FCM marker importance for MRD assessment in T-cell acute lymphoblastic leukemia: An AIEOP-BFM-ALL-FLOW study group report.

T-lineage acute lymphoblastic leukemia (T-ALL) accounts for about 15% of pediatric and about 25% of adult ALL cases. Minimal/measurable residual disease (MRD) assessed by flow cytometry (FCM) is an important prognostic indicator for risk stratification. In order to assess the MRD a limited number of antibodies directed against the most discriminative antigens must be selected. We propose a pipeline for evaluating the influence of different markers for cell population classification in FCM data. We use linear support vector machine, fitted to each sample individually to avoid issues with patient and laboratory variations. The best separating hyperplane direction as well as the influence of omitting specific markers is considered. Ninety-one bone marrow samples of 43 pediatric T-ALL patients from five reference laboratories were analyzed by FCM regarding marker importance for blast cell identification using combinations of eight different markers. For all laboratories, CD48 and CD99 were among the top three markers with strongest contribution to the optimal hyperplane, measured by median separating hyperplane coefficient size for all samples per center and time point (diagnosis, Day 15, Day 33). Based on the available limited set tested (CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99), our findings prove that CD48 and CD99 are useful markers for MRD monitoring in T-ALL. The proposed pipeline can be applied for evaluation of other marker combinations in the future.

Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

Hydrophilic Interaction Liquid Chromatography-Hydrogen/Deuterium Exchange-Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics.

Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

Optimization of Mobile Phase Modifiers for Fast LC-MS-Based Untargeted Metabolomics and Lipidomics.

Liquid chromatography-mass spectrometry (LC-MS) is the method of choice for the untargeted profiling of biological samples. A multiplatform LC-MS-based approach is needed to screen polar metabolites and lipids comprehensively. Different mobile phase modifiers were tested to improve the electrospray ionization process during metabolomic and lipidomic profiling. For polar metabolites, hydrophilic interaction LC using a mobile phase with 10 mM ammonium formate/0.125% formic acid provided the best performance for amino acids, biogenic amines, sugars, nucleotides, acylcarnitines, and sugar phosphate, while reversed-phase LC (RPLC) with 0.1% formic acid outperformed for organic acids. For lipids, RPLC using a mobile phase with 10 mM ammonium formate or 10 mM ammonium formate with 0.1% formic acid permitted the high signal intensity of various lipid classes ionized in ESI(+) and robust retention times. For ESI(-), the mobile phase with 10 mM ammonium acetate with 0.1% acetic acid represented a reasonable compromise regarding the signal intensity of the detected lipids and the stability of retention times compared to 10 mM ammonium acetate alone or 0.02% acetic acid. Collectively, we show that untargeted methods should be evaluated not only on the total number of features but also based on common metabolites detected by a specific platform along with the long-term stability of retention times.

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies - a EuroFlow study.

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

TLR8/TLR7 dysregulation due to a novel TLR8 mutation causes severe autoimmune hemolytic anemia and autoinflammation in identical twins.

Our study presents a novel germline c.1715G>T (p.G572V) mutation in the gene encoding Toll-like receptor 8 (TLR8) causing an autoimmune and autoinflammatory disorder in a family with monozygotic male twins, who suffer from severe autoimmune hemolytic anemia worsening with infections, and autoinflammation presenting as fevers, enteritis, arthritis, and CNS vasculitis. The pathogenicity of the mutation was confirmed by in vitro assays on transfected cell lines and primary cells. The p.G572V mutation causes impaired stability of the TLR8 protein, cross-reactivity to TLR7 ligands and reduced ability of TLR8 to attenuate TLR7 signaling. This imbalance toward TLR7-dependent signaling leads to increased pro-inflammatory responses, such as nuclear factor-κB (NF-κB) activation and production of pro-inflammatory cytokines IL-1ß, IL-6, and TNFα. This unique TLR8 mutation with partial TLR8 protein loss and hyperinflammatory phenotype mediated by TLR7 ligands represents a novel inborn error of immunity with childhood-onset and a good response to TLR7 inhibition.

CD38: A target in relapsed/refractory acute lymphoblastic leukemia-Limitations in treatment and diagnostics.

