Serum Vascular Endothelial Gowth Factor Correlates with Hasford Score in Chronic Myeloid Leukemia.

Introduction and Aim Increased angiogenesis in BM is one of the characteristics of chronic myeloid leukemia (CML) implicated in its progression. Vascular endothelial growth factor (VEGF) one of the most potent regulator of angiogenesis is increased in CML. The prognostic impact of serum VEGF in CML is largely unknown with sparse literature from India. So the present study aimed to measure serum VEGF levels in different phases of CML and to assess its prognostic significance using Hasford score. Methods Forty Ph + patients of CML were enrolled in the study. Complete clinical history and physical examination was done. Hemogram was done by Beckman Coulter LH 500. Peripheral smear (Wright's stain) was done by microscopy. Serum VEGF (plain vial) using ELISA was calculated. Statistical analysis was performed using SPSS software version 20. Results The mean serum VEGF levels were significantly higher in patients than in controls (p < 0.0001). The patients in accelerated/blast phase demonstrated significantly higher levels of serum VEGF (mean 151 pg/mL) than those in the chronic phase (mean 90.87 pg/mL) (p = 0.02). Serum VEGF levels showed a significant positive correlation with the overall Hasford prognostic score (p = 0.023). Conclusion Serum VEGF levels can serve as an independent prognostic marker in CML patients irrespective of phase of CML. Also, S. VEGF levels can be used to monitor patients on imatinib therapy and identify those who might benefit from antiangiogenesis therapy. However, larger studies are needed with a larger number of patients in different phases of CML to validate our findings and thus pave the way for future research.

Serum ferritin <70 µg/L predicts functional iron deficiency in patients with chronic kidney disease.

Anemia is a common complication of chronic kidney disease (CKD) which is treated by erythropoiesis-stimulating agents. However, most of the patients do not respond adequately due to the development of functional iron deficiency (FID). The study was conducted to explore the value of inflammatory markers, high sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) along with serum ferritin (SF) in the diagnosis of FID. Seventy-seven clinically diagnosed patients of CKD (Stage 3, 4, and 5) of either sex, age >18 years with hemoglobin <11 g/dL were included in the study. Complete hemogram with peripheral smear, serum iron, total iron binding capacity, transferrin saturation, SF, transferrin receptors (sTfR), hsCRP, IL-6, and erythrocyte sedimentation rate were estimated and statistically analyzed. sTfR/log ferritin (taken as gold standard) detected 31/77 patients as having iron-deficient erythropoiesis. Nineteen patients were detected as having FID. SF at a cut-off <70 µg/L showed the best sensitivity (83.87%) and specificity (73.91%) in detecting FID in these patients and identified 14/19 cases of FID. The 5 FID cases who were missed had raised hsCRP. The presence of raised hsCRP reduced the sensitivity to 79.16%. SF <70 µg/L emerged as the most sensitive and specific in the identification of iron-deficient erythropoiesis. SF >12 µg/L - SF <70 µg/L was able to identify 14/19 cases of FID. Furthermore, hsCRP further stratified the subgroup of CKD patients in which FID could be detected with higher sensitivity and specificity.

