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2.
Blood ; 140(25): 2722-2729, 2022 12 22.
Article in English | MEDLINE | ID: mdl-35998675

ABSTRACT

Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.


Subject(s)
Antibodies , Purpura, Thrombocytopenic, Idiopathic , Thrombocytopenia , Humans , Antibodies/analysis , Anticoagulants/adverse effects , Cryopreservation , Heparin/adverse effects , Platelet Factor 4 , Purpura, Thrombocytopenic, Idiopathic/chemically induced , Purpura, Thrombocytopenic, Idiopathic/diagnosis , Thrombocytopenia/chemically induced , Thrombocytopenia/diagnosis , Vaccines/adverse effects , Enzyme-Linked Immunosorbent Assay , Blood Platelets
4.
Thromb Haemost ; 116(1): 50-7, 2016 07 04.
Article in English | MEDLINE | ID: mdl-27075008

ABSTRACT

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.


Subject(s)
Activated Protein C Resistance/blood , Factor V/analysis , Protein S Deficiency/blood , Protein S/analysis , Rivaroxaban/therapeutic use , Activated Protein C Resistance/diagnosis , Activated Protein C Resistance/genetics , Blood Chemical Analysis/methods , Blood Chemical Analysis/statistics & numerical data , Factor V/genetics , Humans , Laboratories , North America , Protein S Deficiency/diagnosis
5.
Leuk Lymphoma ; 56(2): 426-33, 2015 Feb.
Article in English | MEDLINE | ID: mdl-24913506

ABSTRACT

Little is known about aberrant antigen expression patterns and their association with cytogenetic aberrations in multiple myeloma (MM). We examined the correlation between flow cytometry and florescence in situ hybridization (FISH) in 167 marrow specimens with MM. Gene expression profiling of CD56, CD117, CD52 and CD20 mRNA in plasma cells (PCs) from patients treated on Total Therapy 2 and Total Therapy 3 trials were also evaluated. Higher expression of CD56 and CD117 was associated with hyperdiploidy. High CD52 mRNA expression was associated with c-MAF and FGFR3 subgroups. Higher expression of CD56 mRNA, but lower Kit expression, were noted in association with FGFR3. In contrast, the c-MAF subgroup showed high Kit expression but lacked NCAM mRNA expression. CKS1B amplification showed positive correlation with CD52 (p=0.0065) but negative correlation with CD20 (p=0.0207). These findings indicate that phenotypic differences in MM are associated with distinct genetic subgroups, which potentially has important diagnostic and prognostic value.


Subject(s)
Antigens, CD/genetics , Chromosome Aberrations , Gene Expression Profiling , Plasma Cells/metabolism , Antigens, CD/metabolism , Antigens, CD20/genetics , Antigens, CD20/metabolism , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Bone Marrow/metabolism , Bone Marrow/pathology , CD52 Antigen , CD56 Antigen/genetics , CD56 Antigen/metabolism , Flow Cytometry , Glycoproteins/genetics , Glycoproteins/metabolism , Humans , In Situ Hybridization, Fluorescence , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Plasma Cells/pathology , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolism
6.
Am J Hum Genet ; 93(5): 812-24, 2013 Nov 07.
Article in English | MEDLINE | ID: mdl-24140114

ABSTRACT

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.


Subject(s)
Common Variable Immunodeficiency/genetics , Germ-Line Mutation , NF-kappa B p52 Subunit/genetics , Signal Transduction , Adolescent , Adult , Amino Acid Sequence , Animals , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Cell Line , Child , Common Variable Immunodeficiency/pathology , Disease Models, Animal , Female , Genetic Testing , Heterozygote , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Microscopy, Confocal , Molecular Sequence Data , NF-kappa B p52 Subunit/metabolism , Pedigree , Phenotype , Young Adult
7.
Am J Clin Pathol ; 140(5): 680-5, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24124147

ABSTRACT

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.


Subject(s)
Cerebral Arteries/pathology , Factor IX/metabolism , Ischemic Attack, Transient/pathology , Adult , Cerebral Arteries/metabolism , Female , Humans , Ischemic Attack, Transient/epidemiology , Ischemic Attack, Transient/metabolism , Male , Middle Aged , Odds Ratio , Retrospective Studies , Risk Factors , Utah/epidemiology , Venous Thromboembolism/epidemiology , Venous Thromboembolism/metabolism , Venous Thromboembolism/pathology , Young Adult
8.
Am J Hematol ; 88(9): 818-21, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23757218

ABSTRACT

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.


Subject(s)
Autoantibodies/isolation & purification , Purpura, Thrombocytopenic/diagnosis , Thrombocytopenia, Neonatal Alloimmune/diagnosis , Thrombocytopenia/diagnosis , Autoantibodies/blood , Autoantibodies/classification , Blood Platelets/immunology , Blood Platelets/pathology , Humans , Immunoassay , Infant, Newborn , Platelet Transfusion , Purpura, Thrombocytopenic/immunology , Purpura, Thrombocytopenic/pathology , Quinine/adverse effects , Sulfonamides/adverse effects , Thrombocytopenia/chemically induced , Thrombocytopenia/immunology , Thrombocytopenia/pathology , Thrombocytopenia, Neonatal Alloimmune/immunology , Thrombocytopenia, Neonatal Alloimmune/pathology
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