Efficiency and Health Economic Evaluations of BD OneFlow™ Flow Cytometry Reagents for Diagnosing Chronic Lymphoid Leukemia.

REASON FOR THE STUDY: To standardize the use of flow cytometry for classifying hematological malignancies and make the results reliable and reproducible across laboratories, the EuroFlow™ Consortium published a comprehensive specification of antibody-fluorochrome conjugates, standard protocols, and algorithms for analysis. The BD OneFlow™ system builds on, and further standardizes, the EuroFlow protocols. We aimed to assess the effects on safety, efficiency, and costs for laboratories of adopting the BD OneFlow reagent tubes (LST and B-CLPD T1) for diagnosing chronic lymphocytic leukemia. METHODS: We compared in-house laboratory processes and results with those using the LST and B-CLPD T1 reagent tubes with, and without, blood film morphology. Outcome measures included concordance in classification results, and efficiency within the laboratory, that is, resource usage, staff time, unwanted events, and cost-consequences. RESULTS: There was 100% concordance between the classifications made with in-house flow cytometry and those with the BD OneFlow reagent tubes. Using BD OneFlow tubes required 13 hours less staff time per month (i.e. for 100 samples) than the in-house process. Sensitivity analyses explored the effects of uncertainties in the price of the BD OneFlow tubes and the prevalence of CLL and identified the thresholds at which laboratories might expect cost-savings from adopting the BD OneFlow system. Laboratory and clinical staff considered the BD OneFlow system to be safe and effective. CONCLUSIONS: Laboratories adopting the BD OneFlow system for classifying patients with suspected CLL can expect safe, efficient processes that can be cost saving if the discount on the list price, and prevalence of CLL (which will both vary between sites and countries), is within the thresholds suggested by the health economics sensitivity analysis. © 2019 International Clinical Cytometry Society.

New graft manipulation strategies improve the outcome of mismatched stem cell transplantation in children with primary immunodeficiencies.

BACKGROUND: Mismatched stem cell transplantation is associated with a high risk of graft loss, graft-versus-host disease (GvHD), and transplant-related mortality. Alternative graft manipulation strategies have been used over the last 11 years to reduce these risks. OBJECTIVE: We investigated the outcome of using different graft manipulation strategies among children with primary immunodeficiencies. METHODS: Between 2006 and 2017, 147 patients with primary immunodeficiencies received 155 mismatched grafts: 30 T-cell receptor (TCR) αß/CD19-depleted grafts, 43 cord blood (CB) grafts (72% with no serotherapy), 17 CD34+ selection with T-cell add-back grafts, and 65 unmanipulated grafts. RESULTS: The estimated 8-year survival of the entire cohort was 79%, transplant-related mortality was 21.7%, and the graft failure rate was 6.7%. Posttransplantation viral reactivation, grade II to IV acute graft-versus-host disease (aGvHD), and chronic graft-versus-host disease (cGvHD) complicated 49.6%, 35%, and 15% of transplantations, respectively. Use of TCRαß/CD19 depletion was associated with a significantly lower incidence of grade II to IV aGvHD (11.5%) and cGvHD (0%), although with a greater incidence of viral reactivation (70%) in comparison with other grafts. T-cell immune reconstitution was robust among CB transplants, although with a high incidence (56.7%) of grade II to IV aGvHD. Stable full donor engraftment was significantly greater at 80% among TCRαß+/CD19+-depleted and CB transplants versus 40% to 60% among the other groups. CONCLUSIONS: Rapidly accessible CB and haploidentical grafts are suitable alternatives for patients with no HLA-matched donor. Cord transplantation without serotherapy and TCRαß+/CD19+-depleted grafts produced comparable survival rates of around 80%, although with a high rate of aGvHD with the former and a high risk of viral reactivation with the latter that need to be addressed.

