This study was conducted on 45 patients recruited from the Internal Medicine Department, between April 2018 and January 2019. The study included 15 multiple myeloma patients, 15 B-non-Hodgkin lymphoma patients, and 15 patients with acute myeloid leukemia (30 male patients and 15 female patients); their age ranged from 38 to 78 years. Patients on treatment were excluded from this study.
The diagnosis of patients was established based on the WHO classification of hematolymphoid tumors , and prognosis evaluation was done according to the Revised International Staging System for multiple myeloma , International prognostic index of B-NHL , and WHO prognostic classification of AML .
All patients were subjected to full history taking; clinical examination with special emphasis on the presence of lymphadenopathy, splenomegaly (SM), and hepatomegaly (HM); signs of anemia and thrombocytopenia; and abdominal ultrasonography for assessing organomegaly.
Five milliliters of the venous blood sample was withdrawn from each participant under complete aseptic conditions for complete blood count (CBC) and chemical test analysis. Also, 5 ml bone marrow (BM) aspirate was collected and divided into an EDTA vacutainer for flow cytometric (FCM) analysis and a heparinized vacutainer for cytogenetics investigations.
CBC was performed on an automatic cell counter Sysmex XS500 (Sysmex, Bohemia, New York, USA) with examination of Leishman-stained smears.
Measurement of serum level of albumin, calcium, and lactate dehydrogenase was performed on automated clinical chemistry analyzer (OLYMPUS AU400), assessment of different fractions of the serum proteins by serum protein electrophoresis.
A flow cytometric immunophenotypic analysis was performed on the BM samples on the same day of their collection, using coulter EPICS-XL four-color flow cytometry (Coulter Diagnostic, Hialeah, FL, USA) using the following panels of monoclonal antibodies (all were supplied by Beckman Coulter, Fullerton, CA, USA).
For the diagnosis of MM: CD19, CD20, CD27, CD28, CD33, CD81, and cyt κ light chain (all are fluorescein isothiocyanate labeled) and CD10, CD13, CD38, CD56, CD117, CD200, and λ light chain (all are labeled by phycoerythrin). Gating was done using phycoerythrin-cyanine-5-labeled CD138 for plasma cells.
For the diagnosis of B-NHLs: CD19, HLA-DR, CD20, CD22, CD23, FMC7, CD103, and κ light chain (all are fluorescein isothiocyanate labeled) and CD5, CD10, CD11C, CD25, CD38, CD52, and λ light chain (all are labeled by phycoerythrin). Gating was done using phycoerythrin-cyanine-5-labeled CD19, and clonality was assessed using κ and λ light chains expressed on CD19+ cells.
For the diagnosis of AML: CD19, CD20, CD33, HLA-DR, CD15, and MPO (all are fluorescein isothiocyanate labeled) and CD34, CD10, TDT, CD38, CD13, CD117, CD5, CD7, CD56, and CD79A (all are labeled by phycoerythrin). Gating was done using phycoerythrin-cyanine-5-labeled CD45.
Cytogenetics investigations included:
Heparinized bone marrow was cultured using complete culture media without phytohemagglutinin for 24 h and 48 h. The cytogenetic preparation and G-banding were done according to routine laboratory procedures according to Verma and Babu .
Patients with one or two independent cytogenetic aberrations were regarded as having simple aberrations, whereas those with three or more independent aberrations were regarded as having multiple aberrations.
Fluorescence in situ hybridization (FISH) technique: using selected probes LSI hTERT (5p15), p53 (17p13.1) gene, LSI IGH (14q32) break apart rearrangement, and LSI 13q14.3 (Vysis, London, UK)
The heparinized BM samples were cultured using complete culture media without phytohemagglutinin, incubated at 37 °C for 2 days, then harvested as described previously by Schlette et al. . FISH was carried out according to the manufacturer’s instructions, and examination of at least 200 interphase cells was conducted using Cytovision software (Leica, NJ, USA).
All of the statistical calculations were made using the excel program and SPSS, version 18 program (SPSS Inc., Chicago, IL, USA). Qualitative data were presented as frequency and percentage, and quantitative parametric data were presented as mean and standard deviation (SD). Association between parameters was done by Pearson chi square. An effect was considered statistically significant at p value less than 0.05 and highly significant at p value less than 0.01.