Issue highlights—November 2022

Abstract

In patients with interstitial lung diseases (ILD) broncho-alveolar lavage (BAL) fluid analysis is important in supporting the diagnosis. The suggested site for obtaining diagnostic BAL is the middle lobe of the patient's lungs when it is washed with isotonic saline and the aspirated solution is analyzed for cell concentration and leukocyte subsets. The procedure for obtaining BAL fluid is quite difficult to standardize and the washing and harvesting procedure is usually carried out in five fractions. In ILD like sarcoidosis, idiopathic pulmonary fibrosis, or eosinophilic ILD, BAL fluid analysis can aid in the diagnosis while in other diseases like primary alveolar proteinosis a therapeutic BAL is applied as drug treatment in these cases, which often fail.

In this issue of Clinical Cytometry two manuscripts from the same group deal with flow cytometric BAL analysis. In the manuscript of Eidhof et al. (2021) the authors compare three methods to stabilize BAL cells for flow cytometric analysis. This procedure has a large practical significance, as cells in the low protein content BAL fluid are quite vulnerable. Samples should be kept on ice and transported to the laboratory and stained within 4 h, otherwise they tend to lyse. In their article, the authors present the comparison of native lavage cells versus stabilized cells utilizing Transfix, Streck Cell Preservative and an in-house method that primarily consists of formaldehyde fixation in buffered conditions containing serum proteins. All three stabilization methods influence the side scatter (SSC) but this does not affect gating. They found a 7-day stability with Transfix and a 28-day stability with their in-house method that can considerably expand the list of laboratories where samples can be transported under the above conditions.

In their other paper, this group by Mulder et al. (2022) demonstrate the results of their external quality flow cytometric studies. Starting from year 2000 with only 12 participating laboratories they are now involving 42 flow labs. The external QC programme required on one hand the actual analysis of the cytometric items that consisted of leukocyte count, leukocyte differentiation, lymphocyte subsets, T-cell subsets related to peripheral blood and CD103 expression on CD4+ cells with microscopic analysis. On the other hand, the participants received relevant clinical information about each case and were able to report their interpretive comments and their diagnosis as free text. The best matches with the diagnosis were obtained for sarcoidosis, eosinophilic ILD and extrinsic alveolitis, that would advance the diagnosis of these patients.

In the past decade the implementation of immunotherapy in the treatment of numerous hematological disorders has become a daily practice. The utilization of rituximab has been incorporated into several treatment protocols making it impossible to use anti-CD20 for the identification of malignant B-cells. Similarly upon daratumumab treatment the identification of malignant plasma cells is not feasible with anti-CD38 labeling. In this Issue of Clinical Cytometry (Mikhailova et al., 2022) Mikhailova and coworkers investigated the applicable B-lineage markers in case of minimal residual disease detection in children suffering from B-cell precursor acute lymphoblastic leukemia after CD19 targeting. The pan B-cell surface marker CD19 molecule can be targeted either with the bispecific antibody blinatumomab or with T-lymphocytes harboring a chimeric antigen receptor against CD19. These treatments result in a loss of CD19 by tumor cells. Identifying residual blasts in ALL can be a real challenge even without blinatumomab treatment since marker expressions may considerably vary in these cases in subsequent time points mostly affected by bone marrow regeneration (Veltroni et al., 2003) or phenotype alterations induced by the administered drugs (Gaipa et al., 2005). In their recent report on a large cohort of over 500 children with ALL the authors investigated what B-cell markers are applicable in the identification of B-cells when the backbone marker CD19 is not detectable. They are suggesting a panel of useful antibodies composed of CD10, CD22, CD24 and cytoplasmic CD79a and also propose an algorithm for the detection of the residual BCP-ALL after CD19 targeting. The caveats in the analysis of such labelings are also described to avoid interference from basophils and plasmacytoid dendritic cells that may be positive for CD22 or mature neutrophils that may interfere with CD10 and CD24 labelings. Such thoughtful analysis is extremely important to reliably identify blast cells in pediatric ALL samples since the results are key parameters in prognostication and in deciding further treatment.