Elisa Y. S. Kimura

Expression levels of CD47, CD35, CD55, and CD59 on red blood cells and signal-regulatory protein-α,β on monocytes from patients with warm autoimmune hemolytic anemia

BACKGROUND: Animal models have shown that CD47‐deficient mice develop severe autoimmune hemolytic anemia (AIHA) because the binding of red blood cell (RBC) CD47 to signal‐regulatory protein (SIRP‐α) on macrophages contributes to the inhibition of phagocytosis. In contrast, complement‐inhibitory proteins such as CD35, CD55, and CD59 may protect RBCs against the lysis by complement.

Expression of Fas and Fas ligand on spleen T cells of experimental animals after unmodified or leukoreduced allogeneic blood transfusions.

BACKGROUND: The clonal deletion seen in recipients of allogeneic blood transfusion (ABT) refers to the removal of lymphocytes that promote the clearance of transfused alloantigens. Interactions between Fas (CD95) and FasL (CD95L) are involved in the clonal deletion of T cells and in the down regulation of the cytotoxic T‐cell activity.

Molecular Basis of KELnull Phenotype in Brazilians

Background: KELnull (K₀) persons can produce clinically significant anti-KEL5 antibody after transfusion and/or pregnancy, requiring K₀ blood transfusion when indicated. 37 K₀ alleles have been reported in studies over different populations, but none in Amerindian-Caucasian descendants from South America. The aim of this study was to identify the molecular basis of K₀ phenotype in Brazilians. Methods: We investigated three K₀ samples from different Brazilian blood banks (Recife, Manaus, and Vila Velha) in women with anti-KEL5. KEL antigen typing was performed by serologic techniques, and the K₀ status was confirmed by flow cytometry. PCR-RFLP and DNA sequencing of the KEL coding and exon-intron regions were also performed. Results: RBCs of the 3 patients were phenotyped as KEL:-1,-2,-3,-4,-7. The 3 patients had the same KEL*02/02 genotype and were negative for KEL*02.03 and KEL*02.06 alleles. The Recife K₀ patient was homozygous for IVS16 + 1g>a mutation (KEL*02N.31 allele). The flow cytometry with anti-KEL1, anti-KEL2, anti-KEL3, anti-KEL4, and anti-CD238 confirmed the K₀ phenotype. In addition, we found the c.1042C>T mutation (KEL*02N.04 allele) in both the Manaus K₀ and the Vila Velha K₀ patients. Conclusion: This report represents the first study of K₀ molecular basis performed in Amerindian-Caucasian descendants from South America.

Multiples aberrant phenotypes in multiple myeloma patient expressing CD56-, CD28+,CD19+

Recent immunophenotypic studies demonstrated that the expression of aberrant antigens in myeloma plasma cells (MPCs) is a common feature. The most frequent aberrant antigens in MPCs are: CD56 (75%) and CD117 (63%). Several reports suggest that multiple myeloma (MM) patients with CD56 negative MPCs present with aggressive disease and have a worse outcome.(1-3) Van Camp et al. were the first to demonstrate that in the context of high-dose chemotherapy and autologous stem cell transplantation, CD56 negative cases behaved similarly to CD56 positive cases.(4) These results are encouraging, but we would like to mention that other immunophenotypic and genetic characteristics have also demonstrated clinical impact. In particular, the rare phenotype, CD19+CD56-, which is generally found in normal plasma cells,(3) responds poorly to combination chemotherapy. Here, we describe a case in which plasma cells expressed the CD19+CD56phenotype as well as several other aberrant antigens (CD20+, CD22+, CD28+, CD33+, CD117+, HLA-DR+). A 60-year-old man was admitted with back pain and weight loss over a 4-month period. Laboratory tests showed: red blood cell count: 2.8 x 1012/L; hemoglobin: 7.5 g/dL; platelet count: 161 x 109/L; and leukocyte count 3.5 x 109/L. The creatinine level was normal, but serum ionic calcium was 1.30 mg/dL. The serum total protein concentration was 15.8g/dL and albumin 2.06 g/dL. Serum s2microglobulin was 7.2 mg/L, and monoclonal component was 7.69 in gamma globulin. IgG was 9160 mg/dL, IgA was 12 mg/dL and IgM < 4 mg/dL. Bence Jones kappa protein was found in urine. A bone marrow (BM) biopsy revealed a hypercellular marrow with 95% of dysplastic plasma cells. There were extensive osteolytic bone lesions in the thoracic spine and bone fractures in L5. A diagnosis of multiple myeloma (IgG kappa, stage IIIA) was made. Four-color flow cytometry of fresh BM cells revealed that the myeloma cells expressed CD38, CD138, CD19, CD20, CD22, CD33, CD28, CD117, HLA-DR but no CD56 or CD45 (Figure 1). The cells were positive for cytoplasmic immunoglobulin kappa light chain. Cytogenetic studies revealed a hyperdiploid karyotype and no rearrangement of the IgH gene or deletion of 13q14. The patient was treated with one cycle of the VAD (vincristine 0.4 mg/day, doxorrubicine 9 mg/day and dexamethasone 40 mg days 1-4, 9-12, and 17-21) regimen, and two cycles of the VMMD (vincristine 1.5 mg/day i.v, day 1; ranimustine 50 mg/day, day 1; dexamethasone 40 mg days 1-4, 9-12, and 17-21 and melphalan 8 mg p.o, days 1-6) and MP (melphalan 10 mg and prednisolone 50 mg p.o days 1-4) regimes, without any significant improvement. The patient died due to systemic infection. Figure 1 Four-color flow cytometry of fresh bone marrow cells showing myeloma cells expressing CD38, CD138, CD19, CD20, CD22, CD33, CD28, CD117, HLA-DR but no CD56 or CD45 Sahara et al. were the first to describe a MM case with the CD19+CD56-phenotype that was refractory to combination chemotherapy. They also reported that the lack of CD56 was related to aggressive disease and significantly shorter survival than CD56+ cases(5). According to Lin et al., these CD56 negative cases should receive intensive treatment that can overcome the poor response(3). However, it would be interesting to evaluate the cases reported by them in the context of other prognostic features: according to other aberrant antigens, such as CD28+, and myeloid associated antigens. Interestingly, they did not find any association of CD56 negative cases with genetic risk groups, including deletion 13q, p53 gene mutation and IgH translocations. Our case presents the rare CD19+CD56-phenotype with several other aberrant antigens (CD20+, CD22+, CD28+, CD33+, CD117+, HLA-DR+). The CD28 antigen is not expressed on normal B cells or plasma cells and according to Shapiro et al., its presence is related to advanced disease. Conversely, this patient presented a hyperdiploid karyotype but no other genetic alteration with a defined prognosis group as reported by Kyle and would therefore be classified in a good prognosis group.(2,6) Mateo et al. reported that non-hyperdiploid MM cases are associated with positivity for CD28 and CD20 and negative for CD117, in contrast to the case presented here. These findings highlight the need of a more comprehensive evaluation that should include DNA ploidy status, IgH gene rearrangements, del 13q and del 17p.(7) However, the CD56 expression pattern could be used as a rapid prognostic risk factor to direct treatment and predict disease outcome.

