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Myeloperoxidase in myelodysplastic syndromes
results, used a case control design, which was prone to spectrum bias,10 or yielded imprecise diagnostic accuracy estimates due to relatively limited sample sizes.
Degranulation of mature granulocytes is a classical dys- plastic feature of MDS,11-13 and this can be analyzed using various methods, including hemogram automaton, cyto- morphological evaluation, and flow cytometry (side scat- ter). Degranulation is associated with myeloperoxidase (MPO) cytoplasmic expression, an enzyme synthesized during myeloid differentiation that constitutes the major component of neutrophil azurophilic granules.14 MPO expression may be studied by immuno-cytochemical staining,11,15 although this approach is limited by the mod- erate sensitivity and subjective nature of cytomorphologi- cal evaluation of PB in routine practice.
Flow cytometric analysis of MPO expression in BM neutrophil granulocytes has been occasionally used to identify MDS patients and to discriminate between low- versus higher-risk patients with MDS.16 However, the accu- racy of flow cytometric analysis of neutrophil MPO expression in PB for the diagnosis of MDS has not been studied.
The present study aimed to assess the performance of flow cytometric analysis of MPO expression in peripheral blood mature granulocytes to rule out a diagnosis of MDS and/or chronic myelomonocytic leukemia (CMML).
Methods
Study design
Using a retrospective case control study design,17 we assessed the diagnostic accuracy of various parameters of neutrophil MPO expression in PB measured by flow cyto- metric analysis and defined a threshold that identified patients who were unlikely to have MDS or CMML. We then assessed the diagnostic accuracy of this threshold in a prospective validation cohort of consecutive patients referred for suspicion of MDS. The protocol for this study was approved by the Comité de Protection des Personnes Sud Méditerranée I, Marseille, France.
Study sites
The flow cytometric analysis protocol was jointly devel- oped and pre-tested at three university-affiliated hospitals in France: Clermont-Ferrand, Saint-Etienne, and Grenoble. Participants in the retrospective case control and prospec- tive validation studies were enrolled at two study sites: Clermont-Ferrand and Grenoble. The index test and refer- ence standard were performed at the site of enrollment.
Participants
In the retrospective case control study, cases were adults with established diagnosis of MDS or CMML, as defined by current guidelines.1,2,4,5,18 They were retrospectively identified by screening the electronic laboratory record using the MDS and CMML diagnosis codes. Controls were individuals referred to the hematology laboratory with normal values for the routine blood cell count. Exclusion criteria for both cases and controls were acute leukemia and admission to the intensive care unit. Cases and controls were matched on gender. The study sample was restricted to controls aged 50 years or older because all cases were over this age.
The prospective validation cohort consisted of consecu-
tive adults who were referred for suspected MDS. Suspicion of MDS was based on medical history and PB cytopenia. All patients enrolled in the validation cohort study were prospectively evaluated for the reference stan- dard and index test.
Index test
Peripheral blood samples were stored at 4°C overnight and processed within 24 hours (h) of collection. We used material remaining after a routine blood cell count with the Sysmex XE-5000 and Sysmex XN-10 automated hematology analyzers (Kobe, Japan).
The blood sample was stained according to the manu- facturer’s recommendations with a panel of antibodies conjugated to fluorochromes. CD64 FITC (clone 10.1), CD15-PerCPCy55 (clone HI98), CD11b-APC (clone D12), CD16-APCH7 (clone 3G8), CD14-V450 (clone MfP9), and CD45-V500 (clone HI30) antibodies were added. Aliquots were stained for 15 minutes (min) at room temperature. The fixation and permeabilization phases were performed using the BD IntraSureTM Kit (BD Biosciences, San Jose, CA, USA) and MPO-PE was added (clone 5B8) during the permeabilization phase. All antibodies, BD FACSTM Lysing Solution and BD IntraSureTM Kit were obtained from BD Biosciences (San Jose, CA, USA).
At least 10,000 neutrophils were acquired on a 3-laser, 8-color BD FACSCanto-IITM flow cytometer (BD Biosciences, San José, CA, USA) and analyzed using BD FACSDiva Software at each study site. The gating strategy is presented in Figure 1.
Myeloperoxidase expression in the PB neutrophil popu- lation within an individual subject was expressed as medi- an, mean, and robust coefficient of variation (RCV).19 The RCV was calculated as the robust standard deviation divided by the median. The robust standard deviation is a function of the deviation of individual data points to the median of the study population.20 The RCV was expressed as a percentage and reflected the variability in MPO expression in the PB neutrophil population within an indi- vidual subject (Figure 2).
The FranceFlow standard operating procedure was used to standardize instrument settings. Rainbow calibration particles (BD SpheroTM, BD Biosciences, San Jose, CA, USA) were analyzed daily and photomultiplier tubes were adjusted if needed (Online Supplementary Table S4).
In the retrospective case control study, flow cytometric analysis was performed within six months of MDS diag- nosis and could not be blinded to patient status for logis- tical reasons. In contrast, flow cytometric analysis was performed within 24 h of BM aspirate and was blinded to the reference standard in the prospective validation cohort.
Reference standard
The reference diagnosis of MDS was established according to current guidelines,1,2,4,5 based on clinical data, peripheral blood cytopenia, cytomorphology of PB and BM aspirate, and cytogenetic analysis. Peripheral blood cytopenia was defined using standard laboratory values: hemoglobin concentration <12 g/dL (females) and <13 g/dL (males), platelet count <150x109/L, and/or absolute neutrophil count <1.8x109/L.18
Bone marrow cytomorphology was evaluated prospec- tively by experienced hematopathologists who were blinded to the index test results. The criteria for MDS
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