Transporte y manejos pre-sacrificio

6.6.1 Measurement of plasma ACE activity

Plasma ACE activity (measured in our Chemical Pathology laboratory) was estimated using 5ml venous blood anticoagulated with lithium heparin. Samples for ACE activity were centrifuged at room-temperature, plasma separated into cryotubes, snap-frozen in liquid nitrogen and stored at -40°C. ACE activity was determined by an automated method, in which hydrolysis of a synthetic TRIS buffered substrate, furanacrylol-1-

phenylalanyl-glycylglycine by ACE produced a furanocrylol blocked amino acid and a dipeptide. The resultant decrease in absorbance at 340 nm is a measure o f ACE activity.

6.6.2 Measurement of Plasma PAI-1 antigen

An enzyme-linked immunosorbant assay (ELISA) was used to measure the levels of PAI-1 antigen (Imulyse, Biopool, Umea, Sweden).

ELISA uses monoclonal antibody to the analyte which is conjugated to peroxidase. Peroxidase is an enzyme which catalyses a change in the substrate ortho- phenylenediamine di-hydrochloride (OPH) resulting in the development of a yellow colour in the reaction medium which can be measured by colorimetry. The intensity of the colour change is a function o f the concentration o f the analyte present in the sample. The testing wells are coated with an anti-PAI-1 monoclonal antibody, raised in mouse, which binds all the PAI-1 present in the sample. A second anti-PAI-1 monoclonal antibody is added which binds to an exposed site o f the PAI-1 molecule already bound to the first antibody. This second antibody is conjugated to peroxidase. The excess antibodies are then washed away and the peroxidase substrate OPH is added, resulting in colour change proportional to the concentration o f PAI-1 antigen in the original sample.

Substances such as rheumatoid factor which bind to immunoglobulin may bind non- specifically to monoclonal anti-PAI-1 antiboby. This would result in increased peroxidase activity and a falsely high estimate o f PAI-1 antigen concentration. To increase the specificity of the assay the Immunological Specificity and Accuracy Control (IS AC) principle is used. Ten micolitre o f each sample to be measured is placed in two monoclonal antibody coated wells. To one well (the A-well) is added an excess of the coating monoclonal antibody to quench PAI-1 binding so that only non-specific binding occurs to the well coat. To the other well (N well) a non-reactive mouse monoclonal antibody is added so that both specific binding o f PAI-1 and non-specific binding will occur to the well coat. PAI-1 concentration can then be measured specifically from the difference in colour change between A and N wells.

Method fo r 40 samples

250 pi o f coating antibody solution was diluted to 25ml with O.IM NaHC03 buffer and 200pl was then added to each well. The plate was covered and incubated at 25°C on a

plate shaker for 2 hours. The solution was then discarded by inverting and tapping the plate. The wells were then washed 3 times with PET buffer (P B S buffer substance with EDTA and Tween 20).

200pl o f coating antibody solution was diluted to 10ml with PET buffer and mixed. 150 |il was added to each A well. 200pl o f non-reactive mouse monoclonal antibody solution was diluted to 10ml with PET buffer and mixed. ISOpl was added to each N well.

50pl of PAI-1 standard concentrations of 50,25, 12.5 and 0 ng/1 were prepared by mixing a standard PAI-1 plasma o f 50ng/ml with PAI-1 depleted plasma in the proportions 4:0, 2:2, 1:3 and 0:4. Plasma samples known to contain more than 50ng/ml PAI-1 were diluted with PET buffer. 10 pi o f each standard and each sample were added to paired A and N wells. The samples were covered and incubated at 25°C on the plate shaker for 1 hour. 80pl o f conjugated antibody solution was diluted with 6ml PET buffer and mixed and then 50pl of this solution was added to each well prior to further incubation at 25°C for 1 hour on a plate shaker. The contents o f the plate were then discarded and the wells washed 4 times with PET-buffer.

The OPH substrate was dissolved in 3ml o f water for 30 minutes before use and then diluted further to 24ml with water. Immediately before use lOpl o f 30% hydrogen peroxide was added. 200pl of the substrate mix was added to each well before incubation for 15 minutes at 25°C on a plate shaker. The reaction was stopped by the addition o f 50pl 4.5M sulphuric acid.

Absorbance at 492nm was measured using a micro-test plate spectrophotometer and the difference in absorbance between the N and the A well (AA) for each standard and sample calculated. AA was plotted against concentration for the standard samples to obtain a calibration curve from which the PAI-1 concentration o f the other samples was read. Where samples had been diluted the concentration was multiplied by the dilution factor.

The coating anti-PAI-1 mouse monoclonal antibody (Biopool MAI-12) binds to all known forms o f PAI-1 and the conjugated mouse monoclonal anti-PAI-1 antibody (MAI-11) binds to both latent and active PAI-1 and to a lesser extent complexed PAI-1. The Imulyse PAI-1 assay is therefore claimed to measure active and latent PAI-1 with poor detection o f PAI-1 complexed with either t-PA or urokinase. The stated coefficients o f variation (CVs) for this assay at 20ng/ml are within-assay 5% and

between-assays 9%. In the laboratory where these tests were performed the CVs were within-assay 8.4% and between-assays 10.5%. As this assay can measure PAI-1 antigen in the range 0.9-50ng/ml, samples with PAI-1 antigen concentrations greater than 40ng/ml were diluted 1:1 with PET-buffer, re-assayed and the resulting concentration multiplied by two.

6.6.3 Measurement of other haemostatic factors

Fibrinogen was measured by the Clauss method, vWF was measured by an ELISA (with antibodies from Dako, Sweden), FVIIiC was assayed on the ACL 3000 (Instrumentation laboratory, Warrington, UK) using FVIIiC deficient plasma rabbit thromboplastin as reagents.