Cytometry Part B: Clinical Cytometry最新文献

Background

The human leukocyte antigen B27 (HLA-B27), associated with chronic inflammatory diseases is thus widely performed for diagnostic purposes. Genotyping by molecular biology is the current gold standard for HLA-B27 typing, but cheaper and faster flow cytometry methods have been developed.

Methods

In this report, we compare analytical performances of three CE-IVD flow cytometry kits: IOTest™ and DuraClone B27™ from Beckman Coulter and BD HLA-B27™ from BD Biosciences on a Navios™ cytometer as compared to molecular biology.

Results

Routine analyses could conclude for HLA-B27 in197 patients (23.2%) and was not conclusive for 66 patients (7%, 8%). The experience revealed the needs to complete IOTest™ with a lineage marker (like CD3-APC) and a standardization procedure in detection of fluorescence. Comparative analysis considering 23/44 non-conclusive samples as negative, pointed out a 100% sensitivity and specificity of IOTest™ in determining genetically proved HLA-B27. Specificity of the BD HLA-B27^TM kit was 94% (two false positives) sticking to the manufacturer instructions but raised to 100% and 94% sensitivity with a cutoff at 8.5 (225 on FACSdiva's scale).

Discussion

The DuraClone B27™ specificity was poor using the manufacturer cutoff but raised to 100% with a 8.0 cutoff instead of 15.

Conclusion

The three flow cytometry kits displayed satisfying performances but need adjustments. The DuraClone B27™ kit seems to be the best while the IOTest™ kit is not conclusive in 8% of cases. In most cases the use of molecular biology can be spared, but genotyping remains essential for patients whose HLA-B27 status cannot be determined with confidence by flow cytometry.

Background

Surface immunoglobulin (sIg) light chains are not always detected on mature B cells. This may present as a challenge for clonality determination in clinical flow cytometry.

Methods

To explore the mechanism and diagnostic significance of sIg negative mature B cells, we retrospectively studied 14 cases of sIg negative reactive B-cell lymphocytosis and 89 cases of sIg negative mature B-cell lymphomas. The expression patterns of sIg and cytoplasmic immunoglobulin (cIg) light chains were studied by flow cytometry using both monoclonal and polyclonal antibodies.

Results

These 14 cases of sIg negative reactive B-cell lymphocytosis were proven to be polytypic based on cytoplasmic light chain studies. In 89 cases of sIg negative mature B-cell lymphomas, we described four distinct patterns of abnormal light chain expression including partial or complete loss of sIg or cIg, suggesting different underlying mechanisms.

Conclusions

This study represents the first reported series of body or cystic fluids where reactive B cells do not have detectable sIg, arguing strongly against making a diagnosis of B-cell lymphoma based on lack of sIg in mature B cells. Since the lack of sIg does not always predict clonal/neoplastic mature B-cell proliferation, further cIg evaluation should be performed when sIg expression is not detected in mature B cells. The lack of both sIg and cIg in mature B cells may serve as a reliable surrogate clonality/neoplastic marker.

Background

Myelodysplastic syndromes (MDS) represent a diagnostic challenge. This prospective multicenter study was conducted to evaluate pre-defined flow cytometric markers in the diagnostic work-up of MDS and chronic myelomonocytic leukemia (CMML).

Methods

Thousand six hundred and eighty-two patients with suspected MDS/CMML were analyzed by both cytomorphology according to WHO 2016 criteria and flow cytometry according to ELN recommendations. Flow cytometric readout was categorized ‘non-MDS’ (i.e. no signs of MDS/CMML and limited signs of MDS/CMML) and ‘in agreement with MDS’ (i.e., in agreement with MDS/CMML).

Results

Flow cytometric readout categorized 60% of patients in agreement with MDS, 28% showed limited signs of MDS and 12% had no signs of MDS. In 81% of cases flow cytometric readouts and cytomorphologic diagnosis correlated. For high-risk MDS, the level of concordance was 92%.

