European flow cytometry quality assurance guidelines for the diagnosis of primary immune deficiencies and assessment of immune reconstitution following B cell depletion therapies and transplantation.

Over the last 15 years activity of diagnostic flow cytometry services have evolved from monitoring of CD4 T cell subsets in HIV-1 infection to screening for primary and secondary immune deficiencies syndromes and assessment of immune constitution following B cell depleting therapy and transplantation. Changes in laboratory activity in high income countries have been driven by initiation of anti-retroviral therapy (ART) in HIV-1 regardless of CD4 T cell counts, increasing recognition of primary immune deficiency syndromes and the wider application of B cell depleting therapy and transplantation in clinical practice. Laboratories should use their experience in standardization and quality assurance of CD4 T cell counting in HIV-1 infection to provide immune monitoring services to patients with primary and secondary immune deficiencies. Assessment of immune reconstitution post B cell depleting agents and transplantation can also draw on the expertise acquired by flow cytometry laboratories for detection of CD34 stem cell and assessment of MRD in hematological malignancies. This guideline provides recommendations for clinical laboratories on providing flow cytometry services in screening for immune deficiencies and its emerging role immune reconstitution after B cell targeting therapies and transplantation.

Kinetics of CD169, HLA-DR, and CD64 expression as predictive biomarkers of SARS-CoV2 outcome.

INTRODUCTION: Discriminating between virus-induced fever from superimposed bacterial infections is a common challenge in intensive care units. Superimposed bacterial infections can be detected in severe SARS-CoV2-infected patients, suggesting the important role of the bacteria in COVID-19 evolution. However, indicators of patients' immune status may be of help in the management of critically ill subjects. Monocyte CD169 is a type I interferon-inducible receptor that is up-regulated during viral infections, including COVID-19. Monocyte HLA-DR expression is an immunologic status marker, that decreases during immune exhaustion. This condition is an unfavorable prognostic biomarker in septic patients. Neutrophil CD64 upregulation is an established indicator of sepsis. METHODS: In this study, we evaluated by flow cytometry the expression of cellular markers monocyte CD169, neutrophil CD64, and monocyte HLA-DR in 36 hospitalized patients with severe COVID-19, as possible indicators of ongoing progression of disease and of patients' immune status. Blood testings started at ICU admission and were carried on throughout the ICU stay and extended in case of transfer to other units, when applicable. The marker expression in mean fluorescence intensity (MFI) and their kinetics with time were correlated to the clinical outcome. RESULTS: Patients with short hospital stay (≤15 days) and good outcome showed higher values of monocyte HLA-DR (median 17,478 MFI) than long hospital stay patients (>15 days, median 9590 MFI, p= 0.04) and than patients who died (median 5437 MFI, p= 0.05). In most cases, the recovery of the SARS-CoV2 infection-related signs was associated with the downregulation of monocyte CD169 within 17 days from disease onset. However in three surviving long hospital stay patients, a persistent upregulation of monocyte CD169 was observed. An increased neutrophil CD64 expression was found in two cases with a superimposed bacterial sepsis. CONCLUSION: Monocyte CD169, neutrophil CD64, and monocyte HLA-DR expression can be used as predictive biomarkers of SARS-CoV2 outcome in acutely infected patients. The combined analysis of these indicators can offer a real-time evaluation of patients' immune status and of viral disease progression versus superimposed bacterial infections. This approach allows to better define the patients' clinical status and outcome and may be useful to guide clinicians' decisions. Our study focused on the discrimination between the activity of viral and bacterial infections and on the detection of the development of anergic states that may correlate with an unfavorable prognosis.

Assessing SARS-CoV-2-specific T-cell reactivity in late convalescents and vaccinees: Comparison and combination of QuantiFERON and activation-induced marker assays, and relation with antibody status.

