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
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.
Arambarri M, Oriol A, Sancho JM, et al. Interference in blood coagulation
tests on lipemic plasma. Correction using n-hexane clearing. Sangre
StatSpin [package insert]. Norwood, Mass.: StatSpin Inc.
Tiffany O. Fluorometry, nephelometry and turbidimetry. In: Tietz
NO, ed. Textbook of Clinical Chemistry. Philadelphia, Pa.:
WB Saunders; 1986:78-97.
Dot Adcock, MD
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
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.
Robert S. Makar, MD, PhD
- Arambarri M, Oriol A, Sancho JM, et al. Interference
in blood coagulation tests on lipemic plasma. Correction using
n-hexane clearing. Sangre (Barc). 1998; 43: 13-19.
- NCCLS. Collection, Transport, and Processing
of Blood Specimens for Coagulation Testing and General Performance
of Coagulation Assays; Approved Guideline—Third Edition.
NCCLS document H21-A3. NCCLS: Wayne, Pa.; 1998.
Massachusetts General Hospital
Member, CAP Coagulation Resource Committee