Nahla M. Heikal

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs(∗)7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853(∗)), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.

Elevated Factor IX Activity Is Associated With an Increased Odds Ratio for Both Arterial and Venous Thrombotic Events

OBJECTIVES Elevations of factor IX (FIX) are thought to contribute to thrombotic risk, but this has not been well characterized. We retrospectively sought to determine whether elevated FIX levels are a risk factor for thrombosis in 81 adult subjects younger than 65 years (mean, 47 years) who were referred for evaluation of a hypercoagulable state. METHODS Patients were classified by arterial transient ischemic attack/stroke (TIA/stroke, n = 62) or venous thromboembolism (VTE, n = 19) events. FIX activity testing was performed on all 81 subjects and a reference group of 40 healthy individuals. RESULTS Thirteen (21%) of 62 subjects with TIA/stroke and 5 (26%) of 19 subjects with VTE had elevated FIX activity. Odds ratios for TIA/stroke and VTE in subjects with elevated FIX activity were 3.7 (95% confidence interval [CI], 0.76-17.65) and 6.8 (95% CI, 1.18-39.07), respectively. CONCLUSIONS Our findings suggest an association between elevated FIX levels and both arterial and venous thrombotic events.

Laboratory testing for platelet antibodies

Laboratory testing for immune‐mediated thrombocytopenias involves identification and classification of antibodies present in patient sera or attached to patient platelets. This article summarizes the available types of platelet antibody testing and applications in disorders such as neonatal alloimmune thrombocytopenia, post‐transfusion purpura, multiple platelet transfusion refractoriness, immune thrombocytopenia, and drug‐induced thrombocytopenia. Am. J. Hematol. 88:818–821, 2013.

Evaluating eosin-5-maleimide binding as a diagnostic test for hereditary spherocytosis in newborn infants

Objective:Neonates with undiagnosed hereditary spherocytosis (HS) are at risk for developing hazardous hyperbilirubinemia and anemia. Making an early diagnosis of HS in a neonate can prompt anticipatory guidance to prevent these adverse outcomes. A recent comparison study showed that a relatively new diagnostic test for HS, eosin-5-maleimide (EMA)-flow cytometry, performs better than other available tests in confirming HS. However, reports have not specifically examined the performance of this test among neonates.Study design:We compared EMA-flow cytometry from blood samples of healthy control neonates vs samples from neonates suspected of having HS on the basis of severe Coombs-negative jaundice and spherocytes on blood film. The diagnosis of HS was later either confirmed or excluded based on clinical findings and next generation sequencing (NGS) after which we correlated the EMA-flow results with the diagnosis.Result:EMA-flow was performed on the blood of 31 neonates; 20 healthy term newborns and 11 who were suspected of having HS. Eight of the 11 were later confirmed positive for HS and one was confirmed positive for hereditary elliptocytosis (HE). All nine had persistently abnormal erythroid morphology, reticulocytosis and anemia, and eight of the nine had relevant mutations discovered using NGS. The other was confirmed positive for HS on the basis that a parent had HS, and the neonate’s spherocytosis, reticulocytosis and anemia persisted. The 20 healthy controls and the 2 in whom HS was initially suspected but later excluded all had EMA-flow results in the range reported in healthy children and adults. In contrast, all nine in whom HS or HE was confirmed had abnormal EMA-flow results consistent with previous reports in older children and adults with HS.Conclusion:Although our sample size is small, our findings are consistent with the literature in older children and adults suggesting that EMA-flow cytometric testing performs well in supporting the diagnosis of HS/HE during the early neonatal period.

Deparaffinization with mineral oil: a simple procedure for extraction of high-quality DNA from archival formalin-fixed paraffin-embedded samples.

