RESUMEN
INTRODUCTION: Expert guidelines indicate that normalised ratios are preferred to clotting times for lupus anticoagulant (LA) assays to mitigate analytical variation. We investigated the effects of deriving normalised ratios from the reference interval (RI) mean or different normal pooled plasmas (NPP). METHODS: Screen, confirm and mixing tests for dilute Russell's viper venom time and APTT were converted to normalised ratios and interpreted for LA. RESULTS: Of 1000 clinical samples, 824 generated identical interpretations using RI mean or NPP-derived ratios and 57 identified LAs in one or both assays via either denominator. Separate RIs were applied for normalised ratios derived from the NPP or RI mean. Applying percentage correction index (PCI) to screen and confirm assays irrespective of screen elevation increased agreement to 92.5%. Two frozen and one lyophilised NPP were then used to derive ratios for 204 samples and 130 generated identical interpretations with all NPPs, 14 had overall interpretation parity and 19 overall agreement via PCI, giving 79.9% overall agreement. The results derived from each NPP were interpreted against RIs derived from RI means to reflect differences resulting from NPPs with clotting times dissimilar to RI means. CONCLUSIONS: Disparities were largely a function of closeness of NPP clotting times to test RI means and not owing to clotting factor level differences and likely related to manufacturing variables. Diagnostic benefit of normalised ratios can be maximised by matching NPP values to RI means. If RI mean is employed, and it likely requires re-establishing with new reagent batches.
Asunto(s)
Síndrome Antifosfolípido/diagnóstico , Pruebas de Coagulación Sanguínea/estadística & datos numéricos , Pruebas de Coagulación Sanguínea/normas , Inhibidor de Coagulación del Lupus/sangre , Interpretación Estadística de Datos , Errores Diagnósticos , HumanosRESUMEN
Evaluating the factor VIII (FVIII) binding activity of von Willebrand factor (VWF) is an important step in the diagnostic work-up of families affected by apparent mild haemophilia A. In von Willebrand's disease (VWD) type 2N (Normandy), mutations at the N-terminal end of the mature VWF subunit gene prevent the binding of FVIII. Individuals heterozygous for type 2N VWD are generally asymptomatic. Homozygotes and compound heterozygotes present with a clinical picture which mimics haemophilia A, with a markedly reduced FVIII : C activity and VWF within the normal range, but instead of exhibiting X-linked inheritance they show an autosomal recessive inheritance pattern. The distinction between haemophilia A and VWD type 2N has important implications for therapy and genetic counselling. We present a highly specific enzyme-linked immunosorbent assay screening method for the Normandy variant, which measures VWF : FVIII binding activity in parallel with VWF antigen, using monoclonal capture and detection antibodies. The assay is fully automated using a robotic microtitre plate processor, requiring minimal user intervention and providing the capacity to screen large numbers of patients.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Pruebas Genéticas/métodos , Enfermedades de von Willebrand/diagnóstico , Factor de von Willebrand/análisis , Automatización , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática/instrumentación , Factor VIII/metabolismo , Genes Recesivos , Genotipo , Hemofilia A/diagnóstico , Unión Proteica , Enfermedades de von Willebrand/clasificación , Factor de von Willebrand/genéticaRESUMEN
Five patients with severe (type III) von Willebrand disease received, by continuous infusion, a solvent-detergent treated von Willebrand factor high purity concentrate to control haemostasis. The clinical indications for treatment included prophylaxis prior to orthopaedic, abdominal and tympanic membrane surgery, and treatment of epistaxis and trauma-related bleeding. Plasma vWf antigen and activity were normalized sooner than factor VIII:C levels after initial bolus followed by infusion of the concentrate. Haemostasis was established in all five patients. The degree of shortening of the bleeding time correlated with concentrate infusion rate and, therefore, with administered dose of high molecular weight multimers. Concentrate clearance decreased over time with continued infusion. The product was shown to be stable and sterile at 24 h after reconstitution with no evidence of neoantigen expression. This report illustrates the effectiveness of high purity vWf concentrate administered by continuous infusion and shows that high-molecular-weight multimers are required to shorten the bleeding time to within the normal range.
