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Lipaemic samples: Effective process for lipid reduction

using high speed centrifugation compared with


ultracentrifugation
Original professional article:
Goce Dimeski*, Brock W. Jones. Lipaemic samples: Effective process for lipid reduction using high speed
centrifugation
compared
with
ultracentrifugation.
Biochemia
Medica
2011;21(1):86-94.

http://dx.doi.org/10.11613/BM.2011.016
Department of Chemical Pathology, Pathology Queensland, Princess Alexandra Hospital, Brisbane, Australia
*Corresponding author: goce_dimeski@health.qld.gov.au

Abstract

Introduction: Reducing laboratory errors and improving patient safety is receiving a lot of
attention. Lipaemic samples are cause of analytical errors and present challenges for
laboratories, particularly for those without ultracentrifuges. Lipaemia can originate from
physiological (postprandial metabolism), para-physiological causes (e.g. IV administration of
lipids) as well as metabolic disturbances (e.g. hypertriglyceridaemia).
Materials and methods: We have evaluated a procedure with 10 native lipaemic sample
pools (triglyceride concentration range 11.6-42.7 mmol/L) for the ability to reduce lipid
concentration using a high speed micro-centrifuge (double centrifuged at 21.885 x g for 15
min) compared with an ultracentrifuge, and provide accurate results. Results of sodium,
creatinine, urate, total protein, lactate dehydrogenase (LD), magnesium and, cholesterol and
triglyceride analysis on a Beckman DxC800 analyser are presented.
Results: Data from our tertiary level hospital showed ~0.7% of the samples received for lipid
studies have triglyceride levels > 10 mmol/L which can potentially cause analytical
interference. The mean differences from the neat aliquot to the ultracentrifuged and high
speed centrifuged sample pools were: cholesterol 4.9 mmol/L and 3.1 mmol/L; and
triglycerides 17.4 mmol/L and 15.0 mmol/L respectively. The data confirms high speed
centrifugation is almost as effective as ultracentrifugation in lipid reduction.
Conclusion: The procedure utilized in this study using a high speed micro-centrifuge showed
it is effective in reducing lipid levels and provides a suitable alternative to ultracentrifuged
samples to provide accurate results.
Key words: lipaemia; interference; ultracentrifugation; high speed centrifugation
Received: December 22, 2010
15, 2011

Accepted: January

Introduction
Improvement in patient safety through error reduction is rightfully receiving a great deal of
focus in medicine including pathology as it plays a pivotal role in medical decision making
(1). Published data states that up to 80% of patient care decisions are based on pathology data
(2), and in some cases pathology data is the sole information used in the clinical decision
process. With the continuously increasing number of tests, pathology will play even greater
role thus laboratorians will need to become actively integrated in the medical care team and
aid doctors with interpretation of laboratory results to reduce medical errors (3). Various

