Method and reagent to clear turbidity due to elevated triglycerides in a biological specimen

Abstract

Certain bodily fluid samples include triglyceride-rich lipoproteins. These triglyceride-rich lipoproteins may interfere with analysis to determine the concentration of other components in the bodily fluid samples. A method is disclosed to eliminate or reduce the concentration of the triglyceride-rich lipoproteins so that a more accurate analysis can be achieved. A method may involve mixing the sample with the reagent including magnetic beads attached to a complexing agent for the triglyceride-rich lipoproteins. Then the triglyceride-rich lipoproteins may be magnetically separated from the bodily fluid sample and the bodily fluid sample may be analyzed.

Description

TITLEMethod and Reagent to Clear Turbidity Due to Elevated Triglycerides in a Biological SpecimenINVENTORJohn H. ContoisAndre L. AlbertRae- Anne NguyenFIELD OF THE INVENTION

[0001] Methods for clearing lipemia of triglyceride-rich lipoproteins (TRL) turbidity from biological samples prior to analysis are described.BACKGROUND OF THE INVENTION

[0002] The presence of increased triglyceride-rich lipoprotein particles in patient specimens (lipemia) frequently (1) causes assay interference of measurement of various analytes in the clinical laboratory. This is especially acute in Clinical Chemistry assays where photometric measurements are desired. The heterogeneous lipid particles (especially the largest particles: chylomicrons, sample size of 70-1000 nm, and large very low-density lipoprotein particles) cause reaction turbidity in the assay, generating incorrect results in many chemistry and immunochemistry assays. The assay interference mechanisms include light scattering and light absorbance by the lipid particles and lipid reactions with assay reagents. Electrolytes are affected by the volume displacement effect of the lipid particles (1). Various immunoassays (especially homogeneous assays, where analytes are not physically separated after the assay reaction) may also suffer from lipemia interference.

[0003] Lipoproteins also can interfere with antigen-antibody reaction by blocking binding sites on antibodies. This can happen even when antibodies are bound to a solid surface (heterogeneous immunoassays). Depending on the nature of the reaction, the interference can cause either falsely elevated or falsely decreased result.

[0004] Lipemia may also be exacerbated or caused by the disease itself, e.g., multiple myeloma, diabetes mellitus, alcoholism, acute pancreatitis, obesity, kidney failure or hypothyroidism. Additionally, lipemia can be caused by insufficient time between meals or parenteraladministration of synthetic lipid emulsions and specimen collection. The overall frequency of lipemic samples ranges from 0.5-2.5% (2), depending on the type of hospital and proportion of inpatient and outpatient samples with outpatient samples having a higher proportion of lipemia interference.

[0005] An important task of the laboratorian is to identify turbid specimens, remove lipemic turbidity, and report correct analyte results. As today’s clinical laboratories become more automated this task is getting more important. Most analyzers used in clinical labs today have serum indexing, where the optical property of the specimen can be assessed for hemolysis, icterus and lipemia. Serum indices are measured by diluting the sample in a saline or buffer solution, followed by absorbance measurements to provide a semi quantitative assessment of the degree of icterus, hemolysis or lipemia in serum / plasma. Depending on the results of such tests, results can be flagged for follow up, including rejection or mitigation of the interference. However, specimen indexing tests are qualitative and significant differences exist among analyzers in the index results (3).

[0006] Most clinical lab tests use serum or plasma as the specimen. These are prepared by routine centrifugation (15 minutes at approximately 3000-3500 rpms in a fixed angle centrifuge or 10 minutes at approximately 2700-3100 rpms in a swing bucket centrifuge). While such centrifugation separates blood into clear serum or plasma, it fails to clear grossly lipemic samples. CLSI C56-A recommends ultracentrifugation to remove the lipid particles: 12,100 g for 5 min or longer. However, few labs possess ultracentrifuges to perform this mitigation (4).

