Acute panmylelosis with myelofibrosis - A rare case report with review of literature.

ABSTRACT: Acute panmyelosis with myelofibrosis (APMF) corresponds to <1% cases of acute myeloid leukemia, which could be an underestimation due to missed diagnosis. Due to its rapidly fatal course, it warrants a timely and correct diagnosis. We present a case of a 44-year male who came with a short history of fever, generalised weakness, revealed pancytopenia with occasional circulating blast in the peripheral blood smear. Bone marrow aspirate was dry tap,biopsy revealed panmyelosis with myelofibrosis with increased (22%) blasts. Flowcytometric immunophenotyping, cytogenetics and molecular tests were undertaken. Together with clinical details, immunophenotypic profile, cytogenetics and molecular studies, the diagnosis of Acute panmyelosis with myelofibrosis was made and managed accordingly. 32 The WHO 2017 describes APMF as an acute panmyeloid proliferation with increased blasts (≥20% in the bone marrow or peripheral blood) and accompanying marrow fibrosis. APMF is rare with poor prognosis thus, must be differentiated especially from Acute megakaryoblastic leukemia to arrive at the correct diagnosis which will help reduce/prevent the early mortality by providing timely chemotherapy followed by upfront hemopoietic stem cell transplantation.

Clinical and Laboratory Profiles of 17 Cases of Chronic Granulomatous Disease in North India.

A primary immune deficiency disorder is often suspected in children with recurrent deep seated and fungal infections and those admitted to pediatric intensive care units. Chronic granulomatous disease (CGD) is inherited disorder leading to infections caused due to defective superoxide production. Cases referred for testing for a primary immunodeficiency disorder were tested for Dihydrorhodamine 123 (DHR) assay by flow cytometry and nitroblue tetrazolium dye (NBT) slide test. The unstimulated and stimulated samples were tested for oxidative burst activity which gives bright fluorescence due to formation of Rhodamine 123 on flow cytometry and blue formazan pigment in NBT slide test. The test results were reported in real time. From a total of 330 patients screened for chronic granulomatous disease using DHR and NBT slide test, 17 patients (5.1%) were found to have CGD. These included 12 boys and 5 girls. They presented with deep seated infections, recurrent and multiple abscess, recurrent pneumonia and granulomatous lymphadenitis. The causative organisms were Mycobacteriae, Staphylococcus, Burkholderia cepacia, Pseudomonas, Aspergillus and Cytomegalovirus. In 6 out of 17 positive cases family studies were carried out. On follow up five children succumbed to disease, two patients underwent allogeneic bone marrow transplant, the chimerism status was demonstrated by repeat DHR assay at day 50 post-transplant. Rest are in follow up under prophylactic antibiotics and supportive care. As facilities for molecular testing are not easily available for primary immuno deficiency disorders, flow cytometry based clinical laboratories can help to screen for some of the frequently suspected disorders like chronic granulomatous disease. This has aided in paediatric care in our centre.

Evaluation of multiparametric flow cytometry in diagnosis & prognosis of myelodysplastic syndrome in India.

Background & objectives: Diagnosis of myelodysplastic syndromes (MDS) is subjective in low-grade cases with <5 per cent blasts or <15 per cent ring sideroblasts. Flow cytometry (FCM) has been used to diagnose MDS; but, it still has only an adjunctive role. This study was conducted to evaluate the role of FCM to diagnose MDS and correlate the number of aberrancies with revised international prognostic scoring system (R-IPSS). Methods: This study included 44 consecutive clinically suspected cases of MDS with refractory cytopenia(s) and 10 controls. Patients were divided into two groups: (i) proven MDS cases (n=26), and (ii) suspected MDS (n=18). Ogata quantitative approach, pattern analysis and aberrant antigen expression were studied. Results: Ogata score ≥2 correctly diagnosed 80.7 per cent (21/26) while aberrant antigen and pattern analysis with flow score of ≥3 could diagnose 92.3 per cent (24/26) patients with proven MDS. Combination of both with flow score ≥3 could diagnose 100 per cent patients. Eight patients in suspected MDS group with persistent cytopenia on follow up were labelled as probable MDS. Ogata score ≥2 was present in 5 of 8 and pattern analysis score ≥3 was present in six probable MDS patients. Combination of both with flow score ≥3 was present in seven of eight patients. Spearman's correlation between Ogata score and R-IPSS, pattern analysis and R-IPSS and combination of both scores and R-IPSS showed significant positive correlation in proven MDS as well as when proven and probable MDS patients were combined. Interpretation & conclusions: Our results showed that combined Ogata approach and pattern analysis, demonstration of ≥3 aberrancies in >1 cell compartment could diagnose most MDS patients. Patients with high flow scores had high R-IPSS scores. Patient with flow score ≥3 and borderline cytomorphology should be observed closely for the development of MDS.