Extracting DNA from formalin-fixed paraffin-embedded (FFPE) archival samples remains difficult. Successful polymerase chain reactions (PCR) with DNA extracted from FFPE samples is still very low. We extracted DNA from 12 recent and old archival FFPE bone marrow trephine biopsies by use of a simple protocol on the basis of deparaffinization with molecular biology–grade mineral oil followed by DNA extraction with the Qiagen FFPE kit. Comparison of this deparaffinization method with standard protocols, for example, xylene or Hemo-D with subsequent rehydration using graded ethanols, was investigated. The quality and quantity of extracted DNA were tested by a combination of ultraviolet spectroscopy, analysis on a Caliper LabChip GX, and real-time PCR combined with high-resolution melt analysis. Highest quality PCR-amplifiable DNA was obtained by deparaffinization with mineral oil, whereas more variable results were obtained for the other 2 deparaffinization procedures. This result was confirmed by real-time PCR and high-resolution melt analysis. Besides improvements in the quality of extracted DNA, use of mineral oil for deparaffinization has the added benefit of decreased time (20 vs. 75 min) and a significant reduction of hands-on labor (1 step vs. multiple hands-on centrifugation and decanting steps).

Immune Function Surveillance: Association With Rejection, Infection and Cardiac Allograft Vasculopathy

BACKGROUND Rejection, cardiac allograft vasculopathy (CAV), and infection are significant causes of mortality in heart transplantation recipients. Assessing the immune status of a particular patient remains challenging. Although endomyocardial biopsy (EMB) and angiography are effective for the identification of rejection and CAV, respectively, these are expensive, invasive, and may have numerous complications. The aim of this study was to evaluate the immune function and assess its utility in predicting rejection, CAV, and infection in heart transplantation recipients. METHODS We prospectively obtained samples at the time of routine EMB and when clinically indicated for measurement of the ImmuKnow assay (IM), 12 cytokines and soluble CD30 (sCD30). EMB specimens were evaluated for acute cellular rejection, and antibody-mediated rejection (AMR). CAV was diagnosed by the development of angiographic coronary artery disease. Infectious episodes occurring during the next 30 days after testing were identified by the presence of positive bacterial or fungal cultures and/or viremia that prompted treatment with antimicrobials. RESULTS We collected 162 samples from 56 cardiac transplant recipients. There were 31 infection episodes, 7 AMR, and 4 CAV cases. The average IM value was significantly lower during infection, (P = .04). Soluble CD30 concentrations showed significantly positive correlation with infection episodes, (P = .001). Significant positive correlation was observed between interleukin-5(IL-5) and AMR episodes (P = .008). Tumor necrosis factor-α and IL-8 showed significant positive correlation with CAV (P = .001). CONCLUSIONS Immune function monitoring appears promising in predicting rejection, CAV, and infection in cardiac transplantation recipients. This approach may help in more individualized immunosuppression and it may also minimize unnecessary EMBs and cardiac angiographies.

Thymic output: Assessment of CD4+ recent thymic emigrants and T‐Cell receptor excision circles in infants

CD4+ recent thymic emigrants (CD4+ RTEs) constitute a subset of T cells recently generated in the thymus and exported into peripheral blood. CD4+ RTEs have increased copy numbers of T‐cell receptor excision circles (TREC). They are characterized by the expression of CD31 on naïve CD4 T‐cells. We aimed to validate a flow‐cytometry assay to enumerate CD4+ RTEs and assess its performance in relation to TREC measurement.

Protein S testing in patients with protein S deficiency, factor V Leiden, and rivaroxaban by North American Specialized Coagulation Laboratories

In 2010-2012, the North American Specialized Coagulation Laboratory Association (NASCOLA) distributed 12 proficiency testing challenges to evaluate laboratory testing for protein S (PS). Results were analysed to assess the performance of PS activity, PS free antigen, and PS total antigen testing. Statistical analysis was performed on the numeric results and qualitative classification submitted for each method. There were 2,106 total results: 716 results from PS activity assays, 833 results from PS free antigen assays, and 557 results from PS total antigen assays. The three assay types performed well in the classification of five normal samples and nine abnormal samples, although certain PS activity methods were more likely to classify normal samples as abnormal and one PS total antigen assay was more likely to classify abnormal samples as normal. PS activity methods were affected by interfering substances such as heterozygous or homozygous factor V Leiden mutation (underestimation) and the anticoagulant drug rivaroxaban (overestimation). In conclusion, NASCOLA laboratories using a variety of PS assays performed well in the classification of clearly normal and abnormal samples. Laboratories performing PS activity assays should be aware of potential interferences in samples positive for FV Leiden or containing certain anticoagulant medications.

