Challenges in the diagnosis and management of autoimmune hemolytic anemia: A case-based approach. Experience from a tertiary care hospital in the Haryana region.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) is a rare immune disorder which occurs when antibodies are directed against self red blood cells (RBCs) leading to hemolysis. AIHA is widely classified as warm autoimmune hemolytic anemia, cold agglutinin syndrome, mixed AIHA, paroxysmal cold hemoglobinuria and rarely drug induced AIHA. The pathogenesis of AIHA is complex interplay between genetic predisposition, immune dysregulation and enviornmental triggers. A direct antiglobulin test can be used to assess the immunological origin of the hemolysis in order to diagnose AIHA after identifying laboratory and clinical symptoms of hemolysis. OBJECTIVE: The objective is to understand underlying mechanism in AIHAs, and usage of targeted therapies to modulate specific components of the immune response. MATERIALS AND METHODS: We are hereby presenting a case series of 11 clinically suspected cases of AIHA in collaboration with their clinical features, immuno-hematological and other laboratory parameters, Flow cytometric analysis of lymphocyte subset in relevant cases, underlying etiology as well as serological subtype are also included. RESULTS: Majority of the patients were categorized as secondary AIHA (7/11, 63.63%). Out of 11 cases 7 were serologically subtyped as warm AIHA (7/11, 63.63%) ,2 cases were DaaT negative AIHA (2/11;18.18%), 2 cases were characterized as mixed AIHA subtype (2/11, 18.18%). CONCLUSION: Accurate subtyping of AIHA requires a systematic immunohematological approach coupled with comprehensive evaluations of clinical, hematological, and biochemical parameters.

A critical appraisal of mucormycosis in COVID- 19 patients in a tertiary care centre in India.

Background and Purpose: Morbidity and mortality of opportunistic fungal infections in COVID-19 patients are less studied and defined. The patients receiving immunosuppressive therapy, broad-spectrum antibiotics, corticosteroids, and invasive and non-invasive ventilation are the high-risk groups. Materials and Methods: The demographic profile as well as clinical and radiological findings of all the patients with COVID-19 suspected of Mucormycosis (MM) were recorded. The tissue samples from all the patients were sent for microbiological (KOH mount and culture) and histopathological analysis for confirmation of MM. Results: In total, 45 COVID-19 patients suspected of MM were included in the study and MM was confirmed in 42 patients. The mean age of the patients was 50.30±14.17 years with a female: male ratio of 1.1:1. The most common symptom was headache (52.38%) followed by purulent nasal discharge (38.09%) and facial pain in 33.33% of the cases. The ocular symptoms included a diminution of vision (33.33%) and redness of the eye (2.38%). The most common site of involvement was rhino-orbital (42.85%) followed by sinonasal (23.80%) and rhino cerebral (19.04%). Majority (38.09%) of the patients were diagnosed with stage II of Rhino-orbital-cerebral Mucormycosis (ROCM) based on radiology. A history of diabetes mellitus and steroids was present in 97.61% and 85.71% of the cases, respectively. Moreover, KOH was positive for MM in 97.61% of the cases while the culture was positive in only 35.71% of the cases. In addition, on histopathology, MM was confirmed in 64.28 % of the cases. Mixed growth with Aspergillus species and Rhizopus species was observed in 14.28% of the cases in culture and 11.90% of the cases in histopathology test. Furthermore, angioinvasion was found in 23.80% of the cases according to the histopathology test. Conclusion: Based on the results, the most common conditions associated with MM in COVID-19 patients were diabetes mellitus and steroid therapy. A high level of clinical suspicion aided with diagnostic tests, including KOH mount, culture, histopathology, and radiology which helped the early detection of opportunistic fungal infection in COVID-19 patients to ensure timely treatment.

A rare case of complicated malaria presenting as warm autoimmune hemolytic anemia.

Autoimmune hemolytic anemia (AIHA) has been rarely reported worldwide or from India as the underlying cause of anemia in malaria. We hereby present a case of complicated Plasmodium falciparum malaria with concomitant warm AIHA in a 31-year-old male. Direct Antiglobulin Test (DAT) was positive and elution studies showed pan-agglutination reaction. Clinico-hematological and serological follow-up of the patient was done post artesunate treatment until day 9. We suggest that it is important to establish the immune basis of anemia in malaria patients for guiding the treatment plan for the clinicians and providing packed red blood cell transfusion if required.

