Blood Treated with Heat, Oxygen/Ozone and UV Irradiation 3.1 Introduction
3.2 Methods 1 Subjects
3.2.3 Trial design and statistical analysis
By taking blood samples prior to the minor autohaemotherapy treatment regime, each of the individuals acted as their own control. Before / after treatment changes were
measured (see Table 3-1). Results were expressed as mean ± sd. Statistical evaluation was performed using the student's t test for paired samples with P < 0.05 as the minimum level of significance.
C h a p te r 3 S a f e ty o f M i n o r A u to h a e m o th e r a p y w ith B l o o d T r e a te d w ith H O U
Table 3-1. Proocal for autohaemotherapy and blood sampling
Day bleed
vol
Blood tested for: re-inject biochemistry haematology
vol
activity of immune cell mediators (PGI9, NO, IFN-gamma and IL-2)*
Mon 40 ml 10ml 10 ml 10ml 10 ml Wed 20 ml 10 ml 10 ml Fri 20 ml 10 ml 10 ml Mon 20 ml 10 ml 10 ml Wed 10 ml 10 ml Wed(+2h)** 30 ml 10 ml 10 ml 10 ml Fri 10 ml 10 ml 1 week later 10 ml 10 ml
* Details given in Chapter 4.
**30 ml blood were collected 2 hours after the injection o f HOU-treated or sham-treated
C h a p te r 3 S a f e ty o f M in o r A u to h a e m o th e r a p y w ith B l o o d T r e a te d w ith H O U
3.2.4 Methodology
The reagents and equipment used are specified in Appendix 4-4.
Blood (10 ml) was collected into sodium citrate anticoagulant, pharmaceutical grade for
injection (from Quarzlampenfabrik, Dr Muller, Gmbh, Essen, Germany) (2.0 ml of a 3.13 % solution, final blood citrate concentration 0.018 mol/L) and 10 ml of the
anticoagulated blood was placed into the reaction vessel of the Ozon-O-Med equipment (Appendix 2-1). After connecting the thermocouple and gas inlet tube to the machine, a
standard three minutes treatment cycle was initiated, using a pre-set ozone concentration of 15
pg / ml. The med
ical grade oxygen was converted to ozone by silent electrical discharge at ambient temperature, and the mixture of oxygen and ozone was thenbubbled through the sample for 3 min. The temperature of the blood was raised to 42.5° C and the blood was also exposed to UVC irradiation, at a wavelength o f253.7 nm and a dose of 6.0 mJ / cm2, during this treatment. After treatment, the blood was withdrawn
into a syringe, then mixed with 2 ml of a local anaesthetic procaine (Wo), pharmaceutical grade (from Quarzlampenfabrik, Dr Muller, Gmbh, Essen, Germany) and injected
intramuscularly into the gluteal muscle (Appendix 2-2).
A disposable sterile pack is provided for each treatment, containing a syringe, sodium citrate, reaction vessel, gas inlet tube and thermocouple (Appendix 2-2). Each sterile pack is used once only, to exclude cross-contamination of blood during treatment in the Ozon-O-Med equipment.
This procedure was carried out 5 times over a 2 week period. Control subjects had
blood withdrawn, anticoagulated as above but the blood was not treated with ultraviolet light, heat and oxygen / ozone. The untreated blood was reinjected intramuscularly after
C h a p te r 3 S a f e ty o f M in o r A u to h a e m o th e r a p y w ith B l o o d T r e a te d w ith H O U
mixing with 2 ml procaine (1%).