Anita Chopra

Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC & modulating effects of nutritional deficiency anaemias from north India

Background & objectives: The usefulness of cation exchange high performance liquid chromatography (CE-HPLC) as a tool for detection of thalassaemia/haemoglobin variants was evaluated in a prospective study in a tertiary care centre in north India. We also tried to evaluate the effect of concurrent nutritional deficiency on the HPLC pattern in the local ethnic population. Methods: A total of 800 blood samples were analyzed on the Bio-Rad Variant HPLC system by β-thal short program. The retention times, proportion of the haemoglobin (%), and the peak characteristics for all haemoglobin fractions were recorded. Alkaline and acid haemoglobin electrophoresis was performed to document the identities of the haemoglobin variants, wherever necessary. Many cases were subjected to family studies for a definitive diagnosis. Results: Among 800 samples tested, 553 (69.1%) were found to have normal HPLC pattern. Apart from β- thalassaemia, nine additional variants were encountered; HbS (2.8%), HbE (2.5%) and HbD (1.1%) being the most common variants present. Other variants included Hb Q-India, Hb-Lepore, δβ-thalassemia/ HPFH, HbD-Iran, HbJ-Meerut and HbH disease. There was a significant decrease in the level of HbA2 associated with iron deficiency anaemia (IDA) (P=0.004) and increase in megaloblastic anaemia (P<0.001) among subjects with normal HPLC pattern. Interpretation & conclusions: HPLC was found to be a simple, rapid and reliable method for the detection of hemoglobin variants. An accurate diagnosis can be provided in majority of cases by use of retention time, proportion of total haemoglobin, and peak characteristics of HPLC. Haemoglobin electrophoresis and family studies play a valuable role in difficult cases. Concurrent nutritional deficiency also has an effect on HbA2 levels.

Evaluation of Cd64 Expression on Neutrophils as an Early Indicator of Neonatal Sepsis

Background: Enhanced neutrophil CD64 (nCD64) expression is likely to be useful in diagnosis of neonatal sepsis. This study evaluated the diagnostic efficacy of nCD64 expression as an early indicator of neonatal sepsis. Methods: Sixty neonates (culture positive, 24; negative, 36) with suspected sepsis and 30 controls were studied prospectively. CD64 expression was evaluated flow cytometrically on neutrophils and monocytes. Mean and median nCD64 expression, mean and median monocyte CD64/nCD64 (M/N CD64) ratios were computed. Results were correlated with blood culture and other conventional indices of sepsis. Results: The sick neonates had significantly higher mean and median nCD64 expression compared with controls. Monocyte CD64 values did not differ significantly among the groups. Both mean and median M/N CD64 ratios were significantly lower in the former group. Culture-positive neonates had significantly higher mean and median nCD64 values and significantly lower mean and median M/N CD64 ratios than clinically indistinguishable but culture-negative neonates. Both groups were significantly different with respect to these indices from normal controls. Median M/N CD64 ratio was the best discriminant by virtue of highest area under the receiver operator characteristic curve (0.903), with sensitivity and specificity of 91.7% and 88.9%, respectively. Conventional indices were inferior, both singly and in combination. Conclusions: Enhanced nCD64 reported as median M/N CD64 ratio is a highly sensitive marker of culture-positive neonatal sepsis. It additionally identifies a separate group among culture-negative sick neonates and may be useful to guide antibiotic administration especially in these neonates.

Immunophenotypic analysis of T‐acute lymphoblastic leukemia. A CD5‐based ETP‐ALL perspective of non‐ETP T‐ALL

