Cytometry Part B: Clinical Cytometry最新文献

CD38 and CD138 are important diagnostic markers in flow cytometric analysis of plasma cells (PC) in the context of multiple myeloma (MM). Anti-CD38 therapy, such as daratumumab, exacerbates CD38 detection. In addition, CD138 can be degraded and is then no longer easily detectable on the cell surface. Variable heavy domain heavy chain antibodies (VHH) are single variable domain antibody fragments. Clone JK36 consists of two anti-CD38 VHH fragments and allows targeting of a cryptic CD38 epitope that is not accessible to conventional antibodies (CA). Therefore, our aim was to test VHH in comparison to our conventional anti-CD38 antibody (LS198) in MM bone marrow samples after daratumumab therapy (d-t) compared to therapy-naïve (n) and samples with unknown therapy. A total of 111 samples were analyzed (n = 11 n, n = 81 d-t, n = 18 with unknown therapy). While CD38 was equally well detected by VHH and CA in therapy-naïve samples, CD38 could only be detected in 8% of d-t samples with CA but in 91% with VHH. This resulted in an overall significant reduction in the number of detectable PC, and three samples with undetectable PC by CA compared to VHH. Furthermore, CD138 was reduced/degraded in 52% of d-t samples of which 88% had undetectable CD38 by CA. In addition to proper detection of CD38, VHH is also able to determine a potential CD38 reduction of cell surface expression, as shown by a reduction in CD38 median fluorescence intensity (MFI) on d-t compared to n samples. One d-t sample revealed two distinct PC populations differing by dim and bright CD38 expression, only detectable by VHH. Interestingly, samples with unknown treatment history can be grouped into scenarios most likely treated with daratumumab, or rather treatment-naïve, respectively. In summary, VHH provides superior CD38 detection in d-t MM patients, which is vital for diagnostic samples, and it is capable of providing information about CD38 integrity on the cell surface.

Flow cytometric analysis plays an important role in the diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Most CLL cases show a typical immunophenotype characterized by the expression of CD5, CD23, CD43, ROR1, and CD200, along with dim expression of B-cell markers. However, some show an atypical immunophenotype. The immunophenotypic, cytogenetic, and mutational profiles of atypical CLL are not well characterized. In this study, we aim to address these gaps by analyzing a cohort of 270 CLL cases with a focus on those with atypical immunophenotypes. Their detailed immunophenotype as assessed by flow cytometry is presented along with cytogenetics and mutational data. Fifty-three (19.6%) cases exhibited an atypical immunophenotype. The common atypical immunophenotypic features detected included increased CD20 in 17 (32.1%), negative CD43 in 17 (32.1%), negative ROR1 in 16 (30.1%), and increased surface light chain in 11 (20.8%) cases. Trisomy 12 was more frequently detected in atypical versus typical CLL cases (58.5% vs. 20.7%, p < 0.01), including those with decreased to negative expression of CD5, CD23, CD43, and ROR1, and increased expression of CD20 and CD22. Cases with increased CD20 expression more often had mutated IGHV. BIRC3 is the most frequent mutation in the atypical CLL group, and alterations in BIRC3 (p = 0.02), KRAS (p = 0.03), NRAS (p < 0.01), KMT2D (p < 0.01), and SMARCA4 (p = 0.02) were more frequently detected in atypical CLL when compared to typical CLL. In summary, approximately 20% of CLL cases show an atypical immunophenotype, and these cases have cytogenetic abnormalities and mutation profiles that differ in frequency from typical CLL cases. Recognition of the immunophenotype of atypical CLL can be helpful in the diagnosis and differential diagnosis in low-grade B-cell neoplasms.

