Evaluation of Diagnostic Usefulness of CD200 Expression in B-cell Chronic Lymphoproliferative Disorders.

Immunophenotyping by flow cytometry (FCM) is a useful diagnostic tool for the evaluation of mature B-cell neoplasms (MBN). Here, CD200 expression may play a significant role and improve the distinction between various MBNs, but any potential as a prognostic marker is yet to be established. The present prospective study was conducted on all the suspected cases of MBNs. Immunophenotyping was done using a BD FACS Canto FCM using a panel of 4 to 6 color combinations of monoclonal antibodies; CD45, CD34, CD5, CD19, CD20, CD22, CD23, CD79b, FMC7, CD10, CD38, ZAP70, CD200, IgG, IgM, CD25, CD103, CD2, CD3, CD11c as well as κ and λ light chains. CD200 expression was compared in different subgroups. Of the total of 130 cases included in the study, CD200 was positive in 118 cases (90%). CD200 was expressed in 100% of the cases of CLL(86 cases), atypical CLL(06 cases), HCL(14 cases), FL(02 cases), SMZL(04 cases), LPL (01 case), and low-grade NHL (05 cases), with the highest intensity of fluorescence in HCL followed by CLL. All the cases of MCL and PLL were exclusively negative for CD200. In conclusion, the results of the present study support inclusion of this marker in the flow cytometric panels for the differential diagnosis of MBNs.

Coomb's negative cold agglutinin disease: A rare report of an incidentally detected case.

Cold agglutinin disease (CAD) is a rare type of autoimmune hemolytic anemia which usually results due to production of immunoglobulin M-type autoantibody against the I/i and H antigens on red blood cell membrane. They can be idiopathic or may be due to underlying lymphoproliferative disorders or atypical infections. It can have a varied presentation ranging from being incidentally detected to being totally transfusion dependent for a longer or shorter duration. Several factors play a role in determining the ability of cold agglutinins in inducing hemolysis such as antibody concentration and temperature. Here, we present a 54-year-old patient, a known case of chronic obstructive pulmonary disease who was admitted to our hospital in the winter months as a case of alcohol withdrawal syndrome. During the course of the stay, the patient developed respiratory insufficiency and went into Type II respiratory failure and hematological investigations revealed features of CAD.

Isolated CNS Relapse in Acute Lymphoblastic Leukemia (ALL): An Experience from a Tertiary Care Center.

Aims and Objectives In this study the various parameters of acute lymphoblastic leukemia (ALL), including the clinical features, peripheral blood and bone marrow (BM) findings, immunophenotypic and cytogenetic details in ALL cases who had isolated relapse involving the central nervous system (CNS), were studied. Patients/Materials and Methods Duration of the study is from 2015 to 2019 in which 5 ALL cases were presented to this tertiary care center. The presenting symptoms varied from headache, fever, and distension of abdomen. These cases were either on therapy or post completion of chemotherapy. The diagnosis of CNS relapse followed after the examination of cerebrospinal fluid (CSF). Patients also underwent BM examination to rule out systemic relapse. Results Age of patients ranged from 7 months to 42 years. There were three female patients. Two patients had isolated CNS relapse 3.5 years after completing therapy and succumbed to their illness. Two patients had t(9;22) while one patient had t(1;14) cytogenetic abnormality at diagnosis. One patient was diagnosed as T-ALL. Treatment offered was German Multicentre ALL protocol for induction along with 10 cycles of maintenance. Conclusion The most common hematolymphoid malignancy in children namely ALL accounts for 75% of childhood leukemias. Complete remission rates reach up to 70 to 80%. CNS involvement is known to occur in these cases. CNS relapse may occur alone or with systemic relapse. Advances in therapeutic protocols along with CNS prophylaxis have drastically brought down the rates of CNS relapse. It is essential to maintain a high degree of suspicion so that these cases of isolated CNS relapse can be identified at the earliest and definitive therapy can be offered.

