MUSEO TUMBAS DEL SEÑOR DE SIPAN

The method is based on bioluminescence of firefly lantern extracts. The reactions involved are as follows:-

luciferase

Prolu ciferin--- ► Luciferin + light emission ATP — ► AMP

ATP is measured directly, ADP must first be converted to ATP as follows:-

pyruvate kinase

Phosphoenol pyruvate---► Pyruvate A D P ► ATP

Reagents and Buffers

Extracted normal plasma: Platelet poor plasma was extracted in the same way as the patient PRP samples and then centrifuged at 1500g (3000RPM) for 15 minutes before storing at -40°C (see 4.2.3).

Assay buffer: 12.1g Tris (hydroxy methyl amino methane), 0.744g EDTA di-sodium salt, made up to one litre with distilled water, pH 7.75

Bovine Serum Albumin Buffer (BSA Buffer): 1% ESA (Sigma Fraction V powder) was dissolved in assay buffer at a concentration of Img/ml.

Magnesium acetate.4H20: IM, 42.89g made up to 200ml with distilled water. Potassium acetate: 2M, 19.6g made up to 200ml with distilled water.

Phosphoenol Pyruvate (PEP): Trisodium Salt, Sigma. lOOmg of PEP was dissolved in 20ml distilled water. 0.2ml aliquots were stored at -40°C. A working solution was made by combining 0.2ml 100 mM PEP, 5ml Magnesium acetate IM and 3ml Potassium acetate 2M.

Pyruvate Kinase Sigma type II: PK was diluted 1/10 with BSA buffer.

ATP Standard: Supplied with the LKB Monitoring Kit. A vial was reconstituted with 10ml distilled water and stored in 0.5ml aliquots at -40°C (concentration equivalent to lO'^M).

ADP Standard: The sodium salt supplied by Sigma was made up to a concentration of ImM and stored in aliquots at -40°C.

Monitoring Reagent: Supplied with the LKB Monitoring Kit. The vial is reconstituted with 10ml distilled water.

Assay Method

Test, control and normal platelet rich plasma extracts (see 2.3.2.3) were thawed at 37°C and centrifuged at 2000g for 15 minutes at room temperature to remove precipitated proteins. The luminometer (LKB Wallac 1250 Luminometer) and pen recorder (Rikandenki) were allowed to warm up for at least 30 minutes before use. For each platelet extract, 4 tubes were prepared. Two for duplicate measurement of total nucleotide content ie ADP + ATP, and two for duplicate measurement of ATP only. To each tube 400/^1 Assay buffer and 20//1 patient's extract supernatant were added. An ATP standard curve was prepared as follows:- ATP Standard was diluted 1/10 in assay buffer (to give concentration of 10"^M (pM)). From this stock solution a series of dilutions were prepared in duplicate to give the following concentrations of ATP: 400pM (stock), 300pM, 200pM, lOOpM, 50pM and OpM. To each standard, 20/^1 extracted normal platelet poor plasma was added. An ADP

standard was used to check that complete conversion to ATP occurred with PK and PEP: ImM ADP was diluted 1/1000 to a strength of lO'^M in assay buffer. 400^1 ADP was added to two LP3 tubes and 20/d extracted normal plasma added. 40/^1 PEP was then added to ALL tubes and mixed well. To the sample tubes that were for "total nucleotide" and "ADP standard" 10/d pyruvate kinase was added and mixed well. These tubes were then incubated at room temperature for 15 minutes. The luminometer was calibrated and the chart recorder set to Icm/minute. To each tube in turn, 100/^1 of monitoring reagent was added, mixed and the response monitored in the luminometer.

Results were calculated as follows: The peak heights of standards and tests from the recorder tracing were read. A standard curve was drawn on linear graph paper and the test samples were read directly from the standard curve. To correct for the dilution factor, the PRP platelet count and to express result as nmoles nucleotide/10^ platelets, the following calculation was applied to each result.

nmoles A T P /10^ platelets =

pmoles ATP x 10'^ x 10^ (PRP count X (5/11) X 20) x ltf

This calculation was performed for both total and ATP only. The ADP content was then calculated by subtracting the ATP reading from the total, then the ATP:ADP ratio was calculated.

