Issue Highlights—September 2022

Abstract

The second paper addresses difficulties using CD19 to detect B-ALL MRD leukemic cells following CD19-directed treatments, such as blinatumomab and CAR-T cells directed against CD19. In these cases, alternatives other than CD19 antibodies are needed (Mikhailova et al., 2021; Pillai et al., 2019). Ekaterina Mikhailova et al. at the National Medical Research Center of Pediatric Hematology, Oncology, and Immunology in Moscow report here on the use of CD22, CD24, and intracellular CD79a (iCD79a) in combination with CD10 (Mikhailova et al., 2022). Up to 519 samples from children with B-ALL were included in their study in which they found CD22, CD24, and iCD79a are expressed by >95% of leukemic cells. Nevertheless, no single antigen was without limitations. For example, CD22 is low to unexpressed in B-ALL patients with KMT2A rearrangements (Shah et al., 2015); CD24 is expressed on granulocytes thus requiring a marker such as CD66b to exclude them (Zhang et al., 2020); and iCD79a requires intracellular staining which can lead to cell losses and can impact staining intensities (Soh et al., 2020). Consequently, the authors recommend including all of these antigens in a panel with CD10. They describe an algorithm using CD22 and iCD79a as the primary gating antigens, which in their studies would have worked in 79.8% of their patients. If either marker is only partially expressed, then the marker with complete antigen expression is used. Using that strategy, leukemic B cells in 98% of their patients could be defined. For the remaining 2% they recommend using CD24 and CD10.

In the last B-ALL related manuscript from this issue, the immunophenotype of ZNF384 rearrangements in adult B-ALL is described. This rearrangement was first reported in 2002 (Martini et al., 2002) and at least 10 or more fusion partners with ZNF384 have now been found (Hirabayashi et al., 2021). Patients with a ZNF384 rearrangement usually do not have overly aggressive disease and express either a B-ALL or mixed B/myeloid acute leukemia phenotype. Ya-Zhe Wang et al. from the Peking University People's Hospital report here on their retrospective studies of 43 adults with ZNF384 rearranged B-ALL and contrast their findings with other B-ALL patients including groups with BCR-ABL, and KMT2A rearrangements (Wang et al., 2022). They conclude that B-ALL cells from patients with ZNF384 rearrangements generally had significantly lower expression of CD10 and higher levels of expression of CD13, CD33, and CD123. The one exception was CD10 expression on cells from patients with the ZNF384 was higher than on KMT2A rearrangements. Using this information, the authors propose a scoring system with CD10, CD13, CD33, and CD123 to predict ZNF384 rearrangement.

Next, two Original Articles in this issue look at the application of flow cytometry to hemolytic disorders. In the first, Yael Shahal-Zimra, et al. from the Bellinson Hospital in Petah Tikva Israel reports on the refinement of an RBC fragility assay to diagnose Hereditary spherocytosis. In the second, Awirut Charoensappakit from Chulalongkorn University in Bangkok, Thailand describes methodology to distinguish glomerular hematuria from non-glomerular hematuria.

Hereditary spherocytosis is a common inherited congenital hemolytic anemia caused by mutations in several genes coding for structural membrane proteins that lead to a spherical RBC shape. This spherical rather than concave shape interferes with their circulation making them prone to rupture and removal by the spleen (He et al., 2018). Two tests are used for diagnosis, the osmotic fragility test which measures the amount of hemolysis by different osmotic solutions and the Eosin-5'maleimide dye binding test (EMA) which binds to band 3 proteins in RBCs membranes and are deficient in hereditary spherocytosis (More et al., 2020; Yamamoto et al., 2014). Both can be done by flow cytometry and when performed in combination can correctly diagnose 100% of hereditary spherocytosis patients. In this issue, Yael Shahal-Zimra, et al. from the Rabin Medical Center in Pethah Tikva, Israel reports on a more efficient osmotic fragility flow cytometric test and compare it to both the classical osmotic fragility and EMA tests for the diagnosis of hereditary spherocytosis (Shahal-Zimra et al., 2021). Their use of flow cytometry for both diagnostic methods simplify procedures, reduces sample requirements, improves accuracy, and yields more rapid results.

Glomerular disease, associated with proteinuria and hematuria are diagnosed by the morphological quantification of dysmorphic RBC's in the urine (Hamadah et al., 2018). However, the morphological definition used to characterize dysmorphic RBCs has not been standardized resulting in difficulty classifying patients with hematuria. Flow cytometric approaches to distinguish the origin of hematuria have to now shown limited capacity to discriminate glomerular from non-glomerular causes of hematuria (Scharnhorst et al., 2006). Here Awirut Charoensappakit et al. describes a novel flow cytometric approach looking at the relationship between surface phosphatidylserine expression on RBCs and RBC derived micro particles. The authors use sizing beads to define large micro particles in urine samples with CD235a and annexing V and to define RBCs and their expression of phosphatidylserine (Dave et al., 2022; Sutherland et al., 2020). They demonstrate increased numbers of RBC derived micro particles and surface phosphatidylserine expressing RBCs in patients with glomerular hemolysis versus patients with non-glomerular hemolysis. The authors speculate these differences are related to differences between the patients' pathology. RBCs found in the urine of patients with glomerular hemolysis must pass through damaged glomeruli placing them under shear stresses resulting in changes to the RBC membrane cytoskeleton and causing increased expression of surface phosphatidylserine and numbers of RBC derived micro particles. While these results need to be evaluated in a larger study, if substantiated would considerably help in the differentiation between glomerular and non-glomerular hematuria.

