Clinical Flow-Cytometric Testing in Chronic Lymphocytic Leukemia.

Flow-cytometric demonstration of the typical chronic lymphocytic leukemia (CLL) immunophenotype is vital for diagnosis. CLL has a characteristic immunophenotype, expressing CD5, CD19, dim CD20, dim CD22, CD23, bright CD43, dim CD45, dim to negative CD79b, dim CD81, CD200, and dim monoclonal surface immunoglobulin. This characteristic immunophenotype allows a definitive diagnosis and the ruling out of another leukemia or lymphoma. Flow cytometry also provides important prognostic information and accurate assessment of response to therapy. Here we describe optimal specimen collection, red cell lysis, appropriate panel, cell staining, acquisition on a flow cytometer, and analysis for CLL specimens.

Differential Expression of CD43, CD81, and CD200 in Classic Versus Variant Hairy Cell Leukemia.

BACKGROUND: Hairy cell leukemia (HCL) and hairy cell leukemia variant (HCLv) are rare diseases with overlapping clinicopathological features. Features distinguishing HCL from HCLv include expression of CD25, CD123, CD200, annexin-A1, and the presence of BRAF V600E mutation. HCLv typically lacks these markers, but they may occur in a subgroup of HCL patients with an aggressive clinical course. We examined CD43, CD81, CD79b, and CD200 expression in HCL and HCLv. METHODS: Multiparametric flow cytometry (FCM) was performed on blood from 59 HCL and 15 HCLv patients for protocol entry. Mean fluorescent intensity (MFI) of CD43, CD79b, CD81, and CD200 was determined (for CD200, n = 17 and 7, respectively). RESULTS: Median MFI of HCL vs HCLv was 545 vs 272 for CD43, 602 vs 2,450 for CD81, 4,962 vs 1,969 for CD79b, and 11,652 vs 1,405 for CD200, respectively. Analysis of the median differences, HCL minus HCLv (and their 95% confidence intervals and P-values) indicated that CD43 MFI (estimated median difference (95% CI): 212 [72-413; P = 0.0027) and CD200 MFI (9,883 [3,514-13,434]; P < 0.0001) were higher in HCL than in HCLv, while CD81 MFI (-1,858 [-2,604 to -1,365]; P < 0.0001) was lower in HCL than in HCLv. CD79b MFI HCL median was more than double that of HCLv, but the observed difference (1,571 [-739 to 4,417]) was consistent with the null hypothesis of no difference (P = 0.13). CONCLUSIONS: CD200, CD43, and CD81 are likely differentially expressed between HCL and HCLv, reflecting their differing disease biology. Inclusion of these markers in FCM is potentially informative. © 2019 International Clinical Cytometry Society.

Quantification of B-cell maturation antigen, a target for novel chimeric antigen receptor T-cell therapy in Myeloma.

B-cell maturation antigen (BCMA) is expressed by normal and malignant plasma cells and is targeted via anti-BCMA chimeric antigen receptor T-cell therapy (BCMA CAR T-cell therapy) in plasma cell myeloma (PCM) patients. Surface BCMA expression is required for CAR T-cell binding and killing. We determined the incidence and intensity of expression of BCMA in bone marrow PCM cells using flow cytometry (FC) and immunohistochemistry (IHC). PCM BCMA expression was assessed by FC in 70 patients and in 43 concurrent specimens by IHC. BCMA expression was detected in 94% of patients. FC could assess BCMA expression in all specimens and expression was quantifiable (QuantiBRITE system, BD Biosciences, San Jose, CA) in 89% of cases. Expression was highly variable and could be numerically classified into dim, moderate or bright levels of expression. In the 43 specimens assessed successfully by both IHC and FC, FC showed higher positivity rate (97%) than IHC (72%), indicating that FC is more useful than IHC in detection of BCMA (p = 0.002; McNemar's test). We conclude that FC is more sensitive than IHC and can be used to objectively quantify BCMA expression by myeloma cells. IHC is primarily useful when there is significant infiltration of the bone marrow by myeloma and is less sensitive with low numbers of myeloma cells. Furthermore, the ability of FC to differentiate between normal and abnormal plasma cells and to quantify BCMA on these cells, makes it a useful and sensitive tool in screening patients for CAR T-cell therapy and for follow-up post therapy.