Hemostatic Abnormalities in Multiple Myeloma Patients

Background: Multiple myeloma (MM) is a neoplastic plasma cell disorder characterized by clonal proliferation of plasma cells in the bone marrow. Diverse hemostatic abnormalities have been reported in patients with myeloma which predispose to bleeding and also thrombosis. Methods: Complete blood count, biochemical parameters and parameters of hemostasis i.e. platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), factor VIII assay results, plasma fibrinogen, D-dimer and lupus anticoagulant, were assessed in 29 MM patients and 30 age matched controls. Results: The most frequent abnormal screening parameter was APTT. Of the six indicative of a bleeding tendency i.e. thrombocytopenia, prolonged PT, APTT, TT, reduced plasma fibrinogen and factor VIII, at least one was abnormal in 8 (27.6%) patients. Of the four prothrombotic markers, lupus anticoagulant, D-dimer, elevated factor VIII and plasma fibrinogen, one or more marker was present in 24 (82.7%). D-dimer was the most common prothrombotic marker, being elevated in 22 (75.9%) patients. One or more laboratory parameter of hemostasis was abnormal in all 29 (100%) patients. Though thrombotic complications are reported to be less frequent as compared to hemorrhagic manifestations, one or more marker of thrombosis was present in 24 (82.7%) patients. Conclusion: This study provided laboratory evidence of hemostatic dysfunction which may be associated with thrombotic or bleeding complications at diagnosis in all MM patients. Hence, screening for these abnormalities at the time of diagnosis should help improved prognosis in such cases.

Morphological Changes in Bone Marrow Post Imatinib Therapy in Chronic Phase CML: A Follow up Study on Sequential Bone Marrow Aspirates and Biopsies.

INTRODUCTION: Imatinib mesylate is used extensively for first line treatment of Chronic Myeloid Leukemia (CML). However, not many studies have documented morphological changes in bone marrow biopsies produced during Imatinib therapy with reference to myelofibrosis. AIM: To document the morphological changes produced in the bone marrow during Imatinib therapy. MATERIALS AND METHODS: This longitudinal study followed up 75 Philadelphia Chromosome Positive Chronic Myeloid Leukemia with chronic phase(Ph+ CML- CP) patients sequentially, receiving 400-600mg Imatinib over a period of 12 or more months. Haematologic parameters were measured at admission, 2 weeks, 1 month, 3 months, 6 months and 12 or more months. Morphologic changes in bone marrow aspirate and biopsy were evaluated at admission, 6 months and ≥12 months of treatment in accordance with National Comprehensive Cancer Network(NCCN) guidelines. RESULTS: Complete Haematologic Response (CHR) was seen in 47.1%, 80%, 85.4%, 90.4% at ≥1 month, 3 months, 6 months and 12 months respectively after therapy. It was noted that patients not showing CHR by 3 months were less likely to show CHR at 6 months and beyond. Bone marrow aspirates and biopsies showed reduction in cellularity and myeloid precursors with regeneration of erythroid precursors in 70-83% at ≥12 months. A significant decrease in myelofibrosis (p-value< 0.04) was noted as early as 6 months. Mild to moderate hypoplasia was noted in 31.8% of biopsies within 6 months. Pseudo gaucher cells and benign lymphoid nodules were also seen. CONCLUSION: Sequential analysis showed that Imatinib reduced the grade of myelofibrosis significantly (p-value< 0.04). It also prevented development of myelofibrosis in patients who did not have it at presentation. Hence Imatinib is effective when used early in the course of CML-CP.

Effect of iron deficiency anemia and iron supplementation on HbA1c levels - Implications for diagnosis of prediabetes and diabetes mellitus in Asian Indians.

BACKGROUND: We investigated the effect of iron deficiency anemia (IDA) on levels of glycated hemoglobin (HbA1c) and to compare its levels before and after iron supplementations. METHODS: Age and sex matched subjects were enrolled and clustered in 2 groups: IDA (n=62) and healthy controls (HC; n=60). HbA1c levels were estimated by HPLC. Hemogram were estimated by hematology analyser. Serum ferritin (ELISA) and other parameters of iron profile were measured by standard guidelines of ICSH. HbA1c values and iron studies were repeated after 3months of iron supplementation to determine the effect of iron therapy on HbA1c levels. RESULTS: Significantly higher HbA1c levels were observed in IDA subjects compared to HC (5.51±0.696 v/s 4.85±0.461%, p<0.001). A significant negative correlation was observed between HbA1c and hemoglobin, hematocrit, RBC count, MCH, MCHC and serum ferritin in IDA subjects (r=-0.632, -0.652, -0.384, -0.236, -0.192 and -0.441). Significant decline was noticed in HbA1c levels in IDA subjects after iron supplementation (5.51±0.696 before treatment v/s 5.044±0.603 post-treatment; p<0.001). Post treatment, 70% subjects (14/20) with HbA1c in pre-diabetes range normalised to normal glucose tolerance (NGT) range and out of 6 patients with pre-treatment HbA1c in diabetes range, 5 reverted to pre-diabetes range while 1 of them reverted to the NGT range. CONCLUSIONS: Caution must be exercised in interpreting the results of HbA1c in patients of IDA and iron deficiency must be corrected before diagnosing diabetes and pre-diabetes solely on the basis of HbA1c criteria.

