Cytometry Part B: Clinical Cytometry最新文献

The diagnosis of T-cell neoplasms remains one of the most challenging areas in hematopathology due to the immunophenotypic heterogeneity and subtle aberrancies often present in these entities. This “Best Practice” manuscript provides a practical framework for laboratories to design, validate, and interpret immunophenotyping studies of immature and mature T-cell neoplasms. We outline the utility of key antigens in the screening and classification of T-cell lymphomas/leukemia including TRBC1 and TRBC2. Analytical strategies using the “difference from normal” method and template-based gating are discussed, along with validation considerations aligned with CLSI H62 guidelines. By integrating these principles into laboratory workflows, this manuscript aims to standardize and improve the assessment of T-cell neoplasms across diverse clinical settings.

To screen hereditary spherocytosis (HS), we first introduced the novel flow cytometric osmotic fragility test (FC-OFT) based on the two-point kinetic assay principle (FC-OFT_Kinetic). With the introduction of FC systems with automatic tube loaders, we updated the FC-OFT protocol to follow the endpoint assay principle (FC-OFT_Endpoint). This study aims to evaluate the updated FC-OFT protocol (FC-OFT_Endpoint) and compare its assay performance with that of FC-OFT_Kinetic. We investigated factors influencing the FC-OFT_Endpoint assay, optimized its protocol, and defined the cutoff index using 152 negative or artificially positive control samples. We then compared the assay performance with that of FC-OFT_Kinetic in 25 patients with anemia, including 14 with spherocytosis—8, 4, 1, and 1 with HS, autoimmune hemolytic anemia, burn injury, and liver cirrhosis, respectively. To optimize FC-OFT_Endpoint, we adopted phosphate-buffered saline as the red cell suspension medium, 50% deionized water for hypotonic osmotic pressure in adults, and a 3-min standby time. This FC-OFT_Endpoint was more accurate than FC-OFT_Kinetic in identifying spherocytosis in the 25 patients with anemia (p = 0.0313). FC-OFT_Endpoint is a viable alternative to conventional OFT or FC-OFT_Kinetic for HS screening in clinical laboratories, as automatic FC enhances assay performance. These findings warrant validation in future multicenter studies with larger sample sizes.

Flow cytometry is a powerful tool for analyzing diverse cellular properties, making it essential in immunology research, clinical trials, and diagnostics. Integrating artificial intelligence (AI) into flow cytometry has the potential to enhance various aspects of assay development and application, including reagent selection, instrument standardization, panel and assay design, data analysis, quality controls, and knowledge dissemination. This paper provides a review of current AI applications in flow cytometry and explores the potential future directions for AI integration in the field.

CD300e is a marker of mature monocytes in flow cytometry; however, there is limited detailed information on staining patterns in conjunction with other monocyte markers. We evaluated the flow cytometric staining patterns of CD64, CD14, and CD300e in 12 negative and 33 positive peripheral blood specimens and 16 negative and 56 positive bone marrow specimens. The positive cases were involved by myeloid neoplasms (increased blasts and/or abnormal monocytes). Flow cytometry plots were reviewed for each case, the monocyte population was identified by bright CD64 expression, and the monocyte maturation pattern was visualized by CD14 versus CD300e plots. Peripheral blood and bone marrow differential counts were collected. A total of 39% (22/56) of the positive bone marrow cases showed a different maturation pattern from the negative bone marrow cases. Of the positive peripheral blood cases, 28/33 (85%) showed a CD14 by CD300e pattern different from that observed in the negative peripheral bloods. When the subset of bone marrow cases involved by monocytic neoplasms was evaluated, there was no significant difference between monocyte percentage by flow cytometry versus morphology and between blast plus promonocyte percentage by flow cytometry versus morphology. We conclude that isolation of monocytes by bright CD64 expression and low side-scatter and subsequent evaluation of the CD14/CD300e maturation pattern may help identify myeloid neoplasms. Quantification of CD64 + CD14- and/or CD64 + CD300e- cells by flow cytometry may aid blast/blast equivalent identification/quantification.

Immunophenotyping by flow cytometry is a valuable test providing important information in a timely manner. In clinical laboratories, it is performed using validated antibody panels designed to ensure consistent and accurate results. However, unforeseen situations, such as unique or unusual immunophenotypes, or supply chain issues, may necessitate ad hoc modifications to these panels. This manuscript provides guidance for performing minor modifications, such as substituting or adding one or two antibodies, while maintaining the integrity of the assay. These modifications are intended for rare clinical situations and are not substitutes for the full validation protocols outlined in CLSI H62. An example of this would be a patient with a rare, but not uncommon, situation in which a B cell lymphoma lacks expression of CD19, CD20, and surface light chains, such that the lineage of the neoplastic cells cannot be determined without a straightforward addition or substitution of another marker into a laboratory's available panel. The recommendations and best practices herein aim to optimize patient care by allowing laboratories to adapt to unique clinical scenarios without compromising assay performance and are not a way to permanently modify the assay. Key considerations include assessing the impact on fluorescence compensation, antibody binding, assay sensitivity, and overall assay performance. The manuscript provides limitations for the extent of modifications, examples, and troubleshooting strategies to ensure reliable results when ad hoc changes are made. Proper documentation with review and approval by laboratory medical directors is recommended to mitigate risks associated with these modifications.