Improved harmonization of eosin-5-maleimide binding test across different instruments and age groups.

The eosin‐5′maleimide (EMA) binding test has been studied extensively for the detection of hereditary spherocytosis (HS). Its performance characteristics have been compared to NaCl‐based or glycerol lysis‐based red cell osmotic fragility tests and cryohemolysis. HS samples are also better identified when both mean channel fluorescence (MCF) of EMA relative to controls and the coefficient of variation (CV) are analyzed.

Pegylated interferon Alfa-2a and hydroxyurea in polycythemia vera and essential thrombocythemia: differential cellular and molecular responses

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative neoplasms (MPNs) with high risk of thromboembolism and tendency to transform to myelofibrosis and acute leukemia. Pegylated interferon-α (PegINFa) is an effective treatment and unlike hydroxyurea (HU), it is reported to improve cytogenetic abnormalities, decrease JAK2V617F and CALR allelic burdens and achieve molecular remission [1–3]. We reported a return to polyclonal hematopoiesis in some females after PegINFa therapy [3], and unlike HU, PegINFa promoted proliferation and differentiation of PV and ET hematopoietic stem cells in vivo [4]. Cytokines, such as TNFα and interleukins are found to be elevated in MPN [5, 6] and are implicated as drivers of clonal expansion of JAK2V617F bearing cells [7]. Genome wide association studies have shown IL28 SNP and treatment with PegINFa were associated with sustained virologic response in hepatitis C [8] and suggested favorable hematological response in 20 PV/ET patients [9]. Given these observations, we analyzed the effect of PegINFa and HU treatment on molecular response, clonal hematopoiesis (females), TNFα transcript levels, and IL28B haplotype as correlated with PegINFa response. We included 82 patients diagnosed with high-risk PV (n= 45) and ET (n= 37) (per WHO 2008) treated from 2008 to March 2017. Mutational analysis for JAK2V617F and cMPL was performed by quantitative allele-specific-PCR [10], and CALR mutations by semi-quantitative fragment analysis [11]. In females, SNPs of 5 X-chromosome genes (G6PD, MPP1, FHL1, BTK and IDS) were genotyped and in heterozygotes transcriptional allelic frequencies of granulocytes were determined by quantitative allele-specific PCR. TaqMan Gene Expression Assays were used to determine TNFα and cytokine mRNAs and plasma levels of cytokines were measured. Relative TNFα expressions and JAK2V617F allelic burden were compared using paired ttest in GraphPad Prism (La Jolla, CA). As many of these patients participated in the MPD-RC trials the hematologic and clinical responses of these patients will be reported by the MPD-RC group. JAK2V617F mutation was detectable in 70 patients, CALR mutation in 11 ET, cMPL G1544T in one ET (Supplementary Table 1). In total 31 patients (27 PV and 4 ET) in the PegINFa group, and 19 patients (17 PV and 2 ET) in the HU group had JAK2V617F mutant allelic burden >10% at baseline and with at least one 6-month follow-up allelic burden value (Supplementary Figure 1). Overall molecular response rate was 14 (45%) in PegINFa group and 17 (77.2%) in the HU group. No complete molecular response rates were noted during the entire follow-up. Among 48 PegINFa treated patients, 29 had prior HU, and in this subset of HU refractory/intolerant patients, only 3 (16.6%) had molecular response. In all patients whose JAK2V617F allelic burden decreased to <5%, the allelic burden was retested using quantitative digital PCR, and their low These authors contributed equally: Tsewang Tashi, Sabina Swierczek, Soo Jin Kim.