Safety and efficacy of splenectomy in immune thrombocytopenia.

BACKGROUND: Immune Thrombocytopenia (ITP) is characterized by low platelet counts. Splenectomy has been in practice for the treatment of ITP since the early 20th century. We aimed to analyze the data of ITP patients from our hospital who underwent splenectomy and further present the long-term outcome and safety profile in these patients. METHOD: This study was a single-center, registry based study conducted at a tertiary care hospital in Northern India. Patients aged 18 years or more, who underwent splenectomy after at least one line of therapy, were included in the study. The primary outcome was the overall response rate (ORR) at one month after splenectomy. Secondary outcomes were sustained response, relapse-free survival, factors affecting the ORR, and adverse events after splenectomy. RESULTS: Forty-five patients of ITP were included in the study. Thirty-six patients underwent splenectomy in the first half (2001-2010), of the study period. The median age of the patients was 38 (19-56) years. The median duration from diagnosis to splenectomy was 1.76 (0.47-2.58) years. The median number of therapy received before splenectomy was 3 (1-6). The overall response rate (ORR) post-splenectomy at day 30 was 89.2% with 61.8% complete response (CR). The ORR was 88.5% at 1-year, with 48.8% CR. The relapse-free survival (RFS) at 5-years was 57.38% (95% Confidence Interval 40.59-71.02%), There was no effect of duration of disease, age, gender, and prior therapy received, on the ORR at one-month. At one year, the platelet response was significantly better in patients who had a CR at one-month than patients who had a partial response at one month. The relapse-free survival was better in patients who achieved CR after 1-month of splenectomy. During the median follow-up of 5.02 (1 month-20 years) years, there were five cases of overwhelming post-splenectomy infection (OPSI). There was no recorded incidence of perioperative mortality, deep vein thrombosis, or mesenteric thrombosis. DISCUSSION: Despite the variation in outcome from different studies, splenectomy gives the best possible long-term treatment-free remission amongst all the available second-line agents. It is also, one of the most financially affordable therapies. Despite advantages, the number of ITP patients undergoing splenectomy has been on the decline and largely attributable to the newer and more effective second-line therapies. There is no pre-surgery variable predicting the ORR after splenectomy. CONCLUSION: Splenectomy in ITP offers a long-term sustained response at an economical cost.

Safety and efficacy of azathioprine in immune thrombocytopenia.

BACKGROUND: Immune thrombocytopenia (ITP) is a benign hematological disorder characterized by low platelet counts in peripheral blood and spectrum of various bleeding manifestations. Azathioprine is one of the effective, readily available, and affordable immunosupressants available for ITP management in developing countries. We aimed to study the efficacy and long-term safety profile of our patients with ITP who were treated with azathioprine. METHOD: This was a retrospective, single-center study conducted at a tertiary care hospital in Northern India. The patients who had received at least one line of therapy before receiving azathioprine were included in this study. All patients received oral azathioprine at a dose of 1 mg/kg/day (50 mg or 100 mg tablet formulations were used), which was increased up to 2 mg/kg/day depending upon the response and adverse effects. RESULT: Sixty-three patients were analyzed. Their median age was 28 years (range 15-68); 29/63 patients (46.03%) were females. The median duration from diagnosis to azathioprine initiation was 539 days (323 days-980.5 days). The patients included in the study had received a median of 3 (range 1-6) prior lines of therapies; 38/63 patients (60.32%) had received ≥3 prior therapies. Six patients (9.5%) had relapsed after splenectomy, and 16 patients (25.4%) had relapsed after receiving rituximab. The mean baseline platelet count was 10000/µL. The median time to response was 95 days (90 days-not reached) and the cumulative overall response rate (complete and partial response) at day 90 was 38.1%. Only one patient achieved complete response with azathioprine in our study. The cumulative rate of relapse at five years was 21.2%. Twenty-six patients stopped azathioprine after achieving some response (CR/PR) with Azathioprine for a median duration of 1067.5 days (range: 236 days-2465 days). They were followed up for a median of 870 days (range: 392 days-1928 days), and twelve of them relapsed. Twenty-six patients (26/63, 41.27%) reported one or more adverse events while on azathioprine. Leucopenia was the most frequent adverse event, followed by anemia and hepatobiliary laboratory abnormalities. Serious adverse events (grade ≥3 CTCAEv4) were noted in three patients (4.7%). One patient succumbed to severe sepsis multiorgan dysfunction while being on treatment. CONCLUSION: We conclude that azathioprine has a good response rate in chronic ITP patients. It is well-tolerated with minimal and manageable side effects.

