COVID-19 vaccinated children, adolescents, and young adults with acute lymphoblastic leukemia show spike reactive antibodies and multifunctional T-cells.

Little is known about the efficacy of COVID-19 vaccines during acute lymphoblastic leukemia therapy (ALL); data for COVID-19 vaccine immune responses in pediatric leukemia remain sparse. We conducted a single center study of patients aged 5-25 years undergoing ALL chemotherapy who received COVID-19 vaccination. Twenty-one patients were enrolled; efficacy was evaluable in 20. Twenty were vaccinated while receiving chemotherapy. Twenty received the BNT162b2 mRNA vaccine. Spike reactive antibodies (S-IgG) and/or T-cells (SRT) were detected in 16 of 20 (80%) vaccinated patients; 13 (65%) and 9 (45%) were positive for S-IgG and SRT, respectively. Six (30%) showed both spike reactive B and T-cell responses. Eleven of the 13 with S-IgG positivity were negative for anti-Nucleocapsid IgG, an antibody profile consistent with a vaccine induced immune response. All 13S-IgG+ patients showed neutralizing antibodies. SRT included CD4+ (7) and CD8+ (6) T-cells; both CD4+ and CD8+ SRT were seen in 4. SRT were multifunctional (producing multiple cytokines) in most patients (8 of 9); 4 showed SRT with triple cytokine and B-cell co-stimulatory responses, indicating a multimodal adaptive immune response. Immune responses were seen among patients vaccinated in the settings of lymphopenia (6 of 12) intensive chemotherapy (3 of 4), and Peg allergy (6 of 8). Sequencing revealed public CD4+ and CD8+ TCR sequences reactive to epitopes across the spike protein. In conclusion, COVID-19 vaccination induced B and/or T-cell responses in a majority of children and young adults undergoing ALL chemotherapy.

Implementation and assessment of an electronic clinical decision support tool to notify clinicians of critical hyperbilirubinemia in preterm neonates less than 35 weeks' gestation.

OBJECTIVES: Critical hyperbilirubinemia in preterm neonates, a condition requiring greater attention, is treated with phototherapy or exchange transfusion when bilirubin results exceed gestational age and age-specific medical decision levels (MDLs) to prevent bilirubin-induced neurologic damage. Conventional evaluation involves multiple manual steps and is poised to inconsistencies and delays. METHODS: We designed and implemented an electronic clinical decision support (CDS) tool to identify and alert neonatal intensive care unit clinicians of critical hyperbilirubinemia with a SmartZone alert. We evaluated the performance of our manual evaluation workflow, the accuracy of the electronic CDS tool, and the outcome of the electronic CDS tool to reduce the time to place orders for interventions. RESULTS: Among the 22 patients who met the criteria to have phototherapy ordered before implementing the electronic CDS tool, 20 (90%) had phototherapy ordered. Fourteen (70%) phototherapy orders were placed less than 24 hours, 4 phototherapy orders were placed 24 to 72 hours, and 2 orders were placed more than 72 hours after bilirubin results exceeded the corresponding MDLs. Among the 15 patients who met the criteria to have phototherapy ordered after implementing the electronic CDS tool, all (100%) received phototherapy orders, with 14 (93%) placed less than 24 hours and 1 order placed less than 48 hours. The electronic CDS tool identified all eligible patients correctly. The proportion of phototherapy ordered less than 24 hours increased from 70% to 93% after the implementation of the electronic CDS tool. CONCLUSIONS: The electronic CDS tool promoted more appropriate and timely intervention orders to manage critical hyperbilirubinemia in preterm neonates.

COVID-19 outcomes in children, adolescents and young adults with cancer.

Pediatric oncology patients are at risk for poor outcomes with respiratory viral infections. Outcome data for COVID-19 in children and young adults with cancer are needed; data are sparse for obese/overweight and adolescent and young adult subgroups. We conducted a single center cohort study of COVID-19 outcomes in patients younger than 25 years with cancer. Candidate hospitalization risk factors were analyzed via univariable and multivariable analyses. Eighty-seven patients with cancer and COVID-19 were identified. Most were Hispanic/Latinx (n = 63, 72%). Forty-two (48%) were overweight/obese. Anticancer therapy included chemotherapy only (n = 64, 74%), chimeric antigen receptor T-cells (CAR-T, n = 7), hematopoietic stem cell transplantation (HSCT, n = 12), or CAR-T and HSCT (n = 4). There was no COVID-19 related mortality. Twenty-six patients (30%) required COVID-19 related hospitalization; 4 required multiple hospitalizations. Nine (10%) had severe/critical infection; 6 needed intensive care. COVID-19 resulted in anticancer therapy delays in 22 (34%) of 64 patients on active therapy (median delay = 14 days). Factors associated with hospitalization included steroids within 2 weeks prior to infection, lymphopenia, previous significant non-COVID infection, and low COVID-19 PCR cycle threshold value. CAR-T recipients with B-cell aplasia tended to have severe/critical infection (3 of 7 patients). A COVID-19 antibody response was detected in 14 of 32 patients (44%). A substantial proportion of COVID-19 infected children and young adults with cancer require inpatient management; morbidity may be high in B-cell immunodeficiency. However, a majority of patients can be taken through chemotherapy without prolonged therapy delays. Viral load is a potential outcome predictor in COVID-19 in pediatric cancer.