A Prospective, Cross Sectional Study of PNH Clone in MDS Patients Using High Sensitivity Flowcytometry: A Single Center Experience.

Subclinical PNH can be present in patients with bone marrow failure like aplastic anemia and myelodysplastic syndrome (MDS). Such clone may have prognostic and therapeutic implications. In literature around 1-10% MDS cases have shown a PNH clone, however, data from India is relatively scarce. A high sensitivity PNH assay was employed using a single tube combination of FLAER, CD157, CD64, CD15 and CD45 antibodies in adult patients of MDS at presentation. A clone size of  > 0.01% was taken as significant. A total of 30 patients were included. PNH clone was present in 30% cases. Correlation done between PNH clone size and LDH values showed moderately positive correlation (r = 0.735, p = 0.001, r2 = 0.541). As per this study a LDH cut off of 247 IU is likely to predict a PNH clone (> 1%) with moderate sensitivity and specificity. High sensitivity PNH assay is able to detect small PNH clone. Calculating the cut-off of LDH to predict PNH positivity can help us judiciously prescribe this test in MDS patients in resource constrained settings.

Aspirin and Clopidogrel Resistance in Indian Patients with Ischemic Stroke and its Associations with Gene Polymorphisms: A Pilot Study.

INTRODUCTION: Antiplatelet resistance is one of the urgent issues in current stroke care. One-third to one-half of the patients who experience a recurrent stroke is already on antiplatelet medications. We studied resistance to aspirin and clopidogrel in Indian stroke patients and its association with gene polymorphisms. METHODS: Platelet function testing by light transmission aggregometry was performed on 65 patients with ischemic stroke who were stable on dual antiplatelet therapy (clopidogrel 75 mg OD and aspirin 75 mg OD) along with 65 age-matched controls. Aspirin resistance was considered as mean platelet aggregation ≥70% with 10 µM adenosine diphosphate (ADP) and ≥20% with 0.75 mM arachidonic acid. Clopidogrel resistance was defined as <10% decrease from the baseline in platelet aggregation in response to ADP 10 µM and semi-response as <30% decrease from the baseline. Polymorphisms CYP2C19 * 2 and GPIIb/IIIa (PLA1/A2) were genotyped by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: We found 64.6% (42/65) patients with inadequate response to clopidogrel (15.4% [10/65] resistant and 49.2% [32/65] semi-responders) and 4.6% (3/65) patients with inadequate response to aspirin (3.1% [2/65] resistant and 1.5% [1/65] semi-responder). The frequency of CYP2C19*2 mutant genotype was significantly higher in clopidogrel nonresponders compared to responders (P = 0.014). Clopidogrel nonresponsiveness was much higher in small vessel stroke. CONCLUSION: Unlike aspirin, a high proportion of nonresponders to clopidogrel was identified. In an interim analysis on 65 Indian patients, a significant association was found between CYP2C19*2 and clopidogrel nonresponsiveness.

Platelet function tests: A 5-year audit of platelet function tests done for bleeding disorders in a tertiary care center of a developing country.