estimates have been provided on the sources and errors rates in chemical pathology. It is well
accepted of the three phases of the pathology testing cycle: the analytical phase is the least
error prone while the pre-analytical is the most error prone (4,5). A lot of these errors can be
linked to the analytical sample integrity of which lipaemia is a contributor. Even though
lipaemic samples are not frequently encountered, these samples continue to provide
challenges for a lot of laboratories, more specifically in establishing good practices for
processing lipaemic samples and providing accurate results for best patient outcomes (6).
The lipid induced interferences can originate naturally in the patient sample as a result of
health-related factors e.g. dietary, diabetes mellitus, alcohol abuse, hypothyroidism,
pancreatitis, drug-induced such as oral contraceptives etc (7), from lipid emulsions used for
nutritional need in critically ill patients such as newborns (8) and surgical patients (9), and in
treatment of lipophilic drug overdoses (10). A review of literature shows it is sparse on
lipaemic studies, and there was no published data indicating the extent of the problem. Unlike
haemolysis and bilirubin studies lipaemic studies are often left in the too hard basket. This is
because of the lack of readily available standardized materials like those used for bilirubin or
haemoglobin to mimic many of the properties of native lipaemic samples to produce the
lipaemic interferences complicates the evaluation of lipaemia (11). Lipaemia presents other
unique problems which include differences in the final lipid composition from patient to
patient, the volume of single native sample normally being too small to do various studies,
and samples not being able to be frozen for future studies. An additional complication is
increasing hyperlipidaemia is associated with increased haemolysis strawberry milk
appearance, possibly the result of increased erythrocyte membrane fragility induced by
alterations in membrane lipid content (12). Hence, artificial compounds i.e. lipid emulsion
e.g. Intralipid are used to perform lipaemic index and analyte interference studies by
manufacturers and laboratories. Lipid emulsions consist predominantly of small, relatively
dense, phospholipid-rich liposomes and triglyceride-rich artificial chylomicrons and no
complex mixture of lipoproteins (13). Thus, because of these composition differences there is
poor association between lipaemic index and triglyceride concentrations
(14,15). Triglycerides are usually the most abundant lipids in lipaemia.
Sample turbidity from lipaemia affects most significantly the photometric assays (end point,
rate, nephelometric or turbidimetric) due to increased light scatter and absorption of the light
by the lipids (mainly chylomicrons and very low density lipoproteins) (16), and this includes
coagulation assays (17). Furthermore, the extent of the effect will be dependent on the
sampling mechanism, the lipid composition of the sample and whether the sample is well
mixed to minimise chylomicrons settling out at the top of the sample. No blanking method
can overcome all the interferences from lipaemia. Such samples have the potential to equally
affect all assays including immunoassays by binding of lipophilic compounds (e.g. drugs) and
blocking/masking the binding sites with reagents to produce timely and measurable end
points (6). The increase in the non-aqueous phase leads to volume displacement errors or
exclusion effect with methods that do not measure activity of the analyte (e.g. indirect ion
selective electrodes used exclusively on laboratory analysers) (18). Unlike haemolysis the
interference of lipaemia can be reduced to eliminate the interference by removal of most of
the interfering lipids. Methods for removal include ultracentrifugation (the gold standard),
high speed centrifugation and lipid clearing agents e.g. LipoClear and n-hexane. The CLSI
Interference testing guidelines recommend clarifying the sample using ultracentrifuge (19)
but not many laboratories are equipped with such centrifuges. However, just about all
laboratories (large or small) possess high speed micro-centrifuges. We are aware many
laboratories use high speed micro-centrifuges for lipid reduction without having the
effectiveness of the procedure confirmed. One such study found the recovery from high speed

centrifugation (10.000 x g) of samples was unacceptable for total bilirubin and CRP (20). The
same study found LipoClear treatment produced unacceptable recovery with GGT, CK-MB,
total cholesterol, HDL-cholesterol and CRP (20). In this study we compare a procedure with
one such high speed micro-centrifuge with an ultracentrifuge for effectiveness in lipid
reduction and suitability for routine use in native lipaemic patient samples using a set of
analytes we have observed over years to be most affected by lipaemia on our Beckman
DxC800 analysers.
Methods and materials
Samples

