Clinico-Hematological Profile of Paroxysmal Nocturnal Hemoglobinuria in Indian Patients: FLAER Flow Cytometry Based Experience from an Indian Tertiary Care Centre.

PNH is a rare disease with wide spectrum of intra-vascular hemolysis and thrombosis to sub-clinical PNH clones. We aimed to study the clinico-hematological profile and clone size on granulocytes and monocytes of PNH patients classified as per International PNH Interest Group recommendations. A retrospective analysis of clinico-hematological profile of 112 PNH clone positive patients by FLAER based flow cytometry between January and September 2017 done and classified into classical PNH, PNH with aplastic anemia or myelodysplastic syndrome (PNH-AA/MDS) and sub-clinical PNH clones (PNH-sc). Of 112 patients, majority were PNH-sc (62) followed by PNH-AA/MDS (34) and classical PNH (16). The commonest clinical feature was anemia in all 3 groups followed by jaundice (87.5%) in classical PNH and fever in PNH-AA/MDS (64.7%) and PNH-sc (48.4%). Thrombosis was present in 25% (4/16) classical PNH and 2.9% (1/34) of PNH-AA/MDS. The mean hemoglobin, reticulocyte count and LDH was higher in classical PNH. Bone marrow was predominantly hypercellular in classical PNH (11/16) and hypocellular in PNH-AA/MDS (31/34) and PNH-sc (50/62) with dyserythropoiesis predominantly in PNH-AA/MDS (83.8%) and PNH-sc (74.1%). Marrow iron was reduced in 62.2% classical PNH contrary to increased in PNH-BMF (58%) and PNH-sc (91%). The mean clone size in PNH-sc was significantly lower with > 50% in 16.2% patients. Three patients with MDS-MLD and MDS-MLD-RS in PNH-sc had > 80% clone on granulocytes and monocytes. Most PNH patients in Indian setting are PNH-sc with significantly lower clone, however, a clone size > 50% is not uncommon in Indian PNH-sc.

Approach to the Diagnosis of Amyloidosis.

Amyloidosis is heterogeneous group of disorder characterized by extracellular deposition of misfolded insoluble proteinaceous material with cross beta pleated sheet structure leading to organ dysfunction. This disease is rare and indeed heterogeneous, as it may be hereditary (familial amyloidosis), secondary to spectrum of inflammatory conditions (AA amyloidosis) or member of plasma cell neoplasm family (AL amyloidosis). AL amyloidosis is the most common type of amyloid, however, is rarely accompanied by multiple myeloma or other lymphoproliferative disorder. This disparity in its origin and presentation needs to be addressed by exhaustive battery of investigation tools, to arrive at right diagnosis with correct typing. This is of utmost importance in guiding the treating physicians to choose appropriate therapeutic options. This review deals with diagnostic approach to amyloidosis and its various subtypes.

Niemann-Pick disease, type B with TRAP-positive storage cells and secondary sea blue histiocytosis.

We present 2 cases of Niemann Pick disease, type B with secondary sea-blue histiocytosis. Strikingly, in both cases the Pick cells were positive for tartrate resistant acid phosphatase, a finding hitherto described only in Gaucher cells. This report highlights the importance of this finding as a potential cytochemical diagnostic pitfall in the diagnosis of Niemann Pick disease.

Utilisation of Flow-cytometry in the Diagnosis of Auto Immune Haemolytic Anaemia.

Auto Immune Haemolytic Anaemia (AIHA) is one of the most common types of acquired haemolytic anaemias. Its main cause is auto-antibody mediated rapid destruction of Red Blood Cells (RBCs). Demonstration of a positive Direct Antiglobulin Test also known as Coomb's test, against these autoantibodies is the crucial serological assay in the diagnosis of AIHA. This routinely used test has the disadvantage of low sensitivity and does not detect low levels of red cell auto antibodies leading to false negative results sometimes. Flow cytometry can effectively diagnose such patients with low levels of autoantibodies. This study was carried out in a tertiary care center, where patients with suspected AIHA were studied during 2 years period. Blood samples of suspected patients of AIHA were tested by both Gel Card Test and by Flow-cytometry for detection of RBC bound IgG. A total of 50 patients with suspected diagnosis of AIHA were studied by flow-cytometry as well as by Gel card test for detection of RBC bound IgG. Out of these 50 cases, 41 cases have turned out to be positive and 9 were negative by flow-cytometry. By Gel card test, out of 50 cases, 34 were positive and 16 were negative. Therefore, there were 7 cases which were negative for RBC bound IgG by Gel card test and these were positive by flow-cytometry. Flow-cytometry is a reliable and more sensitive method and can be used as a new routine diagnostic technique for AIHA.