2.3.3.9 Flow Cytometry

Frozen fixed samples (see 23.23) were thawed rapidly at 3TC. 50ju\ of sample was mixed with 50/il of HEPES buffered saline and \2fû of a florescein isothiocyanate conjugated monoclonal antibody (CD62 D-Selectin, Dako), which measures degranulation through recognition of the an a-granule membrane antigen CD62 (also known as GMP140 and P-selectin), and incubated at room temperature for twenty minutes. The samples were analysed using a Coulter Epics Profile II flow cytometer (Coulter Electronsic Ltd, Luton). Up to five thousand platelets were analysed from each sample and the results expressed as percentage of positive cells. The method of the assay was based on that of Warkentin et al and Janes et al^^^’

166)

2.3.3.10 Activated Clotting Factors

Factor Vila

An assay to measure FVIIa activity was established and validated with the help of Dr Donagh O'Brien from Northwick Park Hospital, London.

Reagents and Buffers

0.1 M TBSA: NaCb 5.844g/l, Tris HCl 6.055g/l, buffered to pH 7.4 with IM HCl (approx 20mls), BSA l.Og/1.

Recombinant factor Vila: Reconstituted to Img/ml with H2O, gift of Novo Nordisk, Bagsvaerd, Denmark.

Soluble TF2 2 0 : Stock concentration 200pM, was a kind gift from Dr Donagh O'Brien.

Phospholipid: Diagen Platelet Substitute, Bell and Alton (Diagnostic Reagents Ltd).

Factor VII deficient plasma: Diagen 25 mM CaCl2: BDH.

Assc^ Method

Coagulation was measured using the ACL300R (IL).

A standard curve (0.1 - 500ng/ml) was prepared by diluting recombinant factor Vila in TBSA buffer. Patient samples were diluted 1:5 in TBSA buffer. The diluted standard and test samples, together with an equal volume of factor VII deficient plasma (final volume of ISOpl), were mixed together and placed in an ACL SOOpl cuvette.

75pi of sample mixture was incubated with 75pi of combined Tp2 2o/phospholipid (equal volumes of 20nM TF22 0 (diluted in TBSA) and phospholipid) for 60 seconds at 37°C. Coagulation was then initiated by the addition of 37pl of 25 mM CaCl2. Coagulation times were subsequently recorded and converted to factor Vila concentration (ng/ml) by comparison to a standard curve.

Factor XII a

Factor Xlla determinations were performed using an ELISA kit method (Shield Diagnostics). All reagents were reconstituted as per kit instructions. 100/^1 of standard (0, 1, 5, 10 and 20ng/ml), control or patients samples were then added to the wells. The plate was sealed, briefly shaken and incubated at room temperature for 60 ± 10 minutes. The plate was washed five times, as described above, with kit

w ash buffer. lOQwl o f d ilu ted peroxid ase-conju gate so lu tio n w as ad d ed to ea ch w ell. T h e p la te w as again sea led and in cu b ated for a further 60 ± 5 m in u tes at room tem peratu re. Just b efo re en d o f in cu b ation th e substrate so lu tio n w as recon stitu ted . T h e p la te w as th en w ashed for a fin al fiv e tim es. 100//1 o f substrate so lu tio n w as ad d ed to ea ch w ell at tim ed intervals. T h e p la te w as th en in cu b ated for 15 m inu tes, at room tem peratu re, in th e dark, after w hich th e rea ctio n w as stop p ed by th e ad d ition o f 50//1 o f stop pin g solu tion to ea ch w ell at th e sam e interval. T h e p la te w as th en read w ith in 3 0 m inutes at 550nm . T h e p la te read er softw are calib rated a factor X H a standard curve (m ean o p tica l density against n g /m l o n a lin ear sca le) and th en calcu lated th e m ean test and con trol resu lts from th e curve.

2 3 .4 S ta tistic a l A n alysis

C oagu lation factors and p la telet counts in p atien ts w ere com p ared by th e Students M est for norm ally distributed variab les and th e M ann-W hitney U -te st for n on - norm ally distributed variab les. D istrib u tion w as a ssessed through com p arison o f m ean s and m ed ians and m anual in sp ection o f data. R esu lts are p resen ted as m ean s or m ed ian s as appropriate w ith co n fid en ce lim its for param etric com p arisons. T w o- ta iled tests w ere u sed and a sign ificance le v e l o f 0.05 assum ed.

N orm al ranges from E u rop ean and P apuan con trols w ere d erived by calcu latin g th e m ean v a lu e ± 2 standard deviations.