A commentary on the effect of lipid emulsions on pathology tests (2009)

Lipaemia is a well recognised cause of interference with a wide range of laboratory tests. It causes analytical challenge for all laboratories irrespective of the lipid origin (native or commercial lipid preparations). The most commonly reported interferences are with optical methods and indirect ion selective electrode (ISE) systems. The mechanism of interference in optical methods: end point, rate, nephelometry, turbidimetry and coagulation tests is due to the scattering of light rays away from the original path and absorption of the light by the lipids (mainly chylomicrons and very low-density lipoproteins) [1]. In general the observed effect is negative: false decrease in concentration of the analyte being measured. The concentration of lipids where interference may begin to be observed is method and instrument dependent. For example, lipid concentrations do not have to be grossly elevated to prevent any results being attainable as Arambarri et al. found in samples with an elevated concentration of triglycerides > 9.79 mmol.l−1, the ACL-3000 analyser (Instrumentation Laboratory UK Ltd, Warrington, Cheshire, UK) failed to give any coagulation results [2] Other plausible interference mechanisms include lipophilic analytes, reagents not being released freely for reactions or analytes being prevented from binding with reagents to produce timely and measurable end points. The increase in non-aqueous phase (lipids or protein) will affect all methods that do not measure activity of the analyte. Lipids decrease the apparent concentration of the analyte by reducing the aqueous component of the sample, since the volume taken by lipids in plasma or serum is included in the calculation of the analyte’s concentration. This results in artefactually lower electrolyte concentrations in lipaemic serum or plasma samples when measured by indirect ISEs (e.g. Roche ISE system, Roche Diagnositcs Ltd, Mannheim, Germany), in contrast to direct ISEs (blood gas analyser). With indirect ISE, a fixed volume of the sample is aspirated and mixed with a diluent (Roche system 1:31 sample:diluent ratio) for analysis, which means the electrolytes are measured in the total volume of plasma, not just the plasma acqueous phase, as is with direct ISE. In normal plasma samples, solids represent approximately 7% of the total volume. Indirect ISE systems are standardised using normal concentration of solids. When the concentration of the solids in plasma is significantly changed (> 7 or < 7%) the ratio of solids to water alters, thus the discrepancies occur between direct and indirect ISE measurements. The indirect ISEs are able to measure a greater concentration range, and can be used for electrolytes not only in plasma and serum, but also other body fluids e.g. urine. The direct ISEs are almost exclusively used in point-of-care systems e.g. blood gas analysers or whole blood analysers. With direct ISEs there is no sample pre-dilution, and these are not affected by variation in concentration of the solids (lipids and proteins). The methods are more accurate, especially when the concentration of solids is further outside the usual reference interval [3]. The limitation of this technique is a narrower measuring range, e.g. the Siemens Rapidlab (Siemens Diagnostics, Medfield, MA, USA) 1265 blood gas analyser measuring ranges are: Na+ 70–200 mmol.l−1; K+ 0.5–20.0 mmol.l−1 and Cl− 40–160 mmol.l−1. The effects of increased lipids on Na+, K+, and Cl− on indirect ISEs compared with direct ISEs and with ultracentrifuged (lipid levels normalised) samples are linear and can be estimated by regression equations. Hyperlipidaemia was demonstrated to decrease Na+ and Cl− by 1 mmol.l−1 and K+ by 0.04 mmol.l−1, respectively per 10 mmol.l−1 total lipids (cholesterol plus triglycerides) on the Roche ISE system [4]. Chlyomicrons and other larger lipid molecules tend to separate on standing, forming a layer at the top of the specimen. Analyser probes only descend a few millimetres below the meniscus to aspirate aliquots for tests. Thus, if the sample is inadequately mixed the probe will aspirate from the lipid rich layer and interference effects will be exaggerated. The manuscript results suggest that the elevated serum lipid content is the causative factor, yet the link is unclear. In my experience, serum lipid concentrations from native samples, or from commercial lipid preparations, have never been high enough to completely prevent measurement of electrolyte concentrations by blood gas analysers, as are demonstrated in these cases. To demonstrate the lipid effect, pooled