Daratumumab, an anti-CD38 antibody, is used experimentally in the treatment of relapsed acute lymphoblastic leukemia (ALL). We treated five patients suffering from relapsed ALL with daratumumab. Four patients had T ALL, three of whom achieved complete remission (CR) after treatment and underwent stem cell transplant (SCT). Two of them had a second relapse and died 6 and 8 months after SCT, respectively. One transplanted T ALL patient remained in CR2 15 months after relapse. In the remaining T-ALL patient, the disease progressed under daratumumab treatment, and the patient died early after the first relapse. The B-cell precursor ALL patient with a second CD19-negative relapse, whose disease turned out to be resistant to the combination of daratumumab with chemotherapy, later achieved CR3 with inotuzumab ozogamicin, underwent SCT and remained in CR3. Leukemia burden should be monitored after daratumumab, and care should be taken not to misclassify leukemic cells with false negativity of surface CD38; using an antibody reacting with nondaratumumab epitopes is advantageous.

DUX4r, ZNF384r and PAX5-P80R mutated B-cell precursor acute lymphoblastic leukemia frequently undergo monocytic switch.

Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.

Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia.

A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10-5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10-5), if sufficient cells (>4 × 106, preferably more) are evaluated.

Next-generation amplicon TRB locus sequencing can overcome limitations of flow-cytometric Vß expression analysis and confirms clonality in all T-cell prolymphocytic leukemia cases.

T-cell receptor (TCR) ß repertoire analysis can distinguish monoclonal from polyclonal T-cell proliferations and crucially aid in the diagnosis of T-cell malignancies. TCR repertoire can be assessed either by flow cytometry (FCM), or by molecular genetic techniques. We compared the results of parallel analyses of Vß expression by FCM and TRB rearrangements by DNA-based next-generation sequencing (NGS) in 80 diagnostic peripheral blood samples of patients with T-cell prolymphocytic leukemia (T-PLL) for (1) the diagnosis of clonality and (2) the assessment of dominant Vß usage. FCM-based analysis of the surface expression was performed using the IOTest Beta Mark kit. The NGS-based analysis employed the multiplex Biomed-2 VB-JB primers. In all the samples, one or two clonal TRB rearrangements were detected by NGS. Although a dominant Vß domain usage was detected by FCM in only 41/80 (51%) samples, clonality was suspected in all of them. In a total of 12 cases, the FCM missed the clone detected by NGS, despite theoretical coverage by the antibodies, the functionality of the rearrangement, and the expression of TCRαß on the cell surface. Partly overlapping with those cases, FCM discovered predominant Vß usage in the T-PLL population that differed from the one detected by NGS in 10 cases. Overall, the concordant NGS and FCM results were obtained on 61/80 (76%) of samples. We conclude that NGS-based TRB analysis can overcome certain limitations of FCM-based analysis by the identification of both productive and nonproductive rearrangements and by covering the whole Vß spectrum. Currently available FCM analysis of Vß expression lacks this breadth but has advantages, such as parallel immunophenotyping and a more accurate quantification of the Vß usage. © 2018 International Society for Advancement of Cytometry.

Detailed immunophenotyping of B-cell precursors in regenerating bone marrow of acute lymphoblastic leukaemia patients: implications for minimal residual disease detection.

Flow cytometric detection of minimal residual disease (MRD) in children with B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) requires immunophenotypic discrimination between residual leukaemic cells and B-cell precursors (BCPs) which regenerate during therapy intervals. In this study, EuroFlow-based 8-colour flow cytometry and innovative analysis tools were used to first characterize the immunophenotypic maturation of normal BCPs in bone marrow (BM) from healthy children, resulting in a continuous multiparametric pathway including transition stages. This pathway was subsequently used as a reference to characterize the immunophenotypic maturation of regenerating BCPs in BM from children treated for BCP-ALL. We identified pre-B-I cells that expressed low or dim CD34 levels, in contrast to the classical CD34high pre-B-I cell immunophenotype. These CD34-dim pre-B-I cells were relatively abundant in regenerating BM (11-85% within pre-B-I subset), while hardly present in healthy control BM (9-13% within pre-B-I subset; P = 0·0037). Furthermore, we showed that some of the BCP-ALL diagnosis immunophenotypes (23%) overlapped with CD34-dim pre-B-I cells. Our results indicate that newly identified CD34-dim pre-B-I cells can be mistaken for residual BCP-ALL cells, potentially resulting in false-positive MRD outcomes. Therefore, regenerating BM, in which CD34-dim pre-B-I cells are relatively abundant, should be used as reference frame in flow cytometric MRD measurements.