Cytoplasmic Tail Truncation of SARS-CoV-2 Spike Protein Enhances Titer of Pseudotyped Vectors but Masks the Effect of the D614G Mutation.

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein-pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV), and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein's function. Here, we optimized concentration methods for SARS-CoV-2 Spike-pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers but had no impact on sensitivity to convalescent serum. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. IMPORTANCE Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant arising early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration if higher-titer vectors are required.

Significance of minimal residual disease in pediatric mixed phenotype acute leukemia: a multicenter cohort study.

The rarity of mixed phenotype acute leukemia (MPAL) has precluded adequate data to incorporate minimal residual disease (MRD) monitoring into therapy. Fluidity in MPAL classification systems further complicates understanding its biology and outcomes; this includes uncertainty surrounding the impact of shifting diagnostic requirements even between iterations of the World Health Organization (WHO) classification. Our primary objective was to address these knowledge gaps. To do so, we analyzed clinicopathologic features, therapy, MRD, and survival in a centrally-reviewed, multicenter cohort of MPAL uniformly diagnosed by the WHO classification and treated with acute lymphoblastic leukemia (ALL) regimens. ALL induction therapy achieved an EOI MRD negative (<0.01%) remission in most patients (70%). EOI MRD positivity was predictive of 5-year EFS (HR = 6.00, p < 0.001) and OS (HR = 9.57, p = 0.003). Patients who cleared MRD by EOC had worse survival compared with those EOI MRD negative. In contrast to adults with MPAL, ALL therapy without transplantation was adequate to treat most pediatric patients. Earlier MRD clearance was associated with better treatment success and survival. Prospective trials are now necessary to validate and refine MRD thresholds within the pediatric MPAL population and to identify salvage strategies for those with poor predicted survival.

Myeloid lineage switch following chimeric antigen receptor T-cell therapy in a patient with TCF3-ZNF384 fusion-positive B-lymphoblastic leukemia.

A pediatric patient diagnosed initially with B-lymphoblastic leukemia (B-ALL) relapsed with lineage switch to acute myeloid leukemia (AML) after chimeric antigen receptor T-cell (CAR-T) therapy and hematopoietic stem cell transplant. A TCF3-ZNF384 fusion was identified at diagnosis, persisted through B-ALL relapse, and was also present in the AML relapse cell population. ZNF384-rearrangements define a molecular subtype of B-ALL characterized by a pro-B-cell immunophenotype; furthermore, ZNF384-rearrangements are prevalent in mixed-phenotype acute leukemias. Lineage switch following CAR-T therapy has been described in patients with KMT2A (mixed lineage leukemia) rearrangements, but not previously in any patient with ZNF384 fusion.

Flow Cytometry Assays in Primary Immunodeficiency Diseases.

Inborn errors of immunity are the cause of the primary immunodeficiency diseases, an extremely diverse group of genetic defects that are inherited in Mendelian fashion and result in the impairment of development and/or function of key components of the immune system. Since the last publication of this chapter in 2011, there have been approximately 100 new primary immunodeficiency diseases officially classified by the "Expert Committee for Primary Immunodeficiency" who met in 2015 and the numbers will continue to rise with the continued evolution and widespread adoption of genomic technologies. The ultimate diagnostic modality involves the identification of a mutation in a gene whose product is known to be involved in immunity. DNA sequencing is however still a rather time-consuming technology. Flow cytometry applications have evolved that are rapid, specific, and relatively inexpensive to screen for abnormalities associated with primary immunodeficiency diseases. The numerous flow cytometry procedures that have been developed to detect abnormalities in peripheral blood cells of primary immunodeficiency patients can barely be covered in an entire book, let alone one chapter. Instead of attempting to cover each disease with a specific assay or test, we will review four procedures each covering one of the three following broad forms of immune abnormalities observed in primary immunodeficiency, i.e., immune subset abnormalities, immune marker abnormalities, and immune function abnormalities.

Glypican 3 as a Serum Marker for Hepatoblastoma.