INTRODUCTION: The platelet function disorders remain largely undiagnosed or incompletely diagnosed in developing nations due to lack of availability of tests like lumiaggregometry, granule release assay or molecular testing. We performed a retrospective analysis of all the platelet function test (PFT) carried out in past 5 years by Light transmission aggregometery (LTA) using a panel of agonist. The indications and the test results were analyzed by two hematopathologist with the aim to look into the present diagnostic facilities or lack of it for correct diagnosis. This is essential for better management and genetic counselling. MATERIALS AND METHODS: The PFT was performed both on patients and healthy unrelated age specific controls by light transmission aggregometry on Chronolog platelet aggregometer using platelet rich plasma. The panel of agonists included ADP (10µm/l and 2.0 µm/l), epinephrine (10.0 µm/l), collagen (2µg/ml), arachidonic acid (0.75 mM) and ristocetin (1.25 mg/ml & 0.25 mg/l). RESULTS: The 5 years records of 110 cases were audited, 101 of these were tested for clinical bleeding , 35 adults and 66 children. The adults included 29 women and 6 men, 17 to 82 years of age. The children were 16 years to 3 months of age, 30 girls and 36 boys. Platelet function test abnormality was found in 31.6% (32/101) cases ,a majority remained undiagnosed of these about 21% had clinically significant bleeding.The cases diagnosed included Glanzmann Thromboasthenia-11 , von Willebrand Disease-6, Bernard Soulier'syndrome-1, storage pool disorder-6, mild defect of Epinephrine-3, isolated defect with collagen in1. CONCLUSION: An epidemiologically large proportion of platelet function disorders amongst people living in developing nations remain undiagnosed. This lacunae needs to be highlighted and addressed on larger scale. The options available are to increase the available armamentarium of tests or international collaboration with a specialized laboratory to aid in complete diagnosis.

C-Window Peaks on CE-HPLC are Extremely Rare in Northern India, and Only Infrequently Represent HbC.

Hemoglobin C (HbC, HBB:c.19G > A) is a structural variant that has been reported rarely from India. This was a retrospective review of all high performance liquid chromatography (HPLCs) submitted over a 14 year period to a tertiary care center in North India with an aim of finding hemoglobins that elute in the C-window. Of the 32,364 HPLCs screened, 6 cases showed peaks in the C-window. Of these 6 cases, only two cases contained hemoglobin C. These was one case each of HbC/ß thalassemia and compound heterozygosity for HbC and HbD. There were 4 cases which showed very similar red cell indices and chromatograms with multiple peaks eluting in D-window, C-window and an additional peak with a retention time of 4.74 min. These four cases were compound heterozygous for an α chain variant HbQ-India and a ß-chain variant HbD.

Comparison of the characteristics of two hemoglobin variants, Hb D-Iran and Hb E, eluting in the Hb A2 window.

BACKGROUND: Cation exchange-high performance liquid chromatography (CE-HPLC) is most commonly used to evaluate hemoglobin (Hb) variants, which elute in the Hb A2 window. This study aimed to assess prevalence of an uncommon Hb variant, Hb D-Iran, and compare its red cell parameters and peak characteristics with those of Hb E that commonly elutes in the Hb A2 window. METHODS: Generally, we assess abnormal Hb using CE-HPLC as the primary technique along with alkaline and acid electrophoresis. All cases with Hb A2 window >9%, as assessed by CE-HPLCs during 2009-2013, were selected. RESULTS: Twenty-nine cases with Hb D-Iran variant were identified-25 heterozygous, 2 homozygous, 1 compound heterozygous Hb D-Iran/ß-thalassemia, and 1 Hb D-Iran/Hb D-Punjab. Overall prevalence of Hb D-Iran was 0.23%. Compared to patients with Hb E, those with Hb D-Iran had significantly higher Hb (12.1 vs. 11.3 g/dL, P=0.03), MCV (82.4 vs. 76.4 fL, P=0.0044), MCH (27.9 vs. 25.45 pg, P =0.0006), and MCHC (33.9 vs. 33.3 g/dL, P=0.0005). Amount of abnormal Hb (40.7 vs. 26.4%, P=0.0001) was significantly higher while retention time (3.56 vs. 3.70 min, P=0.0001) was significantly lower in Hb D-Iran than in Hb E. CONCLUSION: Hb D-Iran peak can be easily missed if area and retention time of the Hb A2 window are not carefully analyzed. To distinguish between variants, careful analysis of peak area and retention time is sufficient in most cases and may be further confirmed by the second technique-alkaline electrophoresis.