Loss of B cells and their precursors is the most constant feature of GATA-2 deficiency in childhood myelodysplastic syndrome.

GATA-2 deficiency was recently described as common cause of overlapping syndromes of immunodeficiency, lymphedema, familiar myelodysplastic syndrome or acute myeloid leukemia. The aim of our study was to analyze bone marrow and peripheral blood samples of children with myelodysplastic syndrome or aplastic anemia to define prevalence of the GATA2 mutation and to assess whether mutations in GATA-2 transcription factor exhibit specific immunophenotypic features. The prevalence of a GATA2 mutation in a consecutively diagnosed cohort of children was 14% in advanced forms of myelodysplastic syndrome (refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and myelodysplasia-related acute myeloid leukemia), 17% in refractory cytopenia of childhood, and 0% in aplastic anemia. In GATA-2-deficient cases, we found the most profound B-cell lymphopenia, including its progenitors in blood and bone marrow, which correlated with significantly diminished intronRSS-Kde recombination excision circles in comparison to other myelodysplastic syndrome/aplastic anemia cases. The other typical features of GATA-2 deficiency (monocytopenia and natural killer cell lymphopenia) were less discriminative. In conclusion, we suggest screening for GATA2 mutations in pediatric myelodysplastic syndrome, preferentially in patients with impaired B-cell homeostasis in bone marrow and peripheral blood (low number of progenitors, intronRSS-Kde recombination excision circles and naïve cells).

Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry.

Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood.

Refractory cytopenia of childhood is the most common type of childhood myelodysplastic syndrome. Because the majority of children with refractory cytopenia have a normal karyotype and a hypocellular bone marrow, differentiating refractory cytopenia from the immune-mediated bone marrow failure syndrome (very) severe aplastic anemia can be challenging. Flow cytometric immunophenotyping of bone marrow has been shown to be a valuable diagnostic tool in differentiating myelodysplastic syndrome from non-clonal cytopenias in adults. Here, we performed the first comprehensive flow cytometric analysis of immature myeloid, lymphoid cells and erythroid cells, and granulocytes, monocytes, and lymphoid cells in bone marrow obtained from a large prospective cohort of 81 children with refractory cytopenia. Children with refractory cyotopenia had a strongly reduced myeloid compartment, but not as severe as children with aplastic anemia. Furthermore, the number of flow cytometric abnormalities was significantly higher in children with refractory cytopenia than in healthy controls and in children with aplastic anemia, but lower than in advanced myelodysplastic syndrome. We conclude that flow cytometric immunophenotyping could be a relevant addition to histopathology in the diagnosis of refractory cytopenia of childhood. (The multi-center studies EWOG-MDS RC06 and EWOG-MDS 2006 are registered at clinicaltrials.gov identifiers 00499070 and 00662090, respectively).

Short-Term Stability of Serum and Liver Extracts for Untargeted Metabolomics and Lipidomics.

Thermal reactions can significantly alter the metabolomic and lipidomic content of biofluids and tissues during storage. In this study, we investigated the stability of polar metabolites and complex lipids in dry human serum and mouse liver extracts over a three-day period under various temperature conditions. Specifically, we tested temperatures of -80 °C (freezer), -24 °C (freezer), -0.5 °C (polystyrene box with gel-based ice packs), +5 °C (refrigerator), +23 °C (laboratory, room temperature), and +30 °C (thermostat) to simulate the time between sample extraction and analysis, shipping dry extracts to different labs as an alternative to dry ice, and document the impact of higher temperatures on sample integrity. The extracts were analyzed using five fast liquid chromatography-mass spectrometry (LC-MS) methods to screen polar metabolites and complex lipids, and over 600 metabolites were annotated in serum and liver extracts. We found that storing dry extracts at -24 °C and partially at -0.5 °C provided comparable results to -80 °C (reference condition). However, increasing the storage temperatures led to significant changes in oxidized triacylglycerols, phospholipids, and fatty acids within three days. Polar metabolites were mainly affected at storage temperatures of +23 °C and +30 °C.

Exploring the Impact of Organic Solvent Quality and Unusual Adduct Formation during LC-MS-Based Lipidomic Profiling.