Asunto(s)
Enfermedades de von Willebrand/tratamiento farmacológico , Factor de von Willebrand/administración & dosificación , Adolescente , Adulto , Tiempo de Sangría , Niño , Preescolar , Femenino , Humanos , Lactante , Infusiones Intravenosas , MasculinoRESUMEN
The surface phenotype of neoplastic plasma cells from peripheral blood of plasma cell leukaemia patients and bone marrow of patients with myelomatosis was investigated with two monoclonal antibody panels including 50 selected from the B cell panel of the IVth International Workshop on Leucocyte Differentiation Antigens. The majority of myelomas expressed CD24 (HB8 epitope only), CD38, CD44, CD54, and the antigen recognized by the monoclonal antibody 8A. A range of other antigens may also be expressed including CD10, CD32 (FcR II), CD19, CD20 and MHC Class II. Antigens expressed by myeloma plasma cells can be considered in three groups: (a) antigens associated with lymphocyte and plasma cell differentiation: (b) antigens which are not lineage specific: and (c) molecules concerned with lymphocyte recirculation and intercellular adhesion (CD44 and CD54). The significance of CD44 and CD54 expression by plasma cells and the potential interaction of plasma cells with T lymphocytes and monocytes is discussed.
Asunto(s)
Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/inmunología , Leucemia de Células Plasmáticas/inmunología , Mieloma Múltiple/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Superficie/inmunología , Humanos , Linfocitos/inmunología , Células Plasmáticas/inmunología , Células Tumorales CultivadasRESUMEN
A new IgG lambda myeloma plasma cell line known as EJM was established from a peritoneal effusion from a patient with extramedullary myeloma. The EJM cells have a plasmablastic morphology with abundant rough endoplasmic reticulum and grow in liquid culture with a doubling time of 72 h and a labelling index of 36%. In addition to cytoplasmic IgG lambda, the cells are positive for CD9, 20, 32, 38, 44, 54, 71, 78, MHC Class II DR, DP and DQ. Studies on the control of the cell line proliferation by cytokines have demonstrated stimulation with interleukin 6. In contrast interferon alpha produces marked inhibition of proliferation in doses of greater than 100 units/ml. The culture conditions and the importance of accessory cells and cytokines in supporting myeloma plasma cell growth in vitro are discussed.
Asunto(s)
Inmunoglobulina G/análisis , Cadenas lambda de Inmunoglobulina/análisis , Mieloma Múltiple/patología , Células Plasmáticas/inmunología , Líquido Ascítico/patología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Técnicas Citológicas , Femenino , Humanos , Cariotipificación , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/inmunología , FenotipoRESUMEN
Two new cell lines with the phenotype of terminally differentiated B cells have been derived from the presentation bone marrow of a patient with plasma cell leukaemia. They express the same immunoglobulin (A1-kappa) as the original bone marrow cells. JJN-1 is an hypodiploid, slow-growing line with a plasmacytic morphology, which grows in medium with 15-20% fetal calf serum. When JJN-1 was stimulated with a supernatant ('ESG') containing B cell stimulatory factor 2 (BSF-2/IL-6), a hypotetraploid sub-line, JJN-2, was selectively stimulated. JJN-2 is dependent on ESG for survival. The stimulatory effect of ESG can be completely abrogated by an anti-BSF-2 monoclonal antibody. However, purified BSF-2 alone only produces sub-maximal stimulation of the lines. Both lines show complex karyotypic abnormalities, including 14q- and del(6q). JJN-1 and JJN-2 may be useful for the study of late B cell differentiation and for use as immunogens for the generation of anti-plasma cell monoclonal antibodies.
