Flow cytometric assessment of CD30 expression in adult patients with acute leukemia Amal M Elafifi, Rasha M Said, Haitham M AbdElbary, Rasha Abdulrahman, Kouthar Ali The Egyptian Journal of Haematology 2019 44(1):1-5 Background CD30, a member of tumor necrosis factor receptor superfamily, was originally identified as a cell-surface marker of Reed–Sternberg cell in classical Hodgkin lymphoma. CD30 is also expressed by several types of T-cell and B-cell non-Hodgkin’s lymphoma, such as anaplastic large cell lymphoma and primary mediastinal large B-cell lymphoma, and Epstein–Barr virus driven clonal lymphoproliferative disorder, as well as in reactive conditions such as infectious mononucleosis. Patients and methods A cross-sectional study was conducted at Clinical Hematology Department in Ain Shams University Hospital during a period from November 2016 to August 2017. A total of 20 new cases of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and 30 refractory/relapsed cases of AML and ALL either T or B, were enrolled in this study. CD30% expression was assessed by flow cytometry on bone marrow sample or peripheral blood. Results CD30 with cutoff more than 20% (+ve) was seen in 46% of cases, whereas cases with cutoff less than 20% (−ve) represented 54% of all leukemia cases. CD30 expression was higher in ALL, especially in T–acute lymphocyctic leukemia (T-ALL), with mean value of 44.564±27.158, with significant increase relapsed T-ALL (P=0.031) followed by B-ALL (23.988±15.678). CD30 expression in relapsed AML and ALL showed an increased percent but not yet statistically significant. Significant correlation was found in risk parameters as in white blood cells (WBCs) (>100 000) as well as Platlets (PLT) (<30 000) and CD30 expression in patients with T-ALL, with P values of 0.038 and 0.021, respectively, and nonsignificant difference between lactate dehydrogenase (LDH) and minimal residual disease in T-ALL and all risk parameters in B-ALL. Receiver operator characteristic (ROC) curve revealed that the accuracy of sensitivity and specificity was 69.9%. Conclusion CD30 has been shown to be a significant diagnostic tool in cases of acute leukemia especially in newly and relapsed T-ALL; moreover, it can be labeled to be a targeted therapy. Drug trials using monoclonal antibodies to CD30 as treatment in relapsed/refractory cases with special concern to response and survival rate are needed.

CYP2B6 polymorphism and lipoprotein lipase expression in chronic lymphocytic leukemia: impact on the outcome of fludarabine–cyclophosphamide regimen Ahmed M.L Bedewy, Waleed R El-Bendary The Egyptian Journal of Haematology 2019 44(1):6-13 Background Chronic lymphocytic leukemia (CLL) is the most common type of leukemia and has a highly variable clinical course. Cyclophosphamide (CPA)-containing regimens are the standard of care for patients lacking the 17p deletion. CYP2B6 is a polymorphic cytochrome P450 isoform that converts CPA to its active form. Lipoprotein lipase (LPL) catalyzes the hydrolysis of triacylglycerol. Remarkably, a growing body of data emphasizes its role in the biology of different tumors. Objectives This study aimed to study CYP2B6 polymorphism and LPL expression in fludarabine cyclophosphamide (FC)-treated CLL patients lacking 17p deletion. Methods 46 treatment-naïve CLL patients negative for 17p deletion and indicated to receive chemotherapy were enrolled. CYP2B6 genotyping and lipoprotein lipase mRNA expression were assayed by Realtime PCR. FC-protocol was given then treatment-related toxicities, response, and event free survival were traced. Results CYP2B6*6 allele was associated with lower rates of treatment-related anemia and hospital admission. The response to FC was affected only by CYP2B6 polymorphism. The event-free survival of responders was significantly higher in patients having low LPL expression. Conclusion CYP2B6*6 infers lower CPA efficacy with lower treatment-induced side effects and increased risk of nonresponding to FC chemotherapy in CLL. LPL expression is a predictor of outcome in CLL, indicating poor survival.

