Specialeprojekt, Rebecka Nørregård (KU-LIFE), Mads Kjelgaard-Hansen (KU-LIFE), Mads Bertelsen

Clinical chemistry is commonly used in veterinary practice as a diagnostic tool and for monitoring progression of diseases. For proper interpretation of measurements obtained from clinical chemical analysis of blood samples, knowledge about normal biological variation is needed. Examples of numbers calculated from studies on biological variation which are used to help the assessment of measurements of clinical chemistry are the index of individuality (IoI) and the critical difference (CD). The index of individuality is used for evaluating how useful population based reference values are to the majority of the population. High IoI values (>1.4) indicate good use of the reference values where as a low IoI (<0.6) indicates that a majority of the population would not befit from reference values to detect small pathological changes. Critical difference is a value used to tell whether differences between serial blood samples from an individual are due to changes in health or if it the differences can be explained by natural biologic variation.

Multiple studies have been conducted on biological variation in humans, but only few in nonhumans and just one in reptiles. This in spite of reptiles having become relatively common to keep as pets and at zoos. To be able to enhance diagnostics and thereby hopefully the health of these animals, more information on natural biological variation is needed. Since one of the most commonly kept reptiles is the green iguana (Iguana iguana), this species would be appropriate for studies on biological variation in reptiles.

The study was conducted by collecting blood from 12 green iguanas at Copenhagen zoo for six consecutive weeks. Heparin stabilized plasma was obtained and stored at -70˚C until analyzed at a single occasion at The Central Laboratory at LIFE, Copenhagen University. Subsequently, components of analytic, intra and inter individual variation was estimated using nested analysis. From these components of variation, IoI and CD was calculated for the different clinical chemical parameters. The parameters measured in this study were albumin, total protein, glucose, alkine phosphate, ALT, total bilirubin, fructoseamine, cholesterol, creatinine, BUN, lipase, phosphate, bile acid, amylase, GGT, Ca, Mg, Na, K and uric acid.

Three iguanas were omitted from the study and components of variance were not calculated for ALP, total bilirubin, GGT, bile acid and amylase as these parameters weren’t normally distributed and thus couldn’t be examined for outliers. For ALT, albumin, cholesterol, fructose-amine, potassium and total protein IoI’s were <0.6 and for the other parameters it was between 0.6 and 1.4. No parameter had an IoI of >1.4. Calculation of index of individuality was not possible in magnesium and sodium as the nested analysis yielded negative values for the intraindividual variance in these parameters. The critical differences were calculated as percentages and were as follow: ALT 66%, albumin 24%, cholesterol 43%, fructose amine 20%, potassium 39%, total protein 23%, BUN 118%, calcium 14%, creatinine 52%, glucose 43%, lipase 13%, phosphate 46% and uric acid 113%.

Proper comparison with previously studies on biological variation is hard because of differences in study designs. The IoI’s calculated in this study fits well with the fact that most IoI’s from previous studies are either low or intermediate for most parameters, however wide ranges of IoI’s has been proposed. In this study there were indications of factors such as body mass may have influenced the outcome of IoI calculation of some parameters. As there were no correction made for these factors IoI’s of this study should be taken as estimates of whether IoI were high or low rather than as absolute values. Comparison with results from a previous study on Dumeril’s monitors shows similar IoI’s for most analytes but there were marked differences in IoI’s for potassium and calcium. Values for critical differences were rather similar in the both reptilian species. Even if CD’s were similar in the two species, further studies in reptilians are needed to investigate whether intraindividual variation changes with disease, for these values to be confidently used.