Background The diagnosis of CLL is supported by a typical morphology and immunophenotype and usually does not present difficulties. Nevertheless, some patients with CLL can show an atypical phenotype, this raising the possibility of a lymphoproliferative disorder other than CLL. It has been recently shown that the expression of CD200 could be a rather consistent marker for CLL. Methods The expression of CD200 was investigated in 120 consecutive patients with B‐cell chronic lymphoproliferative disorders (B‐CLPD) (65 cases diagnosed as typical CLL, 16 atypical CLL, and 39 non‐CLL before entering the study) by using multiparametric flow cytometry with four color combinations. CD200 was analyzed as percentage of positive cells (≥30%) and MFIR expression. ROC curves were used to determine the cut‐off for the CD200 MFIR. Matutes score (MS) was used as comparator. Results All 81 (100%) patients classified as CLL and 25 of 39 (64.1%) classified as non‐CLL expressed high CD200 expression (≥30%). CD200 expression showed a high sensitivity (100%) and a low specificity (35.9%), and the accuracy was similar to that of Matutes score markers (range: 79.2%–86.7%); except SmIg that was 59.1%. The addition of CD200 to the Matutes score correctly identified 74 of 81 (91.4%) CLL cases including 9 of 16 atypical CLL cases. As per non CLL cases, 37 of 39 (94.9%) were correctly diagnosed by the modified system. Altogether, CD200 improved the diagnostic accuracy of Matutes score from 86.7% to 92.5% (P < .01). Conclusion These results show that CD200 is a valuable, albeit not specific, CLL diagnostic marker. © 2018 International Clinical Cytometry Society

Background: B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disorder with respect to its clinical course. Accurate identification of prognostic factors is becoming increasingly important in order to determine those patients requiring aggressive treatments. Two of the most predictive outcome markers are the Ig VH mutational gene status and ZAP-70 expression. In earlier reports, both parameters have shown a high degree of concordance. To assess the value of these determinations in clinical practice, we simultaneously analyzed Ig VH mutations and ZAP-70 expression in a consecutive series of B-CLL.Methods: Fifty-three consecutive B-CLL cases were included in the study. ZAP-70 expression was investigated by flow cytometry. Positivity was established using two methods: comparing ZAP70 expression in B-cells with T-cells using cytoplasmic CD3 (ZAP-70/T) and with NK-cell reactivity (ZAP-70/ NK). The complete immunophenotype was recorded in each case. Ig VH mutational gene status was determined employing purified RNA from peripheral blood samples. Retrotranscribed DNA was PCRamplified, direct-sequenced, and compared with available public databases. VH3.21 family use was also recorded.Results: Using a T-cell marker, 58% of patients were ZAP-70+ and 42% were ZAP-702. NK-cell comparisons gave only 6% of ZAP-70 positivity in B-CLL, and in six cases the absence of a clearly defined NKcell population precluded the ZAP70 analysis. Twenty-four (45%) patients had mutated Ig VH genes and 29 (55%) had unmutated Ig VH genes. The results showed a statistical association between ZAP-70/T expression and VH mutational status. Despite this, in 30% of cases there was a discordant result.Immunophenotypic analysis showed no major differences in Matutes'score between mutated and nonmutated cases. Only FMC7 was more commonly expressed in the unmutated B-CLL cases. VH3.21 was present in 7.5% of cases, mostly having an unmutated pattern.Conclusions: ZAP-70 reactivity using a T-cell marker as a control allows to identify the majority of patients with an unmutated Ig VH genotype. Parallel analysis revealed that discordances with Ig VH analysis are quite common with currently employed flow cytometry reagents and techniques.

Antibodies against CD66 identify antigens from the carcinoembryonic antigen (CEA) family of proteins, which belong to the immunoglobulin gene superfamily. Despite being usually restricted to cells of myeloid or monocytic origin, CD66 expression has also been reported in blasts from children with B-cell lineage acute lymphocytic leukaemia (ALL). An analysis of the CD66 expression was undertaken in a series of acute leukaemia patients. Antigenic expression was analysed using triple combinations of monoclonal antibodies (mAbs) in forty-five patients. The CD66 Kat4 fluorescein isothiocyanate clone was purchased from Dako (Glostrup, Denmark). CD66 was expressed in 2 of 29 patients with AML (acute myeloblastic leukemia) (6.8%) and in 8 of 12 patients with B-cell lineage ALL (66.7%; P<0.001); in blast crisis (BC) of chronic myelocytic leukaemia (CML), CD66 was expressed in two patients with lymphoid BC but not in the two with myeloid BC. The co-expression of CD66 with other myeloid antigens was observed in all CD66+ ALL/ Ly-BC cases tested: CD 13 in six patients, CD33 in seven and CD117 in two patients. The CD66 expression is more frequent in ALL than in AML. Furthermore, we analysed minimal residual disease (MRD) in eight patients in complete remission. CD66 expression was associated with an abnormal B-cell differentiation pattern and with increases in CD34/CD19+ cells in all but one case. These findings suggest that an aberrant expression of CD66 could be used to investigate MRD in ALL. The association between CD66 reactivity and bcr-abl in adult ALL remains to be investigated.

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Acute promyelocytic leukaemia (APL) is a unique clinicobiologic entity that can be successfully treated with All-trans Retinoic Acid ATRA-based regimens. Some cases of acute myeloid leukaemia (AML) with nucleophosmin (NPM) mutations have an immunophenotype that is similar to APL. The objective of the study is to compare antigenic expression in a group of APL patients with that in AML patients with NPM mutations and an APL-like immunophenotype (CD15- CD34- HLA-DR-). A consecutive series of 40 APL and 12 NPM patients with an APL-like phenotype were included in the study. Immunophenotypic patterns were investigated by multiparametric flow cytometry. Promyelocytic leukaemia-retinoic acid receptor-α transcript type, NPM and FLT3 mutations were investigated using conventional methods. Statistically significant differences were found between APL and NPM-mutated AML in CD33, CD13, CD2 and CD110 reactivity. CD2 expression was absent in every patient with NPM-mutated AML. In addition, mean fluorescence intensity and the coefficient of variation (cv) of CD33 and CD13 showed statistical differences between the two groups for CD33 (p = 0.007) and a trend to significance for CD13 (p = 0.05). Furthermore, among 45 evaluable patients, CD110 expression statistically differentiates between the two groups: [2/33 (6%) in the APL group and 8/12 (66.6%) in the NPM-mutated AML (p = 0.014)]. However, these traits were subtle, raising the possibility of practical diagnostic challenges. In conclusion, CD110 and CD33 reactivity may be useful to distinguish APL from NPM-mutated AML with CD15, CD34 and HLA-DR negativity. Nevertheless, cytogenetic and molecular characterization is necessary to establish the accurate diagnosis of AML.