Dual pathogen valvular endocarditis in a case of severe aortic aneurysm.

Infective endocarditis (IE) is the heart valve or endocardium infection. We report a rare case of polymicrobial endocarditis, namely invasive Aspergillus spp and Acinetobacter baumannii, in a 36-year-old male with a medical history of degenerative disease of the aorta with abdominal aortic and ascending aortic aneurysms with a fulminant clinical course and fatal outcome. The treatment was challenging due to multiple comorbidities. The autopsy revealed dual pathogen endocarditis due to Acinetobacter baumannii sepsis and invasive Aspergillus spp mycosis. This report emphasizes that polymicrobial endocarditis (PE) is an infrequent finding with a poor prognosis requiring high clinical suspicion.

Aberrant promoter hypermethylation regulates thrombomodulin in high altitude induced deep vein thrombosis.

BACKGROUND: DNA methylation regulates gene expression by inhibiting transcription factor binding to promoter and regulatory regions. Acute hypoxia during altitude exposure is associated with decreased natural anticoagulants and morbid thrombotic events. Thrombomodulin (TM) is a high affinity thrombin binding receptor protein, vital for vascular homeostasis. The purpose of this study is to determine gene expression regulation via methylation of TM gene in high altitude hypoxia induced deep vein thrombosis (DVT) patients. MATERIALS AND RESULTS: Percent 5-methyl cytosine analysis showed increased methylation in high altitude DVT patients (HAP) as compared to high altitude control (HAC) and seal level control (Control) subjects, while TM protein and mRNA levels were decreased in high altitude DVT patients as compared to other two groups. Bisulfite sequencing analysis indicated increased methylation in TM promoter in high altitude DVT patients compared to high altitude controls. Flow cytometry analysis showed decreased TM expression in hypoxia induced primary human umbilical vein endothelial cells (HUVECs). Treatment with specific DNA methyltransferase (DNMT) inhibitor-decitabine during hypoxia, restored TM expression. in vitro global methylation assay showed increased methylation in hypoxia group. Specific concentration of decitabine in hypoxia decreased global methylation showing a direct correlation between DNMTs and methylation. Selective dose of decitabine restored TM levels in HUVECs. DNMT1 and DNMT3B proteins showed to mediate the overall expression of TM. CONCLUSION: TM emerged as a potential candidate for methylation in high altitude DVT patients, regulated by hypoxia-induced epigenetic mechanism. Hypoxia culminates in methylation of DNA sequences in the promoter region of TM gene and increased the expression of DNMT1 and DNMT3B per se in primary HUVECs. Critical DNA methylation events were found to be compromised in high altitude DVT patients.

An Unusual Clinical Manifestation of Plasmablastic Lymphoma in a Renal Transplant Recipient.

Plasmablastic lymphoma is a rapidly progressive CD20 negative large cell non-Hodgkin lymphoma with poor outcome. It occurs mostly in immunocompromised individuals and has a predilection for extranodal sites. They need to be differentiated from other entities sharing similar morphological features like poorly differentiated carcinoma, Burkitt's lymphoma, Alk positive large B cell lymphoma, Diffuse large B cell lymphoma, and anaplastic myeloma. EBV negativity in recipients, type, intensity, and duration of immunosuppressives used are certain risk factors in development of posttransplant lymphoproliferative disorders. High index of suspicion can help clinch the diagnosis early and prevent catastrophic consequences. Our renal transplant recipient presented with complaints of pain abdomen and malena for which he underwent exploratory laparotomy. Diagnosis was established on histopathology and timely treatment initiated reverted the disease.

Epidemiology and pathophysiology of vascular thrombosis in acclimatized lowlanders at high altitude: A prospective longitudinal study.

