Q & A
Q. What can you tell me about lipemic specimens pertaining to the coagulation section? Are additives available to break up the lipemia as there are in chemistry?
A. Lipemia refers to an abnormally high concentration of lipids in the blood. The lipids that cause the greatest interference in laboratory testing are large lipid particles, particularly lipoproteins and chylomicrons. These particles increase sample turbidity and interfere with instrument systems that function based on light detection or scatter.
Using optical clot detection methods, as is commonly used in determining prothrombin time and activated partial thromboplastin time, lipemia may result in artificial prolongation of clotting times. Lipemia interference is quite variable among instruments. Many coagulation instruments employ an optical detection, or OD, method to measure the optical density of a sample. If a sample is lipemic and the baseline OD is too high, the instrument will not report a result. If the baseline is within the tolerance limit for the instrument’s optics and the change in absorbance of a clot reaction can be detected, a result is reported. Even though a sample is lipemic, some institutions will report the instrument result as long as it can be duplicated within five percent. Mechanical or electromechanical means of clot detection are not affected by lipemia.
Methods such as turbidimetry and nephelometry, which measure scattered light, may also show interference by lipemia. Turbid samples cause attenuation of the intensity of light passed through a sample due to scatter, reflectance, or absorption. Turbidimetry is the measure of incident light beam attenuation through a sample, usually measured at 180° using a spectrophotometer. Nephelometry measures light scatter of insoluble antibody antigen complexes at a given angle, usually at 90°. Nephelometry and turbidimetry are used most commonly in the chemistry lab, but they may also be used for coagulation testing. Immunoturbidimetric methods are commonly employed for automated, quantitative D-dimer testing and von Willebrand factor activity and antigen. Interference by lipemia is dependent on the dilution of samples used in the reaction. In general, the higher the dilution that can be afforded, the lesser the chance of lipid-based interference. For example, samples diluted 1:20 may show lipid interference, which may be avoided using dilutions such as 1:400.
Ultracentrifugation is frequently used to clarify lipemic samples in the chemistry lab, as centrifugation at very high relative centrifugal force will cause sedimentation of large particles. There is concern, however, that when applied to coagulation samples, ultracentrifugation may result in sedimentation of fibrinogen or factor VIII when complexed to von Willebrand factor. This is because of the relatively large molecular mass of these protein complexes, which cause them to sediment at the high centrifugal forces required to clarify lipemic samples. Ultracentrifugation can be accomplished conveniently using the Airfuge ultracentrifuge (Beckman Coulter, Brea, Calif.).
A study we conducted at Kaiser Permanente Rocky Mountain Region Reference Laboratory compared APTT and PT results in very lipemic samples using a mechanical method of clot detection and a photo-optical method of clot detection following ultracentrifugation. These samples resulted in "no clot detected" using the photo-optical method. Although only about 10 very lipemic samples were evaluated, we found no difference in APTT and PT results when comparing the ultracentrifuged samples read by the photo-optical method and the mechanical methodology.
Other means to clarify plasma by adding exogenous materials to clear or digest the lipemic particles have not been well studied in samples for coagulation testing. A thorough literature search revealed only one referenced article, reported in Spanish. In this study, lipid was added to plasma samples such that results could not be determined using a photo-optical clot detection system. Subsequently, n-hexane was added to plasma samples to clear the lipid, and the resultant PT, APTT, and fibrinogen times were not significantly different from the values of the plasma samples before the Intralipid lipid was added. Adding Lipoclear reagent, a nonionic material, reportedly clears lipemia in 95 seconds when used in conjunction with a StatSpin centrifuge. However, we have not found any published data that compares Lipoclear-treated to untreated samples for coagulation markers. In practice, you would have to determine experimentally whether your coagulation analyte of interest is recovered from such pretreatment of lipemic samples. This would be best done using a spike-recovery experiment or using samples containing high concentrations of the analyte of interest.
One way to avoid grossly lipemic samples is to ask that patients fast for 12 hours before sample collection. If this is impractical, a mechanical-based means of clot detection should be available when samples are grossly lipemic.
Dot Adcock, MD Rajeev Ramanathan, PhD Esoterix Coagulation
Rajeev Ramanathan, PhD
A. Optical instruments used to detect clotting may have difficulty with interfering substances such as hemoglobin, bilirubin, and lipids. Little information regarding the handling of lipemic specimens is available. Extraction of lipids from blood specimens with n-hexane before coagulation testing has been described in the literature1 but is not common practice in clinical laboratories. According to the NCCLS guideline on processing blood specimens for coagulation testing,2 clotting of lipemic specimens ideally should be measured by mechanical or electromechanical methods.
At our institution, a specimen that is flagged as lipemic by our optical instrument (MDA 180, BioMérieux, Marcy l’Etoile, France) is visually inspected to see if it appears lipemic, with the lipids evenly distributed in the plasma phase, and to determine if its optical properties are within instrument specifications. If the specimen passes this review, the sample result is reported with a comment that lipemia is present. If the specimen fails this review, an aliquot is centrifuged in an Eppendorf microfuge at maximum speed to separate the lipid phase from the plasma phase. The cleared plasma sample is then again assayed by the optical instrument and the result reported with a comment that lipemia is present. If the lipids cannot be removed, the specimen is assayed with a mechanical instrument.
Massachusetts General Hospital
Member, CAP Coagulation Resource Committee