Hepatoblastoma (HB) is the most common primary liver cancer in children. The conventional serum marker for HB, alpha-fetoprotein (AFP), has its limitations. Novel serum markers need to be explored. Glypican 3 (GPC3) has been reported to be an excellent histological immunomarker for HB. However, the clinical value of serum GPC3 in patients with HB is unknown. A total of 184 serum samples were tested for both GPC3 by ELISA, and AFP by immunometric assay. Of these, 134 were from 32 patients with HB at three treatment stages, 30 from age-matched patients with benign hepatobiliary disorders (BHD) and 20 from age-matched "normal controls"(NC). We found that the GPC3 levels in HB pretreatment group were significantly higher than those in NC group and HB remission group but not statistically different from those in BHD group and HB during treatment group. In contrast, AFP showed significant differences among different groups. The areas under the receiver operating curve (AUROC) value, sensitivity and specificity of GPC3 for HB pretreatment group versus all controls were all significantly lower than those of AFP. Serum GPC3 levels were not associated with prognostic parameters. We concluded that GPC3 is inferior to AFP as a serum marker for HB.

Clinical Significance of Isolated Myeloperoxidase Expression in Pediatric B-Lymphoblastic Leukemia.

Objectives: Diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) requires immunophenotypic evidence of B-lineage and absence of specific myeloid or T-lineage markers. Rare cases of otherwise typical B-ALL express myeloperoxidase (MPO) detectable by flow cytometry with an absence of other myeloid markers, but the clinical significance of this finding is not well studied. Methods: A retrospective cohort analysis of flow cytometry and clinical data was performed to investigate the clinical outcome of this specific group of patients. Results: Twenty-nine cases of otherwise typical B-ALL that expressed MPO by flow cytometry (B-ALL-isoMPO) without expression of other myeloid markers were identified. The B-ALL-isoMPO group had a significantly increased incidence of relapse (univariate log rank P = .0083; multivariate hazard ratio, 2.50; 95% confidence interval, 1.07-5.85; P = .034) and significantly worse event-free survival by univariate analysis (log rank P = .0066) compared with a reference group of patients with B-ALL from the same time period (n = 264). Conclusions: To our knowledge, this is the first report to document the clinical outcomes in a group of pediatric patients with B-ALL that expresses MPO in the absence of other myeloid markers. This group had an increased rate of relapse and a worse event-free survival than the patients with B-ALL who did not express MPO.

A Rapid High-Performance LC-MS/MS Method for Therapeutic Drug Monitoring of Voriconazole, Posaconazole, Fluconazole, and Itraconazole in Human Serum.

BACKGROUND: To achieve therapeutic efficacy and prevent toxicity simultaneously, therapeutic drug monitoring has been increasingly adopted for antifungal agents with narrow therapeutic indexes. We herein report the development and validation of an accurate, simple, fast, and cost-effective clinical test with high-performance LC-MS/MS to simultaneously quantify voriconazole, posaconazole, fluconazole, and itraconazole in human serum. METHODS: Mixed with extraction solution and internal standard, 100 µL serum samples were centrifuged for protein precipitation. Diluted supernatant was injected onto a Phenomenex® Luna C8 (2) 50 × 2 mm (3 µm) column and was analyzed with a Prominence Shimadzu high-performance liquid chromatography (HPLC) system coupled with a SCIEX QTRAP 4000 mass spectrometer in a positive ionization mode with multiple reaction monitoring. The total analytical run time was 3 min. RESULTS: The assay is linear for voricoanzole (0.01-10 µg/mL), posaconazole (0.02-40 µg/mL), fluconazole (0.2-200 µg/mL), and itraconazole (0.02-20 µg/mL). The intraday CVs ranged from 1.9% to 3.8% (n = 20); the interday CVs ranged from 2.7% to 5.4% (n = 20). Method comparison study (n = 39 or 40) demonstrated good correlation with reference laboratories (R >0.99) with average biases ranging from -7.2% to 17.5%. The recoveries for each analyte were above 90%, and matrix effects ranged from 95% to 112%. CONCLUSIONS: The method is acceptable for routine therapeutic drug monitoring of these antifungal agents in clinical laboratories for better therapeutic outcome.

A sensitive LC-MS/MS method for the quantification of urinary 8-iso-prostaglandin F2α (8-iso-PGF2α) including pediatric reference interval.

BACKGROUND: Oxidative stress has been implicated in numerous diseases, including arthritis, atherosclerosis, Alzheimer's disease, cancer, diabetes, hypertension, and inflammation. 8-iso-prostaglandin F2α, a member of the F2 isoprostane family, has been well-accepted as a valuable biomarker for the assessment of oxidative stress. METHODS: We report the development and validation of an ultra-sensitive LC-MS/MS assay for urinary 8-iso-PGF2α measurements in pediatric population. RESULTS: The assay was linear from 0.024 to 20nmol/l (R(2)=0.99). Recoveries were above 85% and matrix effects were below 5%. The variability was determined at nmol/l concentration: the intra-day variability (%CV) ranged from 3.9% to 4.5% (n=20); and the inter-day variability ranged from 4.3% to 5.7% (n=20). The accuracy of our laboratory developed test was evaluated with a clinical reference laboratory (n=39), and a correlation coefficient of 0.9257 was observed. Reference interval were established to be <0.5ng/mg creatinine in a group of pediatric population (2months-18years, n=123). CONCLUSIONS: The precision of the assay will allow for accurate assessment of oxidative stress, and is acceptable for patient testing, particularly in pediatric population.