The samples used in this study were received in several laboratories for routine analysis. The
samples were selected for the study based on the triglyceride level alone in order to cover the
highest triglyceride concentration range possible. The samples with similar triglyceride
concentration levels were pooled to provide sufficient volume for analysis: neat, post
ultracentrifugation and post high speed centrifugation. All samples were native serum, and no
samples from patients receiving nutritional lipid supplement (total parenteral nutrition
TPN) were used in preparing any of the pools. The samples of interest were stored at 2-8 C
and analyzed within two weeks of commencing the pooling to minimise any deterioration in
sample quality. To our observation over many years TPN samples containing lipid emulsions
do not exceed 20 mmol/L triglyceride concentrations, most times they are < 10 mmol/L. In
total 10 such sample pools were analysed for sodium (indirect ion selective electrode),
creatinine (Jaffe), urate (uricase)), total protein (biuret), lactate dehydrogenase (LD) (lactate
to piruvate), magnesium (calgamite), cholesterol (cholesterol esterase) and triglycerides
(glycerophosphate oxidase).
Samples were mixed by multiple inversions before analysis to eliminate the chylomicrons
and other large molecules from forming a layer at the top of the sample. The sampling probes
of analysers only descend 2-3 mm below the meniscus to aspirate the test aliquot, and if the
aliquot is aspirated from a sample that is not well mixed (lipid rich component) the
interference will be exaggerated (6).
Equipment
Each sample pool was split into three aliquots: neat, ultracentrifugation and high speed
centrifugation. The ultracentrifugation was performed in a Beckman Coulter Airfuge for 15
minutes at 107.000 x g. The lipid reduced aliquot was then carefully transferred without
disturbing the lipid layer into an appropriate aliquot tube for analysis. The high speed
centrifugation was performed in a Heraeus Biofuge Primo micro-centrifuge for 15 minutes at
21.885 x g in two Micro tubes (1.5 mL Ref 72.690.001 Sarstedt). The lipid cleared sample,
infranatant (lower part of the sample) was transferred into clean Micro tubes using glass
pipettes by first expelling the air out of the pipette and then immersing it to the bottom of the
Micro tube to aspirate ~2/3 of the sample/infranatant and prevent any bubbles being released
during the pipette descent through the supernatant and re-mixing the sample. Furthermore,
aspiration was carried out slowly to minimise intermixing between the lipid poor infranatant
and lipid rich supernatant layers. This procedure was repeated post re-centrifugation, and
~2/3 of the clear infranatant was aspirated from the two Micro tubes into an appropriate
aliquot tube for analysis.
The neat, ultracentrifuged and high speed centrifuged aliquots were analysed on a Beckman
DxC800 general chemistry analyser (Beckman Coulter, Fullerton, CA, USA) at the same time

on the same analyser in the same rack and the same order for each sample pool. The total
analytical imprecision expressed by the between-run coefficient of variation (CV) for the
analytes was as follows: Na+ (1.0% at 132 mmol/L and 0.9% at 150 mmol/L), creatinine
(4.9% at 68 mol/L and 1.8% at 491 mol/L), urate (1.9% at 0.23 mmol/L and 1.5% at 0.49
mmol/L), total protein (1.8% at 41 g/L and 1.6% at 67 g/L), LD (2.7% at 156 U/L and 2.3%
at 432 U/L), Mg (2.5% at 0.84 mmol/L and 2.3% at 1.63 mmol/L), cholesterol (1.7% at 3.0
mmol/L and 2.3% at 6.5 mmol/L) and triglycerides (3.6% at 1.0 mmol/L, 2.7% at 2.0 mmol/L
and 0.5% at 8.3 mmol/L).
Statistical analysis
The mean and standard deviation were calculated for each analyte for each of the three
aliquots. From the imprecision data the least significant change (LSC) (2.77 x analytical
coefficient of variation) was calculated to determine the maximum allowable difference in
test results between the different aliquots.
Results
A search of our laboratory information systems showed very small percentage of all the
samples requested for lipid analysis had elevated lipids. Currently only ~0.7% and 0.2% of
samples requested for lipid studies have triglyceride concentrations > 10.0 mmol/L and >
20.0 mmol/L respectively in our tertiary level referral hospital.
The procedure evaluated in this study using the Biofuge Primo high speed micro-centrifuge
showed it is capable of providing a sample with significantly decreased lipid levels close to
those obtained by ultracentrifugation (Table 1). The mean differences from the neat aliquot
for the ultracentrifuged and high speed centrifuged sample pools were: cholesterol 4.9
mmol/L and 3.1 mmol/L; and triglycerides 17.4 mmol/L and 15.0 mmol/L respectively. The
data confirms when several re-centrifugation steps are performed high speed centrifugation is
almost as effective as ultracentrifugation in lipid reduction. Although pools one, two, three
and eight had triglyceride levels > 10.0 mmol/L, no analyte results were suppressed due to
interference. The removal of the majority of the chylomicrons during the high speed
centrifugation is likely to have removed of the interference.
Table 1. Comparison of cholesterol and triglycerides concentrations decrease post ultracentrifugation and high
speed centrifugation.

Table 2. Results of analytes affected by lipaemia compared between neat, ultracentrifuged and high speed
centrifuged aliquots.