Background: Previous literature suggests that thrombosis is more common in lowlanders sojourning at high altitude (HA) compared to near-sea-level. Though the pathophysiology is partly understood, little is known of its epidemiology. To elucidate this, an observational prospective longitudinal study was conducted in healthy soldiers sojourning for months at HA. Methods: A total of 960 healthy male subjects were screened in the plains, of which 750 ascended, to altitudes above 15,000ft (4,472m). Clinical examination, haemogram, coagulogram, markers of inflammation and endothelial dysfunction, were studied at three time points during ascent and descent. The diagnosis of thrombosis was confirmed radiologically in all cases where a thrombotic event was suspected clinically. Subjects developing thrombosis at HA were labelled as Index Cases (ICs) and compared to a nested cohort of the healthy subjects (comparison group,(CG)) matched for altitude of stay. Findings: Twelve and three subjects, developed venous (incidence: 5,926/105 person-years) and arterial (incidence: 1,482/105 person-years) thrombosis at HA, respectively. The ICs had enhanced coagulation (FVIIa: p<0.001; FXa: p<0.001) and decreased levels of natural anticoagulants (thrombomodulin, p=0.016; tissue factor pathway inhibitor [TFPI]: p<0.001) and a trend to dampened fibrinolysis (tissue plasminogen activator tPA; p=0.078) compared to CG. ICs also exhibited statistically significant increase in the levels of endothelial dysfunction and inflammation markers (vascular cell adhesion molecule-1[VCAM-1], intercellular adhesion molecule-1 [ICAM-1], vascular endothelial growth factor receptor 3 [VEGFR-3], P-Selectin, CD40 ligand, soluble C-reactive protein and myeloperoxidase: p<0.001). Interpretation: The incidence of thrombosis in healthy subjects at HA was higher than that reported in literature at near sea-level. This was associated with inflammation, endothelial dysfunction, a prothrombotic state and dampened fibrinolysis. Funding: Research grants from the Armed Forces Medical Research Committee, Office of the Director General of Armed Forces Medical Services (DGAFMS) & Defence Research and Development Organization (DRDO), Ministry of Defence, India.

Dual pathogen valvular endocarditis in a case of severe aortic aneurysm

ABSTRACT Infective endocarditis (IE) is the heart valve or endocardium infection. We report a rare case of polymicrobial endocarditis, namely invasive Aspergillus spp and Acinetobacter baumannii, in a 36-year-old male with a medical history of degenerative disease of the aorta with abdominal aortic and ascending aortic aneurysms with a fulminant clinical course and fatal outcome. The treatment was challenging due to multiple comorbidities. The autopsy revealed dual pathogen endocarditis due to Acinetobacter baumannii sepsis and invasive Aspergillus spp mycosis. This report emphasizes that polymicrobial endocarditis (PE) is an infrequent finding with a poor prognosis requiring high clinical suspicion.

Survival Outcomes of Newly Diagnosed Multiple Myeloma at a Tertiary Care Center in North India (IMAGe: 001A Study).

PURPOSE: The outcomes of patients with myeloma from developing countries are often lacking because of poor record maintenance. Publications from such settings are also limited because of the retrospective nature of the data collection. Information technology can bridge these gaps in developing countries with real-time data maintenance. We present the real-time survival data of the patients with myeloma from a tertiary care center in North India using one such indigenously built software. PATIENTS AND METHODS: These are real-time data of all patients with myeloma presenting to a tertiary care center from North India. The patient characteristics (demographics, baseline disease characteristics, risk stratification, and outcomes) were recorded contemporaneously. The survival of the study population was analyzed and grouped based on various disease characteristics at diagnosis. RESULTS: The median age of the study population (N = 696) was 65.9 (34.9-94.9) years with male predominance (65%). The median follow-up was 3.7 years (0-18.6 years) with the median overall survival (OS) not achieved. The OS of the study population at 1, 3, and 5 years was 94% (n = 558), 87.5% (n = 394), and 83.1% (n = 267), respectively. Most of the patients presented in advanced stages based on International Staging System (III:70%). On Kaplan-Meier analysis, the presence of weight loss (P = .01), renal dysfunction (P = .047), and anemia at diagnosis (P = .004) had a significant impact on survival. On Cox proportional model univariate analysis, the presence of renal dysfunction, anemia, and weight loss had the significant hazard ratio of 1.68 (1-2.82, P = .049), 3.18 (1.39-7.29, P = .0063), and 2.81 (1.22-6.42, P = .014), respectively, whereas on multivariate analysis of hypercalcemia, renal disease, anemia, and bone disease (CRAB) features, only anemia was found to have a significant hazard ratio of 2.56 (1.01-6.47, P = .046). CONCLUSION: The real-world data show OS comparable with the published western literature. Only anemia was found to have significant impact on survival. The use of such software can aid in better data-keeping in resource-constrained settings.