T-cell receptor αß+ and CD19+ cell-depleted haploidentical and mismatched hematopoietic stem cell transplantation in primary immune deficiency.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is used as a therapeutic approach for primary immunodeficiencies (PIDs). The best outcomes have been achieved with HLA-matched donors, but when a matched donor is not available, a haploidentical or mismatched unrelated donor (mMUD) can be useful. Various strategies are used to mitigate the risk of graft-versus-host disease (GvHD) and rejection associated with such transplants. OBJECTIVE: We sought to evaluate the outcomes of haploidentical or mMUD HSCT after depleting GvHD-causing T-cell receptor (TCR) αß CD3+ cells from the graft. METHODS: CD3+TCRαß+/CD19+ depleted grafts were given in conditioned (except 3) children with PIDs. Treosulfan (busulfan in 1 patient), fludarabine, thiotepa, and anti-thymocyte globulin or alemtuzumab conditioning were used in 77% of cases, and all but 4 received GvHD prophylaxis. RESULTS: Twenty-five patients with 12 types of PIDs received 26 HSCTs. Three underwent transplantation for refractory GvHD that developed after the first cord transplantation. At a median follow-up of 20.8 months (range, 5 month-3.3 years), 21 of 25 patients survived and were cured of underlying immunodeficiency. Overall and event-free survival at 3 years were 83.9% and 80.4%, respectively. Cumulative incidence of grade II to IV acute GvHD was 22% ± 8.7%. No case of visceral or chronic GvHD was seen. Cumulative incidences of graft failure, cytomegalovirus, and/or adenoviral infections and transplant-related mortality at 1 year were 4.2% ± 4.1%, 58.8% ± 9.8%, and 16.1% ± 7.4%, respectively. Patients undergoing transplantation with systemic viral infections had poor survival in comparison with those with absent or resolved infections (33.3% vs 100%). CONCLUSION: CD3+TCRαß+ and CD19+ cell-depleted haploidentical or mMUD HSCT is a practical and viable alternative for children with a range of PIDs.

Identification of Heterozygous Single- and Multi-exon Deletions in IL7R by Whole Exome Sequencing.

PURPOSE: We aimed to achieve a retrospective molecular diagnosis by applying state-of-the-art genomic sequencing methods to past patients with T-B+NK+ severe combined immunodeficiency (SCID). We included identification of copy number variations (CNVs) by whole exome sequencing (WES) using the CNV calling method ExomeDepth to detect gene alterations for which routine Sanger sequencing analysis is not suitable, such as large heterozygous deletions. METHODS: Of a total of 12 undiagnosed patients with T-B+NK+ SCID, we analyzed eight probands by WES, using GATK to detect single nucleotide variants (SNVs) and small insertions and deletions (INDELs) and ExomeDepth to detect CNVs. RESULTS: We found heterozygous single- or multi-exon deletions in IL7R, a known disease gene for autosomal recessive T-B+NK+ SCID, in four families (seven patients). In three families (five patients), these deletions coexisted with a heterozygous splice site or nonsense mutation elsewhere in the same gene, consistent with compound heterozygosity. In our cohort, about a quarter of T-B+NK+ SCID patients (26%) had such compound heterozygous IL7R deletions. CONCLUSIONS: We show that heterozygous IL7R exon deletions are common in T-B+NK+ SCID and are detectable by WES. They should be considered if Sanger sequencing fails to detect homozygous or compound heterozygous IL7R SNVs or INDELs.

Dendritic cell analysis in primary immunodeficiency.

PURPOSE OF REVIEW: Dendritic cells are specialized antigen-presenting cells which link innate and adaptive immunity, through recognition and presentation of antigen to T cells. Although the importance of dendritic cells has been demonstrated in many animal models, their contribution to human immunity remains relatively unexplored in vivo.Given their central role in infection, autoimmunity, and malignancy, dendritic cell deficiency or dysfunction would be expected to have clinical consequences. RECENT FINDINGS: Human dendritic cell deficiency disorders, related to GATA binding protein 2 (GATA2) and interferon regulatory factor 8 (IRF8) mutations, have highlighted the importance of dendritic cells and monocytes in primary immunodeficiency diseases and begun to shed light on their nonredundant roles in host defense and immune regulation in vivo. The contribution of dendritic cell and monocyte dysfunction to the pathogenesis of primary immunodeficiency disease phenotypes is becoming increasingly apparent. However, dendritic cell analysis is not yet a routine part of primary immunodeficiency disease workup. SUMMARY: Widespread uptake of dendritic cell/monocyte screening in clinical practice will facilitate the discovery of novel dendritic cell and monocyte disorders as well as advancing our understanding of human dendritic cell biology in health and disease.

Respiratory Health and Related Quality of Life in Patients with Congenital Agammaglobulinemia in the Northern Region of the UK.

