Techniques for Artificially Hemolysing EDTA samples
- Can the Roche hemolysis index be used for automated determination of cell-free hemoglobin? A comparison to photometric assays
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.
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
- Arora, et.al found a shortening in both PT and APTT in patient samples, but no difference in normal volunteer samples, after hemolysis. They do not have data on how many of the patient sample population was on heparin or other anticoagulants, but they hypothesize that hemolysis may release platelet activating factor 4 and neutralize the heparin and thus shorten the coagulation assay time. Their conclusion was not to reject hemolyzed samples if the patient has no known history/is for a routine checkup. In case the patient is on anti-coagulants, it may be prudent to reject and ask for a new sample.
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.
- A practical cut off for free haemoglobin is set at 10g/L. This cut off basically means 80% of the hemoglobin in a routine sample, assuming 12g/L as being an average hemoglobin concentraion in a healthy patient.
- Primary to their system is a baseline estimation of the H-Index. If this cannot be done by instrumentation, it must be done by the visual scale and degree of hemolysis (i.e H-Index) must be established as the first step.
- Let us assume that, based on the H-index/amount of haemoglobin, it is determined that a test result (going by the kit insert), will be affected, i.e analytical variation bias will be introduced. We now need to know whether this H-Index is below the threshold for clinically significant variation or not.
- This brings us to the concept of “Reference Change Value”. It is calculated per analyte. Lets take the example of K+ / potassium. Assume we prepare serially diluted hemolyzed aliquots of a plasma. We assign an H-Index/hemoglobin concentration to each aliquot. We proceed to measure the potassium on each aliquot. At a certain dilution , the value of potassium will have changed by an amount, that lies outside the “RCV”(the amount of change in a value that can be considered to be within normal clinical variation). The H-index at which this happens, is considered to be the clinically significant H-Index for potassium.
- So two set points of H-Index are established per analyte. An H-index above which there is analytical variation (this is easy to take from the manufacturer sheets). And another H-index at which we have established the analyte to deviate clinically significantly. Now the entire algorithm for reporting actions is based on where the H-index lies between these two set-points.
- If the H-index is lower than analytical cut-off : No problems, simply report the test.
- If the H-index is between analytical and clinical cut-off : Report with comment saying that recollect sample.
- If the H-index is above clinical cut-off OR > 10g/L , simply ask for a fresh sample, DO NOT REPORT.
Definition of Hemolysis
The following paper, defines hemolysis as free hemoglobin > 0.5 g/L.
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:
- Additive — as result of greater concentrations of analytes in cells than in plasma (e.g. potassium, phosphate, folate, lactate dehydrogenase)
- Spectral (haemoglobin absorbs at 415, 540 and 570 nm) — leading to interference with alkaline phosphatase, gamma-glutamyl transferase, total and direct bilirubin
- Chemical (cross-reactivity) — e.g. errors in creatine kinase due to red cell adenylate kinase
- Enzymatic — release of enzymes which degrade analytes (e.g. insulin)
- Dilutional (gross haemolysis releasing cell fluid and content) — leading to false lowering of results e.g. sodium, chloride etc.
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.
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.