Lupus anticoagulants and anticardiolipin antibodies in Indian women with spontaneous, recurrent fetal loss.

Spontaneous and recurrent pregnancy loss are common complications of pregnancy resulting from varied causes including antiphospholipid syndrome (APS). Treatment of women with APS increases the chance of a subsequent successful pregnancy. The study aimed to find the prevalence of lupus anticoagulants (LA) and anticardiolipin antibodies (ACAs) in women with spontaneous/recurrent fetal loss and compare with women with normal obstetric history. Hundred women with spontaneous/recurrent fetal loss and 50 healthy pregnant controls were tested for LA by complete blood counts, Prothrombin time, Activated partial thromboplastin time (APTT), LA sensitive APTT and dilute Russell viper venom time (dRVVT) (screening and confirmatory) and ACAs (ELISA). LA was detected in 15 % patients using dRVVT confirmatory test and ACA in 5 %, all controls being negative. Twenty one % patients were detected by LA sensitive APTT (sensitivity 92.9 %, specificity 100 %) and 100 % with dRVVT screening test (sensitivity 98.8 %, specificity 100 %). We recommend that screening for antiphospholipid antibodies must be done in women with spontaneous/recurrent foetal loss even in the absence of other clinical manifestations using a combination of tests.

Comparative evaluation of bone marrow aspirate with trephine biopsy in hematological disorders and determination of optimum trephine length in lymphoma infiltration.

INTRODUCTION: Bone marrow examination is an indispensable diagnostic tool to evaluate neoplastic and non neoplastic hematological diseases. AIMS: To compare bone marrow aspirate with trephine biopsy in hematological disorders. To determine the optimum trephine preprocessing length in lymphoma infiltration. METHODS: Diagnostic comparison was done between simultaneous bone marrow aspirates and trephine biopsies in 449 patients. Biopsies were fixed in formalin, decalcified in 5.5% EDTA and routinely processed. Concordance rates and validity parameters for aspirate were calculated. Three deeper sections of trephine biopsy, cut at 0.1-0.2 mm intervals, were assessed for lymphoma involvement. Proportion of biopsies showing marrow infiltration by lymphoma cells was plotted against trephine length and correlation was assessed. RESULTS: Aspirate had a high sensitivity for acute leukemia (89.4%) and multiple myeloma (88.5%), moderate for NHL (67.6%) and nonhematopoietic metastases (58.3%) and low for aplastic anemia (38.5%) and Hodgkin lymphoma (5%). Aspirate has no role in granulomatous myelitis and myelofibrosis. Lymphoma positivity increased with trephine length, with maximum positivity (68.9%) seen in 17-20 mm group and no further gain beyond 20 mm. (lymphoma positivity ≤16mm=40.3% and ≥17mm=66.1%, p=0.0011). CONCLUSION: Aspirate has a high specificity; its sensitivity depends upon the type of disease. Apart from few conditions, in which aspirate alone is sufficient, biopsy is mandatory in most. Preprocessing trephine length of 17-20 mm examined at multiple deeper levels was found optimal for assessing lymphoma positivity.

Alloimmunization to red cells in thalassemics: emerging problem and future strategies.

