Instrumental bias in blood gas analysis of tonometered whole blood.

My purpose in investigating the effect of hemolysis on the accurate assessment of catecholamines and DOPA concentrations by the “Cat-A-Kit” radioenzymic method was to provide a basis for rejecting hemolyzed specimens, and thereby reduce the ever-growing backlog of specimens awaiting analysis in my freezer. I was surprised and disappointed to find that I could not discard hemolyzed samples as being “unsuitable for analysis.” I chose freezing as the means of inducing hemolysis because this best approximates the conditions encountered in the clinical setting, i.e, erythrocyte disruption from physical trauma (shaking of the blood-collection tube, use of a small-bore collection device, excessive vacuum, or excessively hard or prolonged centrifugation). Hemolysis induced by hypotonic solutions does not represent this situation, nor is it physiological. There are several differences between the Cat-A-Kit method (1) and that of Brown and Jenner (2) used by Causon et al., including the specific activity of the labeled compound, the enzyme preparation, the extraction solvents used, and, perhaps most important, the concentration of nonisotopic O-methylated metanephrines added as carrier proteins. Saar et al. (3) demonstrated that the extraction efficiency of 3Hlabeled methylated derivatives depends on the carrier concentration. The method of Brown and Jenner involves only 0.5 g of carrier per assay tube or only 3% of the lowest concentration investigated by Saar et al. Because this low concentration of added carrier reduces the extraction efficiency of 3H-labeled metanephrines, and because the extraction of both ‘Cand 3H-labeled metanephrines should be equivalent, then the ratio of [3Hlmetanephrines to [‘4C]metanephrines recovered (the basis for the estimation by Causon et al. of endogenous catecholamine concentrations from standard) will change with that reduced efficiency. The Cat-A-Kit method involves about 40 zg of each derivative (metanephrine and normetanephrine) per incubation volume, which is nearly the amount of carrier substance (45 tog) described by Saar et al. as “optimal for sensitivity.” Possibly the addition of hemolysate to the incubation mixture may only further diminish the poor recovery and variability observed by Causon et al. Increasing the concentration of carrier may solve this problem, and I would be interested in seeing the results of a similar study after this change. In my original paper I did not mean to imply that all radioenzymic methods were unaffected by specimen hemolysis. Surely, this must be determined for each method. However, I maintain that when sample hemolysis is believed to be caused by collection or processing techniques but is not associated with patient’s stress, the specimen should be considered acceptable for measurement of free or sulfate-conjugated catecholamines or DOPA with the Cat-A-Kit.