[0007] A commercial lipid clearing agent, LipoClear®, was previously available to clear lipemia from clinical specimens. A lipemic sample was pipetted into a tube coated with a non-ionic polymer reagent, mixed well, and incubated for 5 min at room temperature. The sample was then centrifuged, and the clear liquid used for analysis. According to the manufacturer's claims, LipoClear cannot be used to clarify samples intended for lipid or coagulation testing, determination of monoclonal gammopathy immunoglobulins, proteins, phosphorus or electrolytes (5). However, a study compared the efficacy of ultracentrifugation with LipoClear for 26 common analytes and found that the latter was not suitable for lipemia removal from samples designated for glucose, sodium, potassium, chlorides, phosphates, magnesium, CK-MB, ALP, GGT, total protein, albumin, CRP and troponin T measurements (6). The authors recommended that high speed centrifugation should be used for lipemia removal instead forglucose, potassium, phosphates, magnesium, CK-MB, ALP, GGT, albumin, CRP and troponin measurements.

[0008] There is a need for a lipemia clearing method that is generally applicable to a wide variety of samples or specimen and that can effectively remove triglyceride-rich lipoproteins (TRL) from the samples prior to analysis.REFERENCES

[0009] Nikolac N. Lipemia: causes, interference mechanisms, detection and management. Biochemia Medica 2014;24:57-67. http: / / dx.doi.org / 10.11613 / BM.2014.008.

[0010] Soleimani N, Mohammadzadeh S, Asadian F. Lipemia interferences in biochemical tests, investigating the efficacy of different removal methods in comparison with ultracentrifugation as the gold standard. J Anal Methods Chem 2020;2020. https: / / doi.org / 10.1155 / 2020 / 9857636.

[0011] Farrell CJ, Carter AC. Serum indices: managing assay interference. Ann Clin Biochem 2016;53:527-38.

[0012] Clinical Laboratory Standards Institute. Hemolysis, icterus, and lipemia / turbidity indices as indicators of interference in clinical laboratory analysis; approved guideline. CLSI C56-A document. Wayne, Pennsylvania, USA: Clinical Laboratory Standards Institute; 2012.

[0013] Iris Sample Processing. LipoClear® package insert. 54-004752-001 Rev. C. Westwood, MA, USA: IRIS International, Inc.; 2010.

[0014] Saracevic A, Nikolac N, and Simundic AM. The evaluation and comparison of consecutive high-speed centrifugation and LipoClear® reagent for lipemia removal. Clinical Biochemistry 2014; 47: 309-314.SUMMARY OF THE INVENTION

[0015] Embodiments include a novel lipemia clearing reagent that can be used with a variety of analytical systems in removing triglyceride-rich lipoproteins (TRL) specifically from patient samples and methods of removing TRLs from the patient samples or specimen. One embodiment comprises adding lipemic samples to a sampling container, wherein the sampling container comprises an anti-apolipoprotein or other lipid reacting or complexing reagent. Thereagent may be a dried reagent, a liquid reagent, or a solubilized reagent. The sampling container may be a tube, for example.

[0016] The method comprises mixing the sample with the reagent. For example, in one embodiment comprising a dried reagent, the sample may be vortexed vigorously for a time sufficient (for example, 30-60 seconds) to resolubilize the dried reagent and then allowed to react or complex with the lipids. In one specific embodiment, the mixed sample was allowed to incubate for 15 minutes at room temperature. An embodiment may comprise adding magnetic capture beads followed by another incubation period. For example another 15 minutes incubation at room temperature to bind the lipid particles to the magnetic beads.

[0017] The lipid particles bound to the beads may then be magnetically separated, and the lipid free sample can be safely analyzed for interference-free results. Alternatively, the lipid particles may be filtered or centrifuged from the sample.

[0018] Alternatively, the magnetic beads can be conjugated directly to the complexing reagent.

[0019] Sun Diagnostics Assurance™ Lipemia Clearing Reagent uses purified goat anti-human apolipoprotein B (apo B) to immunoprecipitate apo B-containing lipoproteins including chylomicrons, VLDL, and LDL up to at least 1500 mg / dL triglycerides. The procedure is simple: 1 mL sample is added to the anti-apo B-coated centrifugal tube, vortexed briefly, and incubated five minutes at room temperature. Drying the antibody reagent onto the tube removes the need to correct for dilution. A small amount of anti-goat IgG or Protein G-conjugated magnetic beads is then added to the samples and mixed on a vortex mixer. The samples are then subjected to a magnetic field in a tube holder to pull the captured TRLs to the side and allowing the clear serum to be removed.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Fig. l is a flow diagram of an embodiment of the method of using a reagent to clear turbidity due to elevated triglycerides in a biological specimen.DETAILED DESCRIPTION

[0021] To accurately perform certain assays that rely on light absorbance or light scattering, the samples must have sufficient clarity. Lipemia is the presence of a high concentration of lipids (orfats) in the blood. When blood is lipemic, it causes the serum- or plasma-containing products to have a milky appearance.

