Letter to the Editor

Ann Lab Med 2023; 43(5): 512-514

Published online September 1, 2023

Copyright © Korean Society for Laboratory Medicine.

The Issue With Osmotic Shock Hemolysate Preparation Procedure

Tejas Kalaria , M.D., FRCPath

Clinical Biochemistry, New Cross Hospital, Black Country Pathology Service, The Royal Wolverhampton NHS Trust, Wolverhampton WV10 0QP, UK

Correspondence to: Tejas Kalaria, M.D., FRCPath
Clinical Biochemistry, New Cross Hospital, Black Country Pathology Service, The Royal Wolverhampton NHS Trust, Wolverhampton WV10 0QP, UK
Tel: +44 1902 307999 (ext. 8261), E-mail:

Received: January 3, 2023; Revised: February 21, 2023; Accepted: March 22, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Dear Editor,

Bias in hemolysis analyte results due to an increase in the analyte concentration caused by intracellular release (e.g., potassium, phosphate, magnesium, AST, ALT, lactate dehydrogenase, folate, or urea) or a decrease in the concentration because of dilution by intracellular contents (e.g. sodium, chloride, or albumin) is often significant before spectrophotometric or chemical interference by Hb or other intracellular contents is manifested [1]. Therefore, the hemolysate preparation method selected for interference studies must provide an accurate representation of the real analyte changes due to hemolysis.

The osmotic shock hemolysate preparation procedure, originally developed to study analytical interference from dissolved Hb [2], is commonly used in interference studies. In a popular adaptation of this procedure [3], after three saline washes, the red blood cells are hemolyzed by adding an equal volume of deionized water (DIW) followed by a freeze–thaw cycle. The obtained hemolysate is added to serum to prepare samples with a desired Hb concentration. 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 scenario of intracellular release of an analyte, using a modeled sample with a serum urea concentration of 6.0 mmol/L, is illustrated in Fig. 1A. The concentration of urea in red blood cells is approximately 1.2 times the concentration in serum and was considered 7.2 mmol/L in this case [5]. Modeled analyte concentrations in samples prepared for paired interference testing using the osmotic shock hemolysate preparation procedure were compared with those in a sample prepared by the freeze–thaw or mechanical hemolysis method. As the sample composition does not change beyond changes due to hemolysis in the latter two methods, equivalence of the final analyte concentrations was assumed, and the methods are presented together. As equal volumes of washed red cells and DIW are used in the osmotic shock procedure, for simplicity of comparison, Hb, 150 g/L; hematocrit, 50%; and complete hemolysis were assumed. Contributions from white blood cells and platelets were ignored to simplify the explanation. In the prepared sample, the urea concentration increased by 7% compared to that in the control in the osmotic shock method and by 1.0% compared to that in the base pool in the other two methods.

Figure 1. Effect of the hemolysate preparation method on the analyte concentration in the prepared sample explained by modeled samples with (A) urea and (B) sodium concentrations of 6.0 mmol/L and 140 mmol/L, respectively. The models consider the addition of 100 μL (10%) of hemolysate to 900 μL (90%) of serum, and the volume contributions of the individual fractions to the prepared sample are presented left to the bars. The numbers in the bars indicate the analyte concentrations (mmol/L) in the respective fractions, and the numbers in brackets within the bars represent the contributions of the respective fractions to the final concentration in the prepared sample after accounting for dilution.
Abbreviations: RBC, red blood cell fraction; DIW, deionized water; N/A, not applicable.

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]. 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].

The modeled examples in Fig. 1 explain the reason for the different results better than the experimental studies did. The difference introduced by the DIW in the osmotic shock procedure is not accurately compensated when comparing with the base pool or control with added saline or DIW. This applies not only to the abovementioned analytes, for which hemolysis leads to a change in the concentration through intracellular analyte release or dilution with cellular contents, but also to analytes for which the intracellular concentration is the same as the serum concentration. This can be envisaged by substituting analytes and concentrations in the model. This model considers the actual analyte concentrations in prepared samples and disregards spectrophotometric, chemical, or physical interference by Hb and/or other intracellular contents that could further affect the measured results. In summary, in vitro hemolysis of samples is not accurately represented by the osmotic shock procedure because of analyte concentration changes by the DIW and saline used.

Per commonly used definitions of interference [3, 7], a real method-independent change in the analyte concentration is not classified as interference but is often termed influence [8]. The effect of hemolysis, e.g., in the measurement of sodium using an indirect ion-selective electrode, could be a combination of interference (an electrolyte exclusion effect due to released intracellular proteins and lipids in the case of sodium) and influence (a decrease in the serum sodium concentration because of the release of sodium-depleted intracellular contents) [9]. Therefore, in addition to selecting an appropriate hemolysate preparation method, the results should be presented as an “effect of hemolysis” rather than “interference by Hb.”

Kalaria T conceived the model and wrote the manuscript.

The author declares no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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