Iron-deficiency anemia as a risk factor for dyslipidemia in Egyptian patients Maram M. M. Aboromia, Alyaa A El-Sherbeny, Emad A. Abd El-Hady The Egyptian Journal of Haematology 2019 44(1):14-20 Introduction Iron is an important component of some enzymes involved in hepatic lipid metabolism. Several factors such as increased erythropoiesis leading to increased cholesterol demand may lead to hypocholesterolemia combined with anemia. Objective The aim of this study was to investigate the relationship between iron-deficiency anemia (IDA) and dyslipidemia in nonobese patients and to show the effect of iron therapy on their lipid profile. Patients and methods This study was conducted on 60 nonobese individuals selected from Ain Shams University Hospital. It comprised 40 patients with IDA (equally divided according to sex), who were assessed initially and after receiving 3 months of oral iron therapy regarding lipid and iron profiles, in addition to 20 normal individuals without anemia as a control group. All patients were submitted to full history and clinical examination in addition to fasting lipid profile and iron study profile. Results We found that patients with anemia especially male ones had higher lipid parameters compared with healthy nonanemic controls. However, after iron therapy, we found significant increase in hemoglobin (HB) levels and decrease in cholesterol, triglyceride, low-density lipoprotein, and very low-density lipoproteinlevels compared with pretherapy values. In patients with anemia, we found statistically significant negative correlations between the preiron therapy cholesterol levels on one hand and both of HB and ferritin on the other hand. Treatment of iron-deficiency corrected dyslipidemia significantly in the form of significant negative correlation between serum cholesterol level and HB. Conclusion IDA may be associated with increased cholesterol levels. When a course of iron therapy is taken, significant beneficial changes in lipid profile may occur, but the exact mechanism is still unclear. As serum lipid profile is affected by many factors, hence the variations of lipid concentration in IDA may not be related to iron deficiency by itself.

Thrombophilic gene polymorphisms are associated with deep venous thrombosis in a cohort of Egyptian patients Hisham H Essa, Hady Gobran, Sherif M Sabry, Dina H El-Dahshan The Egyptian Journal of Haematology 2019 44(1):21-27 Background There is several evidence suggesting, that inherited thrombophilia increases the susceptibility to venous thromboembolism (VTE). Aim is to detect prothrombotic genes polymorphisms possible association with the risk of occurrence of venous thrombosis. Methods A group of eleven gene polymorphisms related to the risk of thromboembolic events were studied; including Factor V Leiden(FVL) 1691G>A, Methylene tetrahydrofolate reductase gene (MTHFR) C677>T and others. Genotyping was done using PCR, and identification of corresponding bands using the StripAssay in 493 patients with clinical symptoms of deep venous thrombosis (DVT). Five hundred age and sex matched healthy individuals were included as a control group. Results Genotyping of DVT patients showed significantly higher prevalence of FVL 1691G>A, MTHFR C677>T, MTHFR 1298A>C, Prothrombin 20210G>A, PAI 4G, FVIII V34L, B Fibrinogen 455G>A, HPA-1, ACE and APO E e4 gene polymorphisms (p<0.001). Seven and 11 fold increased susceptibility to DVT among patients with MTHFR C and MTHFR A mutations (OR 7.318& 11.23) respectively. Moreover a strong association was detected between DVT and mutations in genes: PAI 4G (OR 46.987), B Fibrinogen (OR 3.331), Ace (OR 187.663) and Apo E (OR 386.361). Conclusion Individuals carrying thrombophilic genes polymorphisms are at a greater risk for venous thrombosis.

Asymptomatic gallstones in patients with sickle cell disease: to wait or to operate? Mohamed A.M El-Menoufy, Hany M El-Barbary, Salah M Raslan The Egyptian Journal of Haematology 2019 44(1):28-33 Background Cholelithiasis (gallstone disease) is found in more than one-half of the patients with sickle cell disease (SCD) by the age of 30 years. Complications of symptomatic cholelithiasis may precipitate to recurrent sickle cell crises, which add to the disease morbidity. Laparoscopic cholecystectomy (LC) is the treatment of choice in patients with symptomatic disease, but the best option for asymptomatic ones is still a source of debate. Aim To compare the outcome of LC in asymptomatic and symptomatic cholelithiasis in patients with SCD. Patients and methods The study was performed on 42 patients with SCD having cholelithiasis. A total of 30 (72%) patients with asymptomatic cholelithiasis underwent prophylactic LC and were compared with those [12 (28%) patients] who were operated owing to symptomatic cholelithiasis. Results The percentage of cholelithiasis was significantly higher in the 12–30 years age group (69%) than in the age groups younger than 12 years and over 30 years (P=0.013). Nine of the 12 (75%) symptomatic patients required emergency cholecystectomy. The mean time of LC procedure and the mean postoperative hospital stay were significantly longer in symptomatic patients when compared with asymptomatic patients (P=0.01 and 0.02, respectively). After LC, a significant reduction in hospital admission rates compared with the rate of admissions during the period before the operation was observed in both groups of patients. Conclusion Elective (prophylactic) LC is recommended to patients with SCD having asymptomatic cholelithiasis because waiting for the appearance of symptoms worsens the postoperative outcome.

