Alex Freire Sandes

CD200 has an important role in the differential diagnosis of mature B-cell neoplasms by multiparameter flow cytometry.

Multiparameter flow cytometry is a useful tool for the diagnostic evaluation of mature B‐cell neoplasms (MBN). Recently, it has been shown that CD200 may improve the distinction between chronic lymphocytic leukemia (CLL; CD200+) and mantle cell lymphoma (MCL; CD200−), but the role of CD200 expression in atypical CLL and other MBN remains to be established.

Combined flow cytometric assessment of CD45, HLA-DR, CD34, and CD117 expression is a useful approach for reliable quantification of blast cells in myelodysplastic syndromes†

Quantification of bone marrow (BM) blasts by cytomorphology is essential for the diagnosis of myelodysplastic syndromes (MDS). Owing to its subjectivity and the potential impact of dysplastic features on accurate identification of blast cells, more objective approaches are required, multiparameter flow cytometry (MFC) being a particularly promising approach in this regard. However, no consensus exists about the optimal combination of markers and strategy to be used.

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes

Background Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes. Design and Methods We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated. Results Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n=5), CD42a (n=1) and CD61 (n=2), together with reactivity for CD34 (n=1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n=16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival. Conclusions Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders.

Diagnostic screening of paroxysmal nocturnal hemoglobinuria: Prospective multicentric evaluation of the current medical indications.

Although consensus guidelines have been proposed in 2010 for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometry (FCM), so far no study has investigated the efficiency of such medical indications in multicentric vs. reference laboratory settings.

First proposed panels on acute leukemia for four‐color immunophenotyping by flow cytometry from the Brazilian group of flow cytometry‐GBCFLUX

Multiparameter flow cytometry is a highly sensitive, fast, and specific diagnostic technology with a wide range of applicability in hematology. Although well‐established eight‐color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four‐color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource‐restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease‐oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable, and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient‐specific needs. The proposed panels will be subsequently validated in an interlaboratory study to evaluate its effectiveness on the diagnosis and classification of AL. (Assoc editor comm. 2).

Improving the outcomes of elderly patients with acute myeloid leukemia in a Brazilian University Hospital

OBJECTIVE: To evaluate the outcomes of acute myeloid leukemia patients who were older than 60 years of age at the time of diagnosis following the implementation of a treatment algorithm based on age, performance status, and cytogenetic results. METHODS: We retrospectively compared the results of 31 elderly acute myeloid leukemia patients (median age of 74 years) who were treated according to the new algorithm. RESULTS: Fifteen patients with a good performance status and no unfavorable karyotypes were treated with either intensive cytotoxic chemotherapy (<70 years, nine cases) or adapted etoposide, 6-thioguanine and idarubicine (>70 years, six cases); 16 cases with a poor performance status or unfavorable cytogenetics received supportive care only. Six patients achieved a complete remission and two achieved a partial remission after chemotherapy. There were three toxic deaths during induction, two in the adapted etoposide, 6-thioguanine and idarubicine group and one in the intensive cytotoxic chemotherapy group. The overall median survival time was 2.96 months, 1.3 months in the supportive care group, and 4.6 months in the treatment group. CONCLUSIONS: Our results illustrate the importance of treatment guidelines adapted to local resources in an attempt to improve the survival of elderly acute myeloid leukemia patients in developing countries.

Association of myelodysplastic syndrome with CD5+, CD23+ monoclonal B-cell lymphocytosis.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License.

Normal variation of bone marrow B-cell precursors according to age - reference ranges for studies in myelodysplastic syndromes in Brazil: Normal Variation of Bone Marrow B-Cell Precursors

Normal B lymphoid maturation occurs in bone marrow (BM) throughout life, but immature B‐cell progenitors (BCPs) are more numerous in children than in adults. To assess the normal values according to age became important as BCPs are decreased in myelodysplastic syndromes and have been considered an important diagnostic and prognostic feature in these clonal disorders.