serum was mixed in different ratios with commercial lipid preparations: Clinoleic 20% (Baxter Healthcare Ltd., Thetford, Norfolk, UK), which is what used in my hospital (Table 1). The samples were analysed on a Beckman D×C800 (Beckman Coulter Diagnostics, Fullerton, CA, USA), a Roche Modular DP (Roche Diagnostics, Mannheim, Germany) and Siemens Rapidlab 1265 blood gas analyser (Siemens Diagnostics, Medfield, MA, USA). Aliquots of the sample preparations were also ultracentrifuged (Airfuge; Beckman Coulter Diagnostics, Fullerton, CA, USA) for 15 min at 107 000 g to provide lipid normalised aliquots. The results clearly indicate patient samples need to be significantly diluted by lipid preparations before unmeasurable results will be encountered. The linearity of both the Beckman and Roche triglyceride assays is ∼ 11.3 mmol.l−1. The Clinoleic lipid emulsion was diluted with normal saline, and the final triglycerides concentration was ∼ 430 mmol.l−1 (Beckman D×C800). Although the ultracentrifuged pooled serum/lipid emulsion aliquots appeared very clear, like the pooled serum, the triglycerides are not completely removed. Ultracentrifugation is very effective in removing only the large lipid molecules such as chylomicrons and very low-density lipoproteins. With the smaller chain fatty acids, a smaller percentage is removed by ultracentrifugation, hence resulting in unmeasurable triglycerides levels. Alternatively, during the aspiration of the infranate (lipid reduced component), some of the lipid rich component can be aspirated and contaminate the infranate. This is most likely to occur in grossly lipaemic samples, and such samples may need re-ultracentrifugation. From the data in the manuscript and the results in Table 1, there are two possible explanations for the ‘no results’ scenario and both are linked to sample collection procedures. The first consideration is that the samples were collected from the drip arm, rather than the opposite arm, or appropriate procedure was not followed, discontinue the IV infusion for set time such as 10 min, discard adequate blood volume (∼ 5 ml), and then collect samples for analysis. The manuscript results are suggestive of Intralipid constituting > 50% of the total sample. This is supported by the fact that no results were obtained for the electrolytes on the blood gas analyser. However, as the blood gas analyser model is not indicated, it is not possible to verify the measuring range for the electrolytes on the analyser used in the authors’ laboratory. If the Intralipid constitutes > 50%, the serum lipid (it is assumed this is the triglyceride level) might have been incorrectly estimated, that is the results obtained need to be checked for flags e.g. ‘20.7 LIMTH’ along with the triglyceride reaction curve. If the results did display any flags, the sample should be diluted to bring the triglycerides concentration into the measurable range (< 11.3 mmol.l−1), and the final results correctly multiplied. The second consideration is that if there were no flags with the triglycerides results, the reaction curve for the triglycerides was correct, and results were properly multiplied for the dilution factor, then the artefactual effect on the analytical results is unlikely to be solely due to Intralipid interference. It would be important to determine whether other IV fluids were infused at the same time, resulting in further dilution of the Intralipid/blood samples, bringing the concentration of the analytes below the measurable range, and decreasing the serum lipid concentration down to the published levels. To confirm the effect on paracetamol, pooled serum containing paracetamol was prepared with one part Clinoleic or saline and 4 parts of the pooled serum. The results were: 1/5 Clinoleic no results, 1/5 Clinoleic ultracentrifuged 185 mg.l−1, and the 1/5 in saline 158 mg.l−1 (Beckman D×C800). The CV of the paracetamol assay at this level is ∼ 3%. Beside within assay variation, some of the difference can be due to the pipetting accuracy in preparing the dilutions. Based on the information presented in the manuscript and from the experiments, an ultracentrifuge would have provided minimal additional information to aid both the clinical and laboratory staff in determining the cause of the artefacts. In conclusion, the inability to obtain results is in part Intralipid induced artefact, but the results do not appear physiologically possible, thus the sample collection technique must be questioned. My sincerest thanks to Robert Flatman and his staff, Sullivan and Nicolaides Pathology, Brisbane for analysing the samples on their Roche Modular DP system.