bax positive cells x Bax immunostaining intensity). The mean BBPR was found to be significantly higher in indolent lymphomas (2.64 ± 1.3) as compared to aggressive lymphomas (0.47 ± 0.9) (P<0.01). The expression of P53 and PCNA in 35 biopsies of Non Hodgkin Lymphomas (NHL) was found to increase from low to high grade tumors. CONCLUSIONS: A significant correlation was found between BBPR and predicted biological behavior of indolent and aggressive lymphomas. This indicates the important role of Bcl-2 and Bax in biological behavior of lymphomas. Furthermore, P53 and PCNA expression were found to increase from low to high-grade tumors suggesting their prognostic value in NHL.