Detection of CD5 in B-cell chronic lymphoproliferative diseases by flow cytometry: a strong expression in B-cell chronic lymphocytic leukemia

<jats:p>PURPOSE: CD5 is a T cell marker, aberrantly express in B cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL). Other chronic B cell malignancies including hairy cell leukemia (HCL) and B cell prolymphocytic leukemia (B-PLL) are CD5 negative or express this antigen in a weak way. In this study, CD5 expression was investigated in leukemic cells from 42 patients with chronic B cell lymphoproliferative disease. METHODS: We studied the CD5 expression in leukemic cells from 42 patients with chronic B-cell malignancies by flow cytometry. Demographic features such as age, sex and clinical date were also analyzed. RESULTS: There were 22 males and 20 females. The immunophenotyping showed that 35 cases were B-CLL, 3 B-PLL and HCL and one patient was MCL. CD5 expression was present in all B-CLL and MCL. Low expression of CD5 was observed in one patient with B-PLL and negative in all cases of HCL. CONCLUSION: Our date demonstrated that CD5 expression can help distinguish among B-CLL from HCL and B-PLL, but is similar expressed in MCL.</jats:p>

section_content: CD5 is a T-cell marker that also is expressed in B1 subset of human B lymphocytes. Mature B cell malignancies, such as B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) are mostly CD5+. Other B cell chronic lymphoproliferative disorders such as B cell prolymphocytic leukemia (B-PLL) and hairy cell leukemia (HCL) are CD5 negative or expressed low level of this antigen 1, 3. 

This immunological characteristic of B-CLL and MCL is an important parameter to distinct between these entities and other chronic B-cell lymphoproliferative diseases such as B-PLL and HCL 4. 

Although the biologic function of CD5 is unknown, it is believed that in T cell subsets this antigen is an important molecule in the mechanism of cellular activation. In B1 lymphocytes or resting cells, it has also suggested that CD5+ B-cell may have a role in autoimmunity. They are increased in some people with autoimmune diseases secreting immunoglobulin that reacts with autoantigens such as rheumatoid factor and single and double-stranded DNA (1,5). Some reports have suggested an association between B-CLL CD5+ and autoimmune disorders. 

The objective of this study was designed to evaluate the diagnostic value of CD5 expression in leukemic cells from peripheral blood of 42 patients with B-cell chronic lymphoproliferative diseases.

section_name: Methods

section_content: Peripheral blood from 42 patients with chronic lymphoproliferative disease (CLD) diagnosted at the Instituto de Onco-Hematologia de Natal (IOHN) were studied. Informed consent was provided by all participants and they study was done with in the guiderlines of the Declaration of Helsinki. Blood samples were collected in tubes containing EDTA. 

We also evaluated clinical and hematological date at diagnosis and the patients were grouped as male vs female, age and clinical features. 

All cases of CLD were analyzed and classified when possible according to standard cytomorpholologic and immunophenotypic criteria 4,6,12

.
section_name: Samples of Patients

section_content: Ten milliliters (mL) of peripheral venous blood was collected into vacutainer tubes containing EDTA and mixed immediately. 

The white blood cells count (WBC), hemoglobin dosage and platelet count was accomplished in the hematological analyzer (Cell-Dyn 3000). The cytomorphologic analysis was accomplished in blood films stained by Leishmann. There were counted 100 leukocytes and the result scored in percentage. Cytomorphologic alterations were written down properly in the record of results.
section_name: Hematological analysis

section_content: The Immunophenotyping was performed by only and dualcolor flow-cytometry analysis of peripheral blood samples. Surface markers were identified by using monoclonal antibodies (MoAb) against CD3, CD10, CD19, CD45, CD14, CD16/56, CD8, CD5, CD7, CD22, CD23, CD25, CD38, HLA-DR, anti immunoglobulin heavy chain (IgH), kappa (?) and lambda (?) light chains of immunoglobulin. All MoAb were purchased from Becton-Dickinson Immunocytochemistry System (San Jose, CA). 

One hundred microliliters (100 ?L) of total blood cells previously homogenized, were incubated with the 20 ?L of MoAb for 30 minutes at room temperature and in darkness. 

Then, the suspension was homogenized and increased to the same 1ml of a 1 in 10 dilution of Becton Dickinson's FACS lysing solution in destilled water, having new incubation for more 10 minutes in the room temperature. After this period, the cellular suspension was centrifuged by 5 minutes at 1500 rpm, the supernadant fluid discarded and the cell pellet was ressuspended in cold phosphate buffered saline (PBS, pH 7. 2) and centrifuged again at 1. 500 rpm, being this last stage accomplished twice again. At last, the cell pellet was resuspended in 1 mL of cold 1% of paraformaldehyde in PBS and the cell suspensions were kept on the dark in cold until flow cytometric analysis. 