Apart from the urate result, all other results from the tested analytes indicate the difference
between the high speed centrifuged and ultracentrifuged aliquots were within the LSC limits
(Table 2). The total protein results in the neat sample aliquots were suppressed, no result was
obtained due to interference.
Discussion
The data indicates < 1% of samples requested for lipid studies contain high enough
triglyceride concentration (> 10 mmol/L) to potentially cause interferences in laboratory
assays. When compared with the total number of samples received in the laboratory for
biochemical analysis this represented one per ~4100 for the 2009-2010 period. For this study
even with the aid of several other large laboratories outside our network it took more than a
year to accumulate sufficient volume of fresh samples to prepare the 10 pools.
The lipaemic index along with the cholesterol and triglyceride levels data in Table 1 indicates
the high speed centrifugation process evaluated here is effective in lowering lipid levels to
allow sample analysis and produce results that are within LSC limits as confirmed in Table 2.
Although the urate difference is outside the LSC limit, it is not considered clinically
significant, < 10% difference (21). The effectiveness of the process in obtaining the clearest
sample will also depend on the lipid levels and technique of aspirating the infranatant. For
samples with higher lipid levels (e.g. triglyceride levels > 50.0 mmol/L) 3 or 4
centrifugations may be necessary provided there is sufficient sample available. Perhaps a
request to the clinical unit to collect more blood tubes may overcome such a problem. This
high speed centrifugation procedure appears to be superior to the one utilized by Vemeer et al
(20), and this can be attributed to the much higher centrifugation speed and the double
centrifugation process used in our study. A limitation of the study is not having performed a
wider range of analytes due to insufficient sample volume (e.g. immunoassays) and suitable
sample type (e.g. coagulation tests).
The Beckman DxC800 analyser measurable lipaemic index range is 0-10. Three of the pools
with triglyceride levels > 30.0 mmol/L exceeded the upper limit, which made the lipaemic
index less suitable than the triglyceride level for lipaemic interference studies. Review of the
Beckman individual method inserts provides interesting information. For sodium, urate and
LD the lipid interference studies used Intralipid concentration up to 500 mg/dL (in terms of

triglycerides this equates to ~5.6 mmol/L), and it is reported there was no significant impact
on any of these analytes. Our data showed these analytes are affected when the triglyceride
concentration increases. For creatinine, it is stated lipaemic index up to 8 produced no
significant impact, whereas with magnesium Intralipid concentration of 150 mg/dL produced
an increase of ~0.16 mmol/L, and for total protein lipaemic index of 4 produced a decrease
by 0.4 g/L, which is in agreement with our findings. Simply there is no standardized process
with the lipaemic interference studies, and no triglyceride levels are provided for more direct
comparison with our findings.
Native lipaemic samples in hospital environments most often originate from emergency
departments, diabetic endocrinology and gastrointestinal units, lipid clinics etc which often
require results urgently. To minimise delays in turn-around times of critical analytes such as
sodium we analyse lipaemic samples first on blood gas analysers (bench top or portable) and
report the electrolytes while the sample is ultracentrifuged. Most laboratories rely on the
lipaemic index and manufacturer method recommendations for acceptable limits that are
almost always established by using emulsions spiked samples in performing interference
studies to determine if a sample will undergo ultracentrifugation or treatment. Others rely on
measuring the triglycerides level, the most abundant lipid component in lipaemic samples
before making a decision as how the sample will be processed because emulsions do not
behave in the same way as native lipaemic samples. Many other laboratories use a
combination of the two plus visually inspect the turbidity level, and this may be impractical
in laboratories with large numbers of samples. It should be pointed that different analytical
methods use different reagents and wavelength parameters, and will be affected differently by
lipaemia levels and other non-lipaemic causes which includes lipaemic index methods. For
example in most samples with triglyceride concentration > 11.0 mmol/L total protein on the
Beckman DxC800 analysers will fail to produce a result. The Beckman lipaemic index
method has been reported to produce high values due to non-lipaemic causes, precipitation of
paraproteins (IgM kappa and lambda) in the Beckman serum index diluent (22). This requires
samples with elevated lipaemic levels to be visually inspected for true lipaemia before
reporting any results including the lipaemic index. In our experience this false high lipaemic
index values in samples that are not visibly lipaemic can be correctly obtained by the use of
normal saline as the index diluent.
Our practice with lipaemic samples presenting for the first time is to have lipids automatically
performed even if they have not been requested by agreement with the hospital as a proactive
service to aid clinicians with accuracy in patient care decision. Such lipaemic samples are
indicative of serious and clinically important patho-physiological changes. This is now a
standard procedure throughout our entire network of 33 laboratories servicing all the public
hospitals in our state. Results reported from ultracentrifuged or lipid reduced samples are
appropriately annotated to ensure clinicians are aware highest quality results have been
reported. However, it is not advocated every laboratory implement such steps until it is
authorized by laboratory management and clinical governance bodies of their hospital or
health care institution.
One final point is the high speed centrifuges are not recommended for centrifugation of
whole blood samples but serum or plasma only. Ideally laboratories will need to validate this
procedure with there respective micro-centrifuges.
Conclusion