T‐cell antigens [CD5,CD1a,CD8] define early T‐cell precursor acute lymphoblastic leukemia (ETP‐ALL). To understand immature T‐ALL of which ETP‐ALL is part, we used these antigens to subcategorize non‐ETP T‐ALL for examining expression of myeloid/stem cell antigens (M/S) and clinical features. Using CD5 (+/−) to start categorization, we studied 69 routinely immunophenotyped patients with T‐ALL. CD5− was a homogenous (CD8,CD1a)− M/S+ ETP‐ALL group (n = 9). CD5+ cases were (CD8,CD1a)− pre‐T‐ALL (n = 22) or (CD8,CD1a)+ (n = 38) thymic/cortical T‐ALL; M/S+ 20/22 (90.91%) in former and 22/38 (57.89%) in latter (P = 0.007). ETP‐ and pre‐T‐ALL together (CD1a−,CD5−/+ immature T‐ALL group) were nearly always M/S+ (29/31; 93.55%). In multivariate analysis, only ETP‐ALL predicted poor overall survival (P = 0.02). We conclude (i) CD5 negativity in T‐ALL almost always means ETP‐ALL. CD1a and CD8 negativity, as much as CD5, marks immaturity in T‐ALL, and the CD5+/−/CD1a−/CD8− immature T‐ALL group needs further study to understand the biology of the T‐ALL–myeloid interface. (ii) ETP‐ALL patients may be pre‐T‐ALL if CD2+; CD2+, conversely, CD5−/CD1a−/CD8− pre‐T ALL patients are ETP‐ALL. (iii) Immunophenotypic workup of T‐ALL must not omit CD1a, CD5, CD8 and CD2, and positivity of antigens should preferably be defined as recommended for ETP‐ALL, so that this entity can be better evaluated in future studies of immature T‐ALL, a group to which ETP‐ALL belongs. (iv) ETP‐ALL has poor prognosis.

A 24‐week outcome following buprenorphine maintenance among opiate users in India

Aims: To evaluate the outcome at 24 weeks following a community‐based treatment programme using buprenorphine maintenance among male opiate users in Dimapur, Nagaland ( India). Design: Quasi‐experimental prospective follow‐up study. Participants: Fifty‐four male current opiate users were recruited following fulfilment of inclusion criteria from a community. All users seeking treatment met the DSM III‐R criteria for opioid dependence. Methods: All subjects received buprenorphine (1.2–1.8 mg sublingually per day) from a community clinic and attended psychosocial sessions. Measurements included an assessment of demographic and clinical variables, Addiction Severity Index (ASI), retention in treatment, drug use at baseline and follow‐up at 24 weeks. Findings: The mean age of sample was 26.3±4.1 years, with a mean duration of opioid use of 4.0±3.8 years. The retention rate was 81.5% at 24 weeks. Scores on the Addiction Severity Index decreased and injecting use reduced. No adverse events were reported. Conclusion: Buprenorphine was found to be effective with greater retention rates and less opioid use. Results support the theory that community‐based setting to provide maintenance treatment with very low staff investment from a community clinic can be initiated and replicated safely and effectively in India. However, future work on evaluation of higher doses is recommended.

Effect of glucocorticoids on von Willebrand factor levels and its correlation with von Willebrand factor gene promoter polymorphism.

von Willebrand factor (vWF) gene promoter polymorphism at nucleotide positions −2659 and −2525 influences corticosteroid-mediated increase in vWF level and possibly hypercoagulability of the blood. This study was undertaken to correlate plasma vWF levels with plasma cortisol and single nucleotide polymorphisms (SNPs) at positions −2659 and −2525 in Cushing syndrome patients in India. Samples from 22 endogenous and 28 exogenous Cushing syndrome patients and 50 normal volunteers were analyzed to determine vWF level and SNPs. vWF level was raised in 40.91% of endogenous and 14.29% of exogenous Cushing syndrome patients. A positive correlation was seen between plasma cortisol and vWF level (P = 0.01) with 1 &mgr;g/ml rise of plasma cortisol causing a 2.52 IU/dl increase in vWF level. Allele frequency of SNPs −2659A, −2659G, −2525A and −2525G was 0.78, 0.22, 0.84 and 0.16, respectively, making cosegregation of −2659A and −2525A the most frequent in our population. Haplotype 1 (−2659A, −2525G) was most frequently associated with higher level of vWF. Patients with haplotype 2 (−2659G, −2525A) had normal vWF. vWF promoter polymorphisms influence corticosteroid-mediated increase in vWF especially in patients with haplotype 1.

