Impact of Hemolysis on Clinical Chemistry and Coagulation Studies

Hemolysis Chart
Hemolysis Chart

Techniques for Artificially Hemolysing EDTA samples

The paper illustrates a method to prepare hemolyzed controls using EDTA samples from routine laboratory patients.

Self-made controls (n = 2) were prepared using fresh EDTA blood. EDTA blood, 120 g/L, artificially hemolyzed by making a 1:5 dilution with distilled water then vortex mixed at room temperature, then further diluted to the desired concentrations with pooled human plasma without pathological serum indices (a 1:20 dilution, resulting in 1.2 g/L High L1 control and a 1:50 dilution, resulting in a 0.48 g/L Low L2 control). These self-made controls were stored at -20°C until use.

2. Hemolyzed Samples Should be Processed for Coagulation Studies: The Study of Hemolysis Effects on Coagulation Parameters

The blood samples were collected in citrated vacuum containers in the proportion of 1:9 parts of sodium citrate of 3.2 gm/dl concentration. After collection the tube was gently mixed by inverting it 4–6 times. Samples were run for PT and aPTT on ACL Elite pro, fully automated coagulometer run on the principle of light scattering by clot formation. Reagents supplied with the machine were used. After the test; the samples were subjected to in vitro hemolysis.

The samples were hemolyzed by rapid aspiration of about one ml of blood into syringe with a 23 G needle followed by strong expulsion back into the test tube, repeated for 5 times.

Then the samples were kept at room temperature for 3 h to simulate specimen transportation time.

Impact on Coagulation Assays

Proposed Algorithm for Management of A Hemolysed Sample for Clinical Chemistry Assays:

A very detailed paper “Practical recommendations for managing hemolyzed samples in clinical chemistry testing” , makes several key observations.

Definition of Hemolysis

The following paper, defines hemolysis as free hemoglobin > 0.5 g/L.

Visual assessment of hemolysis affects patient safety

Hemolysis (concentration of free hemoglobin >0.5 g/L)

It also defines the following three tests as having the maximum susceptibility to variations, using visual scales as estimates for hemolysis, and does not advocate use of visual charts especially for risky tests.

Tests with the highest combination of risk and occurrence rate were troponin T, potassium and total bilirubin.

Mechanisms of Hemolysis

The following paper outlines mechanisms by which hemolysis affects analyte results:

Consensus Statement for the Management and Reporting of Haemolysed Specimens

Alternative methods of measuring Hemolysis

a.The following paper suggests using spectrometric measurement of hemoglobin, by using the wavelength of 540 nm, the major absorption wavelength for hemoglobin. They also suggest indirectly measuring hemolyssi by measuring the iron concentration.

Identification of a Hemolysis Threshold That Increases Plasma and Serum Zinc Concentration

b. Another method that can be used, although not yet proven or sufficiently researched is the optical density measurement using a simple ELISA plate reader.

The following paper uses this method, although in the context of measuring the hemolysing capacity of certain drugs.

A Simple Microassay for computing the hemolysis potention of drugs

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