Not every laboratory can afford or will be equipped with an ultracentrifuge for the very small
number of lipaemic samples that may be received per year. The procedure utilized in this
study using a high speed micro-centrifuge showed it is effective in reducing lipid levels and
produce a suitable alternative to ultracentrifuged samples to provide accurate analytical
results. This procedure may serve as a standardized guideline in laboratories with high speed
micro-centrifuges to improve the handling of lipaemic samples and minimise analytical
errors.
Acknowledgement
We thank Robert Flatman and his staff, Sullivan and Nicolaides Pathology, Brisbane and
Barbara Mottram and her staff, Mater Pathology Services , Brisbane for supplying lipaemic
samples.
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Lipemia interferences in routine clinical biochemical tests


Original article:
Pilar Calmarza1*, Jos Cordero2. Lipemia interferences in routine clinical biochemical tests. Biochemia
Medica 2011;21(2):160-6. http://dx.doi.org/10.11613/BM.2011.025
1

Laboratory of Clinical Biochemistry, University Hospital Miguel Servet, Zaragoza, Spain


Research Unit, Hospital General Yage, Burgos, Spain.
*Corresponding author: mpcalmarza@salud.aragon.es
2

Abstract
Introduction: Lipemic specimens are a common and frequent, but yet unresolved problem in
clinical chemistry, and may produce significant interferences in the analytical results of
different biochemical parameters.
Material and methods: The aim of this study was to examine the effect of lipid removal
using ultracentrifugation of lipemic samples, on some routine biochemistry parameters.
Among all the samples obtained daily in our laboratory, the ones which were visibly muddy
were selected and underwent to a process of ultracentrifugation, being determined a variety of
biochemical tests before and after ultracentrifugation. A total of 110 samples were studied.
Results: We found significant differences in all the parameters studied except for total
bilirubin, glucose, gamma-glutamyl transferase (GGT) and aspartate aminotransferase (AST).
The greatest differences in the parameters analyzed were found in the concentration of
alanine aminotransferase (ALT) (7.36%) and the smallest ones in the concentration of glucose
(0.014%). Clinically significant interferences were found for phosphorus, creatinine, total
protein and calcium.
Conclusion: Lipemia causes clinically significant interferences for phosphorus, creatinine,
total protein and calcium measurement and those interferences could be effectively removed
by ultracentrifugation.
Key words: lipemia; ultracentrifugation; biochemical tests; interferences
Received: March 7, 2011
8, 2011

Accepted: May

Introduction
There is firm evidence that results of laboratory tests provide a substantial contribution to
clinical practice. While laboratory errors are traditionally identified with analytical problems,
an extensive scientific literature now attests that most errors (up to 80-90%) seem to occur
from the extra-analytical phase.
According to the most representative studies, preanalytical errors represent more than half of
the total errors which occur in the clinical laboratory (1,2) and within this type of errors, for
its importance, it is remarkable the quality of the samples to analyze (3,4). Thus, the
hemolysis, hyperbilirrubinemia and turbidity of serum can greatly affect the accuracy of
many laboratory tests (5).

