LABORATORY METHODS

Blood was taken for the measurement of plasma elastase, white cell and differential counts and neutrophil free radical production. Measurement of free radical production was performed by assessing superoxide release and hydrogen peroxide generation by neutrophils in response to stimulation.

White cell counts and differentials These were performed using a portion of the EDTA sample run through a Coulter® MAXM counter.

Plasma elastase A 4ml sample was taken into EDTA and gently mixed. The sample was stored on ice until it could be separated, between 15-20 minutes post­ sampling. This was achieved by centrifugation at 1400g for lOmins. Plasma was removed, aliquoted into 250j l i1measures and frozen at -80°C. Elastase was measured

using an ‘in-house’ ELISA assay, based on the method of Browser and Harpel^^^. Plates were coated with lOOpl sheep anti-human elastase (IgG, Binding Site, Birmingham, UK) diluted 1 in 1000 in carbonate/bicarbonate buffer and incubated for 24 hours at room temperature. On the following day, plates were washed four times with PBS/Tween and blocked with lOOpl 1% BSA in PBS for one hour. The plates were washed again, and

lOO^il standards (Merck elastase calibrators, diluted 1 in 100 in PBS/Tween) and plasma samples (diluted 1 in 20 in PBS/Tween) added, in triplicate. The range of standards was 0 to 320ng/ml. The plates were incubated for two hours, washed, then lOOpl peroxidase conjugated anti-human a-1 antitrypsin (Binding Site, Birmingham, UK), diluted 1 in 1000 in PBS/Tween, added. After one hours’ incubation, the plates were given a final wash prior to addition of the substrate solution. A lOOpl ahquot of 0.5mg/ml OPD, in phosphate citrate buffer containing sodium perborate (Sigma Chemical Co, Poole, UK) was then added to each well, the plates covered in foil and incubated for 15 minutes. The reaction was stopped by the addition of SOpl 4M sulphuric acid and the plates read at 492nm.

Superoxide release This was determined using a microplate technique based on a superoxide dismutase inhibitable reduction of ferricytochrome A 4.5ml blood sample was gently mixed with 0.5ml of Heparin and placed on ice. The sample was then mixed with 0.5ml of 6% Dextran and left for 45 minutes at room temperature to sediment the red blood cells. The white blood cells form a “buffy-layer” between the red cells and the supernatant. This layer is removed and spun at lOOg for 12 minutes to pellet the leucocytes. The supernatant was discarded and the cells resuspended in distilled water for 30 seconds. This effects hypotonic lysis of the residual red cells. Iso-osmolarity is restored with 0.6% KCl. The suspension was then spun at 160g for 4 minutes and the process of hypotonic lysis repeated. Finally, cells were resuspended in 2ml Hanks buffered Saline. Cell viability was assessed using Trypan blue exclusion technique, and revealed>90% viability for this technique. A 20pl aliquot cell suspension is added to 380pl of acetic acid and methyl violet. A neutrophil count was then determined for each

sample using a Neubauer counting chamber; number of cells in the outer four squares x 0.05 = cells X 10^/ml. counting technique. Samples were then diluted with Hanks buffered Saline to give a fixed count of 2 x 10^ cells /ml. Neutrophil superoxide release is then determined using a microplate technique based on a superoxide dismutase inhibitable reduction of ferricytochrome c. Two reaction solutions are mixed:

Solution 1 (-SOD): 64pl Distilled water

320pl cytochrome c (Sigma Chemical Co, Poole, UK) 4.75ml Phorbol myristate acetate (Sigma Chemical Co)

Solution 2 (+SOD): 64pl Superoxide dismutase (Sigma Chemical Co) 320pl cytochrome c (Sigma Chemical Co)

4.75ml Phorbol myristate acetate (Sigma Chemical Co)

A microplate is set up with 85|l i1of solution 1in each of the wells in the top four rows,

and 85j l a1of solution 2 in the lower four. A 74|l i1aliquot of cell suspension is added to

each well. The plate is covered and incubated at 37°C with constant rotation. Phorbol myristate acetate (PMA) causes release of the superoxide radical, which acts to reduce ferricytochrome c from an orange to a pink colour. Superoxide dismutase breakdown superoxide and prevents the colour change. The colour is measured at 550nm using a microplate reader. The difference between stimulated and unstimulated samples is used as a measure of superoxide production.

Hydrogen peroxide generation This was measured using a flow cytometric technique to measure intracellular hydrogen peroxide formation^^^. A 2.7ml blood sample was gently mixed with 0.3 ml of Heparin and placed on ice. The sample was then mixed with 0.3ml of 6% Dextran and left to sediment for 45 minutes at room temperature. The sediment was removed and cell viability assessed. This separation technique gave >95% viability by Trypan blue exclusion test. Neutrophil counts were determined and samples diluted to give a cell count of 2 x 10^/ml. Two 0.5ml samples of each cell suspension are incubated for 15 minutes with 0.5ml Dichlorofluoroscin diacetate (DCFH-DA, Kodak, Liverpool, UK) to give a final concentration of 9.6pg/ml. PMA, final concentration 60ng/ml, is added to one of the duplicates, an equivalent volume of Hanks to the other, and the suspensions incubated for a further 15 minutes. DCFH-DA is deacetylated by the neutrophil membrane to produce dichlorofluoroscin, a non-fluorescent compound. In the presence of hydrogen peroxide, this compound is oxidised to the fluorescent dichlorofluoroscein. PMA is a powerful neutrophil stimulant inducing formation of hydrogen peroxide. The fluorescence of the neutrophil population in each sample is then measured using a Coulter EPICS® XL Flow Cytometer. Neutrophils are gated for based on forward and side scatter, and the Mean Fluorescence Intensity (MFI) is determined for this region. The difference in MFI between the two samples is used as a measure of hydrogen peroxide formation.

Thromboxane B2 Samples were collected as above into tubes containing citrate and aspirin, to prevent in-vitro formation of thromboxane A2. As above, the tubes

were stored on crushed ice both pre- and post-sampling. At intervals, between 5 and 20 minutes following collection, samples were centrifuged at 2 500g at -4°C. The central

part of the supernatant was separated into aliquots of 0.5mls and stored at -80°C. Thromboxane B2 is stable metabolite of Thromboxane A2, a product of arachidonic acid,

and was measured using a commercially available ELISA kit (ACE'^'^, Cayman Chemical Co, Ann Arbor, MI, USA). All specimens were run in duplicate and the mean value determined.