Multiparametric flow cytometry is a highly valuable method for the assessment of measurable residual disease (MRD) in multiple myeloma patients. The aim of the study was to evaluate a one-tube MRD panel on a DxFlex flow cytometer including all EuroFlow recommended immunophenotypic markers (i.e., cytoplasmic light chain kappa and lambda, CD19, CD27, CD38, CD45, CD56, CD81, CD117, CD138) extended by CD200 to a total of 11 fluorochromes in one tube. Bone marrow aspirates from clinical routine underwent an ammonium chloride-based bulk lysis, followed by staining the surface antibodies and, after permeabilization, kappa, and lambda light-chain intracellular staining. We acquired 1 × 10 cells per sample with a DxFlex flow cytometer. We determined the limit of detection (LOD) and lower limit of quantification (LLOQ) as recommended by the International Myeloma Working Group. For the clinical evaluation, 68 samples from 53 patients with multiple myeloma under or after treatment were analyzed with the one-tube MRD panel, and the results were compared with our routine plasma cell panel (RPCP). Six of the 68 samples were additionally sent to another laboratory to confirm our results with the contemporary EuroFlow next-generation flow (NGF) assay. For our novel one-tube MRD panel we determined a LOD of 0.00016% (1.6 × 10) and a LLOQ of 0.00059% (5.9 × 10). Out of 68 specimens, 55 (80.9%) showed concordant results between the MRD- and the RPC-panel. Thirteen (19.1%) specimens showed a distinct population of abnormal plasma cells with the MRD panel not detectable with the RPC panel. The six samples simultaneously measured with our novel one-tube MRD panel and the EuroFlow NGF assay showed concordant results. The novel one-tube MRD panel meets the quality specifications of the International Myeloma Working Group. Our clinical evaluation found higher sensitivity for the one-tube MRD panel when compared to our RPC panel and concordant results with the contemporary EuroFlow NGF assay. Therefore, our novel one-tube MRD panel is well-suited for detecting MRD in multiple myeloma patients.

The International Harmonisation Protocol (IHP) for Reticulocyte Percentage (%Retic) in CLSI-H44-A2 is a microscopic procedure performed on a new-methylene-blue-stained blood film (NMB-IHP). However, IHP-compliant Measurement Procedures (MPs) based on flow cytometry (FCM) are recommended for practical and accurately assigned reference values. Thiazole orange (TO) for nucleic acid staining and CD235a immunostaining for erythrocyte gating are currently widespread for MP using FCM (TO/CD235a-MP); however they have not been validated using NMB-IHP. We recently developed MP using FCM with anti-CD45/CD41a/CD61 antibodies, that are utilized for excluding platelets from the un-nucleated blood cell fraction to select erythrocytes (TO/CD41a/CD61-MP). The aim of this study is to validate TO/CD41a/CD61-MP for measurement of reticulocytes as an IHP-compliant MP. First, TO/CD235a-MP was validated as an IHP-compliant MP by comparing it with NMB-IHP. Then TO/CD41a/CD61-MP was compared with TO/CD235a-MP. The practical utility of TO/CD41a/CD61-MP was evaluated using XN-2000™(Sysmex) and Celltac G + ™(Nihon Kohden) hematology analyzers to assess the accuracy of different nucleic acid staining methods. We first confirmed that TO/CD235a-MP was consistent with NMB-IHP, then evaluated TO/CD41a/CD61-MP using TO/CD235a-MP. Regression analysis demonstrated consistency between TO/CD41a/CD61-MP and TO/CD235a-MP, thereby establishing TO/CD41a/CD61-MP as IHP-compliant MP. In the accuracy assessment, the correlation coefficients to TO/CD41a/CD61-MP were observed to be 0.97 for both hematology analyzers with %Retic ranging from 0.0 to 8.2. TO/CD41a/CD61-MP serves effectively as an IHP-compliant MP. This study provides the first empirical validation of FCM-based MPs.

Measurable residual disease (MRD) testing for chronic lymphocytic leukemia (CLL) is often done on peripheral blood (PB) since the concordance of results with bone marrow is high and testing is less invasive. When analyzing CLL MRD data, one must be aware of small, normal populations in the PB that may be mistaken for residual CLL cells. As part of our CLL MRD assay validation, PB samples were collected from 10 healthy donors and a 2-tube CLL MRD flow cytometry panel was stained for each donor using markers CD19, CD20, BAFF-R, kappa, lambda, CD5, CD200, CD23, CD38, CD81, ROR1, CD79b, CD43, and CD45. Additional markers were utilized to exclude T-cells, NK-cells, and myeloid cells from the analysis. Samples were acquired on the Navios EX flow cytometer, and the data were analyzed using FCS Express software. Once the test was implemented, CLL PB patient samples were monitored. All 10 PBs from healthy donors contained small populations of cells present in the lymphocyte gate which mimicked CLL cells in their expression of CD45, CD19, CD20, CD43 (both positive, although CLL cells showed dimmer positive expression), CD79b, and level of surface light chains, immunophenotypically compatible with plasmablasts. Clinical implementation of the CLL assay revealed 12 out of 77 (16%) CLL PB patient samples demonstrating a small population of plasmablasts. Plasmablasts normally exist in peripheral blood at levels detectable by flow cytometry MRD assays and may be a potential confounder in the identification of MRD in CLL.