OBJECTIVES: Monitoring of SARS-CoV-2 spread and vaccination strategies have relied on antibody (Ab) status as a correlate of protection. We used QuantiFERON™ (QFN) and Activation-Induced Marker (AIM) assays to measure memory T-cell reactivity in unvaccinated individuals with prior documented symptomatic infection (late convalescents) and fully vaccinated asymptomatic donors (vaccinees). METHODS: Twenty-two convalescents and 13 vaccinees were enrolled. Serum anti-SARS-CoV-2 S1 and N Abs were measured using chemiluminescent immunoassays. QFN was performed following instructions and interferon-gamma (IFN-γ) measured by ELISA. AIM was performed on aliquots of antigen-stimulated samples from QFN tubes. SARS-CoV-2-specific memory CD4+CD25+CD134+, CD4+CD69+CD137+ and CD8+CD69+CD137+ T-cell frequencies were measured by flow cytometry. RESULTS: In convalescents, substantial agreement was observed between QFN and AIM assays. IFN-γ concentrations and AIM+ (CD69+CD137+) CD4+ T-cell frequencies correlated with each other, with Ab levels and AIM+ CD8+ T-cell frequencies, whereas AIM+ (CD25+CD134+) CD4+ T-cell frequencies correlated with age. AIM+ CD4+ T-cell frequencies increased with time since infection, whereas AIM+ CD8+ T-cell expansion was greater after recent reinfection. QFN-reactivity and anti-S1 titers were lower, whereas anti-N titers were higher, and no statistical difference in AIM-reactivity and Ab positivity emerged compared to vaccinees. CONCLUSIONS: Albeit on a limited sample size, we confirm that coordinated, cellular and humoral responses are detectable in convalescents up to 2 years after prior infection. Combining QFN with AIM may enhance detection of naturally acquired memory responses and help stratify virus-exposed individuals in T helper 1-type (TH1)-reactive (QFNpos AIMpos Abshigh), non-TH1-reactive (QFNneg AIMpos Abshigh/low), and pauci-reactive (QFNneg AIMneg Abslow).

COVID-19-Associated Pneumonia in a B-Cell-Depleted Patient With Non-Hodgkin Lymphoma: Recovery With Hyperimmune Plasma.

Coronavirus disease 2019 (COVID-19) can have a severe course in immunocompromised hosts and patients with hematological malignancies. In some cases, the bad prognosis is associated with the lack of B lymphocytes, with impaired antibody production and inefficient viral clearance. We report a case of a 67-year-old woman with a story of non-Hodgkin lymphoma treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone), who got a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection while being totally depleted of B cells. This condition has determined a severe and prolonged course of COVID-19, with persistently positive nasopharyngeal molecular swabs and lack of anti-SARS-CoV-2 specific antibodies. The clinical recovery was favored by the administration of convalescent hyperimmune plasma.

Rituximab Therapy for Adults with Nephrotic Syndromes: Standard Schedules or B Cell-Targeted Therapy?

Rituximab is a chimeric anti-CD20 monoclonal antibody. It acts mainly through complement-dependent cytotoxicity on B cells expressing the CD20 marker. In this review, we analyse the efficacy and possible pitfalls of rituximab to treat nephrotic syndromes by taking into account pharmacological considerations and CD19 marker testing utility. Despite the fact that the drug has been in use for years, efficacy and treatment schedules in adults with nephrotic syndrome are still a matter of debate. Clinical trials have proven the efficacy and safety of rituximab in idiopathic membranous nephropathy. Data from observational studies also showed the efficacy of rituximab in minimal change disease and focal segmental glomerulosclerosis. Rituximab use is now widely recommended by new Kidney Disease Improved Outcome (KDIGO) guidelines in membranous nephropathy and in frequent-relapsing, steroid-dependent minimal change disease or focal segmental glomerulosclerosis. However, rituximab response has a large interindividual variability. One reason could be that rituximab is lost in the urine at a higher extent in patients with nonselective nephrotic proteinuria, exposing patients to different rituximab plasma levels. Moreover, the association between CD19+ levels and clinical response or relapses is not always present, making the use of this marker in clinical practice complex. High resolution flow cytometry has increased the capability of detecting residual CD19+ B cells. Moreover, it can identify specific B-cell subsets (including IgG-switched memory B cells), which can repopulate at different rates. Its wider use could become a useful tool for better understanding reasons of rituximab failure or avoiding unnecessary retreatments.