A study on DNA methylation status in promoter region of p15 gene in patients of acute myeloid leukemia and myelodysplastic syndrome.

BACKGROUND: Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are a spectrum of hematological malignancies with a multistep process of accumulated genetic and epigenetic alterations. DNA methylation is most extensively studied epigenetic alteration in malignancies. Recent research studies in the field have brought out translational implications of promoter methylation of tumor suppressor gene p15 in tumors. Therefore, we studied the role of DNA Methylation of p15 gene in AML and MDS. METHODS: The study was carried out in 41 consecutive AML/MDS cases reporting to hematological OPD of a tertiary care center along with 25 age and sex-matched healthy controls. The methylation status in the promoter region of the p15 gene was assessed by methylation-specific PCR (MSP) from blood samples after ethical approval and informed consent of the patients and controls. The association of methylation status was studied with clinical presentations, AML subtypes, and cytogenetics using Chi-square test/Fisher's exact test tools. RESULTS: A total of 41 cases included in the study comprised 33 cases of AML and 08 cases of MDS with an age range between 06 months and 82 years. Of the 41 cases, 29 revealed promoter methylation of the p15 gene, which compared to healthy controls was found statistically significant (p < 0.001). The methylation status did not significantly correlate with AML subtypes or the cytogenetic abnormalities detected in cases. CONCLUSION: The outcome of the study indicates p15 promoter DNA methylation in cases of AML and MDS may identify those individuals who might benefit from the targeted therapeutic approaches.

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.

Proteomic Alterations in Multiple Myeloma: A Comprehensive Study Using Bone Marrow Interstitial Fluid and Serum Samples.

Multiple myeloma (MM) is a plasma cell-associated cancer and exists as the second most common hematological malignancy worldwide. Although researchers have been working on MM, a comprehensive quantitative Bone Marrow Interstitial Fluid (BMIF) and serum proteomic analysis from the same patients' samples is not yet reported. The present study involves the investigation of alterations in the BMIF and serum proteome of MM patients compared to controls using multipronged quantitative proteomic approaches viz., 2D-DIGE, iTRAQ, and SWATH-MS. A total of 279 non-redundant statistically significant differentially abundant proteins were identified by the combination of three proteomic approaches in MM BMIF, while in the case of serum 116 such differentially abundant proteins were identified. The biological context of these dysregulated proteins was deciphered using various bioinformatic tools. Verification experiments were performed in a fresh independent cohort of samples using immunoblotting and mass spectrometry based SRM assays. Thorough data evaluation led to the identification of a panel of five proteins viz., haptoglobin, kininogen 1, transferrin, and apolipoprotein A1 along with albumin that was validated using ELISA in a larger cohort of serum samples. This panel of proteins could serve as a useful tool in the diagnosis and understanding of the pathophysiology of MM in the future.

Utility of Lupus Anticoagulant Assays (APTT-LA, KCT, DPT and DRVVT) in Detection of Antiphospholipid Syndrome (APS) in High Risk Pregnancy Cases.