Utility of Labile Plasma Iron Assay in Thalassemia Major Patients.

Labile plasma iron (LPI) levels are proposed as marker of iron overload in thalassemia patients and are also known to be the earliest parameter to indicate efficacy of chelation therapy. It was a prospective study in 35 patients of thalassemia major. Patients were recruited in two groups-group A (n = 13) patients not on chelation therapy and group B (n = 22) patients who were on regular oral chelation therapy. Ten age and gender matched healthy controls were also studied. For all patients, ferritin levels and LPI levels were measured at baseline, 6 months and 12 months. For group B patients paired samples for LPI were taken (before and 2 h after chelator). LPI levels were found to be significantly higher in group B patients versus group A patients versus normal healthy controls at all time-points. (P value-< 0.0001, 0.001) In group A, both LPI levels and ferritin levels follow an upward trend and correlated well with each other (P value-< 0.0001). In group B, the serum ferritin trend was not significant over follow up period of 1 year (P value 0.16), however LPI levels showed a significant decreasing trend on continued chelation (P value 0.0347) In patients on chelation therapy, the immediate change (2 h) in LPI levels on administration of chelators was not found to be significant (P value 0.22). LPI assay appears potentially attractive alternate to serum ferritin and can serve to monitor the trend of iron overload during long-term follow up.

Hairy cell leukemia: clinical, pathological and ultrastructural findings in Asian-Indians.

BACKGROUND: Hairy-cell leukemia (HCL), lymphoproliferative disease of older age, is characterized by projections from surface of abnormal cells. AIM: The aim was to study the clinical presentation and ultrastructural changes in hairy cells (HCs) following cladribine treatment. SETTINGS AND DESIGN: Clinical presentation, peripheral smear, bone marrow aspiration and biopsy of HCL cases diagnosed over a period of three years were reviewed. MATERIALS AND METHODS: Consecutive HCL cases in Hematology clinic of a tertiary care center were enrolled. Tartarate-resistant acid phosphatase (TRAP) test was done to detect HCs and electron microscopy was done to demonstrate initial ultrastructural changes and alterations following cladribine therapy. RESULTS: Fifteen cases of HCL, aged 32-57 years (median 47 years) were studied. The clinical presentation included splenomegaly in 15 (100%), fever in 10 (67%), hepatomegaly and pain abdomen in eight (53%), fatigue in nine (60 %) cases. The commonest laboratory features were monocytopenia in 13 (87%), neutropenia in 12 (80%), anemia in 10 (67 %) and pancytopenia in nine (60%). All patients showed symptomatic improvement on cladribine therapy. Electron microscopy after treatment (three months) showed loss of the finger like projections, characteristic bald lymphocytes, loss of ribosomal lamellar complexes, as well as decrease in mitochondria and vacuoles. CONCLUSIONS: Indian patients with HCL are younger. Cladribine is an effective therapy for these patients and leads to complete response in most of the patients. There is a significant improvement in the ultrastructural features following cladribine therapy.

Laboratory Monitoring of Chronic Myeloid Leukemia in Patients on Tyrosine Kinase Inhibitors.

Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm characterized by translocation of genetic material from chromosome 9 to chromosome 22 to form a fusion gene (BCR-ABL1) that is responsible for abnormal tyrosine kinase activity and alteration of various downstream signaling pathways. In addition to morphological diagnosis of CML phase, it is essential to detect BCR-ABL1 fusion by either metaphase cytogenetics or reverse transcriptase polymerase chain reaction that also determines type of mRNA transcript. Once treatment begins, monitoring the response to Tyrosine Kinase Inhibitor (TKI) using standardized techniques and guidelines is important to check for failure of response and thus, plan timely intervention by increasing the dose of TKI or opting for second line TKIs. The goal is to stop evolution of CML to accelerated phase or blast crisis that has poor response to treatment. Also, it is desirable to achieve good outcomes and even treatment free remission in patients of CML on TKI. Thus, molecular monitoring by reverse transcriptase quantitative PCR (RT-qPCR) is done at regular intervals. There are international recommendations and quality control measures to standardize the reporting of fusion gene transcript levels by quantitative PCR (RT-qPCR) in CML to achieve and maintain sensitivity in molecular detection of CML disease burden. Various state-of-the-art molecular techniques have emerged to accurately determine the number of fusion-gene transcript levels. This review highlights various methodologies and their practical implications in management of CML patients on TKI.