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). With the introduction of FC systems with automatic tube loaders, we updated the FC-OFT protocol to follow the endpoint assay principle (FC-OFT). This study aims to evaluate the updated FC-OFT protocol (FC-OFT) and compare its assay performance with that of FC-OFT. We investigated factors influencing the FC-OFT 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 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, 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 was more accurate than FC-OFT in identifying spherocytosis in the 25 patients with anemia (p = 0.0313). FC-OFT is a viable alternative to conventional OFT or FC-OFT for HS screening in clinical laboratories, as automatic FC enhances assay performance. These findings warrant validation in future multicenter studies with larger sample sizes.

Platelet refractoriness is caused by antibodies against human leukocyte antigens or human platelet antigens. However, readily applicable assays that assist in selecting immunologically compatible platelet units remain limited. We established a flow cytometric platelet crossmatching assay and assessed its clinical utility by interpreting the results in conjunction with post-transfusion corrected count increments (CCI). Platelets were incubated with serum that may contain anti-platelet antibodies. Using flow cytometry, CD41-expressing platelets were gated, and the median fluorescence intensity (MFI) of fluorescein isothiocyanate (FITC)-conjugated anti-human IgG was measured. The MFI ratio was calculated as test sample MFI divided by baseline negative control MFI. The cutoff value and limit of detection (LoD) were estimated. Platelet crossmatching was performed using residual segments of transfused platelet units and patient serum, and the results were retrospectively interpreted in conjunction with CCIs and clinical findings. The MFI ratios were clearly distinguishable among the three groups: high-positive controls, low-positive controls, and known negative samples (p < 0.001). The cutoff value was calculated to be 1.35, and the LoD was 1.53. In total, eight platelet transfusion events in five patients were analyzed. Four cases were interpreted as non-immune refractoriness, and the remaining four showed adequate post-transfusion platelet increments. All corresponding platelet crossmatching results were negative, which was considered appropriate given that the assay is designed to reflect immune refractoriness. The flow cytometric platelet crossmatching assay was established and demonstrated to be applicable. The assay can help predict transfusion outcomes in alloimmunized patients and contribute to the selection of compatible blood units.

Clinical flow cytometry laboratories are facing rising test volumes, greater assay complexity, and increasing requirements for quality control and assay validation. In response, the International Clinical Cytometry Society (ICCS) conducted a workload survey in early 2023 to gather updated information on assay volumes, complexity, staffing, and technology. Data analysis focused on identifying correlations between length of time to introduce new assays and other factors as a means to gain insight about laboratories that seem to be either adapting or struggling. Flow cytometry assays were categorized into 3 levels of technical/interpretative complexity: high (e.g., measurable/minimal residual disease (MRD assays)), moderate (e.g., leukemia/lymphoma assays (Assays), excluding MRD assays), and low (e.g., CD4 count). Annual assays per staff member were calculated according to staff involved in case sign-out (Staff) or other laboratory operations (Staff). Respondents were from 101 laboratories in the United States (69.3%), Canada (4.0%), and other countries (26.7%). Low, moderate, and high technical/interpretative complexity assays were performed in 85.1%, 97.0%, and 47.5% of all laboratories, respectively. Median annual total assays (Assays) per laboratory were 3515 and, based on complexity, were 1518.5 (low), 1808.8 (moderate), and 350 (high). Among all laboratories, the median time (interquartile range) to introduce new Assays was 6 mos. (4-12 mos.), to introduce MRD assays was 11 mos. (5-12 mos.), and to validate/go-live with new cytometers was 8 mos. (4-12 mos.); these times positively correlated with each other. This study confirmed significantly increased workload since the prior ICCS 2013 workload survey with a concurrent decrease in Staff. Faster introduction of new assays correlated with other successes, including quicker validation of and going live with new cytometers. Among all laboratories, those that performed myeloid MRD assays versus those that did not were also found to be faster to introduce new assays. The need for sufficient staffing has been emphasized because laboratories with both higher annual volumes of myeloma MRD assays and higher ratios of Assays per Staff were slower to introduce new assays. "Lack of staff and/or time dedicated or protected for assay development" and, more generally, "staff number" were the most commonly identified major barriers for new assay development, with the former specifically linked to slower introduction of new assays among all laboratories.

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.

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.

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.

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.

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.

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.

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.