A total of 17 immunophenotypic aberrancies were found independently related to MDS/CMML in ≥1 of the subgroups of low-risk MDS, high-risk MDS, CMML. A cut-off of ≥3 of these aberrancies resulted in 80% agreement with cytomorphology (20% cases concordantly negative, 60% positive). Moreover, >3% myeloid progenitor cells were significantly associated with MDS (286/293 such cases, 98%).

Conclusion

Data from this prospective multicenter study led to recognition of 17 immunophenotypic markers allowing to identify cases ‘in agreement with MDS’. Moreover, data emphasizes the clinical utility of immunophenotyping in MDS diagnostics, given the high concordance between cytomorphology and the flow cytometric readout. Results from the current study challenge the application of the cytomorphologically defined cut-off of 5% blasts for flow cytometry and rather suggest a 3% cut-off for the latter.

Background

Achieving minimal residual disease (MRD) negativity following treatment for multiple myeloma (MM) is associated with improved progression free and overall survival. In the UK, MRD assessments in MM are not incorporated into routine clinical use outside trials. Widely used in other haematological malignancies, there is a role for widening the availability of myeloma MRD assays to laboratories outside larger treating centers.

Methods

We set up and assessed concordance of a multicolor flow cytometry (MCF) assay for MM MRD in collaboration with a reference center including validity following delayed processing of samples using an optimized fixation step. We then conducted a real-world snapshot of MRD results in a cohort of newly diagnosed transplant-eligible patients treated with UK standard induction therapies at the time of analysis.

Results

43 MCF MRD samples run in parallel with a reference center showed high correlation and minimal bias. 24 samples were split and processed in duplicate both fixed and fresh, with strong correlation, minimal bias, and no change in plasma cell phenotype by flow markers confirming a 6-day delay in processing did not affect assay performance. A real-world snapshot found 17% (10/58) of patients were MRD-negative post-bortezomib-based triplet induction therapy.

Conclusions

We successfully adopted a reference MCF MM MRD method which was stable for up to 6 days following sample collection potentially allowing broader access of this assay to smaller laboratories which would facilitate further investigation of the prognostic value and clinical utility of MRD assessments outside the trial setting in real-world practice.

Background

Many novel therapies are being evaluated for the treatment of Multiple myeloma (MM). The cell-surface protein B-cell maturation antigen (BCMA, CD269) has recently emerged as a promising target for CAR-T cell and monoclonal-antibody therapies in MM. However, the knowledge of the BCMA expression-pattern in myeloma patients from the Indian subcontinent is still not available. We present an in-depth study of BCMA expression-pattern on abnormal plasma cells (aPC) in Indian MM patients.

Methods

We studied BM samples from 217 MM patients (211-new and 6-relapsed) with a median age of 56 years (range, 30–78 years & M:F-2.29) and 20 control samples. Expression levels/patterns of CD269 (clone-19f2) were evaluated in aPCs from MM patients and in normal PCs (nPC) from uninvolved staging bone marrow samples (controls) using multicolor flow cytometry (MFC). Expression-level of CD269 was determined as a ratio of mean fluorescent intensity (MFI-R) of CD269 in PCs to that of non-B-lymphocytes and expression-pattern (homogenous/heterogeneous) as coefficient-of-variation of immunofluorescence (CVIF).

Results

Median (range) percentage of CD269-positive abnormal-PCs in total PCs was 71.6% (0.49–99.29%). The MFI-R (median, range) of CD269 was significantly higher in aPCs (4.13, 1.12–26.88) than nPCs (3.33, 1.23–12.87), p < .0001. Median (range) MFI of CD269 at diagnosis and relapse were 2.39 (0.77–9.57) and 2.66 (2.15–3.23) respectively. CD269 levels were similar at diagnosis and relapse, p = .5529.

Conclusions

We demonstrated that BCMA/CD269 is highly expressed in aPCs from a majority of MM patients, both at diagnosis and relapse. Thus, BCMA is a valuable target for therapy for Indian MM patients.

Background

High-quality data on bone marrow involvement (BMI) assessed by flow cytometry (FC) in follicular lymphoma (FL) is lacking.

Aims

We set up a prospective protocol with a 10-color tube and acquisition of 500.000 leukocytes on a Nav flow cytometer for evaluation of BMI in FL by FC.

