1131 www.smj.org.sa Saudi Med J 2012; Vol. 33 (10) Immunophenotyping analysis of lymph node biopsies by flow cytometry Raja-Zahratul A. Raja-Sabudin, MBBS, MPath, Azura A. Hamid, MD, MPATH, Nurasyikin Yusof, MBChB, FRCPA, Hafiza Alauddin, MBBS, MPath, Suria A. Aziz, MBBS, MPath, Sivagengei Kulaveerasingam, Dip MLT, Nozi M. Zin, Dip MLT, Siti-Aishah M. Ali, MBBCh, DCP, Rohaizak Muhammad, MBChB, FRCS, Srijit Das, MBBS, MS, Ainoon Othman, MBBS, Dr Med Sc, Noor H. Hussin, MBBCh, Dr Med Sc. T he conventional laboratory diagnosis of lymphoma using tissue biopsies is based on histology diagnosis and immunohistochemistry. However, this method is tedious and involves multiple steps and procedures. In developed countries, immunophenotyping by flow cytometry is readily performed on tissue biopsies such as lymph nodes for diagnostics purposes. However, the scenario is different in Malaysia where flow cytometric analyses of body tissues are not readily available. Fine-needle aspiration of lymph node tissues combined with flow cytometric immunophenotyping (FCI) has been reported to be successful in evaluating sites for lymphomatous involvement in 75-90% of cases. 1,2 Dunphy 3 described the contribution of FCI in a series of 373 tissue specimens (278 lymph nodes and 95 extra-nodal tissue) from patients with suspected lymphoma. e results showed that the FCI data was consistent with the final tissue histological diagnosis in the majority (94%) of the tissue samples. Ravoet et al 4 evaluated the contribution of flow cytometry to the diagnosis of malignant and non malignant conditions in lymph node biopsies in 116 samples. e results showed that flow cytometric analyses of the lymph node biopsies were in agreement with tissue histology in 102 cases (87.9%). In view of the facts mentioned above, we embarked on this study to assess the role of FCI on lymph node biopsies in providing a diagnosis of hematological malignancies, especially the non Hodgkin lymphoma (NHL), in University Kebangsaan Malaysia Medical Centre (UKMMC) setting. is was a descriptive cross-sectional study of lymph node specimens from patients who were subjected to diagnostic lymph node biopsies. All lymph node specimens were processed in the Hematology and Histopathology Laboratories, Diagnostic Laboratory Services Department, UKMMC, Kuala Lumpur, Malaysia, between November 2007 and October 2008. Informed consent was taken from each patient involved in this study. Patients with lymphadenopathies or with suspected hemato-lymphoid malignancies who were subjected to excisional lymph node biopsies for diagnostic purposes in UKMMC were enrolled in this study. e lymph node tissues, which were excised from patient, were cut into halves. One portion was collected into a container containing RPM1 1640 (media culture) (Logan, UT, USA) for FCI, while another portion was collected into a container containing formalin for histopathology examination (HPE). In the flow cytometry laboratory, the lymph node tissues were processed and cell suspensions were prepared. e cell suspensions were then analyzed using FACSCalibur (Becton Dickinson, San Diego, CA, USA), and the data were analyzed using Paint-a-Gate software (Becton Dickinson, San Diego, CA, USA). e findings of flow cytometry analysis were compared with tissue histological diagnosis. Within the 12 months of study, only 11 lymph node biopsies were available for analysis. ere were 9 males and 2 females involved in this study. eir age ranged from 18-74 years old. Most of the cases had presented with multiple lymph node enlargement (7/11; 64%). Out of 11 samples, 9 (82%) were positive to CD45, which was consistent with hemopoietic cells. Another 2 samples (18%) were negative towards CD45 as well as other monoclonal antibodies. In view of their clinical findings, diagnoses of non-hemopoietic neoplasms were made. Five out of 9 cases with CD45 positivity showed strong expression towards CD19 and CD20, screening panel for B lineage. Two cases showed an immunophenotype that was typical of small lymphocytic lymphoma/chronic lymphocytic leukemia (case one and 11 with CD23+, CD5+, CD22+, surface Ig weak with negative FMC7) (Table 1). In another 3 cases, the diagnoses of B-NHL were made as flow cytometric was able to detect the B neoplastic cells. One case showed positivity towards kappa with absent lambda expression (kappa light chain restriction) (case 5), one showed abnormal kappa; lambda ratio (case 6) and another one showed immunophenotypic aberration (case 10) where no expression of surface light chain immunoglobulin was present (Table 1). Two cases (18%) were positive towards T-cell lineage, in which the dominant cell population (>90%) was positive towards cytoplasmic CD3 in the screening panel. One case was diagnosed as T-lymphoblastic leukemia/lymphoma (case 3) as the population was positive towards CD34, intraTdT, in addition to the T-cell markers. Another case (case 8) was diagnosed as T-lymphoproliferative disease as the predominant Brief Communication Immuno20120309.indd 1131 10/14/12 12:07 PM