Laser Doppler flow has been validated both in vitro by measurement of liquid flows through small capillary tubes using a number of vehicles including polystyrene beads and human blood (Stem et al 1977, Watkins and Holloway 1978). In vivo evaluation of LDF in skin and renal cortex has found a linear correlation between xenon washout and radioactive microsphere techniques respectively (Stem et al 1979, Stem et al 1977).
As regards the gut, LDF validation in a feline model has found a linear correlation between blood flow determined by an electromagnetic flow probe and the LDF. However, this relationship only appears to hold tme for flow rates < 50 ml/min/100 g (Ahn et al 1986). Blood flow to the human small bowel determined by *^Kr elimination technique during abdominal procedures was estimated to be 38 ± 4 ml/min/100 g, with 75% of the flow distributed to the mucosa/ submucosa (Hulten et al 1976). Similarly LDF measurements made during laparotomies in 48 patients (Ahn et al 1986). Total intestinal blood flow was measured by isolating a segment of bowel on one supplying artery. The vein draining the isolated segment was cannulated and the venous outflow collected in a graduated cylinder. Total blood flow averaged 38 ± 15 ml/min/lOOg for the jejunum and 30 ± 13 ml/min/lOOg for the ileum (Ahn et al 1986). A linear relationship was found between total gut blood flow and the laser Doppler signal when TBF was <50 ml/min/lOOg, indicating that human intestinal blood flow is within the limits of accuracy idealised by Ahn et al (Ahn et al 1986).
Further support for the validity of LDF was provided by an ingenious probe which could simultaneously measure mucosal blood flow with hydrogen (H%) clearance and LDF; again a linear relationship was found between the two techniques (Kiel et al
1985). Kveitys et al also found a linear relationship between H2 clearance, LDF and
microsphere estimation of cat jejunal blood flow. Although H% clearance appeared to overestimate total gut blood flow, LDF correlated well with both total and
et al compared the ^^Kr elimination technique with laser Doppler estimation of feline gut blood flow and found a linear relationship (Ahn et al 1985).
Additional evaluation of LDF in liver tissue has found that the LDF technique also has the advantage of recording rapid changes in blood flow. In a rat liver model, Almond and Wheatley found that hepatic nerve stimulation resulted in a 51 ± 14% reduction in total blood flo w , which resulted in 12 ± 10% fall in LDF flow and a 14 ±
10% fall in 85Kr clearance determined blood flow (Almond and Wheatley 1992).
However, accurate quantification of tissue blood flow using LDF may not be valid since the laser Doppler signal depends upon the inherent optical property of tissue to reflect light. Therefore differences in the laser Doppler signal for any given flow may be expected to differ not only between tissue, but between individuals for the same tissue and even for different areas within the same tissue. This problem was encountered by Almond & Wheatley in the quantification of the laser Doppler signal in the rat liver model. Although a linear correlation was established between total blood flow and the LDF for each animal, the coefficient of variation of the slopes of the regression lines was 31% for different animals (Almond and Wheatley 1992).
Reproducibility of Laser Doppler Measurements
The variation in repeated measurements of LDF blood flow may be expressed in different ways. Recently, repeated measures analysis of variance with a single factor design was undertaken to evaluate the precision of laser Doppler fiowmetry from different tissues. The length of the 95% confidence interval was determined using the standard formula for a t confidence interval (Line et al 1992). Paired readings provided an unacceptable precision estimate in the tissues examined (skin, gastric mucosa and pig kidney), but performing more than four repeated measures enhanced the certainty of the perfusion estimate. However, comparisons could not be made between tissues, because different probes were used for determining blood flow (Line et al 1992).
Other studies describing the temporal and spatial variation in LDF have employed the coefficient of variation as a quantitative assessment. Kvemebo et al found that in the gastric mucosa the spatial variation was low and reproducibility o f the LDF measurements consistent (Kvemebo et al 1986). The coefficient of variation may be expected to vary not only with the site under investigation but also the technique and equipment employed to evaluate the LDF blood flow. Tenland et al found the coefficient of variation to be 6% for LDF readings made from a stable emulsion (Tenland et al 1983), whilst Allen et al found the coefficient of variation to be 16.9% when using endoscopic LDF for measuring colonic mucosal blood flow (Allen et al 1987). Because biological systems are in a continuous state of flux, variability of this magnitude has been reported for other blood flow assessment techniques, such as isotope clearance (12.9%) (Forrester et al 1980) and 15-60% coefficient of variation for radiolabelled microspheres (Brown et al 1974, Neutze et al 1968).
The spatial variations in LDF assessment from tissues is due to differences in the vascular architecture for any given area of the tissue. The geometry of the Doppler probes determine the depth and thus the volume of tissue under measurement. With
capillaries may be expected to increase due to vascular heterogeneity. A multiprobe which integrates the signal from several adjacent volumes of tissue may reduce this error. This has been demonstrated by Salerud and Nilsson (Salerud and Nilsson
1986), using a probe which can simultaneously analyse seven volumes of adjacent tissue simultaneously, reducing the spatial variation by the square root of the number of scattering volumes compared to standard probes.