Routine investigation for recurrent pregnancy loss includes measurement of antiphospholipid antibodies. The lupus anticoagulant has long been associated with increased risks for thrombosis and adverse obstetric outcomes. But there are some disadvantages with lupus anticoagulant (LAC) tests which includes varied sensitivity of different clot based assays. ISTH recommends only 2 assays (preferably DRVVT and APTT-LA) for the identification of lupus anticoagulant but there are some studies which don't support this contention. Our study analyzed 526 samples from high risk pregnancy cases for APLA by all four LAC tests from tertiary centre of northern India. Among all the cases studies 65 cases were positive for lupus anticoagulant 25 of this became negative after 12 weeks. Among the 40 repeated positive assays, dRVVT could able to diagnose 36 cases followed by APTT-LA which could able to diagnose 28 cases, while KCT could able to diagnose 23 cases and dPT could able to diagnose only 14 cases. There were 12 cases in whom all lupus assays were positive. Our study thus concluded that DRVVT was the most sensitive followed by APPT-LA, KCT, dPT. The combination of dRVVT with APTT-LA or KCT appeared to be superior to other combinations. No individual test per se is 100% sensitive for the diagnosis of APLA in high risk pregnancy cases. Further results confirmed that repeated LAC result is required even in a high-risk setting. Positive LAC assay in majority were not associated with exclusively recurrent pregnancy loss but were associated with sporadic stillbirth and thrombosis.

Comparison of Immunohistochemistry, Cytochemistry, and Flow Cytometry in AML for Myeloperoxidase Detection.

Acute Myeloid Leukemia (AML) as per World Health Organization (WHO 2008) classification is on the basis of the antigenic characterization, enzymes restriction in the neoplastic myeloid cells and the specific translocations/mutations. AML can be assessed and differentiated by flowcytometry (FCM)/immunohistochemistry (IHC)/cytochemistry techniques. Myeloperoxidase (MPO) is an unequivocal marker to differentiate AML from the acute lymphoblastic leukemia. Despite FCM popularity, it has its limitations, in form of 'dry-tap', cost, and inability of being performed by retrospective analysis. IHC, though an old technique has overcome these disadvantages of FCM. Cytochemistry, on the other hand has its own advantages in being cost-effective; technically easy to do while its disadvantages are its inability to be carried out in the old samples, 'dry-tap' conditions in aleukemic leukemia. There has been non-uniformity in the literature among these techniques especially concerning their sensitivity for MPO. A prospective study was done at All India Institute of Medical Sciences New Delhi from 01 July 2014 to 30 Nov 2015 to include 120 diagnosed acute myeloid leukemia cases. Myeloperoxidase stain was done by cytochemistry, immunohistochemistry and flow cytometry and results were compared. There were 28 cases which showed discrepancies. Out of these 28 cases immunohistochemistry showed positivity in majority (22 cases) followed by flow cytometry (14 cases). Therefore it is important to employ more than one technique and IHC must be included for detection of MPO in all suspected cases of AML especially when negative with FCM .

Evaluation of Bone Marrow Microvessel Density in Patients with Aplastic Anemia.

Bone marrow microenvironment plays a crucial role in the growth of hemopoietic cells and bone marrow function, which in turn depends on an intact microvasculature. Our study assesses the microvessel density (MVD) in the bone marrow of aplastic anemia (AA) patients, compares with MVD of controls and MVD among the different types of AA. Bone marrow specimens from 60 patients with AA and 17 controls were studied. There were 33 patients with non severe AA (NSAA), 12 patients with severe AA (SAA) and 15 patients with very severe AA (VSAA). MVD was calculated on sections stained immunohistochemically for CD34. The mean bone marrow MVD in AA group was 1.28 ± 0.36, being significantly lower than that in control group (6.80 ± 1.59, p < 0.001). MVD of SAA and NSAA patients were 1.16 ± 0.35 and 1.49 ± 0.27, respectively, being significantly different (p = 0.003). MVD of VSAA was 0.93 ± 0.25 and the difference with NSAA is significant, however there was no significant difference between SAA and VSAA. Bone marrow MVD is low in AA patients and is likely to have a role in pathophysiology of bone marrow failure. Proangiogenic agents together with specific therapy might accelerate the recovery of hematopoiesis in AA patients.

