Issue highlights—April 2024

This issue of Cytometry Part B, Clinical Cytometry consists of four original articles and four letters to the Editor, bridged by a discussion forum. Although finding common themes between these works is not necessary, some naturally emerged for me under a simple but helpful way of thinking about clinical flow cytometry that I learned from Professor Alberto Orfao's education sessions. To paraphrase, in clinical flow cytometry, we are doing one of three things at any time: identifying, characterizing (as either normal or abnormal) or enumerating cell populations. Fourth, flow cytometry must be interpreted in a broader clinicopathological context. These principles assist in defining the indication and context of use of an assay, which in turn help determine panel design, and other pre-analytical, analytical and post-analytical components. Lastly, this journal recognizes the value of the single case report. While some journals have abandoned them, if well researched, relevant and succinct, they can serve as a useful educational tool or cautionary tale, illustrate the application, strengths or weakness of a guideline, or document rare, interesting cases and other novel phenomena.

Therefore, rather than in order of appearance, I introduce this issue's contents as follows:

Kumar et al. (2024) provide a nice example of improving the identification of plasma cells for later characterization and enumeration, demonstrating substantial improvement in CD138 expression and, ultimately, plasma cell recovery using a gentler “stain-lyse-no-wash” sample preparation technique compared to their standard “(bulk) lyse-stain-wash” method in 36 paired bone marrow samples, with no adverse effect on the intensity of other antigens in the panel. They changed their practice, using this simpler technique for the surface marker tube on 244 additional samples over 6 years, reserving “lyse-stain-wash” preparation for the analysis of cytoplasmic light chains. Whether this can be applied to myeloma measurable residual disease (MRD) assessment remains to be tested.

The study by Ramalingam et al. (2024) and letter from Placek et al. (2024) reinforce that a masterful appreciation of normal B-cell maturation under various clinical conditions is critical for monitoring residual B-acute lymphoblastic leukemia (B-ALL). Ramalingam et al. provide a concise assessment of the immunophenotype of type 0 hematogones (by CD34, CD10, CD45, CD19, CD20, CD22 and CD24 expression) in 61 pediatric patients under various conditions and time points following CD19-targeting, conventional chemotherapy, and hematopoietic stem cell transplantation. While the existence of CD19-negative B-cell precursors (BCP) has been known for some time (Dworzak et al., 1998; Uckun & Ledbetter, 1988), they have, until recently, remained under recognized within the confines of standard B-ALL MRD panels until Cherian et al. developed an adaptation that provides an alternate gating strategy using CD22 or CD24 (Cherian et al., 2018).

Placek et al. (2024) on the other-hand (in their words), “describe a stereotyped dyssynchronous maturational shift [in the immunophenotype of normal BCP] that appears specific to a particular pre-CART [chimeric antigen receptor T-cell] LDC [lymphocyte-depleting chemotherapy] regimen at a particular bone marrow sample time point…most reminiscent of late stage II BCP…[which] appears to be common but not universal to this LDC regimen [fludarabine and cyclophosphamide]…” in six of the seven patients reported in this small series that were known to have received this conditioning. The authors stress that awareness of this otherwise apparent alarming difference from normal should avoid inadvertent interpretation as residual B-ALL.

Slalina et al. (2024) focusing on common variable immunodeficiency (CVID), worked on low-density neutrophils (LDN), a heterogeneous population of neutrophils found in the peripheral blood mononuclear cell (PBMC) layer after density gradient centrifugation, and which have been the subject of active research in immunology, rheumatology, infectious disease, and solid malignancy for more than 30 years. Following density gradient separation, the authors used a robust gating and sorting strategy to identify, characterize and enumerate mature (mLDN) and immature (iLDN) fractions. Further immunophenotypic and functional characteristics, the latter quantified as a rise in mean fluorescence intensity of rhodamine 123 reflecting oxidative burst in response to simulated sepsis and/or intravenous immunoglobulin (IVIg), were compared in vitro between 25 patients with common variable immunodeficiency (CVID) and 27 healthy controls, and for 7 of the patients with CVID pre- and post-IVIg infusion in vivo. A substantial increase in the proportion of mature mLDN and a reduced capacity of this subset to generate an oxidative burst was demonstrated in vitro for patients with CVID compared to healthy controls, as well as some immunophenotypic changes. However, the proportion of mLDN did not increase in 3 out of 7 CVID patients in response to IVIg infusion in vivo, and the authors discuss this. They suggest these neutrophil anomalies could contribute to an increased susceptibility to recurrent bacterial infections in patients with CVID as a result of potential dysregulation of the immune response rather than directly contributing to the pathogenesis of CVID.