Prevalence and Prognostic Impact of CEBPA Gene Mutation (Simplified Assay Technique) in Egyptian Acute Myeloid Leukemia Patients with Normal Cytogenetics.

Mutations of the CCAAT/enhancer binding protein alpha (CEBPA) gene have been associated with a favorable outcome in patients with acute myeloid leukemia (AML), especially in those with a normal cytogenetics. However, few studies were done on Egyptian AML patients and none of them look for easier and less expensive method for CEBPA mutation screening. This study is aimed to investigate the prevalence of CEBPA mutations and its clinical and prognostic impact in Egyptian patients with cytogenetically normal AML (CN-AML). This was done using fragment analysis to assess this method as a cheaper and less laborious screening method compared to sequencing. Fluorescent PCR was done to amplify CEBPA gene in DNA extracted from 40 CN-AML patients. This was followed by fragment analysis of post-PCR products using GeneMapper software for detection of CEBPA mutations. CEBPA gene mutations were found in 7/40 CN-AML patients (17.5 %) and it was significantly associated with lower LDH levels (p = 0.039). All patients with CEBPA mutations achieved clinical remission and none of them showed refractoriness, relapsed, or died by the end of the 2 years study period. Furthermore, those patients demonstrate significantly longer overall and disease free survival than those with wild type CEBPA gene (p = 0.001 and 0.004 respectively). CEBPA mutation has a favorable prognostic impact in CN-AML. Fragment analysis is a good, lees laborious and cheaper method that can be used for CEBPA mutation screening in patients with CN-AML.

Flow cytometric sensitivity and characteristics of plasma cells in patients with multiple myeloma or its precursor disease: influence of biopsy site and anticoagulation method.

Flow cytometry has increasing relevance for prognosis in myeloma and precursor disease (monoclonal gammopathy of unknown significance/smoldering myeloma), yet it has been reported that plasma cell enumeration by flow varies depending on the quality of marrow aspirate and field biopsied in patchy disease. We demonstrated increased sensitivity of flow over immunohistochemistry in abnormal-plasma cell detection in monoclonal gammopathy (n = 59)/smoldering myeloma (n = 87). We prospectively evaluated treatment-na ve smoldering myeloma (n = 9)/myeloma (n = 11) patients for the percentage of abnormal plasma cells/total plasma cell compartment, plasma cell viability/infiltration and flow immunophenotype depending on anticoagulant use, biopsy site and pull sequence in uni-and-bilateral bone marrow biopsies and aspirates. We found no statistical difference regarding the percentage of abnormal plasma cells, their immunophenotype or number/distribution in marrow samples even when obtained by different sequence in aspirates, or anticoagulants (p > 0.05). Our results show that plasma cell enumeration and immunophenotyping by flow cytometry is consistent under different conditions in these populations.

NPM1 gene mutation in Egyptian patients with cytogenetically normal acute myeloid leukemia.

BACKGROUND: Nucleophosmin1 (NPM1) protein encoded from the NPM1 gene is a ubiquitously expressed nucleolar phoshoprotein which shuttles continuously between the nucleus and cytoplasm. NPM1 protein plays an important role in cell proliferation and apoptosis. NPM1 gene mutations at exon 12 represent the hallmark of a large sub-group of cytogenetically normal acute myeloid leukemia (CN-AML) patients worldwide. METHODS: Genomic DNA from 53 CN-AML patients were amplified by PCR and followed by fragment analysis of post-PCR products using GeneMapper software for detection of NPM1 mutations. RESULTS: NPM1 exon 12 mutations were found are 15/53 CN-AML patients (28.3%) including 3 of M1, 3 of M2, 5 of M4, 3 of M5, and 1 of M6 FAB subtypes. The NPM1 mutation was significantly associated with lower relapse rate (p < 0.05). The complete remission (CR) rate was significantly higher in the patients with high NPM1 mutation load (> 50%) than low NPM1 mutation load (< 50%) (87.5% vs. 28.6%; p = 0.02). CONCLUSIONS: The aim of this study was to evaluate the NPM1 gene exon 12 mutation in Egyptian patients with CN-AML and its relation to clinical characteristics and patient outcome and survival.

