Abstract

Hemolysis of serum and plasma samples is a common problem in veterinary diagnostic laboratories. Hemolysis occurs when red blood cells contact foreign surfaces, when small-gauge needles are used to obtain samples, or when samples are handled inappropriately.⁴ Samples taken during field investigation of free-ranging wildlife or by inexperienced phlebotomists, frequently result in erythrocyte disruption. Significant changes in plasma and serum electrolyte and chemistry values have been described in domestic species,³ zoological species,¹ and human beings^2-4. The species-specific effects of hemolysis depend upon variations in the intracellular concentrations of the analytes. Methodology and instrumentation will also have a profound effect of the degree to which hemolysis will affect clinical chemistry. Previous studies have indicated that many of these alterations are related to the severity of hemolysis.

Ten common green iguanas (Iguana iguana) were individually housed at the University of Wisconsin School of Veterinary Medicine animal housing facility and fed a commercially available formulated diet. Animals were anesthetized using 30 mg/kg of ketamine IM in the triceps muscle. Three to 5 cc of non-hemolyzed whole blood was collected from the right atrium using a 1.5-inch 22-ga needle and a 5 cc syringe. Samples were immediately placed in lithium heparin tubes. One aliquot of blood was centrifuged and the non-hemolyzed plasma harvested. A second aliquot was severely hemolyzed by rapid freezing and the plasma harvested. Consistent levels of hemolysis were achieved by adding known quantities of hemolyzed plasma to the original non-hemolyzed samples. The degree of hemolysis in the plasma was determined using a Kodak Interference Guide color chart. Two levels of hemolysis (moderate and severe) were developed, and the hemolyzed and non-hemolyzed samples were frozen at -70°C for eight months. Nine serum analytes were measured using a Kodak Echtachem 500 chemistry analyzer. Mean values and SD for non-hemolyzed, moderately hemolyzed, and severely hemolyzed plasma were compared (Table 1).

Table 1. Effect of hemolysis on plasma electrolytes and chemistries; values are means (n=)

Note: Means with different superscript letters in each row are significantly different at the P level indicated.

The presence and degree of hemolysis must be considered when interpreting clinical chemistries in the common green iguana (Iguana iguana).

As an adjunct to this study, we developed a technique to draw a relatively large sample of blood from the smaller (0.5 kg) iguana. Anesthetized animals were placed in dorsal recumbency and held at the edge of an examination table. A 22-ga 1.5-inch needle on a 5 cc syringe was inserted into the thoracic inlet and advanced towards the opposite pelvic limb. Sampling by this method was quick, effective, and without complication in all animals sampled. To document the site of cardiopuncture, digital subtraction angiograms were performed, and contrast media (iohexol) was found to be present in the right atria before proceeding throughout the circulation.

Acknowledgments

We would like to acknowledge Dr. Chet Thomas, DVM, PhD, for his help with the statistical analysis.

Literature Cited

1.  Ramer J, MacWilliams P, Paul-Murphy J. Effects of hemolysis and frozen storage on serum electrolyte and chemistry values in cotton-top tamarins (Saguinus oedipus). J Zoo Wildl Med. 1995;26:61–66.

2.  Tietz NW, ed. Textbook of Clinical Chemistry. Philadelphia, PA: WB Saunders Co; 1986;478–498.

3.  Willard MD, Tvedten H, Turnward F. Small Animal Clinical Diagnosis by Laboratory Methods. Philadelphia, PA: WB Saunders Co; 1989:112–303.

4.  Yucel D, Dlava K. Effect of in vitro hemolysis on 25 common biochemical tests. Clin Chem. 1992;38:575–577.