Two studies are presented in this issue of Cytometry Part B that evaluate the role of flow cytometric investigation of body fluid at non-hematological sites. Chan et al. report on the efficacy and diagnostic utility of flow cytometry (FC) in the context of routine pathological evaluation for breast implant associated anaplastic large cell lymphoma (BIA-ALCL) over 6-years at a single tertiary referral cancer center (Chan et al., 2024). The bespoke 10-color single tube FC panel was highly specific for BIA-ALCL and demonstrated a high positive and negative predictive value, but with imperfect sensitivity, reinforcing that FC serves as an excellent confirmatory test, but should not be relied upon as the sole diagnostic method considering other available modalities, namely fluid cytology and capsulectomy histology. There were also major learnings from false-negative cases in identifying various diagnostic pitfalls following a blinded expert review of flow cytometry data.

The multi-center study by Debliquis et al. represents a gap-analysis by survey of numerous flow cytometry and clinical practitioners across France and territories, Belgium and Switzerland, undertaken by a network of French-speaking cytometrist devoted to oculocerebral lymphoma (CytHem/LOC French network) who suspected a degree of inter-center heterogeneity in clinical and laboratory practice given varying epidemiology of meningeal involvement (Debliquis, 2024). Real-world practices for the detection of cerebral lymphoma in the cerebrospinal fluid (CSF) by flow cytometry were compared against those recommended by the European Society for Clinical Cell Analysis (ESCCA) and the Italian Society for Clinical Cell Analysis (ISCCA) (Del Principe et al., 2021) as a catalyst for improved harmonization of practice. Heterogeneity uncovered by the survey was addressed in the form of recommendations, with some flexibility against the international guidelines, on the following critical points of discussion: determination of absolute cell levels in CSF, acquisition and interpretation of scarce events, sample stabilization and transit, threshold of positivity, accounting for blood contamination, and fit for purpose antibody panels.

Going beyond “different from normal” to different from ‘normal abnormal’ this manuscript tackles in detail the question of “how different from a usual case of immunophenotypically defined Chronic Lymphocytic Leukemia (CLL) does a case need to become before it is no longer considered CLL?” (Matos, 2024). Commenting on a recent study in this Journal by Sorigue et al. (2019) “Refining the Limits of Borderline Lymphoproliferative Disorders,” Matos suggests that CD5-negative chronic lymphocytic leukemia may be a contradiction in terms and, until a unifying, CLL-defining molecular lesion is found to represent a gold standard, if one exists, CD5-negative CLL as a true entity may remain unprovable. Nevertheless, the World Health Organization (WHO), European Research Initiative on CLL (ERIC) and European Society for Clinical Cell Analysis (ESCCA) continue to accommodate this diagnostic possibility.

Zhang and Guo (2024) describe the first known patient with de novo BCR::ABL1 p210 acute myeloid leukemia (AML) (itself rare) presenting with concomitant NRAS mutation and unusual CD58 expression who did not, therefore, fit within a standard therapeutic protocol and responded favorably to tyrosine kinase inhibitors, azacytidine and venetoclax.

Finally, Altube et al. (2024) express the value of drawing on recent experience published by the MD Anderson Cancer Center on the immunophenotypic features of primary erythroid leukemia (PEL) compared with reactive erythroid precursors (Fang et al., 2022) having faced a diagnostic challenge of their own in distinguishing PEL from erythroid hyperplasia in what was ultimately a very unusual manifestation of chronic myeloid leukemia in blast crisis presenting as PEL with absent CD34 and CD117 expression.

Best wishes.