Attitude changes in prescribing intravenous iron supplementation in different settings at a hospital consortium in Italy.

BACKGROUND: Iron deficiency anaemia is a public health problem. In case oral iron treatment is ineffective, poorly tolerated or contraindicated, the intravenous route becomes the first choice. The aim of the study was to evaluate the shift between ferrous gluconate (FG) and ferric carboxymaltose (FCM) usage at our hospitals over the years. We also performed a cost comparison between pre and post-FCM availability periods, taking into account the acquisition costs of both intravenous iron and red blood cell units (PRBC). STUDY DESIGN AND METHODS: The amount and costs of FG and FCM released by hospital Pharmacy Services from 2010 to 2019 were analysed, along with the number of transfused PRBC units in the same timeframe. RESULTS: Overall, the proportion of FCM usage rose from 8.6 % in 2014 to 71.9 % in 2019, as percentage of total intravenous iron released. After exclusion of haemodialysis, where FG is still widely used, the FCM use in the last four years raised from 12.9% to 92.5%. Despite the higher FCM cost, the mean yearly expenditure for intravenous iron plus PRBC units did not differ between pre- and post-FCM eras (2010-2013, € 2,396,876 € versus 2014-2019, € 2,307,875 - p = 0.234), as a result of a net decrease of PRBC usage, namely from 15,083 to 12,654 (-16.1 %), respectively. DISCUSSION: Intravenous iron has a major role in treating iron deficiency anaemia in several settings. Third generation compounds are paving the way to more updated and safer treatments.

Heparin dosage, level, and resistance in SARS-CoV2 infected patients in intensive care unit.

INTRODUCTION: Patients with COVID-19 frequently exhibit a hypercoagulable state with high thrombotic risk, particularly those admitted to intensive care units (ICU). Thromboprophylaxis is mandatory in these patients; nevertheless, thrombosis still occurs in many cases. Thus, the problem of assessing an adequate level of anticoagulation in ICU patients becomes evident during the COVID-19 pandemic. The aim of this study was to evaluate the heparin resistance and the efficacy of heparin monitoring using an anti-Xa activity assay. METHODS: Thirty-seven heparin-treated patients admitted to ICU for SARS-CoV-2 pneumonia were retrospectively studied for antifactor Xa activity (anti-Xa), activated partial thromboplastin time (APTT), Antithrombin, Fibrinogen, D-Dimer, Factor VIII, von Willebrand Factor, and the total daily amount of heparin administered. The correlation between APTT and anti-Xa was evaluated for unfractionated heparins (UFH). The correlations between the daily dose of UFH or the dosage expressed as IU/kg b.w. for low molecular weight heparin (LMWH) and anti-Xa were also evaluated. RESULTS: Twenty-one patients received calcium heparin, 8 sodium heparin, and 8 LMWH. A moderate correlation was found between APTT and anti-Xa for UFH. APTT did not correlate with coagulation parameters. 62% of UFH and 75% of LMWH treated patients were under the therapeutic range. About 75% of patients could be considered resistant to heparin. CONCLUSIONS: SARS-COV2 pneumonia patients in ICU have frequently heparin resistance. Anti-Xa seems a more reliable method to monitor heparin treatment than APTT in acute patients, also because the assay is insensitive to the increased levels of fibrinogen, FVIII, and LAC that are common during the COVID-19 inflammatory state.

Iatrogenic anaemia and transfusion thresholds in ICU patients with COVID-19 disease at a tertiary care hospital.