[0022] Thus,%ipemia can be problematic in assays which rely on turbidimetric or colorimetric measurement. Turbidity, usually caused by lipids in the sample, can interfere with absorbance and light scattering measurements in such assays.

[0023] Turbidity in the sample may be caused by excess serum triglycerides, mostly in the form of apo B-containing VLDL and chylomicrons (triglyceride-rich lipoproteins or TRLs) often related to recent dietary fat intake or abnormal lipid metabolism. To remove this interference and accurately measure the desired components in the specimen, the laboratory technician must ultracentrifuge the specimen to physically remove interfering lipids or chemically precipitate the interfering lipids.

[0024] LipoClear® was, until recently, available to clear lipemic samples for analysis. LipoClear comprised a liquid polymer reagent that removes lipoproteins from lipemic samples. However, though the sample was cleared of the lipoproteins, there appears to be loss of proteins and other analytes with this method which would then interfere with an accurate analysis.

[0025] An immunological approach to remove TRLs could provide greater specificity for removing only the unwanted lipids without otherwise affecting other unrelated test results.

[0026] Embodiments of the invention comprise a method for clearing a specimen or sample of physical lipemia, and substantially eliminating the interference.

[0027] An embodiment of the method comprises immunoprecipitating apolipoprotein B (Apo B)-containing lipoproteins with a polyclonal anti-apo B antisera or other similar compound and subsequently magnetic separating the resultant lipoprotein-antibody complex. In other embodiments, the method may comprise immunoprecipitating of apolipoprotein B (Apo B)- containing lipoproteins with a polyclonal anti-apo B antisera or other similar compound and subsequently separating of the resultant lipoprotein-antibody complex the lipoprotein-antibody complex by filtrating or centrifugating.

[0028] The other compounds include, but are not limited to, one or more polyclonal or monoclonal antibodies will also work and antibodies to apo E, CII, CIII, or other apoproteins present on TRLs will also work.

[0029] As discussed above, to provide accurate analysis of blood serum samples comprising lipoproteins, it is advantageous to substantially remove the lipoproteins from the blood serumsample prior to analysis. Embodiments of method of preparing a sample or a specimen for analysis by removing lipids may comprise mixing an anti-apolipoprotein reagent to a blood serum sample to prepare a reagent / sample mixture. This method is especially applicable to blood serum samples comprising lipoprotein in a sufficient concentration to interfere with the desired assay procedure. An example of the method and the components of the kit are shown in Figure 1. In Step 1, a biological sample is placed in a tube comprising dried anti-apolipoprotein reagent.

[0030] After mixing the anti-apolipoprotein in Step 2, the blood serum sample may be incubated in the reagent / sample mixture to form a lipoprotein / reagent complex or otherwise bound lipoprotein particle. The lipoprotein may then be separated by any applicable method from the blood serum sample to provide a clarified blood serum sample and lipid particles. The clarified blood serum sample is now ready for analysis.

[0031] In one embodiment, the anti-apolipoprotein is purified goat anti-human apolipoprotein B. However, any appropriate reagent that binds with the apolipoprotein to convert the apolipoprotein into a more easily separable form may be used including, but not limited to, a monoclonal antibody or an antibody to apo E, CII, CIII, or other apoproteins present on triglyceride-rich lipoproteins.

[0032] The anti-apolipoprotein reagent may be a dried anti-apolipoprotein reagent and the dried may be stored in a sample container. The container may be a analysis tube such as a microtube or other test tube.

[0033] As shown in the embodiment shown in Figure 2, the method may further comprise adding magnetic capture beads to the clarified blood serum to bind with the lipid particles. The capture beads may bind with the magnetic capture beads to provide an easily removed lipid particle as shown in Step 3. Therefore, the method may comprise magnetically removing the magnetic capture beads and the bound lipid particles from the sample container. Alternatively, the lipid particles may be removed by filtering to remove the lipid particles or centrifuging to separate the lipid particles from the blood serum sample. The sample / magnetic capture bead mixture may be mixed and incubated for a sufficient time and at a sufficient temperature to allow separation of the lipid particles by a magnet as shown in Step 5 and 6. For example, the incubated tube may be placed in the magnetic separation holder for an effective time to separate the beads and, therefore, the lipid particles as shown in Step 7.