Brain natriuretic peptide as a sensitive biomarker for early detection of cardiac affection in adult Egyptian patients with β-thalassemia Maha T El Zimaity, Haitham M Abdelbary, Haydi S. Mohamed The Egyptian Journal of Haematology 2019 44(1):34-39 Background Cardiac affection by iron overload is the leading cause of mortality and morbidity in patients with β-thalassemia owing to frequent blood transfusion, increased iron absorption, and hemolysis. N-terminal pro-B-type natriuretic peptide is a tool for early detection of cardiac hemosiderosis, as echo findings occur later. Methods We measured N-terminal pro-B-type natriuretic peptide in 45 β-thalassemia transfusion-dependent patients aged from 16 to 45 years and 30 controls of matched age and sex from Clinical Hematology Department, Ain Shams University hospital, which was correlated with both echo findings and serum ferritin level. Patients with congenital, ischemic heart disease and decompensated heart failure were excluded Results Brain natriuretic peptides (BNP) levels were higher in patients with β-thalassemia than the control group, with statistical significance. BNP levels were higher in patients with β-thalassemia major than in those with β-thalassemia minor. BNP level was higher in patients who received more frequent blood transfusion. When we correlated BNP level with the echo findings, statistically significant difference was found in patients with high right ventricular systolic pressure (RVSP) ˃ 35 mmHg but not with diastolic dysfunction; also a positive correlation was found between BNP level and ferritin but not the ejection fraction. Conclusion BNP levels were higher in patients with beta-thalassemia compared to control group and was correlated with the frequency of blood transfusion. BNP levels were correlated with increased RVSP ˃ 35 mmHg but not with early diastolic dysfunction, also BNP levels were correlated with ferritin level but not the ejection fraction.

Lymphoid enhancer factor 1 gene expression in comparison to other prognostic markers in adult B-acute lymphoblastic leukemia Manal A ElSourdy, Mona W Ayad, Amira I Fayad, Sara M Youssef The Egyptian Journal of Haematology 2019 44(1):40-47 Background Activation of the canonical Wnt signaling pathway by the key mediator lymphoid enhancer factor 1 (LEF1) plays an important role in the development of several hematological and nonhematological malignancies. Aims The aim was to study LEF1 gene expression in adult B-acute lymphoblastic leukemia (ALL) and its clinical significance in comparison to other prognostic markers to improve risk-adapted treatment stratification for high-risk patients. Patients and methods The study was conducted on 50 adult cases with newly diagnosed B-ALL and 50 adult healthy controls with matched age and sex. The cases were divided into three groups according to risk assessment: low risk (have no risk factor), intermediate risk group (have one or more risk factors), and high risk (BCR-ABL positive). LEF1 gene expression was analyzed using quantitative real-time PCR and its correlation with clinical characteristics and treatment outcome at day 28 after induction chemotherapy and in 6 months follow-up period was assessed. Results LEF1 gene expression showed statistically significant higher expression in ALL cases compared with control (P=0.001). Higher LEF1 gene expression was detected in the high-risk group compared with comparable gene expression in both low- and intermediate- risk groups (P=0.05). Statistically significant positive correlation was detected between LEF1 gene expression and BCR-ABL positive cases. Statistically significant positive correlation was detected between the non-remission group and high LEF1 gene expression (Z=3.026, P=0.002). Conclusion The high LEF1 gene expression at diagnosis may be used in identifying patients with high risk of treatment failure. The high LEF1 gene expression can be a prognostic marker in adult B-ALL patients.

CD34 expression and FLT3 mutation are independent poor prognostic factors in normal karyotype acute myeloid leukemia Mohamed A Elakkad, Ayman F Abdelhalim, Hossam E Salah, Ahmed Y Amer The Egyptian Journal of Haematology 2019 44(1):48-53 Background The prognostic impact of CD34 expression on malignant myeloid leukemia blast cells is still under debate. FLT3 mutation is a well-known poor prognostic factor in acute myeloid leukemia (AML). The aim of the study was to assess CD34 expression in this subgroup of AML patients to detect the association of CD34 expression with FLT3-internal tandem duplication (ITD) mutation and its impact on the outcome. Patients and methods This prospective study, carried out at Zagazig University Hospitals, included 33 de-novo AML patients with a normal karyotype. CD 34 expression was detected by flow cytometry using FITC florescent monoclonal antibody and it was considered positive if a cutoff level of 10% expression was exceeded. FLT3-ITD mutation was detected by PCR. Results CD34 was positive in 20 cases while 13 cases were negative. Twenty-one cases were FLT3-ITD wild type while 12 cases were mutated. FLT3 mutation was significantly linked to CD34 reactivity (P=0.048). CD34-positive cases were associated with a significantly less complete remission (CR) achievement rate (33 and 80% in CD34-positive and CD34 negative, respectively, P=0.03). Those with positive CD34 had significantly lower overall survival compared with CD34-negative cases. Multivariate cox regression survival analysis showed that positive CD34 was a predictor of poor survival and higher risk of mortality (HR=1.3 and P=0.027, confidence interval, 1.1–2.9). Conclusion CD34 expression is a poor prognostic biomarker in normal karyotype AML. It is associated with and further worsens the poor prognosis in FLT3-mutated cases. Its role in different subgroups of AML layered by different genetic aberrations needs further study.