Acute promyelocytic leukemia (APL) is a medical emergency that needs immediate diagnosis and treatment. Podoplanin, a transmembrane glycoprotein that binds CLEC-2 on platelets, was recently demonstrated to be abnormally expressed in leukemic blasts in APL, as opposed to other forms of AML, in a study using thawed primary cells. This study aimed to explore and validate the diagnostic accuracy of measuring podoplanin expression by flow cytometry in the differential diagnosis of APL and other forms of acute myeloid leukemia (AML) as part of the diagnostic work-up of all cases suspected of AML in an academic hematology center. Podoplanin expression was measured by flow cytometry in bone marrow samples obtained at disease presentation from all consecutive adult patients suspected of AML. Results from 24 APL patients were compared with those from 23 non-APL AML patients matched by age and sex. Markedly higher PDPN expression was observed in APL patients when compared to other AML patients, with an area under the curve of 0.92 (95%CI: 0.82-1.0, p < 0.0001) for the percentage of positive cells. Combining an optimal cutoff of 7.66% for PDPN-positive blasts and 1691 for the mean fluorescence index of PDPN expression, APL was identified with a sensitivity of 87.5% and a specificity of 100%. Moreover, PDPN expression presented a negative correlation with platelet count and fibrinogen levels. PDPN expression measured by flow cytometry can accurately differentiate between APL and other forms of AML in a real-world clinical setting, contributing to the diagnosis of this form of acute leukemia. If confirmed in larger prospective studies, the negative association of PDPN expression with fibrinogen and platelet counts supports the concept that this biomarker can potentially contribute to the clinical characterization of APL.

Two types of plasmacytoid dendritic cell (pDC) proliferation disease are acknowledged so far by the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) and mature pDC proliferation associated with myeloid neoplasms (MPDCP) in which pDC is part of the malignant clone. We aim to investigate pDC proliferation associated with non-myeloid acute leukemia (AL). A retrospective analysis of all cases admitted in our center with a diagnosis of non-myeloid AL from September 2020 to April 2023 was performed to select cases with pDCs greater than 2% of bone marrow by flow cytometry (FCM). We conducted comprehensive analyses encompassing FCM immunophenotyping, histopathological examination, morphological assessment, cytogenetic studies, and molecular genetic profiling across all cases. Proliferation of pDCs was detected in 10 (0.88%) out of 1140 non-myeloid AL patients by FCM, including 4/944(0.42%) cases of B lymphoblastic leukemia (B-ALL), 3/95 (3.16%) cases of T lymphoblastic leukemia (T-ALL) and 3/101 (2.97%) cases of acute leukemia of ambiguous lineage (ALAL) (p = 0.009). The 4 cases of B-ALL were all Philadelphia Chromosome positive (Ph+). PDCs in 3 out of 10 patients expressed CD56 (37.5%), 8/10 expressed CD7 (80%), 9/10 expressed CD303 (90%), all expressed CD304 (100%), and 5 of 8 evaluable cases were positive for CD34 (62.5%). In cases in which pDCs expressed CD7 and/or CD56, the blast cells all expressed CD7 and/or CD56 as well; the pDCs in all B-ALL patients expressed CD19. FCM dot plot in 2 of the B-ALL-pDC showed a spectrum from blast cells to pDCs: CD303 and CD304 gradually emerged as CD34 disappeared. Among the 8 patients who underwent bone marrow biopsy, pDCs exhibited two distinct distribution patterns: pure interstitial infiltration in 6 cases (75%) and focal/scattered clusters against an interstitial background in 2 cases (25%). NRAS showed recurrent mutations at identical genomic positions. Each NRAS variant (c.35G>A and c.38G>T) was detected twice across three patients. FCM could effectively detect pDC proliferation in non-myeloid leukemia, which occurred at a significantly higher incidence in T-ALL and ALAL than in B-ALL. In B-ALL, it was associated with the Ph + subtype. PDCs and blast cells shared some lymphoid antigens that were uncommon in AML-pDC or BPDCN. In the bone marrow, pDCs were predominantly characterized by an interstitial infiltration pattern.