Recent Advances in Molecular Diagnosis and Prognosis of Childhood B Cell Lineage Acute Lymphoblastic Leukemia (B-ALL).

B cell lineage acute lymphoblastic leukemia is the most common leukemia occurring in children and young adults and is the leading cause of cancer related deaths. The 5 year overall survival outcome in children with B-ALL has improved significantly in the last few decades. In the past, the discovery of various genetic alterations and targeted therapy have played a major role in decreasing disease-related deaths. In addition, numerous advances in the pathogenesis of B-ALL have been found which have provided better understanding of the genes involved in disease biology with respect to diagnostic and prognostic implications. Present review will summarize current understanding of risk stratification, genetic factors including cytogenetics in diagnosis and prognosis of B-ALL.

Transcriptome Profiling Reveals the Endogenous Sponging Role of LINC00659 and UST-AS1 in High-Altitude Induced Thrombosis.

BACKGROUND: The pathophysiology of deep vein thrombosis (DVT) is considered as multifactorial, where thrombus formation is an interplay of genetic and acquired risk factors. Little is known about the expression profile and roles of long noncoding RNAs (lncRNAs) in human subjects developing DVT at high altitude. METHODS: Using RNAseQ, we compared peripheral blood mRNA and lncRNA expression profile in human high-altitude DVT (HA-DVT) patients with high-altitude control subjects. We used DESeq to identify differentially expressed (DE) genes. We annotated the lncRNAs using NONCODE 3.0 database. In silico putative lncRNA-miRNA association study unravels the endogenous miRNA sponge associated with our candidate lncRNAs. These findings were validated by small-interfering RNA (siRNA) knockdown assay of the candidate lncRNAs conducted in primary endothelial cells. RESULTS: We identified 1,524 DE mRNAs and 973 DE lncRNAs. Co-expressed protein-coding gene analysis resulted in a list of 722 co-expressed protein-coding genes with a Pearson correlation coefficients >0.7. The functional annotation of co-expressed genes and putative proteins revealed their involvement in the hypoxia, immune response, and coagulation cascade. Through its miRNA response elements to compete for miR-143 and miR-15, lncRNA-LINC00659 and UXT-AS1 regulate the expression of prothrombotic genes. Furthermore, in vitro RNA interference (siRNA) simultaneously suppressed lncRNAs and target gene mRNA level. CONCLUSION: This transcriptome profile describes novel potential mechanisms of interaction between lncRNAs, the coding genes, miRNAs, and regulatory transcription factors that define the thrombotic signature and may be used in establishing lncRNAs as a biomarker in HA-DVT.

Indian Society of Hematology and Blood Transfusion (ISHBT) Consensus Document on Hematological Practice During COVID-19 Pandemic.

The SARS-CoV-2 (COVID-19) pandemic is a worldwide public health emergency with widespread impact on health care delivery. Unforeseen challenges have been noted during administration of usual haematology care in these unusual COVID-19 times. Medical services have been overstretched and frontline health workers have borne the brunt of COVID-19 pandemic. Movement restrictions during lockdown prevented large sections of population from accessing health care, blood banks from holding blood drives, and disrupted delivery of diagnostic hematology services. The disruption in hematology care due to COVID-19 pandemic in India has been disproportionately higher compared to other subspecialities as hematology practice in India remains restricted to major cities. In this review we chronicle the challenges encountered in caring for hematology patients during the COVID-19 pandemic in India and put forth recommendations for minimizing their impact on provision of hematology care with special emphasis on hematology practice in lower and middle income countries (LMICs).

Posttransplant lymphoproliferative disorder in renal transplant recipients: Experience from a Tertiary Care Center.