Flow cytometric detection of minimal residual disease in B-lineage acute lymphoblastic leukemia by using "MRD lite" panel.

BACKGROUND: Normal B lymphoid precursors that express CD19, CD10, and/or CD34 are highly sensitive to corticosteroids and after two weeks of remission-induction therapy, they form less than 0.01% of the bone marrow population. More than 0.01% of such cells indicate minimal residual disease (MRD). MRD "lite" panel uses only three antibodies, namely CD19, CD10, and CD34 for MRD detection in cases of B-lineage acute lymphoblastic leukemia (B-ALL) expressing CD19, CD10, and/or CD34 by flow cytometry. METHODS: Fifteen cases of B-ALL were studied for MRD at Day 19 of remission-induction therapy by employing a simplified MRD detection protocol using a 3-color fluorochrome conjugated antibody panel (CD19, CD10, and CD34) on bone marrow aspirate samples. RESULTS: All cases at diagnosis expressed CD19, CD10, and CD34. Of fifteen patients, five (33.33%) were MRD negative with less than 0.01% of mononuclear cells and remaining ten cases (66.66%) were MRD positive, with the level of 0.01% to less than 0.1% cells. CONCLUSION: The MRD assay used in this study is a simplified method for detecting MRD at Day 19 of remission-induction therapy for B-lineage ALL. This MRD assay is an effective and useful methodology in cases of B-ALL expressing CD19, CD10, and/or CD34 by flow cytometry.

Leukemic Transformation of Severe Aplastic Anemia Following Matched Allogenic Stem Cell Transplantation, Transplanted Again in CR 1.

Aplastic anemia (AA) is a life-threatening bone marrow failure disorder, if untreated, is associated with very high mortality. Allogenic bone marrow transplantation (BMT) is the standard of care for severe aplastic anemia (SAA) patients those who are younger than 40 years of age. The development of secondary malignancies in post-BMT setting for AA is a rare, however, well documented phenomenon. Among the secondary malignancies, development of acute myeloid leukemia is even rarer entity. Here we report a case of acute myeloid leukemia following human leucocyte antigen (HLA) matched sibling peripheral blood stem cell transplant (PBSCT) in a case of SAA. The patient achieved complete remission (CR) following chemotherapy and in CR1, a second HLA matched PBSCT from a different donor was offered. The patient is presently in remission at day +180 post-PBSCT.

Transfusion-acquired Hemoglobinopathies: A Report of Two Cases.

Transfusion-acquired hemoglobinopathy occurs when a carrier of hemoglobinopathy with no significant abnormalities donates blood, and the blood is transfused to a recipient. This process can lead to spurious results in the recipient without any clinical abnormality or infrequently can result in disastrous situations. The incidental finding of such posttransfusion related abnormal peaks in hemoglobin high-performance liquid chromatography (Hb HPLC) may cause diagnostic dilemmas and result in unnecessary laboratory testing. Here, we report two such cases of transfusion-acquired hemoglobinopathies, which were subsequently resolved by the abnormally low percentage of the Hb variants, transient nature of the peaks, and parental Hb HPLC.

Can threshold for MPO by flow cytometry be reduced in classifying acute leukaemia? A comparison of flow cytometric and cytochemical myeloperoxidase using different flow cytometric cut-offs.