Tracking post-infectious fatigue in clinic using routine Lab tests.

BACKGROUND: While biomarkers for chronic fatigue syndrome (CFS) are beginning to emerge they typically require a highly specialized clinical laboratory. We hypothesized that subsets of commonly measured laboratory markers used in combination could support the diagnosis of post-infectious CFS (PI-CFS) in adolescents following infectious mononucleosis (IM) and help determine who might develop persistence of symptoms. METHODS: Routine clinical laboratory markers were collected prospectively in 301 mono-spot positive adolescents, 4 % of whom developed CFS (n = 13). At 6, 12, and 24 months post-diagnosis with IM, 59 standard tests were performed including metabolic profiling, liver enzyme panel, hormone profiles, complete blood count (CBC), differential white blood count (WBC), salivary cortisol, and urinalysis. Classification models separating PI-CFS from controls were constructed at each time point using stepwise subset selection. RESULTS: Lower ACTH levels at 6 months post-IM diagnosis were highly predictive of CFS (AUC p = 0.02). ACTH levels in CFS overlapped with healthy controls at 12 months, but again showed a trend towards a deficiency at 24 months. Conversely, estradiol levels depart significantly from normal at 12 months only to recover at 24 months (AUC p = 0.02). Finally, relative neutrophil count showed a significant departure from normal at 24 months in CFS (AUC p = 0.01). Expression of these markers evolved differently over time between groups. CONCLUSIONS: Preliminary results suggest that serial assessment of stress and sex hormones as well as the relative proportion of innate immune cells measured using standard clinical laboratory tests may support the diagnosis of PI-CFS in adolescents with IM.

Clinical laboratory immunology: an indispensable player in laboratory medicine.

OBJECTIVES: Clinical laboratory immunology affects practically every aspect of medicine. Accordingly, appropriately trained, board-certified clinical laboratory immunologists are key contributors to the diagnosis and management of patients with various immune-mediated conditions. This review highlights the availability of postdoctoral level training programs for clinical laboratory immunology and identifies possible career tracks. METHODS: Fundamental elements for doctoral level clinical laboratory immunologists are identified and the critical components of diagnostic immunology training as well as career opportunities in and out of academia are described. RESULTS: Relative to other disciplines in laboratory medicine, little emphasis has been given to clinical laboratory immunology in medical, graduate, and postgraduate training. Formal postgraduate fellowship programs and board certification examinations are available, yet there remains a significant lack of awareness in the medical education community about the value and necessity of training in this field. CONCLUSIONS: It is anticipated that sharing this knowledge will increase awareness of the discipline of clinical laboratory immunology at the postdoctoral level with implications for the practice of laboratory medicine.

Major histocompatibility complex class II deficiency complicated by Mycobacterium avium complex in a boy of mixed ethnicity.

Major histocompatibility complex class II (MHCII) deficiency represents a rare form of severe immunodeficiency associated with increased susceptibility to viral, bacterial, and fungal pathogens and commonly leads to failure to thrive and early death. This autosomal recessive disorder is caused by mutations in MHCII transcription regulator genes, resulting in impaired expression of MHCII, and it is usually seen in consanguineous populations. Our patient presented at age 15 months with a history of developmental delay, multiple respiratory infections and skin abscesses, and recently, at 5 years of age, he was found to have disseminated Mycobacterium avium complex. His mother is Mexican-American, and his father is Persian. Laboratory investigations showed hypogammaglobulinemia, modest T-lymphopenia, borderline mitogen responses, absent tetanus toxoid and candida antigen lymphoproliferative assays, and absent tetanus toxoid and Haemophilus influenzae type b antibody levels. Flow cytometry demonstrated absent HLA-DR antigen on monocytes and B-cells, and a diagnosis of MHCII deficiency was made. Genetic analysis yielded a homozygous pathogenic class II transactivator (CIITA) mutation. The same mutation was found in both parents. Coincidently, an Xq28 microduplication was identified and likely was the cause of the patient's developmental delay. This patient demonstrated some of the typical features of MHCII deficiency with the addition of several unique findings: disseminated M. avium complex, homozygosity in a CIITA mutation despite remarkably diverse parental ethnicity, and coincident Xq28 microdeletion with mild intellectual disability.