Hematopoiesis and immune reconstitution after CD19 directed chimeric antigen receptor T-cells (CAR-T): A comprehensive review on incidence, risk factors and current management.

Impaired function of hematopoiesis after treatment with chimeric antigen T-cells (CAR-T) is a frequent finding and can interest a wide range of patients, regardless of age and underlying disease. Trilinear cytopenias, as well as hypogammaglobulinemia, B-cell aplasia, and T-cell impairment, can severely affect the infectious risk of CAR-T recipients, as well as their quality of life. In this review, we provide an overview of defects in hematopoiesis after CAR-T, starting with a summary of different definitions and thresholds. We then move to summarize the main pathogenetic mechanisms of cytopenias, and we offer insight into cytomorphological aspects, the role of clonal hematopoiesis, and the risk of secondary myeloid malignancies. Subsequently, we expose the major findings and reports on T-cell and B-cell quantitative and functional impairment after CAR-T. Finally, we provide an overview of current recommendations and leading experiences regarding the management of cytopenias and defective B- and T-cell function.

Artificial intelligence and the blood film: Performance of the MC-80 digital morphology analyzer in samples with neoplastic and reactive cell types.

INTRODUCTION: Implementing artificial intelligence-based instruments in hematology laboratories requires evidence of efficiency in classifying pathological cells. In two-Universities, we assessed the performance of the Mindray® MC-80 for hematology patients with frequent leukemic and dysplastic cells. METHODS: The Mindray MC-80® locates and pre-classifies cells in blood films. In a two-university study, four films were prepared from 591 samples, two each for the analyser MC-80 and the microscope reference method, using reagents from two different manufacturers. We used Microsoft Excel® statistics for imprecision and distributional inaccuracy and a matrix table model (H20-A2 CLSI standard) for sensitivity, specificity and predictive value for atypical cells. RESULTS: The results indicate minimal within-run imprecision (ICSH method) and good intra-method consistency even on duplicate analysis of 413 samples with a high incidence of hematological abnormalities (r = 0.942 or more, except for basophils, r = 0.841, and reactive lymphocytes, r = 0.847). Distributional inaccuracy was also very low compared to the microscope reference, with a pass rate higher than 80% for pathological cells (except 75.1% for reactive lymphocytes). The primary causes of discrepancy were bizarre shapes of dysplastic neutrophils and inconsistent nomenclature for lymphoma cells. Sensitivity for critical samples containing cells typically absent in circulating blood (immature or malignant) was 98.8% for immature granulocytes, 83.8% for all types of neoplastic cells, 93.6% for reactive lymphocytes and 97.5% for nucleated red blood cells. The negative predictive values of MC-80 were 98.8% for immature granulocytes, 88.4% for the different types of neoplastic cells, 97.8% for reactive lymphocytes, and 96.9% for nucleated red blood cells. CONCLUSION: Our study highlights the outstanding diagnostic performance of this artificial intelligence-based blood film analyzer for hematology patients with circulating abnormal cells. We appreciated the morphological harmonization of cells observed on the screen and those seen in the microscope.

Eltrombopag for Low-Risk Myelodysplastic Syndromes With Thrombocytopenia: Interim Results of a Phase II, Randomized, Placebo-Controlled Clinical Trial (EQOL-MDS).

PURPOSE: In myelodysplastic syndromes (MDS), severe thrombocytopenia is associated with poor prognosis. This multicenter trial presents the second-part long-term efficacy and safety results of eltrombopag in patients with low-risk MDS and severe thrombocytopenia. METHODS: In this single-blind, randomized, placebo-controlled, phase-II trial of adult patients with International Prognostic Scoring System low- or intermediate-1-risk MDS, patients with a stable platelet (PLT) count (<30 × 103/mm3) received eltrombopag or placebo until disease progression. Primary end points were duration of PLT response (PLT-R; calculated from the time of PLT-R to date of loss of PLT-R, defined as bleeding/PLT count <30 × 103/mm3 or last date in observation) and long-term safety and tolerability. Secondary end points included incidence and severity of bleeding, PLT transfusions, quality of life, leukemia-free survival, progression-free survival, overall survival and pharmacokinetics. RESULTS: From 2011 to 2021, of 325 patients screened, 169 patients were randomly assigned oral eltrombopag (N = 112) or placebo (N = 57) at a starting dose of 50 mg once daily to maximum of 300 mg. PLT-R, with 25-week follow-up (IQR, 14-68) occurred in 47/111 (42.3%) eltrombopag patients versus 6/54 (11.1%) in placebo (odds ratio, 5.9; 95% CI, 2.3 to 14.9; P < .001). In eltrombopag patients, 12/47 (25.5%) lost the PLT-R, with cumulative thrombocytopenia relapse-free survival at 60 months of 63.6% (95% CI, 46.0 to 81.2). Clinically significant bleeding (WHO bleeding score ≥ 2) occurred less frequently in the eltrombopag arm than in the placebo group (incidence rate ratio, 0.54; 95% CI, 0.38 to 0.75; P = .0002). Although no difference in the frequency of grade 1-2 adverse events (AEs) was observed, a higher proportion of eltrombopag patients experienced grade 3-4 AEs (χ2 = 9.5, P = .002). AML evolution and/or disease progression occurred in 17% (for both) of eltrombopag and placebo patients with no difference in survival times. CONCLUSION: Eltrombopag was effective and relatively safe in low-risk MDS with severe thrombocytopenia. This trial is registered with ClinicalTrials.gov identifier: NCT02912208 and EU Clinical Trials Register: EudraCT No. 2010-022890-33.