Concomitant Presence of Two Distinct Clones of Chronic Lymphocytic Leukemia and Plasma Cell Myeloma in a Patient.

A 74 years old male patient, presented with history of generalized weakness, fatigue, loss of appetite and breathlessness on exertion for past one and a half months. On examination, he was found to have significant pallor and generalized lymphadenopathy (cervical, axillary and inguinal). The skeletal survey showed punched out lytic lesions in skull and pelvic bones. The peripheral smear examination showed lymphocytosis with absolute lymphocyte count of 25,000/µL. The bone marrow aspirates revealed a hypercellular marrow with 74 % lymphocytes & 14 % plasma cells, suggestive of chronic lymphoplasmacytic disorder. The bone marrow biopsy had two morphologically distinct populations of lymphocytes & plasma cells. The immunohistochemical markers on bone marrow biopsy showed hat plasma cells were positive for CD138 with kappa light chain restriction. Flow cytometry showed B cell population with CD19/CD5 co expression, CD5/CD23 coexpression, were positive for CD22, CD20 and negative for FMC-7 and lambda light chain. In addition, plasma cells were also identified as CD45 negative cells and showed CD38/CD138 co-expression with variable CD19 and CD56 positivity. Serum protein electrophoresis revealed M band, serum immunofixation electrophoresis corresponded to IgA -Kappa. The final diagnosis of chronic lymphocytic leukemia with concomittant presence of plasma cell myeloma was concluded. This case imparts an important message to look for presence of coexisting entities in a single specimen and highlights the benefits of testing both plasma cell and B-cell compartments when the clinical features are not entirely consistent Flow cytometry together with protein electrophoresis can help to clinch difficult and rare dual diagnosis. These cases are rare and pose therapeutic challenge.

Compound heterozygous hemoglobin d-punjab/hemoglobin d-iran: a novel hemoglobinopathy.

Cation exchange high performance liquid chromatography (CE- HPLC) is an excellent tool for the diagnosis of various hemoglobin (Hb) disorders. HbD-Punjab is an uncommon structural Hb variant seen in North-India. Rarely, a compound heterozygous state for HbD-Punjab with high HbA2 has been described. We describe an index case whose CE-HPLC showed a compound heterozygous state for Hb-Punjab/HbD-Iran which was confirmed by family study, acid and alkaline electrophoresis and beta gene sequencing. This case highlights the role of alkaline and acid electrophoresis to resolve common peaks that elute with HbA2 on CE-HPLC. To the best of our knowledge, this compound heterozygous state of HbD-Punjab with HbD-Iran has not been reported earlier.

Expression of CD20 in B Cell Precursor Acute Lymphoblastic Leukemia.

CD20 is a B cell differentiation antigen with variable expression in B cell precursor acute lymphoblastic leukemia (BCP-ALL). The significance of CD20 expression has been evaluated in BCP-ALL with conflicting results. There is paucity of data regarding CD 20 expression in BCP-ALL in Indian patients. We retrospectively analyzed 100 patients of BCP-ALL for CD20 expression. CD20 positivity was defined as expression of CD20 to be more than or equal to 20 % in the blast population. 62 % patients expressed CD20 while 38 % patients were negative for CD20. The positivity ranged from negative to dim (35.5 % patients), moderately bright (19.3 % patients) to bright (45.2 % patients). Additional prospective studies are needed to determine the optimal use of rituximab in treatment of CD20-positive BCP-ALL.

A Bi-Lineal Acute Leukemia (T/Myeloid, NOS) with Complex Cytogenetic Abnormalities.

We report an unusual case of bi-lineal mixed-phenotype acute leukemia (T/Myeloid, NOS) with complex cytogenetic abnormalities in a 2-year-old boy. Despite attaining complete remission with therapy, he succumbed to status epilepticus following febrile illness. Flow cytometry represents the current standard of care for the diagnosis of this malignancy and the approach adopted in our case is discussed.