Zoledronic Acid for Treatment of Low Bone Mineral Density in Patients with Beta Thalassemia Major.

To determine the efficacy of zoledronic acid (ZA) in thalassemia major associated low bone mineral density. Prospective, open label, single arm trial. Bone mineral density (BMD) at lumbar, hip and forearm region were performed at baseline and after 1 year of therapy. Initial, 9 patients received a first dose of 4 mg. Due to severe adverse effects, further doses for these patients and all new recruited patients were 1 mg once every 3 months for 4 doses. All patients were receiving 500 mg of calcium carbonate twice daily and 0.25 µg alfacalcidol once daily before and during the entire study period. Dual energy X-ray absoptiometry was performed at baseline and after 1 year. Twenty-seven patients with transfusion dependent thalassemia with a median age 19.5 year (15-38 years) were eligible for ZA treatment. Seven patients had bony pains. Four patients developed grade 4 hypocalcemia (3 developed tetany) and 2 developed infusion related toxicity with initial dose of 4 mg. One mg dose was well tolerated. At the end of 1 year, bone pains had completely resolved. There was significant increase in BMD at lumbar (p = 0.002) and forearm regions (p = 0.04) and intertrochantric area (p = 0.041). The % change in BMD at 1 year was +3.7 ± 3.2%. ZA is an efficacious agent in treatment of low BMD in these patients. ZA produces significant adverse reactions at 4 mg dose but 1 mg dose is well tolerated and is efficacious.

Spectrum of hemostatic disorders in Indian females presenting with bleeding manifestations.

INTRODUCTION: Hemostatic disorders are often missed in women with bleeding particularly menorrhagia. Preexisting hemostatic disorders are now known as common risk factor for postpartum hemorrhage and prolonged bleeding in puerperium. Females with bleeding complaints constitute an important population referred to hematology clinic. Hence, we aim to evaluate the type and frequency of hemostatic disorders among females presenting with bleeding in a tertiary care hospital and a basic hemostatic laboratory. METHODS: Three-year data were retrospectively analyzed for 200 females with various bleeding complaints. Due to resource constraints, a hemostatic workup was done with prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen assay, clot solubility test, mixing studies, specific factor assays, platelet function test, and von Willebrand factor antigen level. RESULTS: A total of 200 females were investigated to identify the cause of their bleeding. Thirty-five of 200 (17.5%) females were found with an underlying bleeding disorder. Of these 35 females, 65.7% presented with bleeding from more than 1 site. Most common bleeding manifestation was spontaneous bruising in 18 of 35 (51.4%) patients followed by petechiae (48.6%). Inherited bleeding disorders were noted in majority. The most common inherited bleeding disorder identified was von Willebrand disease (VWD) in 34.3% females. Second most common disorder was Glanzmann's thrombasthenia accounting for 22.8%. Rare coagulation factor deficiency, such as factors VII, X, and XIII deficiencies, was noted. Three cases revealed acquired causes of coagulation defects. CONCLUSION: Underlying hemostatic defects should be searched for in women with unexplained bleeding complaints. This will not only help in diagnosis but also in proper management for future hemostatic challenges.

Hypercoagulable state in five thalassemia intermedia patients.

Fifty-three patients of thalassemia intermedia and 40 controls were studied for clinical evidence of thrombosis and laboratory evidence of hypercoagulable state. Thrombotic episodes were detected in 5 (9.4%) patients. Two of these 5 patients with thrombosis were splenectomized. Laboratory evaluation showed presence of thrombocytosis in 8 (15%), 5 of these were splenectomized. Platelet hyperaggregation was detected in 12 (22.2%) patients. Although rate of aggregation was slow in 7 (13.2%) patients, degree of aggregation was normal in these 7 patients and platelet hypoaggregation was not detected in any patient. Level of coagulation inhibitors protein C and protein S, and antithrombin III were decreased in 31 (58.4%) patients. There was no correlation between low level of protein C and protein S with hepatic dysfunction and iron overload. Antithrombin III level was decreased only in 8 (15%) patients. There was a statistically significant association between the lower level of this inhibitor and hepatic dysfunction. In conclusion, this study provides evidence for the existence of a chronic hypercoagulable state in patients with beta thalassemia intermedia, and suggests that expression of a procoagulant surface by thalassemia intermedia red blood cells may be the major underlying factor giving rise to platelet and coagulation inhibitor abnormalities in these patients. These alterations are not related to iron overload or hepatic dysfunction.

Is it safe to avoid bone marrow examination in suspected itp?