Prognostic implications of NPM1 mutations and FLT3 internal tandem duplications in Egyptian patients with cytogenetically normal acute myeloid leukemia.

Nucleophosmin (NPM1) and fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) gene mutations represent the most frequent molecular aberrations in patients with cytogenetically normal-acute myeloid leukemia (CN-AML). We analyzed the prognostic impact of these mutations and their interactions in adults with CN-AML. NPM1 mutation (NPM1mut) and FLT3-ITD mutation (FLT3-ITD+) were analyzed by polymerase chain reaction and GeneScan assays of bone marrow samples obtained from newly diagnosed 104 CN-AML patients. FLT3-ITD+ and NPM1mut were detected in 36 (34.6%) and 30 (28.8%) out of 104 subjects, respectively, 16 cases (15.4%) had double NPM1mut/FLT3-ITD+. The incidence of FLT3-ITD+ was significantly higher in the NPM1mut group than in the NPM1 wild (NPM1wt) group (P = 0.018). Statistical analysis revealed that isolated NPM1mut group had a better clinical outcomes in terms of higher complete response (CR) rate (P = 0.01) and a trend towards favorable overall survival (OS) and disease-free survival (DFS) (P = 0.28, 0.40, respectively). In contrast, the isolated FLT3-ITD+ group had an unfavorable outcome in terms of lower CR rate (P = 0.12), shorter OS, and DFS (P < 0.0001 for both). The NPM1mut/FLT3-ITD-group had the best OS and DFS, while the NPM1wt/FLT3-ITD+ group had the worst OS and DFS than other groups (NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD-) (P < 0.0001 for both). Multivariate Cox regression analysis showed that age and FLT3/ITD+ were independent poor prognostic factors for OS (P = 0.006, <0.0001, respectively), while FLT3/ITD+ was independent predictor for DFS (P = 0.04). However, NPM1mut did not have a significant impact on OS and DFS. In conclusion, adult patients with CN-AML carrying isolated NPM1mut and isolated FLT3-ITD+ exhibit different clinical outcomes than those with NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD-. Patients with NPM1mut/FLT3-ITD- had the best prognosis in terms of higher CR, OS, and DFS, while those with NPM1mut/FLT3-ITD+ had the worst CR rate, and NPM1wt/FLT3-ITD+ had the lowest OS and DFS.

Giardia diagnostic methods in human fecal samples: a comparative study.

Several methods were tried for Giardia detection in stool. This study aimed to compare between the results of ordinary microscopy, direct immunofluorescence assay (DIF), and flow cytometry (FC) for the detection of Giardia cyst in human stool samples. The study included 84 children recruited from outpatient clinics of Mansoura University Children Hospital. Fecal samples were processed and examined for Giardia cysts using conventional microscopy, DIF, and FC. Among 84 fecal samples, 40 (47.6%) were diagnosed as Giardia-positive by saline wet mount, while DIF and FC detected 52 (61.9%), and 38 (45%) Giardia-positive cases, respectively. When compared with DIF as a gold standard method, ordinary microscopy had 76.9% sensitivity and 100% specificity while the FC had a sensitivity of 73.1% and 100% specificity, with statistically significant differences between DIF and the other two methods (P < 0.05). DIF was able to detect as few as 500 cysts/g of concentrated stool, yielding a threshold higher than ordinary microscopy (1,800 cyst/g) even after concentration. It is concluded that direct microscopic examination is reliable in Giardia diagnosis as a first choice test. DIF is an excellent technique in clinically suspected cases after negative microscopy. FC was found to be less sensitive to obtain accurate organisms' count but it could be an effective alternative method for the detection of Giardia cysts, especially for large-scale epidemiological studies or extensive surveillance programs as it has the beneficial attribute of speed and do not depend on an experienced microscope viewer. However, DIF remains the gold standard while FC still requires significant technical improvements before it can compete with DIF for Giardia diagnosis.