Asunto(s)
Inmunoglobulina A/inmunología , Interleucinas/farmacología , Leucemia de Células Plasmáticas/inmunología , Antígenos de Superficie/análisis , Linfocitos B/inmunología , Línea Celular , ADN de Neoplasias/análisis , Femenino , Humanos , Cadenas kappa de Inmunoglobulina/inmunología , Interleucina-6 , Cariotipificación , Persona de Mediana Edad , Mitosis/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat kidney mitochondria were found to be essentially similar to those described previously for other mammalian tissues; in particular each enzyme could be activated severalfold by Ca2+ with half-maximal effects (K0.5 values) of about 1 microM and effective ranges of approx. 0.1-10 microM Ca2+. In intact mitochondria prepared from whole rat kidneys incubated in a KCl-based medium containing respiratory substrates, the amount of active, nonphosphorylated pyruvate dehydrogenase could be increased severalfold by increases in extramitochondrial [Ca2+]; these effects could be blocked by ruthenium red. Similarly, Ca2+-dependent activations of NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase could be demonstrated in intact, fully coupled, rat kidney mitochondria by either following O2 uptake (in the presence of ADP) and NAD(P)H reduction (in the absence of ADP) on presentation of non-saturating concentrations of either threo-Ds-isocitrate or 2-oxoglutarate, respectively, under appropriate conditions, or for the latter enzyme only, also by following 14CO2 production from 2-oxo[1-14C]glutarate (in the absence or presence of ADP). Effects of Na+ (as a promoter of egress) and Mg2+ (as an inhibitor of uptake) on Ca2+-transport by rat kidney mitochondria could be readily demonstrated by assaying for the Ca2+-sensitive properties of the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat kidney mitochondria. In the presence of physiological concentrations of Na+ (10 mM) and Mg2+ (2 mM), activation of the enzymes was achieved by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05-5 microM) and with apparent K0.5 values in the approximate range of 300-500 nM. The implications of these results on the role of the Ca2+-transport system of kidney mitochondria are discussed.
Asunto(s)
Calcio/farmacología , Isocitrato Deshidrogenasa/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Cetona Oxidorreductasas/metabolismo , Riñón/enzimología , Mitocondrias/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Piruvato Deshidrogenasa (Lipoamida)-Fosfatasa/metabolismo , Adenosina Difosfato/farmacología , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Activación Enzimática/efectos de los fármacos , Femenino , Concentración de Iones de Hidrógeno , Ácidos Cetoglutáricos/metabolismo , Magnesio/farmacología , Masculino , NADP/metabolismo , Piruvatos/farmacología , Ácido Pirúvico , Ratas , Ratas Endogámicas , Sodio/farmacología , Espectrometría de FluorescenciaRESUMEN
Fresh bone marrow from 43 cases of myeloma and three cases of plasma cell leukaemia has been phenotyped both by indirect immune-rosetting and, on fixed cytospin preparations, by indirect immunofluorescence. Both clustered and unclustered B cell associated antibodies from the IIIrd International Workshop on Human Leucocyte Differentiation Antigens were used. The results confirm the lack of many pan-B antigens on the surface of myeloma plasma cells, i.e. CD19-23, 37, 39, w40. Strong surface reactivity is seen with CD38 antibodies and with one CD24 antibody (HB8). Weak reactions are sometimes obtained with CD9, 10 and 45R. On cytospin preparations CD37, 39 and w40 are sometimes weakly positive, and anti-rough endoplasmic reticulum antibodies are always strongly positive. Specific and surface-reacting antiplasma cell antibodies are still lacking.
Asunto(s)
Antígenos de Neoplasias/análisis , Mieloma Múltiple/inmunología , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos de Superficie/análisis , Citoplasma/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Leucemia de Células Plasmáticas/inmunología , Formación de RosetaRESUMEN
A simple procedure was developed for the estimation of bile acid taurine conjugates in fowl plasma. Laying hens fed a diet containing rapeseed meal (RSM) (400 g kg-1) for 12 weeks had higher bile acid levels (154 mumol litre-1) than hens fed a control soyabean diet (116 mumol litre-1) (P less than 0.01). The incidence of liver haemorrhages was higher (34.8 per cent) in RSM-fed hens than in controls (21 per cent), but the severity of the lesions did not correlate with the bile acid concentration in affected birds. Histological examination of sections from livers of RSM-fed birds did not reveal significant hepatocyte degeneration outside the immediate vicinity of the haemorrhage. Canalicular bile plugs were never seen. The incidence of liver haemorrhages (13 per cent) and plasma bile acids (85 mumol litre-1) were lower in hens fed a diet containing beta-aminopropionitrile (0.5 g kg-1), a known lathyrogen. Acute treatment of hens with the hepatotoxin alpha-naphthyl isothiocyanate over four days induced necrosis of hepatocytes and resulted in elevated bile acid concentrations (262 mumol litre-1) compared with controls (73 mumol litre-1) given arachis oil. It was concluded that laying hens fed high levels of RSM develop cholestasis but the toxic principle is not known.