The establishment of the concentration of lipids which produce significant interference


depends on the analyzer, reagents, analytical method and the concentration of interfering
constituents that we are measuring (6).
Lipemic sera are often found in the practice of clinical laboratories and can cause significant
interferences in the analytical results of different biochemical parameters (7,8). The most
common causes of the occurrence of lipemia are diet, alcohol ingestion, diabetes mellitus (9),
hypertryglyceridemia, chronic renal failure, hypothyroidism, pancreatitis, multiple myeloma,
primary biliary cirrhosis, lupus erythematosus, total parenteral nutrition (10), drugs such as
protease inhibitors (HIV infection), estrogen, oral contraceptives, etc.
Lipemia may interfere with tests which use transmission of light as part of their measurement
system. The interference caused by lipemia is due mainly to three distinct mechanisms: light
scattering, increasing non-aqueous phase and effects of partition between polar and non polar
phases (7).
Regarding routine clinical chemistry analyzers, the partitioning effect is the least frequent
problem of the three potential mechanisms for interference.
The increase in non-aqueous phase will affect all methods that do not measure the activity of
the analyte and leads to volume displacement errors, by reducing the water available in the
volume of the sample. This is important because more analytes are dissolved in the aqueous
phase of serum/plasma.
Lipemia interference is also due to increased light scatter and the absorption of the light by
the lipids (mainly chylomicrons and very low density lipoproteins) in the spectrophotometric
methods. This phenomenon causes a decrease in the intensity of light reaching the solution,
which will be absorbed (11), so the turbidity most likely affects the photometric methods than
the non-photometric methods. Both chylomicrons and VLDL particles produce this
phenomenon, but in both cases the particles are very heterogeneous and there is an enormous
variation in their size and triglyceride content, so the direct measurement of triglyceride
content does not show good correlation with the phenomenon of light scattering. Moreover,
the turbidity of the samples was very weakly correlated with the concentration of
triglycerides present in the sample (12,13). Due to the heterogeneous nature of lipemia there
are difficulties in the simulation in the laboratory of lipemic samples, and presently there are
no standardized materials that simulate lipemia adequately. Glick and collaborators (6) used
already a synthetic emulsion for intravenous administration that is known to simulate lipemic
samples and is called Intralipid, but Bornhorst et al. (14) in a subsequent investigation made
it clear that we must be very cautious in interpreting results interference studies in which
Intralipid was used to simulate lipemia, since these solutions can not accurately mimic
lipemia. This is so because both VLDL and chylomicrons represent a heterogeneous group of
particles that vary greatly in size and number, between individuals and also as to their content
of triglycerides (15). The differences in the size of the particles in lipemic samples can
greatly affect the effect of light scattering and the turbidity (15,16). Neither a single
concentration may provide enough results so it is advisable to use a wide range of
hyperlipidemic samples.
In the present study, sera with a high content of triglycerides (visibly turbid), but in different
concentrations were used and these samples were subjected to an ultracentrifugation process
to clarify the samples. Ultracentrifugation separates lipid complexes, preferentially larger,
less dense (chylomicron) and VLDL particles, being both located on the top and we have
determined the concentration of the different routine biochemistry parameters in the samples
before ultracentrifugation and in the infranadant obtained after ultracentrifugation.

Materials and methods


Samples
This study was performed in the Biochemistry laboratory of the Hospital General Yage of
Burgos (Spain), from January to June of 2009. This is a tertiary hospital that receive on
routine analysis about 500-600 samples daily of which 3-4 samples are visibly turbid (0.60.7%).
Among all of these routine samples, we selected the samples which were visibly turbid and
after their centrifugation (1.500 x g for 15 minutes) three aliquots were obtained, one of
which was used to determine appropriate parameters, another was further subjected to the
ultracentrifugation process, after which the same biochemical determinations from the
previous case were repeated. The third aliquot was frozen at -80 C for possible checks. A total
of 110 samples were studied, although in some of them there was not enough amount of
sample
to
perform
the
biochemical
analysis
of
all
the
different parameters. The process by which samples were subjected involved
ultracentrifugation at 40.000 x g and +4 C, without adjustment of density (d = 1.006 kg/L)
for 18 hours in a Centrikon T-1080 Ultracentrifuge (Kontron AG, Switzerland).
Ultracentrifugation can achieve greater speed of rotation than high speed centrifugation, and
therefore generate higher centrifugal forces.
Methods
The supernatant was carefully separated and biochemical determinations were made in the
infranatant. The analytical parameters determined (in the first aliquot and in the aliquot
subjected to ultracentrifugation) were the following: cholesterol, triglycerides, alkaline
phosphatase (ALP), gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST),
alanine aminotransferase (ALT), lactate dehydrogenase (LD), total bilirubin, total calcium,
creatinine, phosphorus, glucose, iron, urea, uric acid and total protein in a Hitachi Modular D
and P (Roche Diagnostics, Mannheim, Germany).
Analytical methods employed for each of the biochemical parameters that have been determined are presented
in table 1. The maximum number of samples analyzed was 110 for Uric acid, Urea and Total Protein and the
minimum
number
was
93
for
Iron.
Table 1. Analytical methods used in the D, P Modular Analyzer (Roche Diagnostics, Mannheim, Germany)