BACKGROUND: Patients with severe COVID-19 disease frequently develop anaemia as the result of multiple mechanisms and often receive transfusions. The aims of this study were to assess the impact of repeated blood samplings on patients' anaemic state using standard-volume tubes, in comparison with the hypothetical use of low-volume tubes and to evaluate the transfusion policy adopted. STUDY DESIGN AND METHODS: Transfusion data of mechanically ventilated non-bleeding patients with COVID-19 disease hospitalized in ICU for a minimum of 20 days were recorded. The total volume of blood drawn for samplings with standard-volume tubes and the corresponding red blood cell mass (RBCM) removed during hospitalization for each patient were calculated and compared with the hypothetical use of low-volume tubes. RESULTS: Twenty-four patients fulfilled the inclusion criteria. Ten patients were anaemic at ICU admission (41.7 %). Overall, 6658 sampling tubes were employed, for a total of 16,786 mL of blood. The median RBCM subtracted by blood samplings per patient accounted for about one third of the total patients' RBCM decrease until discharge. The use of low-volume tubes would have led to a median saving of about one third of the drawn RBCM. Eleven patients were transfused (45.8 %) at a mean Hb value of 7.7 (± 0.5) g/dL. CONCLUSION: The amount of blood drawn for sampling has a significant role in the development of anaemia and the use of low-volume tubes could minimize the problem. Large high-powered studies are warranted to assess the more appropriate transfusion thresholds in non-bleeding critically ill patients with COVID-19 disease.

ESCCA/ISCCA protocol for the analysis of cerebrospinal fluid by multiparametric flow-cytometry in hematological malignancies.

Central nervous system (CNS) involvement is a serious but often underdiagnosed complication of hematological malignancies. Currently, the gold standard to detect CNS involvement is conventional cytology (CC) whose sensitivity though is lower than 50%. Multiparametric flow cytometry (MFC) demonstrated a superior sensitivity over CC, particularly when low levels of CNS infiltrating cells are present. Although prospective studies are few, a positive finding by MFC appears to anticipate an adverse outcome even if CC shows no infiltration. However, the use of MFC to diagnose CNS involvement presents some pitfalls, due to the typical hypocellularity of cerebrospinal fluids (CSF), and low cell vitality. Furthermore, the threshold to be used for defining the MFC positivity is not universally defined. In this paper, a working group of the European Society for Clinical Cell Analysis-ESCCA-and the Italian Society for Clinical Cell Analysis-ISCCA-will discuss the critical aspects of CSF processing, highlighting difficulties in storage and processing of samples, interpretation and reporting of data.

The ISCCA flow protocol for the monitoring of anti-CD20 therapies in autoimmune disorders.

BACKGROUND: Anti-CD20 monoclonals (MoAbs) are used in a variety of autoimmune disorders. The aim is to eliminate memory B cells sustaining the tissue damage and the production of pathogenic autoantibodies, while preserving naïve cells. The disappearance of memory B cells and the repopulation by naïve cells correlate with good clinical response, while the reappearance of memory B cells and plasmablasts correlates with relapse or resistance to therapy. Anti-CD20 induce extremely low B cell levels, requiring high-resolution techniques. The immune monitoring protocol developed by ISCCA is described and validated, to provide a standardized method for the clinical decision-making process during anti-CD20 therapies in autoimmune diseases. METHODS: A 10-marker, 8-color staining panel (CD20-V450, CD45-V500c, CD4-FITC + sIgM-FITC, CD38-PE, CD3-PerCP Cy5.5, CD19-PE-Cy7, CD27-APC, CD8-APC H7 + sIgG-APC-H7) is used to identify B cells, plasma cells/blasts, naïve and memory B cells, sIgM+ and sIgG-switched memory B cells, T and NK cells, with high-sensitivity analysis (>106 CD45+ cells). RESULTS: After an anti-CD20 dose, the B cell level is about zero in most patients. If B cells remain virtually absent (<0.1/µl), subsetting is not reliable nor meaningful. If B cells raise >0.3-0.5/µl, subsetting is possible and informative, acquiring >1.0-1.5 × 106 CD45+ events. Further testings can follow the quality of B cell repopulation. If B cells become detectable (>1/µl), the prevalence of memory B cells indicates non-responsiveness or a possible relapse. CONCLUSIONS: The ISCCA Protocol is proposed for a standardized prospective monitoring of patients with autoimmune disorders, to assist the safe and rational usage of anti-CD20 therapies.