CLL: chromosomal abnormalities (FISH) and their relation with clinical stage, CD38 and ZAP-70

Chronic lymphocytic leukemia is the most prevalent type of leukemia in the West. It is characterized by an extremely variable clinical course. The aim of the study was to detect the most frequent chromosomal abnormalities in patients with CLL using FISH, and assess them regarding age, gender, clinical stage and CD38 and ZAP-70 expressions. We found 51.7% of the patients with chromosome abnormalities. The most frequent one was del 13q14 in 34.5% of cases. It was associated to other alterations in 17.2%. 17p13 deletions were found in 17.2% and trisomy 12 in 13.8% (in isolation in 6.9% and associated to del 13q14, in 6.9% of the cases). An 11q22 deletion was found in one case associated to a 13q14 deletion. To better evaluate the relationship between chromosome aberrations and other prognostic factors in CLL, two cytogenetics groups were considered: favorable (13q deletion in isolation and no alteration) and unfavorable outcomes (trisomy 12, 17p13 deletion, 11q22 deletion and two simultaneous alterations).The unfavorable alterations were more frequently seen among young individuals (<60y). There were more females (70%) than males in this group (p=0.04). In relation to the Binets staging system, patients with unfavorable cytogenetic alterations, tended to be B and C stages, while in the favorable group prevailed patients in stage A. Additionally, patients with poor prognostic cytogenetics tended to express CD38 and ZAP-70 proteins.

O papel da expressão de Bcl-2 em material obtido por PAAF no diagnóstico de doenças linfoproliferativas B

BACKGROUND: The diagnosis of lymphoproliferative disorders (LPD) is routinely made through histological and immunohistochemical analysis of lymph nodes. Immunophenotyping by flow cytometry (FC) is a sensitive and fast tool, which may be applied in samples obtained through fine needle aspiration for the diagnosis of LPD. Bcl-2 is a proto-oncogene that appears in several LPD and it has a significantly high expression in follicular lymphomas. OBJECTIVES: to diagnose LPD in FNA samples through morphology and flow cytometry immunophenotyping. MATERIAL AND METHODS: Samples from 25 patients with lymphadenopathies and 2 reactive tonsils were studied through morphology and immunophenotyping. The antigens expressions were evaluated by using a screening panel of monoclonal antibodies (CD3, CD4, CD8, CD19, light chains kappa; and lambda), followed by CD5, CD10, CD11c, CD23, CD79b, sIgM, FMC-7 and Bcl-2 when required. The results were compared with histology. RESULTS:Four out of 25 samples were reactive processes and 21were B-LPD. In all cases there was consistency with histological results. The mean fluorescence intensity of Bcl-2 in Follicular Lymphoma (19.92) was higher compared with other lymphoproliferative diseases (11.93) and controls (3.49) (p = 0.032). CONCLUSION: Fine needle aspiration of lymph nodes associated with cytomorphology and flow cytometry immunophenotyping allows a fast differentiation between reactive processes and B lymphoproliferative cases. The high expression of Bcl-2 by cytometry shows its usefulness in the diagnosis of the most frequent type of B-LPD. Fine needle aspiration sampling requires training and more than one aspiration is recommended.