Peripheral Blood Allogeneic Stem Cell Mobilization: Can We Predict a Suboptimal Mobilization?

Allogeneic peripheral blood stem cells mobilization is now the basis of most stem cell transplants. In a very limited number of cases, mobilization is suboptimal leading to further collection procedures, to suboptimal cell doses infusion with delayed engraftment time, increased risks of transplant procedure and of related costs. To date we have no recognized and shared criteria for early estimating the probability of poor mobilization in healthy donors. We then analyzed allogeneic peripheral blood stem cell donations performed at the Fondazione Policlinico Universitario A.Gemelli IRCCS Hospital from January 2013 to December 2021 in order to identify premobilization factors associated with successful mobilization. The following data were collected: age, gender, weight, complete blood cell count at baseline, G-CSF dose, number of collection procedures, CD34+ cell count in peripheral blood on the first day of collection, CD34+ cell dose per kg body weight of recipient. Mobilization efficacy was defined according to the number of CD34+ cells in peripheral blood on day +5 of G-CSF administration. We classified donors as sub-optimal mobilizers or good mobilizers according to the achievement of the 50 CD34+ cell/µL threshold. We observed 30 suboptimal mobilizations in 158 allogeneic peripheral blood stem cell donations. Age and baseline white blood cell count were factors significantly associated with negative or positive impact on mobilization, respectively. We did not find significant differences in mobilization based on gender or G-CSF dose. Using cut-off values of 43 years and 5.5×109/L WBC count, we built a suboptimal mobilization score: donors who reach 2, 1 or 0 points have a 46%, 16% or 4% probability of suboptimal mobilization, respectively. Our model explains 26% of the variability of mobilization confirming that most of the mobilization magnitude depends on genetically determined factors; however, suboptimal mobilization score is a simple tool providing an early assessment of mobilization efficacy before G-CSF administration begins in order to support allogeneic stem cells selection, mobilization and collection. Through a systematic review, we looked for confirmation of our findings. According to the published articles, all the variables we included in our model are confirmed to be strongly related to the success of mobilization. We believe that score system approach could be applied in clinical practice to assess the risk of mobilization failure at baseline allowing for a priori intervention.

Decitabine Versus Hydroxyurea for Advanced Proliferative Chronic Myelomonocytic Leukemia: Results of a Randomized Phase III Trial Within the EMSCO Network.

PURPOSE: Hydroxyurea (HY) is a reference treatment of advanced myeloproliferative neoplasms. We conducted a randomized phase III trial comparing decitabine (DAC) and HY in advanced myeloproliferative chronic myelomonocytic leukemias (CMML). PATIENTS AND METHODS: Newly diagnosed myeloproliferative CMML patients with advanced disease were randomly assigned 1:1 to intravenous DAC (20 mg/m2/d days 1-5) or HY (1-4 g/d) in 28-day cycles. The primary end point was event-free survival (EFS), events being death and acute myelomonocytic leukemia (AML) transformation or progression. RESULTS: One-hundred seventy patients received DAC (n = 84) or HY (n = 86). Median age was 72 and 74 years, and median WBC count 32.5 × 109/L and 31.2 × 109/L in the DAC and HY arms, respectively. Thirty-three percent of DAC and 31% of HY patients had CMML-2. Patients received a median of five DAC and six HY cycles. With a median follow-up of 17.5 months, median EFS was 12.1 months in the DAC arm and 10.3 months in the HY arm (hazard ratio [HR], 0.83; 95% CI, 0.59 to 1.16; P = .27). There was no significant interaction between treatment effect and blast or platelet count, anemia, CMML Prognostic Scoring System, Groupe Francophone des Myelodysplasies, or CMML Prognostic Scoring System-mol risk. Fifty-three (63%) DAC patients achieved a response compared with 30 (35%) HY patients (P = .0004). Median duration of response was similar in both arms (DAC, 16.3 months; HY, 17.4 months; P = .90). Median overall survival was 18.4 months in the DAC arm and 21.9 months in the HY arm (P = .67). Compared with HY, DAC significantly reduced the risk of CMML progression or transformation to acute myelomonocytic leukemia (cause-specific HR, 0.62; 95% CI, 0.41 to 0.94; P = .005) at the expense of death without progression or transformation (cause-specific HR, 1.55; 95% CI, 0.82 to 2.9; P = .04). CONCLUSION: Compared with HY, frontline treatment with DAC did not improve EFS in patients with advanced myeloproliferative CMML (ClinicalTrials.gov identifier: NCT02214407).