Liquid chromatography-mass spectrometry (LC-MS) is the key technique for analyzing complex lipids in biological samples. Various LC-MS modes are used for lipid separation, including different stationary phases, mobile-phase solvents, and modifiers. Quality control in lipidomics analysis is crucial to ensuring the generated data's reliability, reproducibility, and accuracy. While several quality control measures are commonly discussed, the impact of organic solvent quality during LC-MS analysis is often overlooked. Additionally, the annotation of complex lipids remains prone to biases, leading to potential misidentifications and incomplete characterization of lipid species. In this study, we investigate how LC-MS-grade isopropanol from different vendors may influence the quality of the mobile phase used in LC-MS-based untargeted lipidomic profiling of biological samples. Furthermore, we report the occurrence of an unusual, yet highly abundant, ethylamine adduct [M+46.0651]+ that may form for specific lipid subclasses during LC-MS analysis in positive electrospray ionization mode when acetonitrile is part of the mobile phase, potentially leading to lipid misidentification. These findings emphasize the importance of considering solvent quality in LC-MS analysis and highlight challenges in lipid annotation.

Serum TGF-ß1 and CD14 Predicts Response to Anti-TNF-α Therapy in IBD.

Background: Tumor necrosis factor-alpha (TNF-α) agonists revolutionized therapeutic algorithms in inflammatory bowel disease (IBD) management. However, approximately every third IBD patient does not respond to this therapy in the long term, which delays efficient control of the intestinal inflammation. Methods: We analyzed the power of serum biomarkers to predict the failure of anti-TNF-α. We collected serum of 38 IBD patients at therapy prescription and 38 weeks later and analyzed them with relation to therapy response (no-, partial-, and full response). We used enzyme-linked immunosorbent assay to quantify 16 biomarkers related to gut barrier (intestinal fatty acid-binding protein, liver fatty acid-binding protein, trefoil factor 3, and interleukin (IL)-33), microbial translocation, immune system regulation (TNF-α, CD14, lipopolysaccharide-binding protein, mannan-binding lectin, IL-18, transforming growth factor-ß1 (TGF-ß1), osteoprotegerin (OPG), insulin-like growth factor 2 (IGF-2), endocrine-gland-derived vascular endothelial growth factor), and matrix metalloproteinase system (MMP-9, MMP-14, and tissue inhibitors of metalloproteinase-1). Results: We found that future full-responders have different biomarker profiles than non-responders, while partial-responders cannot be distinguished from either group. When future non-responders were compared to responders, their baseline contained significantly more TGF-ß1, less CD14, and increased level of MMP-9, and concentration of these factors could predict non-responders with high accuracy (AUC = 0.938). Interestingly, during the 38 weeks, levels of MMP-9 decreased in all patients, irrespective of the outcome, while OPG, IGF-2, and TGF-ß1 were higher in non-responders compared to full-responders both at the beginning and the end of the treatment. Conclusions: The TGF-ß1 and CD14 can distinguish non-responders from responders. The changes in biomarker dynamics during the therapy suggest that growth factors (such as OPG, IGF-2, and TGF-ß) are not markedly influenced by the treatment and that anti-TNF-α therapy decreases MMP-9 without influencing the treatment outcome.

SARS-CoV-2 Specific Humoral Immune Responses after BNT162b2 Vaccination in Hospital Healthcare Workers.

BACKGROUND: COVID-19 pandemic has led to a loss of human life in millions and devastating socio-economic consequences worldwide. So far, vaccination is the most effective long-term strategy to control and prevent severe COVID-19 disease. The aim of the current study was to evaluate the humoral immune responses raised against the BNT162b2 vaccine in hospital healthcare workers. METHODS: Total number of 173 healthcare workers enrolled in the study. Their blood samples were collected in three different time intervals after the second SARS-CoV-2 vaccination and evaluated by the ELISA method to detect anti-spike protein IgM and IgG antibodies. The baseline characteristics of all participants were collected using questionnaires and were evaluated for finding any significant data. RESULTS: Our results demonstrated that the levels of antibodies were higher in the young group (21-30 years old) and also among male participants. Moreover, the highest levels of antibodies were detected from the group that received the third shot vaccination. CONCLUSIONS: Our results indicate that age, gender and third-dose vaccination can affect the levels of humoral immune responses against the BNT162b2 vaccine in healthcare workers.