Diagnosis of canine B-cell chronic lymphoid leukemia with a CD21 negative phenotype using the LT21 clone CD21 antibody in flow cytometry: a case report

Abstract

Background: Chronic lymphoid leukemia (CLL) is a hematological disorder characterized by the clonal expansion of small mature lymphocytes that accumulate in the blood and bone marrow. CLL can arise from B-, T-, or natural killer cell clones. The cytological evaluation of blood smears is often the simplest and least invasive method for diagnosing lymphoid leukemia. Immunophenotyping is used to further subclassify the type of lymphoid leukemia.

Case presentation: A 15-year-old, 4.4-kg spayed female Shih Tzu was presented to the veterinary medical teaching hospital of Kangwon National University. Despite having a normal appetite and activity level, cervical and inguinal lymph node enlargement was noted on physical examination. Complete blood count revealed severe leukocytosis, severe lymphocytosis, and monocytosis. Splenomegaly, hepatomegaly, and lymph node enlargement were detected on radiographic and ultrasonographic examination. Immunophenotyping was performed using peripheral blood mononuclear cells (PBMCs). The majority of lymphocytes exhibited the following profiles: CD3^-CD79a^- (97.5%), CD4^-CD8^- (98.6%), CD21^-CD79a^- (98.4%), CD34^- (0.1%), CD45⁺ (99.6%), major histocompatibility complex class II⁺ (99.5%), and CD14^- (0.5%). Based on the immunophenotyping results, possible differentials considered included the following: the majority of lymphocytes may be natural killer (NK) cell clones, plasma cell clones, or show aberrant expression or loss of CD21 marker due to the neoplastic nature of the cells. Further flow cytometry was performed using antibodies against CD3, CD5, CD94, and granzyme B. The combined results indicated that the predominant lymphocyte subset in the PBMCs was CD3^-CD5^-CD21^-CD94^-granzyme B^-. To confirm monoclonality and exclude the aberrant loss of CD markers, a polymerase chain reaction for antigen receptor rearrangement (PARR) assay was conducted. The PARR assay, using DNA from blood and lymph node samples, showed B-cell monoclonality. Immunocytochemistry using PBMCs showed that the plasma cell marker Multiple Myeloma Oncogene 1 (MUM1) was not expressed. Therefore, the diagnosis was confirmed to be B-cell CLL.

Conclusion: Immunophenotyping can help subclassify the type of lymphoid leukemia; however, as tumor cells can show aberrant expression or loss of the CD21 marker, combining immunophenotyping with the PARR assay could yield a more accurate diagnosis.

Keywords: B-cell chronic lymphoid leukemia; CD21; Dog; Immunophenotyping; Lymphoproliferative diseases.

Fig. 1

Cytological examination of the blood smear, inguinal lymph node and spleen. (A) Peripheral blood smear (Diff-quick stain, × 1,000, scale bar: 10 μm). (B) Fine-needle aspiration of the inguinal lymph node (Diff-quick stain, × 1000, scale bar: 10 μm). (C) Fine-needle aspiration of the spleen (Diff-quick stain, × 1000, scale bar: 10 μm)

Fig. 2

Immunophenotyping of peripheral mononuclear cells before chemotherapy. Peripheral blood mononuclear cells isolated from the canine patient were immunophenotyped by flow cytometric analysis using FITC-conjugated mouse anti-dog CD3/PE-conjugated mouse anti-CD79A, FITC-conjugated rat anti-dog CD4/PE-conjugated rat anti-dog CD8 monoclonal antibody, FITC-conjugated mouse anti-dog CD21/PE-conjugated mouse anti-CD79A, PE-conjugated mouse anti-canine CD34, FITC-conjugated rat ant-canine CD45, FITC-conjugated rat anti-canine MHC class II, and FITC-conjugated mouse anti-human CD14 antibodies. Nonspecific binding of targeted antibodies to cell surface antigens was estimated using isotype control antibodies. Gating was performed with reference to populations of unstained, single stained, and isotype controls. FITC: fluorescein isothiocyanate; PE: phycoerythrin

Fig. 3

Additional immunophenotyping of peripheral mononuclear cells. Peripheral mononuclear cells isolated from the canine patient were immunophenotyped by flow cytometric analysis using FITC-conjugated mouse anti-dog CD3/Pacific Blue™-conjugated anti-dog CD5/Alexa Fluor^® 647-conjugated mouse anti-dog CD94, and FITC-conjugated mouse anti-dog CD3/PE-conjugated mouse anti-human Granzyme B. The nonspecific binding of targeted antibodies to cell surface antigens was estimated using isotype control antibodies. Gating was performed with reference to populations of unstained, single stained, isotype, and FMO controls. FITC: fluorescein isothiocyanate; PE: phycoerythrin; FMO: fluorescence minus one

Fig. 4

Capillary electrophoresis traces of PARR using DNA samples from a blood smear slide and an inguinal lymph node slide provided by the reference laboratory. Both (A) blood and (B) lymph node samples showed monoclonality in IgH major and IgH minor. PARR performed using IgH major (target Tm: 85ºC and target bp: 120), IgH minor (target Tm: 86ºC and target bp: 120), TCR (target Tm: 83ºC, target bp: 90), and lymphocyte control primers (target bp: 130). bp, base pair; Tm, melting temperature; PARR, polymerase chain reaction for antigen receptor rearrangement; IgH, immunoglobulin heavy chain; TCR, T-cell antigen receptor

Fig. 5

Immunophenotyping of peripheral mononuclear cells performed at day 793 after chronic lymphoid leukemia diagnosis. Peripheral mononuclear cells isolated from the canine patient were immunophenotyped by flow cytometric analysis using FITC-conjugated mouse anti-dog CD3/PE-conjugated mouse anti-CD79A, FITC-conjugated rat anti-dog CD4/PE-conjugated rat anti-dog CD8 monoclonal antibody, FITC-conjugated mouse anti-dog CD21/PE-conjugated mouse anti-CD79A, PE-conjugated mouse anti-canine CD34, FITC-conjugated rat ant-canine CD45, FITC-conjugated rat anti-canine MHC class II, and FITC-conjugated mouse anti-human CD14 antibodies. Nonspecific binding of targeted antibodies to cell surface antigens was estimated using isotype control antibodies. Gating was performed with reference to populations of unstained, single stained, isotype, and FMO controls