Coronavirus disease 2019 (COVID-19): Focus on peripheral blood cell morphology.

Numerous studies have shown peculiar morphological anomalies in COVID-19 patients' smears. We searched all the peer-reviewed scientific publications that explicitly reference the cytomorphological alterations on peripheral blood smears of patients with COVID-19. We extracted data from sixty-five publications (case reports, patient group studies, reviews, and erythrocyte morphology studies). The results show that frequent alterations concern the morphology of lymphocytes (large lymphocytes with weakly basophilic cytoplasm, plasmacytoid lymphocytes, large granular lymphocytes). Neutrophils display abnormal nuclei and cytoplasm in a distinctive cytomorphological picture. Besides a left shift in maturation, granulations can be increased (toxic type) or decreased with areas of basophilia. Nuclei are often hyposegmented (pseudo-Pelger-Huёt anomaly). Apoptotic or pycnotic cells are not uncommon. Monocytes typically have a large cytoplasm loaded with heterogeneous and coalescing vacuoles. Platelets show large and giant shapes. The presence of erythrocyte fragments and schistocytes is especially evident in the forms of COVID-19 that are associated with thrombotic microangiopathies. Such atypia of blood cells reflects the generalized activation in severe COVID-19, which has been demonstrated with immunophenotypic, molecular, genetic, and functional methods. Neutrophils, in particular, are involved in the pathophysiology of hyperinflammation with cytokine storm, which characterizes the most unfavorable evolution.

Digital morphology in hematology diagnosis and education: The experience of the European LeukemiaNet WP10.

Hematological diagnostics is based on increasingly precise techniques of cellular and molecular analysis. The correct interpretation of the blood and bone marrow smears observed under an optical microscope still represents a cornerstone. Precise quantitative and qualitative cytomorphological criteria have recently been codified by up-to-date guidelines for diagnosing hematopoietic neoplasms. Morphological analysis has found formidable support in digital reproduction techniques, which have simplified the circulation of images for educational or consultation purposes. From 2007 to 2019, the Working Group WP10 of European LeukemiaNet (ELN) used, in annual exercises, digital images to support training in cytomorphology and verify harmonization and comparability in the interpretation of blood and bone marrow smears. We describe the design, development, and results of this program, which had 741 participants in-person or remotely, to which 2055 questions were submitted regarding the interpretation of cytomorphological images. We initially used circulation and presentation of digital microphotographs and then introduced a virtual microscopy (VM). Virtual slides were obtained using a whole slide imaging technique, similar to the one largely used in histopathology, to produce digitized scans of consecutive microscopic fields and reassembles them to obtain a complete virtual smear by stitching. Participants were required to identify cells in labeled fields of view of the virtual slides to obtain a morphological diagnosis. This work has demonstrated substantial improvements in diagnostic accuracy and harmonization with the VM technique. Between-observer concordance increased from 62.5% to 83.0%. The integrity of the digitalized film image, which provides a general context for cell abnormalities, was the main factor for this outcome.

Successful "on-demand" plerixafor for autologous peripheral blood stem-cells transplantation for relapsed/refractory germ cell tumors.

BACKGROUND: Germ cell tumors represent, among solid cancers, a potentially curable disease even if up to 20% to 30% of patients (pts) relapse after first-line treatment especially considering intermediate and poor prognosis groups. In this scenario, patients are candidates for high-dose chemotherapy and autologous stem-cells transplantation as second-line treatment even though stem-cells mobilization potential can be affected by several cycles and regimens of chemotherapy. To date, plerixafor is authorized in poor mobilizer adult pts diagnosed with lymphoma or multiple myeloma and in pediatric solid tumors or lymphoma. Therefore, the use of plerixafor in adult pts with relapsing/refractory GCT is still off label. MATERIALS AND METHODS: In our study, we describe mobilization and collection of peripheral blood stem cells for 10 pts with germ cell tumors. Six patients underwent plerixafor administration since classified as poor mobilizers based on WBC count (>5.000/µL) and CD34+ cell count (<15/µL) the day before apheresis procedure. RESULTS: On the first day of apheresis, plerixafor administration in poor mobilizers made possible a remarkable boost of CD34+ cells in such a way to overlap that of good mobilizers' (32/µL vs 35/µL, respectively, P > .05). CONCLUSION: Therefore, in our experience, plerixafor made a good fraction of poor mobilizer patients eligible for mobilization and collection and able to undergo the predicted autologous stem-cells transplantation; thus, the lack of access to the use of plerixafor in this setting of patients risks jeopardizing an effective treatment, especially in case of poor prognosis.