Diagnosis of chronic lymphoproliferative disorders by flow cytometry using four-color combinations for immunophenotyping: A proposal of the brazilian group of flow cytometry (GBCFLUX)

Multiparametric flow cytometry (MFC) is a powerful tool for the diagnosis of hematological malignancies and has been useful for the classification of chronic lymphoproliferative disorders (CLPD) according to the WHO criteria. Following the purposes of the Brazilian Group of Flow Cytometry (GBCFLUX), the aim of this report was to standardize the minimum requirements to achieve an accurate diagnosis in CLPDs, considering the different economic possibilities of the laboratories in our country. Most laboratories in Brazil work with 4‐fluorescence flow cytometers, which is why the GBCFLUX CLPD Committee has proposed 4‐color monoclonal antibody (MoAb) panels.

Brazilian Group of Flow Cytometry (GBCFLUX) panels for acute leukemias--Response to Matos.

We thank Matos D (1) for the comments about the consensus proposal of the Brazilian Group of Flow Cytometry for the diagnosis of acute leukemia (2). Concerning the suggestion to include BDCA-2 (CD303) to confirm the diagnosis of Blastic Plasmacytoid Dendritic Cell Neoplasia (BPDCN), although it is a quite specific marker, we have not included it in our antibody panel because we looked for the minimum requirements needed to achieve the correct final diagnosis, taking into account the cost-effectiveness acceptable for services with different economic possibilities (2). It could be included as an optional marker in cases of doubt. However, in the proposed panel for acute myeloid leukemia we included not only CD123 and HLA-DR that have a typically bright expression in BPDCN, but also CD36, NG2, CD4, and CD56 that compose the phenotype of this entity. This profile allows a strong evidence of BPDCN, as cited by Shi in a recent revision: “the brighter CD123 coupled with the co-expression of CD56 and CD4 is almost pathognomonic of this disease in flow cytometric analysis” (3). We also recommended the morphological analysis and to take into account clinical aspects that frequently are typical in that neoplasm. Thus, we believe that all these features are sufficient for the diagnosis in most BPDCN cases. Also, it should be stressed that the cost-benefit of this one antibody that will be used to confirm a rare disease is not affordable for many Brazilian laboratories. The other suggestion of Matos was to recommend that anti-TCRab and antiTCRgd antibodies should be included in the panel for every (negative or positive surface CD3) case suspect of T cell Acute Lymphoblastic Leukemia (T-ALL). This was a subject of many discussions by GBCFLUX taking into account the normal T-cell ontogeny. In this setting, the TCR ab and gd rearrangement occurs in the thymic cortical maturation phase but they need the membrane CD3 complex to be expressed in the cell surface, what happens only in the medullar phase. Asnafi et al. (4) referred to T-ALL cortical (SmCD3 negative, CD1a1, CD4 and CD8 double positive) cells and the expression of TCRb (pre-TCR ab and gd expression) in the leukemia cells. They also described a T-cell receptor (TCR)based classification of T-ALL that allows division into: “mature” T-ALL, that express SmCD3 and TCRab or TCRgd; “pre-ab” which express cytoplasmic TCRb (cyTCRb) in the absence of SmCD3/SmTCR; and “immature” SmTCR and cyTCRb double negative T-ALL (5). Besides, there are some relevant technical aspects that our group wants to emphasize: (i) the carryover that can occur in tubes containing cytoplasmic markers; (ii) spectral spillover between monoclonal antibodies and fluorochrome combinations used, that can imply in unspecific and/or false positive results for TCR analysis and/or false negative results for SmCD3. Finally, we would like to emphasize that the proposed panels are being used in Brazilian laboratories and will be subsequently validated in a collaborative study to evaluate its effectiveness on the diagnosis and classification of acute leukemia, as has already been done by another group (6). The recommendations will be updated periodically by the GBCFLUX (M.R.V. Ikoma, A.F. Sandes, I. Lorand-Metze, and M. Yamamoto on behalf of GBCFLUX), according to the objectives proposed by the group.