AIMS AND OBJECTIVES: To evaluate the magnitude of red cell alloimmunization in regularly transfused patients with thalassemia major and analyse factors responsible for development of antibodies. MATERIALS AND METHODS: This cross sectional study was conducted on 116 thalassemics receiving regular transfusions. All the patients underwent antibody screening. Patients with positive antibody screen were further tested for antibody identification. The data was analysed to find out the frequency, pattern and factors influencing red cell alloimmunization secondary to multiple transfusions. RESULTS: Mean age of the patients was 14 years (range 1.5-27 years). Red cell alloantibodies were found in 11 patients (9.48%). In four (36%) patients first transfusion was given before 6 months of age and in seven (64%) patients, first transfusion was given after two years of age. The interval between consecutive transfusions varied from 18 to 35 days. The most common antibody was Anti-E found in 4 (36.4%) patients, followed by Anti-K (three patients, 27.2%), Anti-Kp(a) (two patients, 18.2%) and Anti-C(w) (two patients, 18.2%). The interval from first transfusion to antibody development varied from 1.5 to 14 years. None of the eight out of 116 patients, who underwent splenectomy showed any antibody development. CONCLUSIONS: The rate of red cell alloimmunization was found to be 9.48% in thalassemics receiving regular transfusions. The incidence of alloantibody development was higher if first transfusion was received at more than 2 years of age. Early institution of red cell transfusions and Rh and Kell phenotyping followed by provision of matched blood could prevent alloimmunization.

Developmental and neurophysiologic deficits in iron deficiency in children.

INTRODUCTION: Several studies in animals and humans have clearly demonstrated the effect of ID on development, cognition, behavior and neurophysiology. The effect of ID have been shown: on brain metabolism, neurotransmitter function, and myelination. Changes in brain iron content caused by early ID in animals are not reversible by iron therapy, inspite of correction of anemia and other tissue deficits and result in changes in behavior which continue into adulthood. ID has repercussions in the perinatal period, infancy and childhood. Some effects are irreversible while other defects may be corrected: timing of ID in a child may be critical. DEVELOPMENTAL DEFICITS: Children (6-23 months) with moderate to severe anemia (ID) or chronic anemia (>3 months) had lower mental and psychomotor development scores than the nonanemic, and except for some continued to have lower scores in spite of iron therapy for 3 months although anemia was corrected. The deficits persisted on re-evaluation at 5, 11-14, and at 19 years. SCHOLASTIC ACHIEVEMENT: Scholastic achievement is lower and ID children are twice more likely to have problems with mathematics. Ten year follow-up indicated special educational assistance was required for initially anemic children. ID affects WICS items of information, comprehension and verbal performance and full scale IQ. EEG power spectrum had a slower activity suggesting developmental lag compared to iron sufficient children. Treatment with iron improved IQ scores significantly; other studies found differential effects: improvement in cognition and mental scores in older but not in younger children. IQ levels are affected by ID: IQ at 4 years may be predicted by hemoglobin at 5 and 36 months. NEUROPHYSIOLOGICAL DEFICITS: Abnormal Evoked Response Potentials (ERPs):ABRs and VEPs are seen in ID, which persist in children who were anemic in infancy on retesting at 4 years. Differences have been consistently found in ID infants and in older children. Iron supplementation may significantly reduce latencies of some ERPs. ID affects newborn temperament, ERPs and recognition memory. Iron supplementation in infants (<1,301 g) improved neurocognitive and psychomotor development by 5.3 years (median age). Preventive iron supplementation in well nourished infants also show a positive effect on motor development. The changes are usually subtle, however, with prevalence of anemia of 79.2% in children 6-35 months and 57.9% in pregnant women (NFHS-3, 2005-06), the adverse effects of cognitive, development and behavioral defects should not be underestimated.

Iron profile in children with behavioural disorders: a prospective study in a tertiary care hospital in north India.