Materials and methods

FC was compared with a combination of histopathology and IGH gene rearrangement, which were considered the gold standard. We also compared BMI by FC with PET.

Results

Fifty-two patients were included (median 67 years, 54% female). BMI by FC was seen in 35 (67%), with a median involvement of 1.2% (interquartile range: 0.3%–7%) of leukocytes. Comparison with the gold standard revealed two false negatives and two false positives (potentially true involvement undetected by the gold standard). BMI by PET was seen in 14/46 (30%). Immunophenotype of FL in the bone marrow was highly heterogeneous. The most common phenotypic abnormality was dim expression of CD19 (>0.5 log loss in 30% of patients). CD10 was negative in 13 (37%) and incompletely positive (overlap with the negative population) in a further 8 (28%) while entirely positive only in 14 (48%). Other abnormalities (loss of CD20, gain or loss of CD79b, expression of CD43, and substantial loss of CD45) were rare. Computational analysis by means of FlowSOM confirmed the heterogeneous phenotype, with FL from different patients clustering in unrelated metaclusters.

Conclusion

BMI by FL was frequent and immunophenotype was heterogeneous. However, this protocol enabled detection of FL in bone marrow in the vast majority of patients with bone marrow involvement by the gold standard.

Background

The immunophenotype of pure erythroid leukemia (PEL) as determined by flow cytometry immunophenotypic analysis is not well characterized. The immunophenotypic difference between PEL and reactive conditions is under-explored.

Methods

We assessed and compared the immunophenotype of 24 PEL cases and 28 reactive cases containing early erythroid precursors by flow cytometry.

Results

The neoplastic erythroid cells in all PEL cases were positive for CD36 and CD71. CD45 was also positive in all cases, but the expression level was often dimmer than granulocytes. CD117 expression ranged from partial to uniform, and CD235a was often only positive in the CD117-dim to negative cells, corresponding to more differentiated subset. PEL cases frequently (87%) showed decreased or negative CD38 expression, contrasting to reactive early erythroid precursors that showed bright CD38 (p < 0.0001). CD7 (25%) and CD13 (29%) aberrant expressions were only observed in PEL but not in the reactive erythroid cells. Normal early erythroid precursors in all reactive bone marrows showed partial expression of CD4; In contrast, aberrant CD4 expression was detected in 71% PEL cases, either uniformly positive (50%) or completely negative (21%). While normal/reactive bone marrows almost always contained a small subset of CD34-positive early erythroid precursors, the neoplastic pronormoblasts in all PEL cases were CD34 negative. Although not increased in number, CD34-positive myeloblasts were frequently detected in PEL and demonstrated an aberrant immunophenotype in 90% PEL cases.

Conclusions

PEL shows a distinctive immunophenotype which can be distinguished from reactive erythroid precursors by flow cytometry immunophenotyping.

Background

Flow cytometric immunophenotyping is essential for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Most cases have easy to interpret flow cytometry profiles with red cells, neutrophils and monocytes showing complete deficiency of glycophosphatidylinositol (GPI) linked antigen expression. Some cases are more challenging to interpret due to the presence of multiple populations of PNH cells and variable levels of GPI antigen expression.

Methods

We studied 46 known PNH patients, many with complex immunophenotypic profiles using a novel, single tube, multi-parameter 7-color immunophenotyping assay that allowed simultaneous detection and assessment of PNH clones within multiple lineages of peripheral blood leucocytes. Red cell PNH clones were also assessed in total and immature (CD71+) components by CD59 expression.

Results

For individual patients, total PNH clones in each cell lineage were highly correlated. Monocytes, eosinophils and basophils showed the highest proportions of PNH cells. Red cell PNH clones were typically smaller than monocyte and neutrophil PNH clones. In most cases, PNH clones were detectable in minor leucocyte populations where multiple populations of PNH cells were present, variability in the proportions of type II and type III cells was seen across different cell lineages, even though total PNH clones remained similar.

Conclusions

This study shows that PNH patients with multiple PNH clones do not always display the same abnormality across all cell lineages routinely tested. There is no simple explanation for this but is likely due to a combination of complex molecular, genetic and biochemical dysfunction in different blood cell types.