The sixth Original Article addresses an issue of reduced HLADR expression on blood samples collected in BCT tubes for clinical trials evaluating immunostiumulating agents. HLADR expression on blood monocytes is considered a robust marker of immunosuppression in severely injured ICU patients and is being used as a stratification criteria in clinical trials (Eksioglu-Demiralp et al., 2022, Venet, 2021 #29; Payen et al., 2019). Reliable methods to measure HLADR on monocytes in multicenter clinical trials incorporating Quantribright beads and collection in BCT tubes have been developed that enable shipment of samples to a central location for evaluation (Quadrini et al., 2021). However, Sarah Hamada et al. at Edouard Herriot Hospital in Lyon and others at University of Limoges in Limoges, France in this issue reveal that samples collected in BCT tubes have a significantly lower HLADR expression on monocytes than samples collected in EDTA – the anti-coagulant originally used to establish cutoff thresholds for immunosuppression (Hamada et al., 2021). In fact, samples collected in BCT tubes from most patients in their study were below the threshold indicative of immunosuppression but were found above the cutoff if collected in EDTA. The authors caution a larger study to establish a new cutoff threshold for samples collected in BCT tubes is warranted.

Anyone using the AQUIOS for T, B, and NK cell reporting will be interested in the seventh Original Article in this issue. Léa Lemoine et al. from the Edouard Herriot Hospital in Lyon, France report on a decision tree they developed which addresses most AQUIOS automated analysis irregularities (Lemoine et al., 2022). Previously, Degandt et al. reported that a substantial number of T, B, and NK results from the AQUIOS CL had run ‘notifications’ indicating an abnormal cell distribution or population had been detected and the samples either had to be rerun or manually gated resulting in what they describe as ‘revision fatigue’ among operators (Degandt et al., 2018). This, despite the fact that in cross-institutional surveys the AQUIOS has shown better precision and robustness then other IVD/CE approved T, B, and NK methods (Ticchioni et al., 2019). Looking at 862 samples collected over a 10-day period, Lemoine et al. confirmed Degandt et al. observation. Among their 862 analyses, 25.4% showed on one or more dot plot analysis irregularities. These irregularities could be classified into four categories and the authors used these data to develop a decision tree. Applying their decision tree, 94% of the AQUIOS results could be released without additional bench work. The remaining were restrained and run on a convention cytometer.

The remaining articles in this issue include three Letters to the Editor and an eighth Original Article which describe a 4 color protocol to assess viability, acrosome integrity, and mitochondrial activity in boar spermatozoa using Hoechst 33342, peanut agglutinin, propidium iodide, and MitoTracker Deep Red™ (Gonzalez-Castro et al., 2022).

The first Letter to the Editor submitted by Bartosz Grzywacz et al. at the University of Minnesota (Grzywacz et al., 2021) confirms the work of Lyapichev et al. (Lyapichev et al., 2021) regarding the presence of CD7 negative and CD26 negative populations in healthy individuals. They further extend these finding to patients with autoimmune disease, abnormal peripheral blood counts, and circulating clonal B cells. Thus, emphasizing that the presence of CD4+ T cells lacking either CD7 and/or CD26 are not specific to Sezary syndrome and mycosis fungoides and the need to quantify Sezary syndrome and mycosis fungoides by enumerating immunophenotypically aberrant CD4+ T-cells, rather than CD26- or CD7- in isolation. (Craig, 2021; Horna et al., 2021; Illingworth et al., 2021).

The next two Letters to the Editor are Case Reports. The first is a fascinating one from Joseph Rohr et al. (Rohr et al., 2021) describes a newborn with cartilage hair hypoplasia (CHH) a primary immunodeficiency affecting both cellular and humoral immunity (Rohr et al., 2021). Flow cytometric evaluation of the newborn's blood was performed starting at day 5 and week 4. At week 5, the patient received an unrelated bone marrow transplant. Thereafter flow immunophenotyping was performed on months 4 and 5. Interestingly, the patients immunophenotyping findings, which are reported here, appear to recapitulate in part the prenatal generation of thymocytes, referred to as layered immune constitution (Davenport et al., 2020).

Finally, the second Case Study is presented by Eman Abdelghani, Clifford Mueller, and Huifei Liu from the Ohio State University College of Medicine and Esoterix Pathology Practice Group. They describe an abnormal, transient lymphomyelopoiesis resembling a mixed phenotype acute leukemia in a newborn patient with Nooan syndrome (Abdelghani et al., 2022). A number of genetic mutations can result in Noonan syndrome, which may be inherited as an autosomal dominant condition or occur as a new mutation (Roberts et al., 2013). Patients present with mildly unusual facial features, short height, congenital heart disease, bleeding problems, and skeletal malformations. It is defined as a RASopathy, a group of genetic conditions caused by mutations in genes of the RAS-MAPK pathway. A CBC on this patient 14 days after birth showed a leukocytosis, polycythemia, and thrombocytopenia. Flow cytometry detected lymphoblasts (11.6%), myeloblasts (9.8) and monocytes (23.3%) suggesting a congenital mixed phenotype acute leukemia (Porwit & Bene, 2019). Next generation sequencing detected a missense mutation in PTPN11 a signaling molecule in the RAS/MAPK pathway. The patient remained stable and a watch and wait strategy was chosen. At 9 months of age though a persistent monocytosis remained but the CBC had returned to the normal range with low levels of circulating lymphoblasts (0.04%) and myeloblasts (0.2%).