Improperly functioning phagocytic cells contribute to disturbed phagocytosis in chronic lymphocytic leukaemia Myriam AbouSeif, Manal El-Sorady, Amira I Fayad, Omnia F El-Lakany The Egyptian Journal of Haematology 2019 44(1):54-64 Background Patients with chronic lymphocytic leukaemia (CLL) are endangered by fatal infectious complications. CLL cells shape their surrounding microenvironment as immune effectors which manifests as tolerance mechanisms, avoiding self-reactivity and inhibiting phagocytosis. Immune-therapeutic approaches tailoring antitumor potentials of phagocytic cells have been the focus of recent studies. Aim To study whether examining phagocytic cells’ functional status in patients with CLL can provide insights into immune system deregulation responsible for fatal infectious complications. Patients and methods The study was conducted on patients with CLL: 30 de-novo (group IA), 30 treated (group IB), and 20 healthy age-matched and sex-matched volunteers. Phagocytosis by neutrophils and monocytes was studied using phagotest kit, assayed as percentage of cells ingesting FITC-Escherichia coli, and phagocytosis mean fluorescence intensity (MFI), representing individual cellular phagocytic activity. Phagocytic activity was correlated with corresponding leukocytes CD47 surface expression levels. Results There was a decreased percentage of neutrophils and monocytes ingesting bacteria in subgroup IA or IB compared with group II (P=0.043 and 0.015, respectively). Phagocytosis MFI by neutrophils or monocytes in subgroup IA or IB was lower than control group. There was increased CD47 surface expression by lymphocytes, neutrophils, and monocytes at the time of diagnosis, which decreased as the disease progressed; however, it was still higher than the control group. Paradoxical correlation was detected between CD47 surface expression levels and phagocytosis MFI by neutrophils or monocytes (r2=0.606, P=0.02, and r2=−0.419, P=0.6, respectively). Conclusion In CLL, phagocytic cells were improperly functioning and the percentage of functioning cells was decreased. CD47 expression levels by leukocytes varied throughout the course of the disease, reflecting disease progression and paradoxically correlated with phagocytic function.

In-vitro anti-sickling and membrane stability potentials of Mishenland polyherbal extract on sickle red blood cells Musiliu A Oyenike, Helen B Akpan, Olatoye J Otulana, Adebayo K Adefule, Kamoru A Adedokun, Waheed A Oluogun, Musa A Muhibi, Hammed O Ojokuku The Egyptian Journal of Haematology 2019 44(1):65-71 Background Sickle cell disease is a genetic disorder that causes stiff, rod-like sickle-shaped hemoglobin in red blood cells (RBCs) and consequently poses serious health complications. Aim We investigated an in-vitro anti-sickling potential of a novel Mishenland polyherbal formula (MPF) for possible ameliorative effects. Materials and methods Sickling of RBCs induced with 2% sodium metabisulfite was followed by treatment with MPF mixtures in different saline concentrations (7, 9, 14, and 28 mg/ml). The red cell morphology was examined microscopically. Percentage sickling was assessed at 30 min intervals at 37°C for 2 h. The effect of the MPF on membrane stability of RBCs was analyzed using osmotic fragility testing. Results Qualitative phytochemical screening demonstrated the presence of some secondary metabolites namely alkaloids, glycosides, phenols, saponin, tannin, and terpenoids. Sickling of RBCs induced with metabisulfite was inhibited by MPF. This anti-sickling effect was directly proportional to the concentration of the MPF, dose dependently. There was a significant difference (P<0.05) between MPF-treated and untreated sickled red cell counts. Osmotic fragility curves obtained from MPF-treated RBCs showed leftward shifts against the untreated control, indicative of increased RBC membrane stabilization and hemolytic resistance, while the mean corpuscular fragility also showed a significant difference (P<0.05). Conclusion MPF demonstrated significant anti-sickling and erythrocyte membrane stability properties. These effects under hypoxia signified a promising effect of the bioactive components as probable drug candidates against sickling of red cells.