Chronic lymphocytic leukemia (CLL) is characterized by a heterogeneous clinical course  with survival times ranging from months to decades; this heterogeneity reflects the biological diversity of CLL . Neoplastic cells express Certain markers that can help to predict the prognosis of patients with CLL [3,4]. The detection of these markers provides a vital tool that can stratify patients into groups with good or poor prognosis .
The traditional prognostic parameters (clinical stage, pattern of bone marrow infiltration, lymphocyte doubling time, beta-2 microglobulin levels, and lactate dehydrogenase level) are valuable but less accurate in prediction of disease progression [6,7].
Last years, research in prognostic factors in CLL has focused on biological factors such as the variable region of the immunoglobulin heavy chain gene (IgVH) mutational status, CD38 and ZAP-70 expression. The strongest independent prognostic factor in CLL is the presence of IgVH somatic mutations . However, IgVH mutation studies are not cost effective, technically demanding and are not available at most centers. Therefore, there is a need for surrogate marker that is reliable, easily standardized and more suitable for application in clinical laboratories .
The zeta-chain-associated protein kinase-70 (ZAP-70) was first described in 2003  and is present in normal T-cells, NK-cells, and not present in normal B-cells. Published reports demonstrated a strong association between overexpression of ZAP-70 and unmutated IGHV genes in CLL. Also, low expression of ZAP-70 is associated to mutated IGHV genes [5,10,11] and time to disease progression and overall survival (OS) [5, 10–13].
Although there has a consensus on the prognostic significance of ZAP-70, the detection method of ZAP-70 at various laboratories has been a source of discussion . Several methods are used to study ZAP-70 expression in CLL but flowcytometry remains the most suitable, reliable and applicable method into routine use .
Crespo et al.  and Rassenti et al.  demonstrated that patients with more than 20% ZAP-70 positive cells have higher risk of disease progression and lower survival rates compared with those with less than 20% ZAP-70-positive cells, suggesting that ZAP-70 might be more indicative of worse prognosis compared with the mutational status of the IGHV.
In this study, the authors aimed to determine the frequency of ZAP-70 among Sudanese patients with chronic lymphocytic leukaemia and to associate ZAP-70 expression to age, sex, lymphadenopathy, organomegaly, haemoglobin concentration, TWBCs count, platelet count, and modified Rai clinical staging system.
2. Materials and Methods
A total of 93(69.7% males and 30.3% females) newly diagnosed (based on CBC and immunophenotyping), untreated B-CLL patients, attending the Khartoum Oncology Hospital during the period from September 2016 to February 2017, were enrolled in this cross-sectional study. Patients were diagnosed as CLL, in accordance with the International CLL Workshop Criteria  and the Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia . Also, all the patients were staged according to the Rai staging system ; 3 ml of peripheral blood (PB) was collected in EDTA tubes from each patient according to the standard protocol. The PB samples were processed within 6–24 h of collection, preserved at room temperature (22–24ºC). Clinical and demographic data were collected in a predesigned questionnaire. This study was approved by the ethical committee of the Sudan Medical Specialty Board (SMSB) and an informed consent was obtained from all patients before sampling in accordance to the guidelines and requirements of the ethical committee.
Each patient was subjected to a history taking and physical examination that was performed to determine the diameters of lymph nodes. Chest X-ray and abdominal ultrasound were used to evaluate the size of the liver and spleen.
IPT was carried out to confirm the diagnosis of CLL using the following monoclonal antibodies (Mo Ab) (Beckman Coulter); CD45, CD5, CD19, CD20, CD22, CD23, kappa and lambda light chains, FMC7, CD79b. A marker was considered positive at a cutoff level of 20%.
Flowcytometric analysis of intracellular ZAP-70 expression
Four color flowcytometer (COULTER EPICS XL-MCLTM Flowcytometer – Miami, Florida – USA) with SYSTEM II software was used to detect and measure the intracellular ZAP70 expression in PB samples according to the following protocol; First PerFix-nc a fixation and permeabilization kit was used as follows: Pipette 50 μL of blood sample into the bottom of each appropriately labeled tube. Add 5 μL of the FixativeReagent to each tube. Vortex immediately and incubate for 15 min at room temperature (18–25 C). Vortex the fixed specimen again and add 300 μL of the Permeabilizing Reagent to each tube; vortex immediately. Then, immediately add to each tube 20 μl ZAP-70 Isoclonic Control. Vortex immediately and incubate for 10 min at room temperature (18–25 C). Add to each tube 20 μL ZAP-70-PE (PN B57658). Vortex immediately and incubate for 45 min at room temperature protected from light. Add 3 mL of the Final 1 X Reagent (prepared from the 10 X concentrated Final Solution) to each tube; vortex immediately. Pellet the cells at 500 x g for 5 minutes, and completely discard the supernatant by aspiration. Re-suspend the cell pellet in 0.5 mL of the same Final 1X Reagent; the sample is now ready for acquisition on a flowcytometer. The ZAP-70 expression was considered positive at a cutoff level of 20%.