Hemoglobin OIndonesia in India: a rare observation

Dear Editor, Over 1,000 hemoglobin variants have been described [1] but identification of these variants cannot be made by electrophoresis alone. In India, both α and β globin chain variants have been reported but the spectrum and frequency remain unknown. Some of these hemoglobin variants are asymptomatic while others are associated with varying degrees of clinical health problems especially when found in some combinations. We present the clinical and laboratory work-up of a case of Hb OIndonesia (Hb OInd). Chromatography studies revealed an abnormal peak suggestive of an alpha globin chain variant. Molecular study confirmed the diagnosis of Hemoglobin OIndonesia (Hb OInd), rarely reported in Indian population. Further analysis revealed anemia to be due to an iron deficiency. The anemia in this patient was investigated by hemoglobin analysis and iron studies performed and their interaction is outlined in this letter. A 23-year-old woman from the northern Indian state of Uttar Pradesh underwent a routine health check at 14 weeks of gestation. Hematological analysis showed that she was slightly anemic, had a borderline low red blood cell count, and had hypochromic, microcytic red cell indices with a reticulocyte count of 0.5% and was referred to our laboratory for further investigation. Routine screening for hemoglobinopathies by cation exchange high performance liquid chromatography using the beta thalassemia short program on Bio-rad “variant” system revealed an abnormal band (11.5%) that eluted in the C-window with a retention time of 4.88 min along with HbA2 1.9%, HbA 75.5%, and HbF 0% (Fig. 1a). Starch agar gel electrophoresis performed at pH 8.6 showed a band in the S/D/G region. Parental hemoglobin analysis demonstrated a similar band (11.4%) in the father and a normal pattern in the mother (Fig. 1a, b). Fresh whole blood was collected and couriered to Oxford for molecular analysis. DNA was extracted by a phenol chloroform method, and globin genes were amplified by a standard protocol. Sequence analysis revealed a single heterozygous point substitution (GAG to AAG) at codon116 of the alpha 1 globin gene that results in a change of amino acid from glutamic acid to lysine described originally by Lie Injo in 1957 [2]. Further investigation of the anemia revealed serum iron concentration of 34 μg/dL, total iron binding capacity A. Chopra : J. M. Khunger :H. Pati (*) Department of Hematology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India e-mail: harappati@yahoo.co.in

Plasma cell leukemia--a study of 28 cases from India.

Abstract Plasma cell leukemia (PCL) is a rare neoplasm that has not been comprehensively reported in an Indian population. We report the clinico-pathological features of 28 cases studied during 1999–2008. Organomegaly and bleeding tendency was common in primary PCL but not in secondary. Misdiagnosis as acute leukemia or the leukemic phase of lymphoma on the initial peripheral blood smear examination was frequent (31·4% cases) in the primary form of PCL. This is best addressed by an emphasis on the morphological appearances and confirmation by simple serum electrophoresis rather than by more sophisticated testing that may not be widely available. Response to treatment is poor and PCL has a poor prognosis, a situation that may be amenable to improvement by a better understanding of the biology of the disease.

Treatment Seeking Behavior of Inhalant Using Street Children: Are We Prepared to Meet Their Treatment Needs.

Context: There is a lack of evidence for help and treatment seeking behavior of street children using inhalants. Aims: The present study was planned to provide a comprehensive understanding on the patterns, correlates of inhalant use and treatment seeking behavior of street children from Delhi, India. Material and Methods: Participants were a purposive sample of 100 inhalant using street children below 18 years identified with the assistance of five community service organizations/nongovernmental organization working with street children in the city of Delhi. Information on a semi-structured questionnaire with items pertaining to the demographic and drug use parameters was collected by trained research staff in a one-to-one in field setting. Statistical Analysis: All data were entered into SPSS 12.0, data quality checks performed and examined. Results: The sample had an average age of 12.8 ± 2.4 years (range 4-17 years), 96.5% reported regular past month and 87.0% past 24 h use of inhalants. The mean age of onset of inhalant use was 9.3 ± 2.8 years The substances most commonly reported were toluene from eraser fluid (by 83.0%), glues (34.0%) and petroleum products (3.0%); mean frequency of use was 9.8 times in a day. Of the sample, 18% used inhalants when they were alone, and the rest reported using with drug using network friends; 76% reported tolerance and mild withdrawal symptoms such as restlessness, anxiety, craving, irritability and lethargy. A variety of problems and perceived benefits due to inhalant use were reported; 46% inhalant users had never abstained from its use, and 77% reported never having sought any medical help. Conclusions: Study findings provide a better contemporary understanding of inhalant abuse among Delhi street children. This information can assist in the formulation of a needs-based intervention for the inhalant using street children.