Posttransplant lymphoproliferative disorders (PTLDs) are potentially fatal complications arising after solid organ or hematopoietic stem cell transplant. The most crucial factor in pathogenesis of PTLDs is either a primary infection with Epstein-Barr virus or reactivation of its latent state due to immune dysregulation. This complex pathobiology leads to a myriad of clinical manifestations due to uncontrolled lymphoproliferation that may be reactive, polymorphous or monomorphous. We report our experience at a tertiary center of six cases detected over a span of six years. All our patients were proven as high grade B-cell lymphoma on histopathology, which remains the gold standard for diagnosis. Two cases were of primary central nervous system lymphoma, two had disseminated disease, fifth showed allograft involvement, and last case presented with gastrointestinal obstruction. All the patients were managed with reduction of immunosuppression, chemotherapeutic agents, and rituximab. Five patients responded well with a follow-up period of 3-28 months since the time of treatment initiation and had preserved renal function with no episodes of disease recurrence or allograft rejection.

Primary Ewing sarcoma/primitive neuroectodermal tumor of the lung.

We present the autopsy findings and differential diagnosis in a 42year old male who presented with fever and rapidly progressive respiratory symptoms like breathlessness, nonproductive cough and right sided chest pain. Initial imaging workup done at our hospital revealed a large unilateral tumor with tracheal shift. While being evaluated patient developed facial puffiness, tachypnea suggestive of superior vena cava obstruction. Antemortem biopsy of lung mass was attempted twice and that suggested malignant lesion. Unfortunately, the individual had a rapid downhill course following admission. Post mortem examination was conducted that on opening the thoracic cavity revealed total replacement of right lung tissue by a necrotic growth which was deeply adherent to the rib cage. The contralateral lung as well as all other visceral organs were unremarkable grossly. Histopathology confirmed primary Ewing sarcoma of the lung. We hereby, report a rare case of primary lung Ewing sarcoma diagnosed at autopsy.

Real-world experience of eltrombopag in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is characterized by decreased platelet count in the peripheral circulation. The first-line therapy is corticosteroids with 53-80% overall response rate. Eltrombopag has been used as second-line therapy in ITP for over a decade now. The long-term efficacy and safety profile have been widely reported in the western world. However, the data from the resource-constraint settings of the developing world is scarce. We aim to present the real-life experience of efficacy and safety of eltrombopag from the resource-constraint settings. This was a retrospective, single-center study conducted at a tertiary care hospital in Northern India from 2012-2019. On audit of medical records, patients of ITP receiving eltrombopag were screened for inclusion. Patients whose treatment outcomes were not available were excluded. Finally, 53 patients were analyzed using statistical packages of Python v3.7. The patients' median age was 35 years (range 17-78), with 23 (43.4%) being female. The median time to response was 35 days (range 28-50 days) and the cumulative overall response rates (ORR) at day 30, day 60 and day 90 were 41.5%, 69.8%, and 81.1% respectively. A total of 10 patients on eltrombopag relapsed during follow up. The cumulative rate of relapse at one year, three years, and five years were 6.6%, 25.3%, and 47.7%, respectively. There was no significant difference in outcome (response rate or relapse) in any subgroups depending on age, sex, duration of disease, number of prior lines of treatment, splenectomy, or baseline platelet count. Six patients stopped eltrombopag after having a median sustained response for 796 days (range 658-1185), and after a median follow up of 624 days (range 92-1339), they continued to be in remission. Seventeen patients (17/53, 32%) reported one or more adverse events while on eltrombopag therapy. A total of 49 adverse events (n=4, grade ≥3 CTCAEv4) were noted. Anemia was the most frequent adverse event followed by hepatobiliary dysfunction as reflected by deranged AST/ALT or raised bilirubin. The use of eltrombopag among adult ITP patients in resource-constraint settings was well-tolerated and yielded excellent overall response. The benefit was found to be sustained on long-term follow up. However, events like anemia, hepatobiliary, and thrombotic complications merit closer follow up.

Prevalence and Clinical Characteristics of Post-Thrombotic Syndrome in High-Altitude-Induced Deep Vein Thrombosis: Experience of a Single Tertiary Care Center from Real-World Settings.

