The guidelines project is a joint initiative of the Associação Médica Brasileira and the Conselho Federal de Medicina. It aims to bring together information in the scientific literature to standardize conduct in order to help decision-making during treatment. The data contained in the following articles were prepared by and are recommended by the Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular (ABHH). Even so, all possible medical approaches should be evaluated by the physician responsible for treatment depending on patient's characteristics and clinical status.

Description of the method used to gather evidence.

These guidelines are the result of a systematic review centered on the Evidence-Based Medicine movement, where clinical experience is integrated with the ability to critically analyze and rationally apply scientific information, thereby improving the quality of medical care.

Questions are structured using the Patient/Problem, Intervention, Comparison and Outcome (PICO) system, allowing the identification of keywords which were the basis of evidence-based search strategies in the major scientific databases [Medline (via PubMed), EMBASE, Central (Cochrane), Lilacs (via BVS)] and manual searches.

Degree of recommendation and level of evidence

A: Major experimental and observational studies

B: Minor experimental and observational studies

C: Case reports (non-controlled studies)

D: Opinion without critical evaluation based on consensus, physiological studies or animal models

These guidelines are subdivided into five articles:

Part 1: Laboratory workup to confirm diagnosis

Part 2: Classification systems

Part 3: Treatment of low-risk patients without the 5q deletion

Part 4: Treatment of low-risk patients with the 5q deletion

Part 5: Treatment of high-risk disease

Objective

This article presents the guidelines on the examinations that are needed to confirm the diagnosis of myelodysplastic syndromes.

PICO system

Using the PICO system, the P corresponds to patients with suspected myelodysplastic syndrome, I to the indication of examinations, and O to the outcomes (diagnosis).

Thus, 38 studies were found and selected to answer the clinical question (Appendix I).

What examinations are needed to confirm the diagnosis of myelodysplastic syndromes?

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell (HSC) clonal diseases resulting from a sequence of acquired genetic alterations with the formation of an anomalous and genetically unstable clone or clones, with potential to evolve to acute myeloid leukemia.

So far, there is no single reliable biological or genetic marker for diagnosis. Dysplastic alterations of peripheral blood (PB) and bone marrow (BM) are still fundamental for the diagnosis and classification of this group of diseases.

The detection of increased blasts facilitates diagnosis of the most advanced phases of the disease; however, in the early phases with minor morphological abnormalities, diagnosis is mainly based on the exclusion of other nonclonal cytopenias and diseases.

These clonal disorders may occur at any age, but they are more common in adults, with exponential increases after the 5th decade with a mean age at diagnosis of 70 years. Most patients acquire de novo clonal anomalies, with some acquiring somatic mutations after exposure to genotoxic agents, such as chemotherapy or radiotherapy for other neoplasms. Moreover, aging-associated inflammation (inflammaging) contributes to genetic instability and MDS predisposition.

The complete blood count with reticulocyte count and cytomorphological analysis are the vital first steps in the diagnosis of MDS. Isolated or combined, persistent for at least six months and unexplained cytopenias are common. Anemia, the most common cytopenia, is usually macrocytic, associated with a significant reduction in the reticulocyte count. All other causes of cytopenias/dysplasias should be excluded, as well as other clonal diseases and congenital abnormalities. Subsequently, a morphological analysis of BM cytological smears and an investigation of ring sideroblasts by Perls staining are performed. BM biopsy is complementary as it allows a better evaluation of the morphology and distribution of megakaryocytes, topography of other lineages, search for lymphoid aggregates and assessment of the degree of fibrosis by a standardized staining procedure. This is followed by classical and molecular cytogenetic analysis and immunophenotyping1(D).

Cytogenetic analysis plays an important role in determining clonality in patients with suspected MDS. Cytogenetic abnormalities are observed in 50–60% of patients, with the most common anomalies being del(5q), monosomy or deletion of 7q, trisomy 8 and del(20q). The diagnosis of MDS can be made in the absence of morphological dysplasia when there are recurrent cytopenias and cytogenetic abnormalities as described in the WHO 2008 classification (Table 1)17(D). However, nullisomy Y (-Y), 8 trisomy, and del(20q) are cytogenetic abnormalities that, in the absence of dysplasia, are not considered definitive evidence of MDS as they can be seen in other myeloproliferative diseases4,17(D).

In the case of repeated failure of conventional cytogenetic investigations (G-band karyotyping) due to the absence or poor quality of metaphases, fluorescence in situ hybridization (FISH) may complement the cytogenetic analysis.

BM samples from 48 MDS patients (43 low risk) were evaluated by karyotyping and FISH using probes for chromosomes 5, 7, 8, 11, 13 and 20. Eighteen of the 48 samples (37.5%) had an abnormal clone identified by metaphase chromosomal analysis, while 17 of 48 (35.5%) had an abnormal clone detected by FISH analysis. Normal karyotypes were detected in 30 of the 48 patients (62.5%) with 29 of these 30 having a normal FISH result. The sensitivities of the two methodologies (FISH and karyotyping) were very similar and so FISH may be a good tool to study BM aspirate when cytogenetic analysis is not available18,19(B).