Susceptibility tests of oropharyngeal Candida albicans from Egyptian patients to fluconazole determined by three methods

Candida albicans frequently cause oropharyngeal candidiasis in immunocompromised patients. As some of these isolates show resistance against azoles, the clinician is wary of initiating therapy with fluconazole (FZ) until a final susceptibility report is generated. We aimed to evaluate the efficacy of rapid flow cytometry (FCM) and disc diffusion (DD) methods in comparison to reference microdilution (MD) of Clinical and Laboratory Standards Institute (CLSI) method for FZ. Thirty seven Candida albicans isolates were tested by the three methods. By both MD and FCM, 26/37 (70.3%) were sensitive with minimal inhibitory concentration (MIC) ¡Ü 8¦Ìg/ml, 5/37 (13.5%) were susceptible dose dependant (S-DD) with MIC 16-32 ¦Ìg/ml and 6/37 (16.2%) were resistant with MIC ¡Ý64¦Ìg/ml. More than 92% of isolates susceptible to FZ by the MD were susceptible by the DD methods with good agreement (81.08%, P = 0.000). However, 4/5 isolates diagnosed as S-DD by MD were resistant by DD. Interestingly, the MIC by FCM at 4 h showed excellent agreement (95.59%, P = 0.000) to that obtained by MD method at 24 h. Overall, FCM antifungal susceptibility testing provided rapid, reproducible results that are valuable alternative to MD. The DD test is recommended as a simple and reliable screening test for the detection of susceptible Candida albicans isolates to FZ.

Susceptibility tests of oropharyngeal Candida albicans from egyptian patients to fluconazole determined by three methods.

Candida albicans frequently cause oropharyngeal candidiasis in immunocompromised patients. As some of these isolates show resistance against azoles, the clinician is wary of initiating therapy with fluconazole (FZ) until a final susceptibility report is generated. We aimed to evaluate the efficacy of rapid flow cytometry (FCM) and disc diffusion (DD) methods in comparison to reference microdilution (MD) of Clinical and Laboratory Standards Institute (CLSI) method for FZ. Thirty seven Candida albicans isolates were tested by the three methods. By both MD and FCM, 26/37 (70.3%) were sensitive with minimal inhibitory concentration (MIC) ≤ 8µg/ml, 5/37 (13.5%) were susceptible dose dependant (S-DD) with MIC 16-32 µg/ml and 6/37 (16.2%) were resistant with MIC ≥64µg/ml. More than 92% of isolates susceptible to FZ by the MD were susceptible by the DD methods with good agreement (81.08%, P = 0.000). However, 4/5 isolates diagnosed as S-DD by MD were resistant by DD. Interestingly, the MIC by FCM at 4 h showed excellent agreement (95.59%, P = 0.000) to that obtained by MD method at 24 h. Overall, FCM antifungal susceptibility testing provided rapid, reproducible results that are valuable alternative to MD. The DD test is recommended as a simple and reliable screening test for the detection of susceptible Candida albicans isolates to FZ.

Flowcytometric immunophenotypic profile of acute leukemia: mansoura experience.

Acute leukemia (AL) displays characteristic patterns of antigen expression, which facilitate their identification and proper classification. The purpose of this study is to evaluate the diagnostic usefulness of commonly used immune-markers for immunophenotyping of AL and to define the best immune-markers to be used for proper diagnosis and classification of AL. Besides, to recognize the frequency of different AL subtypes and the antigen expression profile in our Egyptian patients. We retrospectively analyzed the immunophenotypic data of 164 de novo AL patients from our institution during 2009 and 2010. Among these patients, 68.9% were classified as acute myeloblastic leukemia (AML) while 31.1% classified as acute lymphoblastic leukemia (ALL). The commonest FAB subtype in AML group was AML-M4/5 (34.5%) which may differ from most published data. As regard ALL, there were 74.5% with B-ALL and 25.5% with T-ALL. It was found that combined use of HLADR and CD34 was much more helpful in distinguishing APL from non-APL AML than either of these antigens alone. It was found that cCD79a and CD19 were the most sensitive marker for B-ALL while cCD3, CD7 and CD5 were the most sensitive antigens for T-ALL. Our analysis of AL phenotypes proved that employed antibody panels are adequate for proper diagnosis and classification of AL. Flowcytometry was found to be especially useful in the identification of AML-M0 and differentiation of APL from non-APL AML. Immunophenotyping results and FAB classification of our AL patients were comparable to internationally published studies apart from predominance of AML-M4/5 and more frequent APL.