Conflicting effects of haemolysis on plasma sodium and chloride are due to different haemolysis study protocols: A case for standardisation

Abstract

Background Haemolysis has been reported as having a positive, negative or no effect on plasma sodium (PNa) and chloride (PCl). We investigated the haemoltytic effect of different haemolysis protocols on PNa and PCl using modelling and laboratory experiments. Methods In a modelling experiment, percentage change and recovery due to dilution in routinely ( in vitro) haemolysed samples were compared against shear stress haemolysis and samples spiked with haemolysate from whole blood freeze–thaw, packed cells freeze–thaw and osmotic shock protocols. The results were compared against a control base pool. Additionally, for the osmotic shock method, results were compared against saline- and deionised water (DIW)-spiked controls. In a laboratory experiment, percentage change and recovery were similarly compared using haemolysate from whole blood freeze–thaw and osmotic shock protocols. PNa, PCl and H-index were measured on the Abbott Architect and haemoglobin on the Sysmex XN-9000. Results In the modelling experiment, the percentage decrease in PNa and PCl was similar in in vitro haemolysis, shear stress haemolysis, whole blood freeze–thaw haemolysis and packed cells freeze–thaw haemolysis and this was lower compared to the osmotic shock method. In the laboratory experiment, the change in PNa compared to the base pool was less ( p < 0.001) per unit increase in H-index in the freeze–thaw method (−0.33 mmol, 95% CI −0.35 to −0.31) compared to the osmotic shock method (−0.65 mmol, 95% CI −0.66 to −0.64). PCl did not change with haemolysis in the freeze–thaw method and changed by −0.21 ± 0.01 mmol per unit increase in the H-index in the osmotic shock method. Recovery of PNa and PCl increased with increasing H-index in both methods. Conclusion The osmotic shock protocol is inappropriate for haemolysis studies because of dilution with DIW used for cell lysis. Recovery calculations may incorrectly compensate for genuine dilution caused by haemolysis.

The Issue With Osmotic Shock Hemolysate Preparation Procedure

“…In the prepared sample, the sodium concentration remained unchanged compared to that in the DIW control in the osmotic shock method, whereas it decreased by 5% compared to that in the base pool in the other two methods. These results are similar to those in previous experimental studies covering mild to gross hemolysis [4,6].…”

“…In addition to changes due to hemolysis, the sample composition is altered by the substitution of the serum with DIW in the added hemolysate; therefore, the analyte result of the test pool is compared with that of a control prepared by adding an equal volume of saline to the same serum pool [ 3 ]. Most other hemolysate preparation methods, e.g., methods that use multiple freeze–thaw cycles or shear stress hemolysis, do not alter the sample composition beyond changes due to hemolysis, and the result of the test pool is compared with that of a non-hemolyzed base pool [ 4 ].…”

“…The difference in the scenario of dilution by cellular contents, taking sodium as an example, is illustrated in Fig. 1B [ 4 ]. For sodium (and chloride), it is preferred to compare the result with a control prepared by adding an equal volume of DIW rather than a control prepared by adding saline [ 3 ].…”

Validation of an automated sample preparation module directly connected to LC-MS/MS (CLAM-LC-MS/MS system) and comparison with conventional immunoassays for quantitation of tacrolimus and cyclosporin A in a clinical setting

“…Recently, a hemolysis method for whole blood that involves freezing at -80 °C for just 10 min and thawing in running water for 5 min was reported and was employed to measure tacrolimus by CLAM-LC-MS/MS [ 20 ]. Furthermore, since there are several hemolysis methods [ 26 ], it will be necessary to optimize and validate the hemolysis method for the measurement of tacrolimus and cyclosporin A by CLAM-LC-MS/MS in clinical practice.…”

Effects of storage temperature, storage time, and hemolysis on the RNA quality of blood specimens: A systematic quantitative assessment

“…Therefore, it can be concluded that for hemolyzed specimens, the method of hemolysis has no effect on total RNA if it does not affect leukocytes. Kalaria et al [ 24 ] also found that different hemolysis research protocols had different effects on a given parameter. They found conflicting effects of hemolysis on plasma sodium and chloride levels due to different hemolysis protocols.…”