Iron deficiency anemia is the most frequent micronutrient deficiency in the developing countries like India especially affecting pregnant women and young children. Iron is an essential element involved in myelin formation, neurotransmitter synthesis and neuro-metabolism. Several behavioural disturbances have been reported in iron deficient children. In the present study, we determined the prevalence of iron deficiency anemia in children with behavioural disorders and assessed the improvement in terms of symptoms (by child behaviour check list), haematological parameters and iron status after treatment with oral iron. In this prospective study, 44 children in the age group of 3-12 years who were diagnosed with behavioural disorders were evaluated. Complete blood counts using automated hematology analyzer and iron parameters (serum iron, total iron binding capacity, % transferrin saturation and serum ferritin) were measured in all the patients to assess the prevalence of iron deficiency in these children. Thirty age matched controls were also studied. Iron deficiency was found in 32 (73%) children, as assessed by transferrin saturation <16% and/or serum ferritin <16 µg/l. Following treatment with iron for 100 ± 10 days, there was a statistically (P ≤ 0.05) significant improvement in the clinical features, haematological profile and iron status. The presence of iron deficiency in children with behavioural disorders and subsequent improvement in clinical features, haematological profile and iron status suggests a possible causal relationship between iron deficiency and behavioural disorders.

HbQ-India associated with microcytosis: An uncommon hemoglobin variant associated with a common hematologic condition.

HbQ-India is a rare alpha chain variant that usually presents in the heterozygous state. Normally, HbQ-India is clinically silent. It becomes symptomatic when present in association with other conditions. We report a case of HbQ-India with concomitant presence of iron deficiency anemia. A 16-year-old female presented with weakness and pallor intermittently for six years. Complete blood count showed severe microcytic hypochromic anemia. Hemoglobin electrophoresis showed a prominent band in the S,D,G region. Tests for sickling were negative. High performance liquid chromatography (HPLC) showed a peak in the unknown window (4.70-4.90 min) suggestive of HbQ-India. Serum iron profile was suggestive of iron deficiency anemia. Based on the above findings, a diagnosis of coexistent HbQ-India-iron deficiency anemia was made. A family study revealed the father as having moderate anemia with similar findings while the mother was normal. Abnormal hemoglobin in the patient was confirmed by molecular diagnosis. HbQ variants are the alpha globin chain variants due to structural mutations (α64 Asp→His) inherited in autosomal dominant fashion. Three molecular variant types have been documented, namely HbQ-India, HbQ-Thailand and HbQ-Iran. Normally, HbQ is clinically silent. Therefore, careful screening of the samples using routine techniques like Hb electrophoresis and HPLC are needed for identification of such abnormal hemoglobin variants like HbQ-India.

Transient thrombocytosis with megathrombocytes in a case of acute myeloblastic leukemia.

Thrombocytosis is commonly seen in reactive conditions and certain neoplastic states, such as chronic myeloproliferative disorders. It is rarely seen in acute leukemia. A 12-year-old girl with acute myeloblastic leukemia (FAB M2) in remission presented with pyoderma. Her hemogram revealed anemia (Hb-6.4g/dl), leucopenia (TLC - 1.2 x 109/L) and thrombocytosis (platelet count- 580 x 109/L). A peripheral blood film showed numerous abnormally large platelets with few atypical cells. The thrombocytosis subsided with the clearance of infection but atypical cells persisted. One month later, she relapsed. Cytogenetic analysis revealed variable results (trisomy 9 and deletion 3). This case has been presented because thrombocytosis is rare in AML and its appearance calls for a close follow-up.

A prospective study of iron status in exclusively breastfed term infants up to 6 months of age.