Statistical analysis
Normality was tested for each variable. Variables that were distributed normally were presented
with arithmetic mean standard deviation. Variable (triglycerides) that was not distributed
normally was presented with median and interquartile range.
To compare the average values of the various parameters measured before and after being
subjected to ultracentrifugation parametric Student t test for paired data (for normally
distributed data) and nonparametric Wilcoxon test (for triglycerides) were used.
Percentage of change was calculated for each analyte before and after centrifugation and
compared to desirable inaccuracy according to data published in the literature (17).
Statistical analysis was performed using SPSS software (SPSS Inc., Chicago, Illinois, USA).
Results
The results of the various serum parameters measured by spectrophotometric methods before
and after samples were subjected to ultracentrifugation are presented in Table 2.
In this table the mean concentration before and after ultracentrifugation and the number of
specimens analyzed is expressed as well as the median, the standard deviation or Q1-Q3 for
each parameter, the percentage change in the mean values after samples being subjected to
ultracentrifugation, the intraindividual variation coefficient and the desirable inaccuracy for
each of the parameters, according to data published in the literature (17).
Aside from the expected changes in the concentration of triglycerides (20.67%) and
cholesterol (7.50 %), the greatest difference in the parameters analyzed was found for ALT
(7.36%). Minor changes were found in the concentration of phosphorus (5.20%), creatinine
(4.25%), GGT (3.04%), urea (2.24%), iron (1.98%), ALP (1.88%), total protein (1.75%), uric
acid (1.47%), total bilirubin (1.24%), calcium (1.16%) and AST (1.14%). Glucose with less
than 1% difference was the least affected parameter.

When the values of various parameters measured before and after ultracentrifugation were
compared, significant differences were observed in all cases except for total bilirubin (P =
0.881), GGT (P = 0.326), glucose (P = 0.972) and AST (P = 0.609).
In the rest of the parameters we obtained significant differences with a significance level of P
<0.001 except for calcium which had a significance level of P < 0.005 and urea with P < 0.05.
Discussion
In this study we have found significant differences before and after ultracentrifugation in
lipemic sera in all parameters studied except for total bilirubin, GGT, glucose and AST.
Studies by Brady et al. and Jabbar et al. show that the majority of analytes are affected by
hyperlipidemia (18,19). In this sense, The Committee for the Implementation of the French
Society of Clinical Biology (SFBC) (20) decided to study the interference problems caused
by visible interferences - bilirubin, hemolysis and turbidity, and their effect in 20
biochemical tests (13 of which were substrates and 7 enzymes). These parameters were
measured in the 15 Autoanalyzers more representatives of those found on the French market.
To cause turbidity in the serum, Intralipid was added. The different tests were not affected
with equal intensity. Substances with higher interference by turbidity were, in this order, uric
acid, iron, total protein and bilirubin.
Also, Steen et al. (21) conducted a multicenter analysis of 16 Dutch clinical laboratories,
evaluating the interference caused by hemolysis, hyperbilirubinemia and lipemia in the
determination of 32 different analytes. On the basis of biological variation these authors
suggested a cut-off value above which, clinically significant interference exists. They found
clinically significant interference from lipemia in 12 of the 32 analytes studied. The
experiment was carried out also with test samples from a pool spiked with 10% Intralipid.
An important concept to consider when we refer to interference in biochemical analysis is
that we must differentiate between analytically significant interferences and clinically
significant interferences. The first are those that according to the IUPAC (International
Union of Pure and Applied Chemistry) produce in an analytical procedure a systematic error
greater than three times the standard deviation found in a study of precision for a given
concentration of the component under study (22) and produce a systematic error greater than
the one justified by the imprecision or the uncertainty of the method.
The clinically significant interferences are those that generally lead to significant errors in
the interpretation of laboratory results. These interferences should be considered as such
when, in relative terms, the value of the interference is greater than half the intraindividual
biological coefficient of variation (17,23).
According to Glick the differences between the samples with or without interference must be
below 3%, when assessing the result of a sample for a short term follows up, and to
monitoring a patient for a long-time this author can accept a maximum range equal to the
objective of inaccuracy of the method, except for ALT, CK and GGT in which the change
should not exceed 10%. When the result is to be used in the screening of population it is
consistent to choose the total error as the limit of permissiveness, except for GGT and
triglycerides in which is preferable to choose higher standards.
In the study under consideration the differences between the two measurements, native and
ultracentrifugated samples did not exceed 10% and the total maximum error allowed has not
been exceeded in any of the techniques.