[0034] The component to conduct the method described herein may be packaged as a kit. An embodiment of the lipoprotein removal kit may comprise a sample container, such as a microtubes, containing dried anti-apolipoprotein reagent and magnetic capture beads. The components of the test kit may be used to clarify a blood serum sample.

[0035] Any of the anti-apolipoprotein reagents may be included in the kit. The kit may comprise the magnetic capture beads in a slurry.EXAMPLE OF A TEST KIT and METHOD

[0036] Examples of a Test Kit contains the following components in sufficient quantity to perform clearance of lipemia from 15 x ImL human serum samples:

[0037] 2.0 mL microtubes with dried anti-apolipoprotein reagent, and magnetic bead slurry

[0038] Magnetic separation holder / rack, pipettes and tips, and vortex mixer also required.

[0039] Procedure:

[0040] • Add 1.0 mL lipemic human serum sample to the reagent tube, cap and vortex vigorously for 30-60 seconds to resolubilize the dried reagent.

[0041] • Incubate for 15 minutes at room temperature.

[0042] • Gently mix the Beads by inversion, or by brief vortex. Add 40 pL of the Beads to the incubated sample. Vortex, and incubate for 15 minutes for at room temperature.

[0043] • Place the incubated tube in the magnetic separation holder for 10-15 minutes at room temperature. If sample is not clear, allow for up to 30 minutes to clarify supernatant. Highly lipemic samples will take more time to clarify than lower lipemic samples.

[0044] • Keep the tube in the magnetic separation holder and harvest the cleared serum from the for subsequent analysis.

[0045] Chylomicrons, VLDL, and LDL particles bind strongly to the antibodies and are bound by the reagents. The resulting cleared serum is free of TRL and suitable for analyses. With a high concentration of lipids, a “pedicle” may form on the surface. Use a narrow-tipped pipet to remove the clear sample.

[0046] In the following experiments leftover, deidentified serum specimens were treated with complexing reagent, as described above. An equal volume of water was added to create a controlsample with an equal concentration of analyte. These data indicate that the lipid clearing reagent removed triglycerides without altering the concentrations of these common analytes.

Claims

CLAIMS1. A method of preparing a sample or a specimen for analysis, comprising: mixing an anti-apolipoprotein reagent to a biological sample to prepare a reagent / sample mixture, wherein the biological sample comprises whole blood, serum, plasma, urine or other body fluid; incubating the reagent / sample mixture to form a lipoprotein / reagent complex; separating the lipoprotein / reagent complex from the blood serum sample to provide a clarified blood serum sample and lipid particles; and analyzing the clarified blood serum sample.

2. The method of Claim 1, wherein the anti-apolipoprotein is purified goat anti -human apolipoprotein B.

3. The method of Claim 1, wherein the anti-apolipoprotein is selected from a monoclonal antibody or an antibody to apo E, CII, CIII, or other apoproteins present on triglyceride-rich lipoproteins.

4. The method of Claim 1, wherein the anti-apolipoprotein reagent is a dried anti- apolipoprotein reagent.

5. The method of Claim 4, wherein the blood serum sample is mixed in a tube with the dried anti-apolipoprotein reagent.

6. The method of Claim 5, comprising adding magnetic capture beads to the clarified blood serum to bind with the lipid particles.

7. The method of Claim 6, comprising magnetically removing the magnetic capture beads and the bound lipid particles.

8. The method of Claim 1, comprising filtering to remove the lipid particles.

9. The method of Claim 1, comprising centrifuging to separate the lipid particles.

10. A test lit, comprising: microtubes containing dried anti-apolipoprotein reagent; and magnetic capture beads.

11. The test kit of Claim 10, wherein the dried anti-apolipoprotein reagent is a purified goat anti-human apolipoprotein B.

12. The test kit of Claim 10, wherein the dried anti-apolipoprotein reagent is selected from a monoclonal antibody or an antibody to apo E, CII, CIII, or other apoproteins present on triglyceride-rich lipoproteins.

13. The test kit of Claim 10, wherein the magnetic capture bead are in a slurry.

14. The test kit of Claim 10, wherein the test kit is used to remove lipoproteins from a blood serum sample.

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