Objectives Myeloperoxidase (MPO) detection either by enzyme cytochemistry (cMPO) or flow cytometry (fMPO) plays a major role in acute leukaemia (AL) diagnosis as per World Health Organization (WHO) 2008 classification. Although 3% cMPO was recommended as positivity, no specific cut-off had been mentioned by WHO for fMPO. Various authors recommend different cut-offs ranging from 3 to 28% for fMPO. The aim of this study was to analyse fMPO cut-offs ranging from 3 to 10% in classifying AL and to assess whether a new cut-off could be suggested. Methods Totally, 216 cases of AL were retrospectively analysed for fMPO ranging from 3 to 10% and compared with gold standard. Presence of cMPO (≥3%) and/or expression of two or more pan-myeloid markers (CD13, CD33, and CD117) in the absence of CD19 and CD3 were kept as gold standard for diagnosis of acute myeloid leukaemia (AML). Results Sensitivities for classifying AL as AML/mixed phenotypic acute leukaemia (MPAL) at 3, 5.4, and 10% were 98.3, 98.3, and 96.6%, respectively, whereas specificities at this cut-off were 22.2, 91, and 71%, respectively. Discussion Only few studies have been done in this aspect to define a consistent cut-off for fMPO for proper classification of acute leukaemias. This was one of the largest and few studies available till date in this regard. Conclusion The newer cut-off for fMPO (5.4%) emerged out from our study with best sensitivity and specificity for accurately classifying AL cases into acute lymphoblastic leukaemia, AML, and MPAL.

Hairy cell leukemia: A decade long experience of North Indian Hematology Center.

INTRODUCTION: Hairy cell leukemia is a rare chronic B-cell disorder that follows an indolent but progressive course. This disorder is characterized by pancytopenia, splenomegaly, bone marrow fibrosis and the presence of atypical lymphoid cells with hairy projections in peripheral blood, bone marrow and spleen. Treatment is mainly with nucleoside analog cladribine, which induces complete remission in up to 85% cases. MATERIALS AND METHODS: This is a retrospective analysis of Hairy cell Leukemia cases diagnosed and treated in the Department of Hematology, All India Institute of Medical Sciences, New Delhi between 2002 and 2013. Various parameters such as clinical features, laboratory parameters including complete blood cell count, bone marrow findings, cytochemistry, immunophenotyping by flowcytometry or immunohistochemistry, treatment protocol and complications secondary to treatment and relapse were reviewed. RESULTS: A total of 35 cases were diagnosed during this period of 12 years of which 27 received cladribine and went in to remission. Median follow-up duration was 26 months. 5 (18%) cases had a relapse and all relapsed cases achieved second remission with cladribine; however, there was no case of second malignancy in our cohort. CONCLUSION: Cladribine has emerged as the treatment of choice for hairy cell leukemia given that the overwhelming majority of patients achieve long-lasting complete remissions. Upon relapse, these patients could be successfully salvaged with cladribine retreatment.

Aberrant myeloid antigen co-expression is correlated with high percentages of CD34-positive cells among blasts of acute lymphoblastic leukemia patients: an Indian tertiary care center perspective.

BACKGROUND: Aberrant myeloid antigen (MA) co-expression and high expression of CD34 antigen on the blasts of acute lymphoblastic leukemia (ALL) patients are independently reported to have a role in pathogenesis and prognosis. This study was conducted to determine whether these two parameters are related. METHODS: A total of 204 cases of ALL were included in an analysis of blast immunophenotypic data. CD34 expression was categorized as low when less than 50% of blasts were CD34-positive (CD34(low)) and as high when 50% or more were CD34-positive (CD34(high)). RESULTS: Of 204 cases of ALL, 163 and 41 were of B-cell origin (B-ALL) and T-cell origin (T-ALL), respectively. Of all cases, 132 (64.7%) showed co-expression of MA and among these, 101 (76.51%) were CD34(high), while the remaining 31 (23.48%) were CD34(low). Of 72 cases without MA co-expression, 25 (34.72%) were CD34(high) and 47 (67.25%) were CD34(low). Furthermore, of 163 cases of B-ALL, 111 showed co-expression of MA and 84 of these were CD34(high). Of 52 cases of B-ALL without MA expression, 22 were CD34(high). Among 41 cases of T-ALL, 21 co-expressed MA, 17 of which were CD34(high). Moreover, all 20 cases of T-ALL without co-expression of MA were CD34(low). These differences were statistically significant. CONCLUSION: We observed a strong correlation between aberrant MA expression and CD34(high) expression on the blasts of ALL. We hypothesize that these different patient subsets may represent unique prognostic characteristics.