In vivo expansion of a CD9+ decidual-like NK cell subset following autologous hematopoietic stem cell transplantation.

Autologous hematopoietic stem cell transplantation (autoHSCT) is a treatment option for hematological disorders and pediatric solid tumors. After an autoHSCT, natural killer (NK) cells are the first lymphocyte subset returning to normal levels. To uncover global changes during NK cell reconstitution after autoHSCT, we performed RNA-sequencing on NK cells before and after autoHSCT. Results showed profound changes in the gene expression profile of NK cells immediately after autoHSCT. Several biological processes including cell cycle, DNA replication and the mevalonate pathway were enriched. Significantly, we observed that following autoHSCT, NK cells acquired a decidual-like gene expression profile, including the expression of CD9. By using multiparametric flow cytometry, we confirmed the expansion of NK cells expressing CD9 immediately after autoHSCT, which exhibited higher granzyme B and perforin expression levels than CD9- NK cells. These results provide insights into the physiopathology of NK cells during their reconstitution after autoHSCT.

Initial study of anaemia profile for primary care centres with automated laboratory algorithms reduces the demand for ferritin, iron, transferrin, vitamin B12 and folate tests.

AIM: To evaluate the influence of an algorithm designed to incorporate reflex testing according to haemogram results for analytical tests ordered to investigate anaemia. METHODS: In 2020, a new request for 'initial study of anaemia' was created in three primary care pilot centres for suspected anaemia or new anaemias. A haemogram was ordered and the remainder of the tests were created in a reflex manner according to an algorithm integrated in the laboratory information system that also generates a comment that is completed and validated by a haematologist. The demand for tests was evaluated over three time periods. RESULTS: Of 396 requests, anaemia was detected in 80 (20.2%), with 26 microcytic anaemias (6.57%), 20 iron deficiency anaemias, 41 (10.3%) normocytic anaemias and 13 macrocytic anaemias (3.28%); 4 with folate deficiency; and 1 haemolytic anaemia. No haematological diseases were detected. Twenty-four (6.06%) cases exhibited microcytosis/hypochromia without anaemia, 12 of which exhibited iron deficiency. Four young women exhibiting within-limit haemoglobin levels had iron deficiency. There were 56 (14.1%) cases of macrocytosis without anaemia.With the new profile of 'initial study of anaemia', the demand for tests was reduced and was significantly lower than in the remainder of primary centres for iron, transferrin, ferritin, vitamin B12 and folate. CONCLUSIONS: A new profile of 'initial study of anaemia' in the request form with algorithms integrated in the laboratory information system enabled submission of orders and decreased the demand for unnecessary iron, transferrin, ferritin, vitamin B12 and folate tests.

Large granular lymphocyte leukaemia study at the University Hospital of Donostia.

INTRODUCTION: Large granular lymphocyte (LGL) leukaemia is considered a mature T-cell or natural killer (NK) cell neoplasm, characterised by a clonal proliferation of LGL. AIMS: To analyse the characteristics and to establish (if possible) the prognostic parameters of these patients diagnosed in a single centre: University Hospital of Donostia. METHODS: We retrospectively studied data about 308 patients with LGL leukaemia diagnosed in our centre. RESULTS: The frequency of T-LGL leukaemia and chronic lymphoproliferative disorder of NK cells was 89% and 6.8% respectively, and no aggressive NK-LGL leukaemia was seen in our population. The median age at diagnosis was 65.7 years and male-to-female ratio was 1.08. 59% of our patients were asymptomatic at the time of diagnosis. Most patients presented lymphocytosis and 63.6% more than 20% LGLs in the peripheral blood count, but it has to be taken into account that these results may be influenced by the selection bias of our study, as we recognised these patients as 'alarms of the laboratory analysers'. Neutropenia was the most common cytopenia, and autoimmune disorders were described in 16.5% of the patients. Only 12 patients (3.9%) required treatment, a much lower percentage that the one reported in the literature, and this is consistent with the fact that patients were less symptomatic than in other series, as we expected. The 5-year and 15-year overall survival was 92% and 87%, respectively. CONCLUSIONS: Our patients may represent the even more benign end of the spectrum of clonal T LGL and NK proliferations.