In another study, 433 BM samples from patients with suspected MDS or AML were submitted to karyotyping and FISH for -5/5q-, -7/7q-, +8 and 20q-. Abnormal karyotypes were identified in 114 cases (26.3%) and abnormal FISH results of at least one locus were found in 94 cases (21.7%). Abnormal results by FISH or karyotyping were detected in 136/433 samples (31.4%). Of the 136 cases with any abnormal finding, 22 (16.2%) were identified only by FISH. The two methods had concordance in 96.5%, 96.5%, 93.9% and 93% of the cases of abnormalities of chromosomes 5, 7, 8 and 20, respectively20(B).

Immunophenotyping by flow cytometry (FCM) allows an analysis of the antigen expression related to lineage and degree of maturation in normal and abnormal hematopoiesis27(B). The expression of this antigen is tightly controlled by genetic and epigenetic mechanisms, leading to a predictable normal pattern at different stages of maturation28(B). In MDS, there is abnormal granulocytic, monocytic, and erythroid differentiation resulting in deviations from the normal pattern. There may be an increase or decrease in antigen expression, asynchronous expression, or aberrant cross-lineage antigen expression. This can be detected by FCM. Thus, this technique is an important ancillary tool in the diagnosis of MDS cases where few morphological abnormalities are found in BM cytology and histology and the karyotype is normal28–31,33(B),32,34(D).

In the last 15 years, numerous papers have been published about this subject, and international study groups, such as the European LeukemiaNet, have standardized this analysis for 4- and 8-color panels32(D),33(B). In contrast to morphology, that is susceptible to subjective evaluation, FCM analysis is an objective method if well standardized. Furthermore, in cytological analysis it is recommended to count 500 cells, while in FCM at least 100,000 cells (and >250 CD34+ cells) should be analyzed for the diagnosis of MDS. Immunophenotyping of BM cells is able to detect numerous maturation abnormalities in the myelomonocytic and erythroblastic lineages as well as analysis of myeloid and lymphoid progenitors34(B).

Increases of myeloid CD34+ cells often presenting aberrant and cross-lineage antigen expressions may be found, especially in refractory anemia with excess of blasts (RAEB)29–31,34–38(B). Moreover, B-cell precursor counts (hematogones type I) are lower compared to the normal number for the age of the patient33,39(B).

No fixed antibody panels or combinations have been recommended32(D),34(B). The analysis performed must always be compared to the findings of age-matched normal BM (BM donors, orthopedic surgery patients, etc.)32(D),29,31,33–35,39(B). FCM abnormalities found should be interpreted in the context of the clinical findings and other laboratory results, such as BM cytology and cytogenetics. There is no single pathognomonic abnormality for MDS. International recommendations establish that all abnormalities should be reported and the diagnosis of MDS is suspected if at least two aberrations are found.

Several efforts have been made to establish diagnostic scores but there is much controversy in the literature concerning this subject and none of the described scores is universally accepted21(D),33(B). The most commonly used in the clinical practice is the Flow Cytometry-based Scoring System (FCSS)40(B) based on several parameters of maturation abnormalities of the myelomonocytic series and increases and phenotypic aberrancies of myeloid progenitors. A simpler score (Ogata score) is based on four parameters: CD34+ myeloblast-related and B-progenitor-related cluster sizes, CD45 expression of myeloblasts and granulocyte side scatter using the values obtained for these parameters in the lymphocyte gate as an internal control. This score has a sensitivity of 70% and a specificity of 93% for the diagnosis of MDS41(B).

In earlier studies there was a concern to compare the findings of FCM with those of BM cytology and cytogenetics28,30,36(B). However, in more recent papers, as FCM has been well standardized, the emphasis is placed on a comparison with normal age-matched BM and how much the variables obtained in MDS differ from normal findings. Thus, it is important that each laboratory working in this field has its own local normal control values.

Nowadays, immunophenotyping in MDS has been well standardized, and based on the knowledge of the normal antigen expression of the several hematopoietic precursors in BM. Usually one or two standard deviations from the normal mean fluorescence intensity is used to say that there is an abnormal expression of an antigen. Also, it is more important to have local normal values of CD34+ cells rather than to use fixed normal values described in the literature, as these values may change with the fluorochromes conjugated to the monoclonal antibodies, the gating strategy used, quality controls of each laboratory as well as patients’ age31(B),32(D). This technique demands good training of the operator but it is easily feasible in a routine laboratory and is useful to discriminate between reactive (non-clonal) peripheral cytopenias and low grade MDS with few BM cell atypias29,33,40,41(B).

FCM analysis of BM cells is also able to provide prognostic parameters, associated with survival, response to treatment and BM transplantation32,36,40(B),42(D). In a cohort of 101 Brazilian patients, the number of CD34+ myeloblast-related cells was an independent prognostic parameter compared to the WHO Classification-Based Prognostic Scoring System (WPSS) and Revised IPSS (IPSS-R), and had higher prognostic value than IPSS.

FCM is considered an ancillary technique in the diagnosis of MDS and should always be reported and interpreted together with the PB values, BM cytology and cytogenetics which remain the gold standard for diagnosis29(B). However, when the karyotype is normal, and cytology is inconclusive, FCM is a valuable technique to confirm the diagnosis4(D).

The authors declare no conflicts of interest.