BACKGROUND: Can exclusive breastfeeding until six months of age maintain optimum iron status in term babies? We evaluated iron status of exclusively breastfed term infants in relation to breast milk iron and lactoferrin. METHODS: In this prospective study in Delhi, India, during the period 2003-2004 normally delivered babies of non-anemic [(Hemoglobin (Hb) = 11 g/dl, n = 68] and anemic (Hb 7 - 10.9 g/dl, n = 61) mothers were followed until 6 months of age. Iron parameters were measured in the cord blood at 14 weeks and 6 months. Breast milk iron and lactoferrin were measured at the same intervals. RESULTS: Iron parameters in babies of both groups were within normal limits at birth, 14 weeks and 6 months. Mean breast milk iron and lactoferrin in non-anemic (day 1: 0.89, 6 months: 0.26 mg/l; day 1: 12.02, 6 months: 5.85 mg/ml) and anemic mothers (day 1: 0.86, 6 months: 0.27 mg/l; day 1: 12.91, 6 months: 6.37 mg/ml) were not different on day one or at other times. No relationship was found between breast milk iron, lactoferrin and iron status of the babies. CONCLUSION: Exclusively breastfed infants of non-anemic and anemic mothers did not develop iron deficiency or iron deficiency anemia by six months of age.

Validation of ESR analyzer using Westergren ESR method.

Erythrocyte sedimentation rate (ESR) is one of the most frequently ordered laboratory test. ESR analyzers were developed to provide a quick and efficient measure of ESR. We compared the results of ESR obtained by an ESR analyzer with those by the Westergren method in a group of 75 patients Linear regression analysis showed a good correlation between the two results (r = 0.818, p < 0.01). The intra class correlation was 0.82. The analyzer method had the advantages of safety, decreased technician time and improved patient care by providing quick results.

Blood collection procedures in hematology: knowledge and practice among laboratory personnel.

Blood collection is an important preanalytical component of haematological testing. This questionnaire based study was conducted on laboratory personnel to elicit their knowledge and practice regarding blood collection procedures for haematological testing. Questionnaire comprised of 37 multiple choice questions, of which 10 questions each were related to essential and desirable knowledge, 10 to practice and 7 to educational and job profile. Ninety four laboratory personnel participated in the study. Analysis was done on SPSS software. Percentages of unsatisfactory scores were 42.6%, 4.3%, 17% and 6% in the essential knowledge, desirable knowledge, practice and total scoring respectively. 91.5%, however, had a satisfactory total score. Importance needs to be given to blood collection procedures both, individually and collectively. The study highlights the deficient areas which need to addressed by all laboratorians. Such studies should also be conducted among nursing and resident staff- the other groups concerned with blood collection.

Diphenylhydantoin-induced pure red cell aplasia.

Pure red cell aplasia is an uncommon complication of diphenylhydantoin therapy. It has not been reported in Indian literature. Awareness of the entity helps in establishing the cause of anaemia in these patients and alerts the physicians to the need of comprehensive haematological monitoring in these patients. A case of 58-year-old male who developed pure red cell aplasia following three months of diphenylhydantoin therapy is reported here.

Impact of iron deficiency anaemia on T lymphocytes & their subsets in children.

BACKGROUND & OBJECTIVES: While there is evidence of an altered immune profile in iron deficiency, the precise immunoregulatory role of iron is not known. Information particular in children who are vulnerable to iron deficiency and infection, is lacking. We undertook this study with the aim of documenting the changes in T cell subsets in children in the age group of 1 to 5 yr with iron deficiency. METHODS: The levels of T lymphocytes, their CD4+ and CD8+ subsets and the CD4 : CD8 ratio were evaluated in 40 iron deficient and 30 healthy children. The impact of oral iron supplementation for three months on the same parameters was also noted in 30 children. RESULTS: Significantly lower levels of T lymphocytes as well as CD4+ cells was observed in the iron deficient children (P<0.01 and 0.002 respectively). The CD4 : CD8 ratio was also significantly lower in this group (P<0.05). Iron supplementation improved the CD4 counts significantly. INTERPRETATION & CONCLUSION: Our study demonstrated quantitatively altered T cell subsets in iron deficiency in children, and a relationship between the severity of haematological and immunological compromise. The clinical and epidemiological implications of this relationship have topical relevance since ID is the most common micronutrient deficiency worldwide.