Test result interference

 

Inaccurate laboratory test results may be due to sample collection (extra laboratory) or due to incorrect laboratory methods. This information concentrates on the problems encountered with suboptimal samples received in the laboratory and inaccurate laboratory results that most commonly originate from interference caused by lipaemia, haemolysis and icterus as well as other causes such as insufficient sample volume and changes caused to molecules due to drug therapy. Interfering substances such as lipids, free haemoglobin and bilirubin will affect the blood results. These substances cause analytical variables as they directly interfere with test performance and results in various ways and these variables are method dependent.

Chemistry tests

Haemolysis interferes with chemistry tests by increasing the absorbance of the light in the spectral range. Many of the chemical reactions are based on measuring a change in absorbance around the optical density of haemoglobin. This can be offset by using kinetic reactions (which measure the rate of change in absorbance) or blanked endpoint reactions (which are the most affected by haemolysis). Free or released haemoglobin can directly inhibit chemical reactions.

The release enzymes found in high concentrations in red blood cells will falsely increase the values of test parameters such as potassium, AST, LDH, magnesium and iron. It is also possible that phosphate can increase with haemolysis as organic phosphates are converted to inorganic phosphates over time with storage. The release of enzymes which participate in chemical reactions can also increase enzyme activity in serum e.g. CK activity.

Haemolysis alters results by diluting serum constituents thereby lowering their serum values. It also interferes with colorimetric quantification based on protein binding or formation of coloured complexes.

When using a refractometer to determine protein levels the haemolysis blurs the line in the refractometer, making it difficult to read. Haemoglobin, as a protein, may also contribute somewhat to the refractive index measurement.

Haematology tests

Haemolysis interferes with haematology tests due to lysis of red blood cells which causes light scatter when analysed by the instrument. This affects the Haematocrit (HCT), packed cell volume (PCV) and red cell count as these values will be falsely decreased.

Automated analysers deliberately lyse RBC to measure haemoglobin and therefore the haemoglobin measurement is the same with or without haemolysis of the sample. Haemolysis causes an elevated haemoglobin reading relative to the HCT and RBC count and parameters related to these measurements such as mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC). These may be falsely elevated and may be inaccurate. The mean cell volume (MCV) will also be inaccurate if the value is calculated using the PCV. In some instances the analyser automatically measures the MCV.

The Platelet count is also affected by haemolysis as ghost RBCs may be counted by the analyser as platelets, falsely increasing the platelet count.

Lipaemia

Lipaemia in blood samples is caused by the increase in triglycerides and are usually post-prandial and can be minimized or avoided by collecting blood from a fasted animal (minimum of, 12 – 14 hours fasting). Lipaemic samples appear milky and fatty similar to a milk shake in appearance due to the high lipid levels in the sample. Lipaemia in a fasted animal indicates the presence of underlying diseases such as diabetes mellitus, pancreatitis, hepatic lipidosis (horses), neoplasia (horses).

Lipaemia interferes with chemistry tests by affecting the light scatter of the molecules leading to increased absorbance readings of some of the analytes, particularly those that are endpoint reactions e.g. total bilirubin. This results in false high concentrations of bilirubin.

The displacement of molecules and therefore the volume may falsely decrease values of some analytes such as electrolytes e.g. sodium chloride and potassium.

Lipaemia in a sample causes haemolysis of erythrocytes. This can affect results of individual tests e.g. the haemoglobin will absorb at wavelengths used to detect reactions in the analyser leading to incorrect results.

The laboratory will try to minimize changes in the results due to lipaemia by refrigerating and centrifuging the plasma/serum sample at high-speed. A fat layer will form on the top of the sample. The plasma below the fat layer is aspirated from the tube and used for analysis.

Chemical clearing methods are unreliable and may affect results in other ways and therefore they are not generally used to clear lipaemic samples. Note that triglyceride measurements must be performed on unadulterated samples, otherwise values will be falsely decreased.

Haemolysis

Haemolysis is usually an in vitro artefact due to poor venepuncture technique, lipaemia, freezing of whole blood samples, delayed separation of serum or plasma from cells, delayed sample submission and certain anticoagulants e.g. fluoride-oxalate. Red blood cells are also more fragile in lipaemic samples and tend to lyse more readily, even if the blood is stored or handled correctly. Haemolysis can occur intravascularly with certain types of heamolytic anaemias caused by infection for example.

Icterus/Jaundice

Icterus has minimal to no effect on haematology results, including plasma protein measured by refractometer. It may interfere with assays such as albumin, cholesterol, glucose and total serum protein when tested by auto analysers. Bilirubin interferes with the spectral properties as it reacts chemically with other reagents resulting in decreasing values particularly of creatinine concentrations in serum. Drug Therapy affects chemical tests resulting in false high or low values. It is advisable to include any medication that the patient is receiving in the history when submitting a sample to the laboratory.