2021 update of the 2012 ICSH Recommendations for identification, diagnostic value, and quantitation of schistocytes: Impact and revisions.

In 2012, the International Council for Standardization in Hematology (ICSH) published recommendations for the identification, quantitation, and diagnostic value of schistocytes. In the present review, the impact of these recommendations is evaluated. This work is based on citations in peer-reviewed papers published since 2012. The first 2012 ICSH Recommendations have also been revised to incorporate newly published data in the literature and current best laboratory practice. Recommended reference ranges have been proposed for healthy adults and full-term neonates of 1% or less schistocytes. More than 1% of morphologically identified schistocytes on the blood film are considered suspicious for thrombotic microangiopathy. For preterm infants, a normal level of 5% or less is recommended. The fragment red cell count (FRC) generated by some automated hematological analyzers provides a valuable screening tool for the presence of schistocytes. Specifically, the absence of FRCs can be used as a valuable parameter to exclude the presence of schistocytes on the blood film. The validity and usefulness of microscope schistocytes and automated FRCs, respectively, are discussed in the context of the laboratory diagnostic tests used for thrombotic microangiopathies.

Cytomorphology of normal, reactive, dysmorphic, and dysplastic megakaryocytes in bone marrow aspirates.

This paper aims to emphasize the importance of applying international consensus guidelines to detect qualitative and quantitative abnormalities of megakaryocytes on smears of bone marrow aspirates (BMA) for a shared and harmonized diagnostic path between different laboratories. Careful evaluation of megakaryocytes on BMA smears represents a cornerstone in the diagnosis of most clonal and nonclonal hematological diseases. Images associated with the detailed morphologic description of normal, reactive, abnormal, and dysplastic megakaryocytes are also reported together with examples of similar cells that, if not promptly identified, can lead to a morphological misdiagnosis.

Inline and offline extracorporeal photopheresis: Device performance, cell yields and clinical response.

BACKGROUND: Extracorporeal photopheresis (ECP) is an effective treatment for graft-vs-host-disease (GvHD). Photopheresis can be performed in offline or inline method. The first uses a conventional cell separator for collection of mononuclear-cells that are photoactivated by a separate device and manually reinfused; the second one involves a dedicated device performing the entire procedure (collection, photoactivation and reinfusion). STUDY DESIGN AND METHODS: The objective was to compare the two methods and cell product features to highlight key process, devices performance, and to evaluate ECP clinical response. Patients developing steroid-resistant GvHD underwent ECP as second-line treatment using either inline (Therakos CellEx) or offline system (Terumo BCT Spectra or Optia and UVA PIT system). Data about patients' features, pre-apheresis blood-count, cell product characteristics and clinical response were collected for analysis. RESULTS: We evaluated 494 procedures performed on 28 patients from April 2018 to March 2019. The offline procedure allows to achieve greater cell yield, it is characterized by larger processed blood volume, longer runtime, and higher ACD consumption. The inline procedure shows shorter runtime, high mononuclear-cells percentage and low percentage of granulocytes in cell product. We observed a significant difference in cell yields between inline and offline system; furthermore we did not find a significant relationship between cell dose and clinical response. CONCLUSION: Inline ECP is fast, highly automated and productive, making it particularly suitable for ECP treatments. Offline ECP collects high cell yields implying longer procedure and greater operator intervention. Our study did not find a significant relationship between cell dose and GVHD response.

How I investigate difficult cells at the optical microscope.

Blood cell morphological identification on the peripheral blood and bone marrow films remains a cornerstone for the diagnosis of hematological neoplasms to be integrated with immunophenotyping, molecular genetics, and histopathology. Although standardization is still far from being achieved, with high interobserver variability, in recent years, several classification approaches, from the 1976 FAB to the 2016 WHO classification, have provided hematologists with detailed morphological descriptions for a large number of diseases. Counting blasts and detecting dysplastic specimens are two cornerstones of morphological diagnosis. This review deals with identifying difficult cells, with particular reference of those with relevant diagnostic implications.