Two children with suspected ITP are described. One child was treated outside with corticosteroids and was diagnosed acute lymphoblastic leukemia. Another child was fresh and diagnosed as acute myeloid leukemia on bone marrow aspirate examination. Both the children had no physical or peripheral smear finding suggestive of leukemia. We suggest that a bone marrow examination is required in developing countries for evaluation of thrombocytopenia before labeling it an immune thrombocytopenic purpura.

Autoimmune hemolytic anemia in children.

The clinical and hematological profile and treatment outcome of children with warm autoimmune hemolytic anemia (AIHA) were assessed using retrospective case record analysis. There were 26 (17 idiopathic; 9 secondary) patients with a median age of 11 years. Pallor (100%), fever (39%), and jaundice (59%) were the main presenting complaints. Jaundice was much more common in idiopathic (70%) compared to secondary (44%). Direct antiglobulin test was negative in 3 patients. Oral prednisolone produced remission in 81% patients. Four patients relapsed after a median period of 7 months (2 months to 2 year) after response. All responded to a second course of steroids in median 14 days. One child required cyclosporin A in addition. No correlation was found between response and parameters such as age, sex, jaundice, low pretreatment hemoglobin, reticulocyte count, total leukocyte count, platelet count, subtype of AIHA, and hepatosplenomegaly. Relapse correlated with increased duration between the onset of symptoms and treatment. This study indicates that oral prednisolone is an effective therapy for autoimmune hemolytic anemia. In refractory cases cyclosporine A may be useful.

AML1-ETO positive AML: first report from India.

Translocation (8;21) is associated with few typical morphological features and favorable prognosis. All patients of AML and MDS with increased blasts (N = 35) according to FAB criteria, presenting (between Jan 2004 to June 2005) to the Department of Hematology, AIIMS were studied. RT-PCR was done for the AML1-ETO fusion transcript in all cases. Overall incidence of AML1-ETO was 28.57% and no correlation was found between AML1-ETO positivity and clinical or hematological parameters except for a direct correlation with absolute blast count (ABC) (a lower ABC in the AML1-ETO positive cases). Interestingly, 1/3 MDS cases were positive for the same fusion transcript and thus, it appears worthwhile to look for AML1-ETO in all cases of MDS with increased blasts. Objective morphological evaluation using a scoring system based on morphological features was not helpful in predicting positivity for AML1-ETO. The effect of this translocation on long-term survival could not be determined by the present study.

Low Dose Rituximab in Chronic ITP: Still an Option in Resource Limited Settings.

The etiology of ITP remains unknown but its pathogenesis consists of loss of tolerance to platelet antigens. There is a complex dysregulation of the immune system involving both the B cells and the T cells. Splenectomy is the standard second line option in steroid refractory chronic ITP patients. However, costs of surgery and reluctance for surgery in severely thrombocytopenic patients on part of surgeons are major obstacles in resource limited settings. Rituximab has been used in both the standard doses of 375 mg/m2 and low doses of 100 mg/m2 with similar results. We studied the utility of low dose Rituximab (@100 mg/m2 weekly × 4 doses) in resource limited settings. Overall response, complete response (CR) and partial response (PR) rates were 47.6% (10/21), 33.3% (7/21) and 14.3% (3/21) respectively. Median time to response in patients achieving CR was 75 days (range 45-185 days) while in patients achieving PR it was 105 days (range 45-165 days). However, there was no significant difference between males and females achieving CR or PR. We also observed that patients who had earlier responded to any form of treatment were more likely to respond to Rituximab treatment. The cumulative relapse free survival (RFS) at 13 months was 78%. By giving lower dose, six times less than conventional dosing dose, we have been able to demonstrate cost effectiveness in our study population. We were able to administer all the doses in day care without any major adverse events leading to further cost savings on in-patient care.

Blood Components Load in Post-operative Neurosurgical Patients Suspected with Disseminated Intravascular Coagulation.

Neurosurgical patients with suspected DIC receive large amount of transfusion support in form of red cell concentrates (RCC), platelet rich plasma (PRP) and fresh frozen plasma (FFP). However, there are very few studies which have studied the effect of blood components load in the outcome of the patient. We conducted a prospective observational study on 61 post operative neurosurgery patients suspected with DIC and had at least one deranged haemostatic parameter namely platelet count, prothrombin time, partial thromboplastin time and thrombin time. Their blood components load was co-related with the outcome and with the hemostatic derangements. Twenty-eight patients died in our study group. 19/28 died patients had DIC. The red cell load was significantly more in patients who died compared to those who were alive (p = 0.041). On the other hand, load of PRP as well as FFP was significantly different between the patients who were alive and dead. This difference was further heightened when the DIC deaths were compared with the other patients. This is especially true for FFP transfusion which was significantly higher in DIC deaths (p = 0.006). Also, the number of FFPs received by neurosurgical patients suspected with DIC was significantly more in patients >2 coagulation abnormalities (p = 0.008). However, no correlation was found between PRP and RCC received and number of coagulation abnormalities present. To conclude, the load of FFP was maximum in patients with DIC deaths and the load of RCC was associated with overall mortality.