Effusion Cytology of Granulocytic Sarcoma in an Unusual Clinical Scenario: A Diagnostic Challenge

Background: Presentation of granulocytic sarcoma (GS) as an effusion is very rare and most cases are misdiagnosed as lymphoma infiltration. Detailed descriptions of cytological features of GS in an effusion have not been published to date. Case Report: We present the case of a 35-year-old male who presented clinically with a mediastinal mass, peripheral lymphadenopathy and pleural/pericardial effusion. His effusion cytology showed an atypical morphology. Immature cells with marked nuclear indentation/convolution, fine nuclear chromatin, conspicuous nucleoli and a moderate amount of cytoplasm were seen, without any granularity. GS was suspected based on the abnormal nuclear contours and fine nuclear chromatin, and was confirmed by lymph node fine needle aspiration and biopsy with immunocytochemistry. Conclusion: Abnormal nuclear contours are a significant marker in effusion cytology and provide an important morphological clue to the diagnosis of a myeloid malignancy in addition to fine nuclear chromatin especially in the absence of cytoplasmic granularity. In this case, these features helped to suspect the diagnosis despite a clinical scenario suggestive of non-Hodgkin’s lymphoma.

Burkitt's Leukemia After Treatment of Primary Mediastinal Nonseminomatous Germ Cell Tumor

A 37-year-old man presented to us in February 2007 with reports of rising total leukocyte count (32 10/L to 92 10/L in 3 days). This had been detected in the course of a routine check after chemotherapy and surgery done for primary mediastinal germ cell tumor (GCT) 3 months earlier, in another hospital. His hemoglobin was 12 g/dL and platelet count was 25 10/L. There was no lymphadenopathy or hepatosplenomegaly. Previous to this the patient had been investigated in February 2006 for a 3-month history of breathlessness, cough, and expectoration. Chest x-ray and contrast-enhanced computed tomogram of the chest had revealed a large mediastinal mass with necrosis (Fig 1, arrows) and small pleural effusion (Fig 1). Computed tomography scan of abdomen and pelvis, and examination and ultrasound of testes were normal. Serum -fetoprotein was 14,571 IU/mL and -human chorionic gonadotropin was 157.6 mIU/mL. Tru-cut biopsy from mediastinal mass showed variegated areas characteristic of mixed GCT: undifferentiated cells in solid sheets with necrosis, and areas showing ectodermal (squamous epithelium), endodermal (mucosal glands), and mesodermal (cartilage and smooth muscle) differentiation as well as an immature component consisting of primitive neuroepithelium (Figs 2A to 2D). A diagnosis of nonseminomatous GCT (embryonal carcinoma and immature teratoma) was made. The patient was treated with etoposide 100 mg/m days 1 through 5 and cisplatin 20 mg/m days 1 through 5 every 3 weeks from April 2006. Bleomycin was omitted due to apprehension about pulmonary toxicity. Five cycles of etoposide and cisplatin had been given until July 2006; at this point serum markers had become normal. Contrast-enhanced computed tomogram chest revealed almost 70% to 80% reduction in the size of the tumor, with a residual mass measuring 9 cm that was invading pericardium and myocardium with effacement of coronary vessels. Fluorodeoxyglucose positron emission tomography showed mildly increased uptake along the margins of the mass. Given that the mass was considered inoperable, the patient was given two courses of chemotherapy with etoposide 100 mg/m days 1 through 5, ifosfamide 2 g/m days 1 through 5 with mesna, and cisplatin 20 mg/m days 1 through 5. Surgical resection R0 type was then carried out in November 2006. Histopathologic examination showed residual immature teratoma. Postoperative computed tomography scan of the chest showed no residual disease and both markers continued to be normal. Our investigations in February 2007 showed 12% blasts in the peripheral-blood smear and 75% blasts in the bone marrow. The blasts had deeply basophilic cytoplasm with oil red-O–positive vacuoles (Fig 3, arrows); they were negative for myeloperoxidase and nonspecific esterase. Mitotic figures were present. The overall morphologic features were those of Burkitt leukemia. Flow cytometry showed blasts to be positive for CD19, CD22, CD 79a, HLA-DR, and CD45, and negative for all T-cell and myeloid markers. Fluorescent in situ hybridization technique was applied on interphase and metaphase cells using LSI MYC and LSI immunoglobulin heavy chain (IgH) dual-color, break-apart probes and CEP 8 probe (Vysis Abbott, Wiesbaden-Delkenheim, Germany). The IgH probe revealed normal copies of red/green signals. The signal pattern of MYC revealed two red/green signals (normal MYC copies; Fig 4, long arrows) and red signal at band 8q24 (Fig 4, short arrow) in 25% of interphase cells (large blast-like cells). Metaphase cells (15 of 15) with red/green signals and a red signal at 8q24 on copies of chromosome 8 indicated three copies of chromosome 8 with break in C-MYC followed by deletion of distal segment of MYC Fig 1. A B