The samples were analysed in Fluorescence Activated Cell Analyser (FACScan, San Jose, CA, USA) with Cell Quest software. The (Cell Quest TM Software, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA), with acquisition of 10. 000 events, being taken into account the parameters Forward Scatter (FSC) in lineal scale that evaluates the cellular size, Side Scatter (SSC) also in lineal scale, which evaluates the cellular complexity, FL1 and FL2 in logaritimical scale detect green and orange fluorescence, respctively which represents the reaction antigen-antibody conjugated fluorescein isohtiocyanate (FICT), phycoerythrin (PE). 

The immunophenotyping was considered positive there were happened more than 25% of positive cells for all MoAb. The Results were supplied in the form of histograms in percentage of the cellular population with positive or negative reaction and fluorescence intensity.
section_name: Flow cytometry analyzis

section_name: Results

section_content: The clinical and demographic characteristics of all patients analyzed in this study were showed in table I. There were 22 males and 20 females and due to the small number of cases, no statistical significance was observed. The WBC was longer when compared with other B-cell malignancies. The lymphadenopaty was the most frequent clinical finding in all diseases, observed in 75% of B-CLL patients and in all cases of B-PLL, HCL and MCL. 

Cytomorphologic alteractions of leucocytes from peripheral blood were also observed in blood film stained. In B-PLL, were observed elevated count of pro-lymphocytes in all cases (³55%) and in HCL was observed the presence of lymphocytes with cytoplasmatic projections (hairy cells). II ). Leukemic cells from B-CLL patients, were characterized by strong coexpression of CD23 and CD5, associated with low expression of sIg and CD22 in all cases (Figure 1 ). In B-PLL, we observed that leukemic cells exhibited high level of sIg and CD22 and low expression of CD5 in one case (Figure 2 ). The immunophenotypic characteristic of leukemic cells of HCL in this study were: CD19+, CD22+, CD23+, CD38+, strong sIg+ and CD5-(Figure 3 ). The leukemic cells from MCL patient were characterized by strong expression of CD5, sIg and and lack of CD23 (Figure 4 ).
section_name: Patients Characteristics

section_content: The CD5 surface marker has a molecular weight of 65. 000 -67. 000 daltons and it is considered to be a pan-T cell marker, pan-thymocyte marker 1,2,5

. A restricted B cell expressing this marker, named B1 lymphocytes which reside in fetal primary follicles and spleen, in small numbers at periphery of the germinal centers of adult lymphonodes and tonsils 1,3,5. 

In pathologic conditions, CD5 was identified in large numbers of B lymphocytes in the peripheral blood of patients with rheumatoid arthritis or after allogeneic bone marrow. The laboratorial criteria for the diagnosis of CLL are well stabilished and include lymphocytosis of 5. 000/?L (5 x 10 9 /L) or more, fewer than 50% of prolymphocytes in peripheral blood and 30% or more of lymphocytes in the bonne marrow 10, 12. The distinctive immunologic properties of B-CLL are expression of weak monoclonal sIg, high affinity for binding mouse erythrocytes, CD23, and CD5 expression. This immunophenotypic pattern of HCL, suggest that cells are in a more advanced maturation stage compared with B-CLL, and HCL cells are also considered activated B-cell due to their strong expression of CD25 and CD38 4, 6, 7, 9. 

In this study, for the differenced diagnosis between HCL and B-CLL, we observed the CD5 expression in B-CLL in associated with weak sIg and CD22 in all cases, and strong coexpression of CD25 and CD38 associated with high intensity of sIg and CD22 and lack expression of CD5 in all cases of HCL. 

Recently, CD103 has been indicated as the most reliable markers to distinguish HCL from other B-cell leukemias 4,7

, but in this study we do not utilize this cell marker. B-PLL is a rare chronic B-cell leukemia characterized by a high WBC count, splenomegaly and more than 55% prolymphocytes in peripheral blood 7, 15. The immunophenotype of leukemic cells from B-PLL are similar to B-CLL, both are CD19+, CD22+ and HLADR+. However, the cells from PLL exhibit strong FMC-7 and sIg and are usually CD5-or express low levels of this antigen 4,7

. 

In this study, the elevated percentage of prolymphocytes in peripheral blood, lack of CD5 in two cases and weakness in one case, associated with a strong expression of sIg and CD22 in B-PLL, can be a differentiate diagnosis between B-CLL and B-PLL. .
section_name: Discussion

section_content: In conclusion, the results of the current study demonstrated that CD5 expression can distinguish the B-CLL of other chronic B-cell malignancies such as HCL and B-PLL but not between B-CLL and MCL.
section_name: Conclusion