INTRODUCTION: Patients with congenital agammaglobulinemia, characterized by a defect in B lymphocyte differentiation causing B alymphocytosis, require life-long IgG replacement. There is scant literature regarding the effectiveness of IgG treatment at preventing mucosal (particularly sinopulmonary tract) infection and whether current management adequately restores "normal" health and quality of life (QoL). We aimed to document infective episodes pre- and post-commencing IgG replacement, determine any change in lung function and structure and assess respiratory status and QoL in a cohort of patients treated in Newcastle. METHODS: Clinical data were extracted from medical records of 15 patients identified from the immunology database, focusing on infective episodes, serial chest CT and spirometry results. Thirteen patients completed a selection of standardized and validated questionnaires assessing physical health, respiratory health and QoL. RESULTS: Pediatric patients on IgG therapy suffered fewer infections per patient year (0.74) than adults (2.13). 6/14 patients showed deteriorating respiratory status despite adequate therapy. Health questionnaires revealed a significant burden of respiratory disease on a patient's life. CONCLUSION: Clinical data showed patients with congenital agammaglobulinemia receiving immunoglobulin therapy retained a higher than average infection rate, most of which affected mucosal barriers. Most patients self-reported worse respiratory symptoms, a lower respiratory-related QoL and a lower general health QoL relative to a healthy population. Most participants had progressive structural lung damage and decreased lung function. These results suggest that current management is not entirely effective at preventing deterioration of respiratory health or restoring QoL.

Defective Leukocyte Adhesion and Chemotaxis Contributes to Combined Immunodeficiency in Humans with Autosomal Recessive MST1 Deficiency.

PURPOSE: To investigate the clinical and functional aspects of MST1 (STK4) deficiency in a profoundly CD4-lymphopenic kindred with a novel homozygous nonsense mutation in STK4. Although recent studies have described the cellular effects of murine Mst1 deficiency, the phenotype of MST1-deficient human lymphocytes has yet to be fully explored. Patient lymphocytes were therefore investigated in the context of current knowledge of murine Mst1 deficiency. METHODS: Genetic etiology was identified by whole exome sequencing of genomic DNA from two siblings, combined with linkage analysis in the wider family. MST1 protein expression was assessed by immunoblotting. The ability of patient lymphocytes to adhere to ICAM-1 under flow conditions was measured, and transwell assays were used to assess chemotaxis. Chemokine receptor expression was examined by flow cytometry and receptor signalling by immunoblotting. RESULTS: A homozygous nonsense mutation in STK4 (c.442C > T, p.Arg148Stop) was found in the patients, leading to a lack of MST1 protein expression. Patient leukocytes exhibited deficient chemotaxis after stimulation with CXCL11, despite preserved expression of CXCR3. Patient lymphocytes were also unable to bind effectively to immobilised ICAM-1 under flow conditions, in keeping with a failure to develop high affinity binding. CONCLUSION: The observed abnormalities of adhesion and migration imply a profound trafficking defect among human MST1-deficient lymphocytes. By analogy with murine Mst1 deficiency and other defects of leucocyte trafficking, this is likely to contribute to immunodeficiency by impairing key aspects of T-cell development and function such as positive selection in the thymus, thymic egress and immune synapse formation in the periphery.

Astute Clinician Report: A Novel 10 bp Frameshift Deletion in Exon 2 of ICOS Causes a Combined Immunodeficiency Associated with an Enteritis and Hepatitis.

ICOS encodes the Inducible T-cell Co-Stimulator (ICOS). Deficiency of this receptor in humans causes a common variable immunodeficiency (CVID) characterised by an absence of class-switched memory B cells and hypogammaglobulinemia. Three pathogenic mutations in ICOS have been described to date in a total of 13 cases. Here we report a novel homozygous 10 base pair frameshift deletion in exon 2 discovered by whole exome sequencing of two siblings from a family of Pakistani origin. Both patients presented in early childhood with diarrhea, colitis and transaminitis and one showed defective handling of human herpesvirus 6. Activated patient CD3(+)CD4(+) T lymphocytes demonstrated a complete absence of ICOS expression and, consistent with previous reports, we detected a reduction in circulating T follicular helper cells. Findings in this kindred emphasise the phenotypic variability of ICOS deficiency and, in particular, the variably impaired antiviral immunity that is a poorly understood facet of this rare disorder.

Immunological characteristics and T-cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T-cell-depleted autologous stem cell transplantation.

Children with systemic Juvenile Idiopathic Arthritis (sJIA), the most severe subtype of JIA, are at risk from destructive polyarthritis and growth failure, and corticosteroids as part of conventional treatment can result in osteoporosis and growth delay. In children where there is failure or toxicity from drug therapies, disease has been successfully controlled by T-cell-depleted autologous stem cell transplantation (ASCT). At present, the immunological basis underlying remission after ASCT is unknown. Immune reconstitution of T cells, B cells, natural killer cells, natural killer T cells and monocytes, in parallel with T-cell receptor (TCR) diversity by analysis of the ß variable region (TCRVb) complementarity determining region-3 (CDR3) using spectratyping and sequencing, were studied in five children with sJIA before and after ASCT. At time of follow up (mean 11.5 years), four patients remain in complete remission, while one child relapsed within 1 month of transplant. The CD8(+) TCRVb repertoire was highly oligoclonal early in immune reconstitution and re-emergence of pre-transplant TCRVb CDR3 dominant peaks was observed after transplant in certain TCRVb families. Further, re-emergence of pre-ASCT clonal sequences in addition to new sequences was identified after transplant. These results suggest that a chimeric TCR repertoire, comprising T-cell clones developed before and after transplant, can be associated with clinical remission from severe arthritis.