We found clinically significant interferences according to biological coefficient of variation


and objectives of inaccuracy (17) for the following parameters: phosphorus, creatinine, total
protein and calcium (Table 2).
Table 2. Variation in the concentration of the different parameters measured by spectrophotometry before
and after samples were subjected to ultracentrifugation

Methods for removal lipids of the samples include ultracentrifugation (the gold standard),
high speed centrifugation and lipid clearing agents, mainly Lipoclear. Vermeer et al. (24)
compared reducing lipemia by high speed centrifugation or treating sera with Lipoclear and
irrespective of the methodology used found excellent recovery in most of cases, but using high
speed centrifugation the recovery was unacceptable for total bilirubin and CRP and using
Lipoclear the recovery was inacceptable for GGT, HDL cholesterol, cholesterol and CRP.
On the other hand, Anderson et al. (25) using a clarifying agent Lipoclear, found no critical
differences in the concentration of the analytes studied before and after treatment with
Lipoclear, except in the concentration of total protein, phosphorus and an expected fall for
cholesterol and triglycerides.
Moreover, there are too few studies using native lipemic samples, but Dimeski et al. (26) in a
recent study evaluated a procedure with 10 native sample pools for the capacity to reduce
lipid concentration using a speed micro centrifuge compared with an ultracentrifuge. It
appears that the high speed centrifugation procedure used by Dimeski et al. (26) is superior to
the one utilized by Vermeer et al. (24), perhaps by the higher centrifugation speed and the
double centrifugation process used by Dimeski. Data obtained by Dimeski et al. (26) confirm
that when several re-centrifugation steps are performed, high speed centrifugation is almost as
effective as ultracentrifugation in lipid reduction.
The main limitation of our study is that we have used ultracentrifugation to reduce lipemia
and many laboratories do not have ultracentrifuges, moreover this is a very long procedure
for treatment of samples that come, in many occasions, from Emergency Services.
Further studies are needed that compare ultracentrifugation methods with high speed
centrifugation methods and the use of Lipoclear. It is necessary to correlate these methods

with serum indices obtained in the autoanalyzers (based on alternative photometric


wavelength measurements). This is so because most laboratories rely on the lipemic index
and manufacturer method recommendations for acceptable limits that are almost always
established by using emulsions spiked samples, and emulsions do not behave in the same way
as native lipemic samples.
Conclusion
Although the percentage change in the concentration of different analytes before and after
ultracentrifugation in hyperlipidemic sera never exceeded the total allowable error, significant
differences in all parameters were found except for total bilirubin, glucose, GGT and AST
and a variation that exceeds the allowed desirable inaccuracy and therefore clinically
significant interferences in phosphorus, creatinine, total protein and calcium.
This gives an idea of the importance of proper treatment of lipemic samples and the
significance of interferences in preanalytical phase and in the whole process of biochemical
analysis of serum samples.
Potential Conflicts of Interest: None declared.
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