Impact of gender parity on preoperative anaemia prevalence and Patient Blood Management practice.

BACKGROUND: Anaemia is a common finding in the preoperative setting, affecting around one-third of patients for whom major surgery is programmed. Moreover, preoperative anaemia has been shown to worsen patient outcome and increase length of hospital stay and costs. In the field of preoperative anaemia correction, a recent Consensus statement suggested reviewing the classic World Health Organization (WHO) criteria in adults by aligning the haemoglobin cut-off to 13 g/dL for both genders. The aim of our study was to assess the differences in terms of prevalence, transfusion rate, transfusion trigger, and blood losses according to gender in a mixed population of surgical patients. MATERIAL AND METHODS: We reviewed data of 610 consecutive patients undergoing elective major surgery at a tertiary care hospital during a 9-month period. Transfusion rate and transfusion triggers were recorded, analysed and stratified by haemoglobin class, with a particular focus on the 12.0-12.9 g/dL range. RESULTS: Since the anaemia threshold was redefined at 13 g/dL for both genders, its prevalence rose from 26.4 to 39.5% (161/610 vs 241/610; p<0.001) in the overall population and from 22.7 to 49.3% (68/300 vs 148/300; p<0.001) in women. Eighty women (26.7%) fell in the haemoglobin 12.0-12.9 g/dL range, and this category was the most represented among transfused women (34.0%). There was no statistical difference in transfusion triggers or overall transfusion rate between genders. Subjects of both genders were transfused at the same haemoglobin level (8.1 g/dL), but women reached the transfusion trigger after less red cell mass loss than men, i.e. 377 mL (249-472 mL) vs 528 mL (356-717 mL), respectively (p<0.001). DISCUSSION: Treatment of pre-surgical anaemia is one of the core principles of Patient Blood Management. Aligning the haemoglobin threshold between genders in the management of pre-surgical anaemia may result in a lower transfusion rate, but in an increased workload for medical staff in the preoperative phase.

Inhibitory anti ADAMTS13 antibodies with a new rapid fully automated CLiA assay.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder characterized by severe ADAMTS13 deficiency. The acquired form is associated with autoantibodies directed against ADAMTS13. Both noninhibitory and inhibitory autoantibodies can be detected by ELISA assay, while only inhibitory autoantibodies are detected by Bethesda assay. Due to its short TAT and good performance, chemiluminescence (CliA) ADAMTS13 activity (HemosIL Acustar) has proven to be a good choice in the diagnosis of TTP in emergency settings. Aim of this study was to analyse the performance of the CliA ADAMTS13 activity assay in detecting inhibitory ADAMTS13 antibodies using the Bethesda assay. METHODS: A method comparison study was performed on 69 stored samples: 11 acute TTPs, 38 TTP follow-ups, 5 TTP relapses, 1 congenital TTP, 10 HUS, 4 suspected TTPs. We retrieved the results of tests previously run in ELISA for both activity and autoantibodies. At the same time, we reran new tests including ELISA and CliA activity, ELISA autoantibodies, and ELISA and CliA Bethesda assays on thawed frozen samples. RESULTS: Very good correlation was observed between ELISA and CliA activity assay results (r = 0.96) and between archived ELISA and CliA activity results (r = 0.93). Agreement between the anti-ADAMTS13 assays ranged from good (k = 0.63) to very good (k = 0.92). CONCLUSIONS: CliA and ELISA Bethesda assays showed very good agreement with samples run at the same time using ELISA ADAMTS13-autoantibody assay. Albeit more expensive, the CliA Bethesda assay identified inhibitory anti-ADAMTS13 within almost the same TAT as ELISA, but with better automation and limited operator involvement.

Decrease of Non-Classical and Intermediate Monocyte Subsets in Severe Acute SARS-CoV-2 Infection.