Low-dose serotherapy improves early immune reconstitution after cord blood transplantation for primary immunodeficiencies.

Cord blood transplantation (CBT) is curative for many primary immunodeficiencies (PIDs) but is associated with risks of viral infection and graft-versus-host disease (GvHD). Serotherapy reduces GvHD but potentially increases the risk of viral infection by delaying immune reconstitution. Because many PID patients have pre-existing viral infections, the optimal dose of serotherapy is unclear. We performed a retrospective analysis in 34 consecutive PID patients undergoing CBT and compared immune reconstitution, viral infection, GvHD, mortality, and long-term immune function between high-dose (n = 11) and low-dose (n = 9) serotherapy. Serotherapy dose had no effect on neutrophil engraftment. Median CD3(+) engraftment occurred at 92.5 and 97 days for high- and low-dose serotherapy, respectively. The low-dose serotherapy group had higher CD3(+), CD4(+), and early thymic emigrant counts at 4 months compared with the high-dose group. GvHD severity and number of viral infections did not differ between serotherapy doses. Survival from the transplantation process was 90.9% for high-dose and 100% for low-dose groups. In conclusion, low-dose serotherapy enhanced T cell reconstitution and thymopoiesis during the first year after CBT with no increase in GvHD.

Phenotypic variations of cartilage hair hypoplasia: granulomatous skin inflammation and severe T cell immunodeficiency as initial clinical presentation in otherwise well child with short stature.

We report a child with short stature since birth who was otherwise well, presenting at 2.8 years with progressive granulomatous skin lesions when diagnosed with severe T cell immunodeficiency. When previously investigated for short stature, and at the time of current investigations, she had no radiological skeletal features characteristics for cartilage hair hypoplasia, but we found a disease causing RMRP (RNase mitochondrial RNA processing endoribonuclease) gene mutation. Whilst search for HLA matched unrelated donor for haematopoietic stem cell transplantation (HSCT) was underway, she developed rapidly progressive EBV-related lymphoproliferative disorder requiring laparotomy and small bowel resection, and was treated with anti-B cell monoclonal antibody and eventually curative allogeneic HSCT. Screening for RMRP gene mutations should be part of immunological evaluation of patients with 'severe and/or combined' T cell immunodeficiency of unknown origin, especially when associated with short stature and regardless of presence or absence of radiological skeletal features.

Graves' immune reconstitution inflammatory syndrome in childhood.

BACKGROUND: The use of hematopoietic stem cell transplantations (HSCTs) as a curative therapy for life-threatening immunodeficiencies has had a profound impact on clinical outcomes. A subset of patients may experience immune reconstitution inflammatory syndrome (IRIS) post-transplant affecting the thyroid gland, but this has received little attention in the pediatric literature. We present the clinical, biochemical, and cytological course of patients with Graves' disease after HSCT in the pediatric population. PATIENTS AND METHODS: Four children (median age 1.5 years, range 2 months-9 years) underwent HSCT. The conditioning regimen included chemotherapy but not radiotherapy. None of the children or their donors had evidence of thyroid disease pre-HSCT or during the follow-up period. Engraftment was uneventful in all, with stable donor T-cell chimerism, and none had evidence of graft-versus-host disease. RESULTS: Patients developed Graves' disease soon after undergoing HSCT, with a median time interval between HSCT and Graves' disease of 22 months (range 16-28 months). Graves' disease was diagnosed on the basis of clinical and biochemical parameters, including a suppressed thyrotropin, raised free thyroxine, and raised thyrotropin receptor antibodies. Three patients were hypothyroid initially (suggestive of a Th1 profile) before Graves' disease (suggestive of a Th2 profile). In three patients, the clinical picture changed rapidly with hypothyroidism abruptly followed by profound thyroid hormone excess. The onset of Graves' IRIS coincided with a rapid expansion in naïve and total CD4. CONCLUSIONS: Immunological dysregulation during T-cell engraftment is the most likely mechanism for developing Graves' IRIS after allogenic HSTC. Clinicians need to be aware that HSCT-engendered immune recovery may result in a particularly aggressive form of autoimmune thyroid disease in children with implications for the developing central nervous system. Careful surveillance of thyroid function post-HSCT is essential.