NK Cell Reconstitution After Autologous Hematopoietic Stem Cell Transplantation: Association Between NK Cell Maturation Stage and Outcome in Multiple Myeloma.

Autologous hematopoietic stem cell transplantation (autoHSCT) is a standard of care for transplant-eligible patients with multiple myeloma (MM). Among factors that influence outcome after autoHSCT, it has been suggested that the number of natural killer (NK) cells plays an important role. However, the impact that different NK cell subsets and their phenotype could have in disease progression after autoHSCT are less clear. For this reason, we have phenotypically and functionally characterized NK cells during immune system reconstitution after autoHSCT in 54 MM patients. Shortly after leukocyte recovery, an extensive redistribution of NK cell subsets occurs in these patients. In addition, NK cells undergo a profound phenotypic change characterized, among others, by their increased proliferative capacity and immature phenotype. Importantly, MM patients who showed lower frequencies of the mature highly differentiated NKG2A-CD57+ NK cell subset at +30 and +100 days after autoHSCT experienced superior progression-free survival and had a longer time to the next treatment than those with higher frequencies. Our results provide significant insights into NK cell reconstitution after autoHSCT and suggest that the degree of NK cell maturation after autoHSCT affects the clinical outcome of MM patients treated with this therapeutic strategy.

A NKp80-Based Identification Strategy Reveals that CD56neg NK Cells Are Not Completely Dysfunctional in Health and Disease.

Natural killer (NK) cells are usually identified by the absence of other lineage markers, due to the lack of cell-surface-specific receptors. CD56neg NK cells, classically identified as CD56negCD16+, are very scarce in the peripheral blood of healthy people but they expand in some pathological conditions. However, studies on CD56neg NK cells had revealed different results regarding the phenotype and functionality. This could be due to, among others, the unstable expression of CD16, which hinders CD56neg NK cells' proper identification. Hence, we aim to determine an alternative surface marker to CD16 to better identify CD56neg NK cells. We have found that NKp80 is superior to CD16. Furthermore, we found differences between the functionality of CD56negNKp80+ and CD56negCD16+, suggesting that the effector functions of CD56neg NK cells are not as diminished as previously thought. We proposed NKp80 as a noteworthy marker to identify and accurately re-characterize human CD56neg NK cells.

Evaluation of the predictive value of the hematopoietic progenitor cell count using an automated hematology analyzer for CD34+ stem cell mobilization and apheresis product yield.

INTRODUCTION: We evaluated the value of hematopoietic progenitor cells (HPCs) counted in Sysmex XN analyzers to predict the mobilization and collection of CD34+ cells in apheresis for stem cell transplantation. METHODS: Eighty patients who underwent stem cell transplantation were enrolled (50 autologous and 30 allogeneic). In the autologous group, patients were considered poor mobilizers when the CD34+ count was <10 × 106 /L or <20 × 106 /L in patients with multiple myeloma who were going to undergo two transplants. ROC curves were generated, and HPC cutoffs were calculated. RESULTS: The correlation between the HPC and CD34+ cell counts was good. Two algorithms were proposed. In the first algorithm, samples collected the day before apheresis, negative and positive HPC cutoffs were selected to detect poor and good mobilization and, therefore, the need or not to administer plerixafor. In the second algorithm, samples collected pre-apheresis, the negative HPC cutoff was an indication to delay apheresis; an HPC higher than the optimal cutoff was an indication to start apheresis. When the HPC values were between these cutoffs, there was an indication to count CD34+ cells for a better decision-making. Finally, in samples collected pre-apheresis, HPC counts could be used to predict patients who would have poor CD34+ cell collections. In the allogeneic group, all the donors mobilized well, and very few needed two apheresis procedures. CONCLUSIONS: The HPC count is useful for decision-making in the management of patients subjected to apheresis procedures to collect peripheral blood stem cells.