Central nervous system (CNS) involvement in multiple myeloma (MM) is a rare but severe complication with a poor prognosis. The identification of malignant plasma cells in cerebrospinal fluid (CSF) is essential for early diagnosis and intervention. However, the sensitivity of traditional diagnostic methods like cytology is low, especially in samples with low-cell counts. This study aimed to develop a multidimensional radar dot-plot analysis using Kaluza software to enhance the sensitivity and specificity of flow cytometry for detecting abnormal plasma cells in CSF. One hundred and twenty-five CSF samples were sent for flow cytometric testing to investigate the central nervous system involvement of MM. Finally, 89 samples from 40 patients were included in our study. Multicolor flow cytometry was performed using an 8-color labeling method, and radar dot-plot analysis was developed using diagnostic bone marrow samples to distinguish normal plasma cells, abnormal plasma cells, and cellular debris. The sensitivity of the novel method was evaluated by diluting myeloma bone marrow cells in pooled CSF samples to simulate low cell counts. Of the 125 CSF specimens, 16 samples from 4 patients showed abnormal plasma cells using both conventional and multidimensional flow cytometry analysis. Discordant results were found in 32 samples (25%), where conventional analysis suggested the presence of abnormal cells, but these were ruled out by multidimensional analysis. Sensitivity testing showed that the multidimensional dot-plot method outperforms conventional two-dimensional dot-plot analysis, as the radar dot plot can be used to identify abnormal cells in samples diluted to 5 WBC/μL, where the cell count of abnormal plasma cells is < 1 cell/μL. Our results showed that the new radar dot-plot analysis can increase the sensitivity and specificity of flow cytometry in MM for the detection of CNS involvement, even in low-cell-count CSF samples, regardless of whether the sample was obtained in a tube containing special reagent or not (TransFix/EDTA CSF Sample Storage tubes). This approach improves diagnostic accuracy, reduces the number of false positive cases caused by antibodies adhering to cell debris, and provides a reliable tool for assessing neurological complications in MM. Further validation is needed in a larger number of cases and testing of the method on different antibody panels.

T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm of immature T cells. Flow cytometry plays a critical role in the diagnosis and management of the disease. It is used to establish the abnormal immature T-cell phenotype and to distinguish the early T-cell precursor (ETP)-ALL from more mature types at diagnosis. The evaluation of mediastinal disease is often complicated by the difficulty of the phenotypic distinction between the normal thymic precursors and the abnormal T lymphoblasts. Follow-up measurements of minimal/measurable residual disease (MRD) are critical for therapy decision-making and prognostication. In the MRD setting, flow cytometry requires a high degree of analytical expertise and assessment of numerous antigens. To address the diagnostic and monitoring challenges, we developed a single-tube 21-antigen assessment with simplified analysis. The assay distinguishes between normal thymic precursors and T lymphoblasts in tissue samples, enables evaluation of ETP versus non-ETP phenotypes, and allows for MRD assessment below 0.01% robust to antigenic changes.

Monitoring subpopulations of CD4⁺ T cells in blood, especially regulatory CD4⁺CD25⁺Foxp3⁺CD127^loT cells, has the potential to identify tolerance to transplants and defects that cause autoimmunity. CD45RA is expressed by naïve/resting CD4⁺, not by activated cells. Staining CD45RA with CD25 or Foxp3 identifies five populations of CD4⁺ T cells, three Treg, and two CD4⁺Foxp3⁻T cells. CD25 is induced on activation of effector cells and is constitutively expressed by Treg. Examining Foxp3⁺ cells in CD4⁺CD25⁺CD127^lo, identified three Treg populations. It is not known how stable these populations of CD4⁺T cells are within individuals and between individuals. Repeated estimations over 3 years in 10 HV showed the proportion of cells in the three Treg populations was stable, whereas the two Foxp3⁻ populations varied. In a larger cohort of 110 samples, females had higher numbers of CD4⁺ cells than males. As a percentage of lymphocytes, there was no sex difference in the proportion of cells in the five populations. With age, there were fewer total Treg, with fewer resting Treg but an increase in activated Treg. Activation of both naïve CD4⁺ T cells and Treg induces expression of chemokine receptors associated with Th1, Th17, and Th2 responses that promote their infiltration into sites of inflammation. Activated Treg expressed CCR4 and, in addition, expressed CXCR3 (Th1), CCR6 (Th17), or neither CXCR3 nor CCR6 (Th2). Some Treg expressed both CCR6 and CXCR3. HLA-DR and CD39 were expressed by activated Treg, and many cells expressed both. There was low PD-1 expression. The stability of the major Treg populations suggested it could be feasible to establish normal ranges for the three Treg populations. Staining for chemokine receptors and Treg effector molecules in activated Treg populations may detect changes in immune homeostasis and tolerance.