Uday, Yanamandra, Revanth Boddu, Suman Pramanik, Kundan Mishra, Rajan Kapoor, Ankur Ahuja, Tathagata Chatterjee, and Satyaranjan Das. Prevalence and clinical characteristics of post-thrombotic syndrome in high-altitude-induced deep vein thrombosis: experience of a single tertiary care center from real-world settings. High Alt Med Biol. 21:319-326, 2020. Background: Exposure to high altitude (HA) is a recognized predisposing factor for venous thrombosis. Post-thrombotic syndrome (PTS) is a significant late complication, occurring in ∼30%-50% of patients of deep vein thrombosis (DVT). There are not many studies about the characteristics of PTS in patients with HA-DVT. Aim: The aim was to study the epidemiology and clinical characteristics of PTS using a noninvasive Villalta score and identify the risk factors for its development in patients with HA-DVT. Methodology: This is a retrospective single-center observational study (n = 47). The diagnosis of HA-DVT was confirmed using color Doppler ultrasonography at HA. The patients were managed with low molecular weight heparin, followed by vitamin K antagonist therapy till normalization of D-dimer and imaging. The therapeutic target range of >80% was ensured. Villalta scale was used for PTS assessment. JMP 15.0 was used for statistical analysis. Results: All study participants were male with a median age of 34 years, of which 46.81% developed PTS with mean Villalta of 5.29 ± 4.25. The most common symptom was pain (87.23%; n = 41), whereas the most common sign was hyperpigmentation (42.5%; n = 20). On multivariate analysis, the time from onset of DVT and the extent of DVT were related to the development of PTS (degree of freedom [dF] = 5, χ2 = 17.34, p = 0.0039) with a likelihood ratio of 4.95 (p = 0.026) and 4.96 (p = 0.026), respectively. The extent of DVT was associated with the severity of PTS (dF = 5, χ2 = 12.6, p = 0.0273) with a likelihood ratio of 5.24 (p = 0.022). Conclusions: PTS develops in approximately half of the patients with HA-DVT. The extent of DVT is a significant risk factor for both development of PTS and its severity, whereas time to assessment of PTS from the onset of DVT was associated only with the occurrence of PTS.

Proteomics and functional study reveal marginal zone B and B1 cell specific protein as a candidate marker of multiple myeloma.

Multiple myeloma (MM) is a plasma cell­associated cancer and accounts for 13% of all hematological malignancies, worldwide. MM still remains an incurable plasma cell malignancy with a poor prognosis due to a lack of suitable markers. Therefore, discovering novel markers and targets for diagnosis and therapeutics of MM is essential. The present study aims to identify markers associated with MM malignancy using patient­derived MM mononuclear cells (MNCs). Label­free quantitative proteomics analysis revealed a total of 192 differentially regulated proteins, in which 79 proteins were upregulated and 113 proteins were found to be downregulated in MM MNCs as compared to non­hematological malignant samples. The identified differentially expressed candidate proteins were analyzed using various bioinformatics tools, including Ingenuity Pathway Analysis (IPA), Protein Analysis THrough Evolutionary Relationships (PANTHER), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Database for Annotation, Visualization and Integrated Discovery (DAVID) to determine their biological context. Among the 192 candidate proteins, marginal zone B and B1 cell specific protein (MZB1) was investigated in detail using the RPMI-8226 cell line model of MM. The functional studies revealed that higher expression of MZB1 is associated with promoting the progression of MM pathogenesis and could be established as a potential target for MM in the future.

Post Transplant Lymphoproliferative Disorder.

Posttransplant lymphoproliferative disorder is an extremely fatal complication arising in transplant recipients as a side effect of immunosuppression. PTLDs are seen after both solid organ and hematopoietic stem cell transplants though the incidence is much higher in the former. Primary Epstein-Barr virus (EBV) infection or reactivation due to a state of immune dysregulation along with intensity of immunosuppression used are of paramount importance in pathogenesis of PTLD. EBV associated PTLDs occur early in the post transplant period whereas late onset lymphomas are usually EBV negative. The uncontrolled B cell proliferation can create a spectrum of histological patterns from nondestructive lesions to destructive polymorphic or more aggressive monomorphic PTLDs. Early detection of seropositivity by serial monitoring in the recipient can prevent PTLD development by starting pre-emptive therapy. The mainstay treatment in established cases remains reduction of immunosuppression. Chemotherapeutic and immunomodulatory agents are added sequentially based on the type of PTLD and based on its response to initial therapy. Despite various treatment options available, the morbidity remains high and achieving state of disease remission without causing graft rejection can be quite challenging. Hence, a better understanding in pathobiology of EBV+ versus EBV- PTLDS may help prevent lymphomagenesis in transplant recipients.