In patients with severe SARS-CoV-2 infection, the development of cytokine storm induces extensive lung damage, and monocytes play a role in this pathological process. Non-classical (NC) and intermediate (INT) monocytes are known to be involved during viral and bacterial infections. In this study, 30 patients with different manifestations of acute SARS-CoV-2 infection were investigated with a flow cytometric study of NC, INT, and classical (CL) monocytes. Significantly reduced NC and INT monocytes and a downregulated HLA-DR were found in acute patients with severe SARS-CoV-2 symptoms. Conversely in patients with moderate symptoms NC and INT monocytes and CD11b expression were increased. © 2020 International Society for Advancement of Cytometry.

Efficacy and safety of high-dose intravenous iron as the first-choice therapy in outpatients with severe iron deficiency anemia.

BACKGROUND: Asymptomatic severe iron deficiency anemia is a common finding in subjects admitted to the outpatient anemia clinic. Although the condition can be easily be reversed with intravenous iron (IVI) therapy and several guidelines have suggested a restrictive threshold for using transfusion in hemodynamically stable patients, transfusion is often the rule in clinical practice. This study describes clinical practice results of IVI therapy without transfusion. STUDY DESIGN AND METHODS: In this multicenter retrospective observational study, data of severely anemic outpatients treated only with high-dose IVI with ferric carboxymaltose were collected. Inclusion criteria were hemoglobin (Hb) level of less than 7.0 g/dL and ferritin level of less than 30 ng/mL or mean corpuscular volume of less than 75 fL. RESULTS: Overall, 303 patients referred to the anemia clinic mainly from primary health care centers (46.2%) or the emergency department (28.7%) met the inclusion criteria. Median (interquartile range [IQR]) age was 47 (37-62) years and 84.5% were female. The median (IQR) Hb concentration at first visit was 6.5 (6.1-6.8) g/dL, 64 patients (21.1%) presented with a Hb level of less than 6.0 g/dL at diagnosis, and 11 of them (3.6%) had extreme anemia (Hb ≤ 5 g/dL). Gynecologic and gastroenteric bleeding were the main cause. After a mean IV administration of 1500 mg of iron, the Hb increased by a median of 5.7 g/dL. Thirteen patients experienced only mild side effects. CONCLUSIONS: In chronic very severe sideropenic anemias, third-generation IVI is effective and safe for quick correction and avoidance of red blood cell transfusion. These results suggest that more specific guidelines for this clinical setting are warranted.

Early intravenous iron administration in the Emergency Department reduces red blood cell unit transfusion, hospitalisation, re-transfusion, length of stay and costs.

BACKGROUND: Moderate to severe iron deficiency anaemia is a common finding in patients admitted to the Emergency Department (ED). According to Patient Blood Management principles, intravenous iron should be the therapy of choice instead of blood transfusion for selected cases affected by chronic iron deficiency anaemia. However, this option is only rarely taken into account by physicians in the ED. As a result, in many circumstances, treatment of iron deficiency anaemia in the ED can differ from that of the Anaemia Clinic. With the aim of reducing inappropriate transfusions, and to implement intravenous iron usage, we shared a specific protocol with the ED. MATERIAL AND METHODS: We reviewed the medical records of all subjects admitted to the ED (n=267, Post-protocol group) with hemoglobin ≤9.0 g/dL and mean corpuscular volume <80 fL in a 13-month period, except if the massive transfusion protocol was activated, and results were compared with an equivalent Pre-protocol historical cohort (n=226). RESULTS: In comparison with the Pre-protocol series, the number of patients transfused did not change, but the appropriateness in terms of transfusion and red blood cell volume transfused improved sharply (87.0 vs 13.3%; p<0.001) with a significant increase in intravenous iron administration (50.2 vs 4.4% of cases; p<0.001). As a positive consequence, both the time spent in the ED by patients who were then directly discharged and costs per subject treated dropped by 37.9% and 59.0%, respectively. Treatment with infusion only in comparison with transfusion only led to a statistically significant Relative Risk reduction in transfusion on the ward and post-discharge transfusion of 55.6% and 44.4%, respectively. DISCUSSION: The implementation of Patient Blood Management principles and early intravenous iron therapy in the Emergency Department have proved to be effective tools to optimise resources both in terms of units transfused and costs.