A Novel Double Heterozygous Hb D-Punjab/Hb J-Meerut Hemoglobinopathy.

A comprehensive laboratory diagnosis of hemoglobinopathies forms an integral part in workup of disorders of globin chain synthesis. Clinical findings, complete blood counts, peripheral smear examination along with hemoglobin (Hb) electrophoresis and/or cation exchange high performance liquid chromatography findings and parental study helps to clinch a final diagnosis. Compound heterozygous hemoglobinopathy presents with variable clinical findings and some of them are picked up on screening tests done as part of routine antenatal workup. Here we report a rare double heterozygous hemoglobinopathy of Hb D-Punjab and Hb J-Meerut in a 35 year antenatal female.

Congenital Hypotransferrinemia, an Unusual Cause of Iron Deficiency Anemia: Report of Two Cases.

Hereditary hypotransferrinemia is a very rare cause of iron deficiency anemia in childhood characterized by microcytic hypochromic anemia refractory to iron therapy and concomitant iron overload. Regular plasma infusion to replace the deficient transferrin molecule is the therapeutic option. We report two cases; both presented with refractory anemia requiring blood transfusions and responded to monthly fresh frozen plasma replacement.

Higher rate of central nervous system involvement by flow cytometry than morphology in acute lymphoblastic leukemia.

INTRODUCTION: Central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is diagnosed traditionally by cytopathology (CP) of the cerebrospinal fluid (CSF). Role of flow cytometry (FC) to diagnose CNS involvement has not been extensively investigated. METHODS: We aimed to detect CNS involvement in 42 ALL patients (33 B-ALL, nine T-ALL) at diagnosis by FC and comparing it with CP and to correlate it with known risk factors for CNS disease like Lactate dehydrogenase (LDH). A receiver operating characteristic curve was used to determine the cutoff of LDH to predict CSF involvement. For the analysis of categorical/quantitative variables, Fisher's exact test was used. For the analysis of continuous variables, Mann-Whitney test was used. A P value of <.05 was taken as significant. RESULTS: CP and FC were positive in five (11.9%) and 11 patients (26.14%) respectively with FC detecting a significantly higher level of involvement (P=.001). All CP-positive cases were FC positive. A LDH value of >472 U/L had a sensitivity of 61% and specificity of 62.5% for diagnosis of CSF involvement by FC. CONCLUSIONS: CSF FC detects CNS disease in ALL patients at diagnosis at a rate double than CP alone and is statistically associated with an elevated LDH level. It should be incorporated in the evaluation of CSF to detect CNS involvement.

Erythrocyte enzyme abnormalities in leukemias.

Red cell enzymes were assayed in a total of 67 patient including 24 patients with AML (19 relapse, 5 remission), 16 patients with ALL (10 relapse, 6 remission), 22 patients with CML and 5 patients with blastic CML. Diagnosis of leukemia was based on clinical presentation, peripheral blood smear and bone marrow examination (as per FAB classification). PK activity was significantly high in case of CML and blastic CML (p<0.01). Red cell HK was high in all leukemia subtypes. There was no alteration in red cell G6PD. Notably there was no PK deficiency in AML or G6PD deficiency in ALL. Activities of G6PD and PK could be correlated in cases of CML, AML, (p<0.05) and ALL (p<0.01) i.e. when there was increased activity of G6PD, PK activity also tended to be higher. HK activity showed a positive correlation with PK and G6PD activity in cases of CML (p<0.05), however in acute leukemia there was no such correlation. Alteration of enzyme activities among red cells in leukemia occurred only during relapse. At the time of remission there has been no significant alteration in any of the enzyme activities. It would therefore, appear that enzyme alterations seen in leukemia patients is due to abnormal pluripotent stem cell that has given to a leukemia cell. The fact that enzyme alterations have primarily occurred at the time of relapse would further substantiate that abnormalities of red cell enzymes may be the result of a derivation some circulating red cells from the abnormal pluripotent stem cell. With the recovery of normal stem cells function during remission, enzyme abnormalities tend to become normal.