The collected data were analyzed using the software program of the Statistical Package for Social science for Windows (SPSS), version 19. Quantitative variables were summarized as mean and median and qualitative variables as percentage. Categorical variables were analyzed using Chi-Square test. P value less than 0.05 was defined as significant.
A total of 93 CLL patients (63 (67.7%) males and 30 (32.3%) females) were involved in this study with a mean ( SD) age 62.29 11.68, median 63 years (range: 36–95). According to Rai staging system, 35 (37.6%) patients were in Stage IV, 28 (30.1%) patients were in Stage III, 17 (18.3%) patients were Stage II, 9 (9.7%) patients were in Stage I, 4 (4.3%) patients were Stage 0; 66 (71.0%) patients presented with lymphadenopathy, 50 (53.8%) with splenomegaly, 23 (24.7%) with hepatomegaly. Anemia was detected in 68 (73.1%) patients and thrombocytopenia in 42 (45.2%) patients (Table 1). Immunophenotyping results revealed characteristic CLL immunophenotype.
We found that the mean ( SD) expression of ZAP70 was 17.06 21.89, with a median of 8.3 (range: 0.03–85.5%). Out of 93 patients, only 21 (22.6%) patients were positive for ZAP70 expressions at cutoff levels of 20%, whereas 72 (77.4%) patients were negative. No significant association was observed between ZAP70 expressions at cutoff levels of 20% and the study variables (Age, Sex, Lymphadenopathy, organomegaly, Hb concentration, TWBCs count, Platelets count, and Rai staging system) (p-value 0.05) (Table 2).
ZAP-70 has a great future as a prognostic marker as it has been found that ZAP-70 is highly predictive of time to treatment in a large cohort of early-stage (Rai 0-1) and untreated CLL [11,19]. The optimal cutoff for ZAP-70 positivity was defined by many authors as 20% [10,12,20,21]. We found that 21 (22.6%) patients were positive for ZAP70 expressions at cutoff levels of 20% and 72 (77.4%) patients were negative. Many published reports indicated ZAP-70 positivity in CLL ranging from 25% to 57% as presented in Table 3; our result is slightly lower.
Our results revealed that there was no significant association of ZAP-70 with age, sex, lymphadenopathy, organomegaly, hemoglobin concentration, TWBCs count, Platelets count, and Rai staging system (p 0.05). Our findings go in line with a study done by Gogia et al. from India who reported no association of ZAP-70 positivity with age, sex, lymphadenopathy, organomegaly, and Rai staging system . A study by Del Poeta et al. and Hus et al. indicated a significant correlation between high ZAP-70 levels and advanced Rai stage and splenomegaly [20 22]. This is in disagreement with our results. On the other hand, El-Kinawy et al. reported that ZAP-70 expression was significantly associated with advanced Rai stages 3 and 4, while it was negatively correlated to Hb levels and platelet counts .
Crespo et al.  found that at a cutoff of 20%, ZAP70 positivity clearly separated CLL patients into two groups; those with 20% ZAP70 had increased survival time and decreased chance of disease progression.
In most laboratories, ZAP70 is considered positive when at least 20% of the CLL-cells have a signal that is greater than the background control signal. Subjectivity in these methods can lead to variable results among research laboratories. To overcome this subjectivity, it has been suggested that mean fluorescence intensity (MFI) values from CLL-cells and background T lymphocytes should be measured rather than calculating the percentage of positive cells . Rossi et al  reported that when using MFI values and calculating the ratio of T-cells to B-cells, those patients with a ratio lower than 3 had a shorter time-to-treatment than ZAP70-negative CLL patients and those estimated to be ZAP70-positive by the T-cell percentage method only .
Unfortunately, prognostic impact of ZAP70 could not be investigated in our study due to lack of follow-up.
Contrast of our results with other workers may be contributed to by many factors such as biology of disease in the Sudanese population, sample sizes, and sensitivity of method.
The present study was aimed to study the prevalence of ZAP-70 in CLL patients and to relate them to the study variables (age, sex, lymphadenopathy, organomegaly, hemoglobin concentration, TWBCs count, Platelets count, and Rai staging system). ZAP-70 positivity was detected in 21 patients (22.6%). There was higher frequency of high-risk group among study population. There was no significant association of ZAP-70 positivity with the study variables. The prognostic value of ZAP-70 should be tested in the setting of a controlled prospective trial. Further studies are strongly recommended to develop a standardized flowcytometry protocol using MFI and T/B ratio method to verify the prognostic impact of ZAP70.
The authors are grateful to those who have contributed so much to this work; Khartoum Oncology Hospital staff and the Flowcytometry Laboratory for Leukemia & Lymphoma Diagnosis.