Flow Cytometric Immunophenotyping of Mature B-cell Neoplasm in Adult Sudanese Patients

Abstract

Flow cytometer (FC) became one of the most pivotal and definitive techniques in the diagnosis and classification of mature B cell neoplasm (MBCN). Since flow cytometer exploits the laser and photomultiplier technology for reliable high quality result with extremely high sensitivity and specificity, we used these important specifications in this cross sectional descriptive hospital based study to study the properties of B cells in the adult Sudanese patients whose have initial diagnosis as mature B cell neoplasm in the period of October 2010 and March 2013. For the studying of these properties, we depended up on the evaluation of immunophenotypic antibodies using in the diagnosis and classification of MBCN against (CD45, HLA, CD34, CD3, CD5, CD23, CD22, CD79b, FMC7, Kappa, Lambda, CD10, CD11c, CD25 and CD103) with calculation of the mean of their flow cytometric parameters for each markers (Percentage, Fluorescence Intensity and Positive Peak Width), and focusing on the substantial feature which important in differentiation between CLL and other NHL. Also we evaluated the best type of sample between venous blood, bone marrow aspiration and lymph node aspiration in the highlighting of accurate result of markers. One hundred and forty-six samples were conducted. (17.7 %) was lymph nodes (LN) samples, (48.8 %) was bone marrow (BM) samples and (33.5%) was venous blood samples. We prepared the lymph node samples which collected as Fine Needle Aspirations in a 3.0 ml PBS (pH=7.2), Mononuclear cells for flow cytometry preparation were separated from LN and BM samples using HISTOPAQUE-1077. Mononuclear cells from LN suspensions, BM suspensions and PB samples were conjugated with fluorescence labelled antibodies in the dark place. Tubes of kappa and lambda were going through washing procedure for 3 times by PBS before adding of Abs. Then all tubes were analyzed by flow cytometer and all flow cytometric parameters were recorded for each marker. Data acquisition and analysis were performed with an EPICS XL Beckman Coulter flow cytometer and SYSTEM II software. Both 3 and 4 color protocols were performed using CD45 and light scatter gating system to identify cell populations and exclude the cells debris. The majority (70%) of MBCN were males and the rest were females (30%). The mean age was (60.7) years. (66.5%) of patients had initial diagnosis as CLL, (28.7%) diagnosed as NHL and (4.8%) were normal samples. The sub-classification of NHL was done and there were (6) cases diagnosed as DLBCL, (13) cases as PLL, (2) cases as MCL, (2) cases as LPL, (1) case as SLVL, (2) cases as FL, (1) case as Hairy cell leukaemia and (17) cases had inconclusive diagnosis. The mean Hb% value for MBCN patients was (84.7 %), (82 X 103) for TWBC mean, (164.4 X 103) for platelets mean and (78.6%) for lymphocytes mean. CD45 showed an important role in the identification of MBCN (p.value = 0.0021). CD45 Min had significant difference between CLL and NHL (p.value = 0.004). CD34 was insignificant for diagnosis of MBCN or differentiation between CLL and NHL (p.value = 0.598). While HLA-DR could differentiate between them (p.value = 0.001). When we used CD20 and CD19 together, they showed very high significant values to differentiate between CLL and NHL. NHL cases showed high CD20 Min than CLL cases (p.value = 0.000), high CD20Min:CD19Min ratio (p.value = 0.000) and low CD20 Pw (p.value = 0.000). The immunophenotyping features of CLL was (s+ve CD45, -ve CD34, +ve HLA, +ve CD19, w+ve/-ve CD20, +ve CD5, +ve CD23, -ve/w+ CD22, -ve/w+ CD79b, -ve/w+ Ig), while the immunophenotyping of NHL was (s+ve CD45, -ve CD34, +ve HLA, +ve VD19, s+ve CD20, -ve or +ve CD5, -ve/rare +ve CD23, s+ve CD22, s+ve CD79b, s+ve Ig). The characteristic markers for diagnosis of hairy group were CD11c, CD25 and CD103 and for follicular lymphoma was CD10. Venous blood and LN samples showed the best results to differentiate between CLL and other NHL. As a conclusion of this study, Flow cytometer have a very distinctive role in the diagnosis of MBCN and also ability to differentiate between CLL and NHL. Diversity of FC parameters can help in the minimization of markers following into the minimization of panel cost without affecting in the result accuracy like using of CD20 & CD19 with their flow cytometric parameters and without the other markers showed a significant role in the differentiation between the two diseases. MBCN immunophenotyping feature of Sudanese patients was not much differing from other immunophenotyping feature in the other world especially when using the scoring system of Matutes as a guide.