Preparation of lipemic plasma or serum samples for the determination of a lipid interference

Abstract

The present invention is in the field of in vitro diagnostics and relates to a method for preparing lipemic plasma or serum samples and the use thereof for establishing a lipid interference in the quantitative determination of the amount or the activity of an analyte in a plasma or serum sample.

Claims

1. A method for preparing a lipemic plasma or serum sample, the method comprising the steps: (a) centrifuging a lipid-containing plasma or serum sample in order to separate a lipid-containing supernatant from a lipid-depleted phase; (b) removing the lipid-containing supernatant and (c) mixing the lipid-containing supernatant with a plasma or serum sample.

2. The method as claimed in claim 1, wherein a first subamount of a lipid-containing plasma or serum sample is centrifuged in step (a) and the lipid-containing supernatant is then removed in step (b), and the lipid-containing supernatant is then mixed with a second subamount of the same lipid-containing plasma or serum sample in step (c).

3. The method as claimed in claim 1, wherein the removed lipid-containing supernatant is mixed with a subamount of the lipid-depleted phase.

4. The method as claimed in claim 1, wherein the lipid-containing plasma or serum sample is centrifuged in step (a) for at least 10 minutes at at least 2000g.

5. A method for establishing a lipid interference in a method for quantitatively determining an amount or an activity of an analyte in a plasma or serum sample, the method for establishing a lipid interference comprising the steps: (a) providing a first assay mix by mixing at least one analyte-specific detection reagent with a nonlipemic plasma or serum sample having an analyte concentration or activity and measuring a first assay result; (b) providing a second assay mix by mixing the same at least one analyte-specific detection reagent with a lipemic plasma or serum sample having the same analyte concentration or activity and measuring a second assay result; (c) establishing a difference between the first and second assay result; and (d) establishing a lipid interference when the difference between the first and second assay result exceeds a predetermined tolerance limit; wherein the lipemic plasma or serum sample has been prepared using a method as claimed in claim 1.

6. The method as claimed in claim 5, wherein the nonlipemic plasma or serum sample has been prepared using a method comprising the following steps: (a) centrifuging a lipid-containing plasma or serum sample and isolating the lipid-depleted phase from the lipid-containing supernatant.

7. The method as claimed in claim 5, wherein the lipemic plasma or serum sample and the nonlipemic plasma or serum sample have been prepared from the same lipid-containing plasma or serum sample as starting material.

8. The method as claimed in claim 5, wherein the nonlipemic and the lipemic plasma or serum sample have in each case an analyte concentration or activity that is reduced or elevated with respect to a norm.

9. A method for establishing a lipid interference in a method for quantitatively determining an amount or an activity of an analyte in a plasma or serum sample, the method for establishing a lipid interference comprising the steps: (a) providing a first assay mix by mixing at least one analyte-specific detection reagent with a first subamount of a nonlipemic plasma or serum sample and measuring a first assay result; (b) providing a second assay mix by mixing the same at least one analyte-specific detection reagent with a lipid-depleted phase of a second subamount of the same nonlipemic plasma or serum sample, which had previously been centrifuged at at least 2000 g for at least 10 minutes, and measuring a second assay result; and (c) establishing a first difference between the first and second assay result; and (d) providing a third assay mix by mixing the same at least one analyte-specific detection reagent with a first subamount of a lipemic plasma or serum sample and measuring a third assay result; (e) providing a fourth assay mix by mixing the same at least one analyte-specific detection reagent with a lipid-depleted phase of a second subamount of the same lipemic plasma or serum sample, which had previously been centrifuged at at least 2000 g for at least 10 minutes, and measuring a fourth assay result; and (f) establishing a second difference between the third and fourth assay result; and (g) establishing a lipid interference when a deviation between the first and second difference exceeds a predetermined tolerance limit, wherein the lipemic plasma or serum sample has been prepared using a method as claimed in claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1Determination of the lipid interference in a method for determining the APTT;

(2) FIG. 2Determination of the lipid interference in a method for determining protein C.

DETAILED DESCRIPTION

(3) The following exemplary embodiments serve to illustrate the method according to the invention and are not to be understood as a restriction.

EXAMPLES

Example 1

Determination of the Lipid Interference in a Method for Determining the APTT

(4) 1a) Inventive Preparation of Lipemic Plasma Samples for the Determination of the Lipid Interference in a Method for Determining the APTT Using Dade Actin Reagent

(5) Use was made of 7 native lipemic citrate plasma samples (triglyceride level >150 mg/dL) from 7 donors. The triglyceride level was determined using the TRIG assay and using the Dimension Vista System (Siemens Healthcare, Newark, USA). About 18 to 20 mL of said samples were centrifuged for 1 hour in an ultracentrifuge (Thermo Scientific Sorval WX Ultra, Rotor T-1250, ThermoFisher, Hanau, Germany) at 33 200 rpm, corresponding to 133 000g. With the aid of a pipette, the uppermost, lipid-rich supernatant was carefully removed while avoiding mixing with the underlying lipid-depleted phase. Depending on the sample, between 0.7 and 3.0 mL of said lipid-rich supernatant were mixed with a noncentrifuged subamount of the native citrate plasma sample from the same donor and, in some cases, with a nonlipemic factor deficiency plasma (triglyceride level typically approx. 100 mg/dL). The total volume of the thus prepared lipemic samples was about 12 mL. The triglyceride level of the thus prepared samples was measured again and was between 234 mg/dL and 1007 mg/dL.

(6) Table 1 contains the final triglyceride concentration of the lipemic sample Nos. L3-L9 prepared according to the invention and three further native lipemic samples (sample Nos. L1, L2 and L10) and also the mixing ratio of the amount of native (noncentrifuged, untreated) plasma sample from the donor, lipid-containing supernatant, deficiency plasma and a heparin solution. The mixing with deficiency plasma or heparin solution was done to expand the APTT measurement range.

(7) Table 2 contains the composition and the final triglyceride concentration of the nonlipemic sample Nos. NL1-NL12, which were likewise used in the interference study.

(8) TABLE-US-00001 TABLE 1 Composition Of The Lipemic Samples Composition Native Lipid- Plasma with a Final Sam- sample containing deficiency of Heparin triglyceride ple amount supernatant FV FVII PC solution concentration No. mL mL mL mL mL mL mg/dL L1 12.0 0.0 0.0 0.0 0.0 0.0 237.5 L2 12.0 0.0 0.0 0.0 0.0 0.0 399.5 L3 11.1 0.9 0.0 0.0 0.0 0.0 731.0 L4 3.0 3.0 0.0 6.0 0.0 0.0 316.5 L5 3.0 2.5 0.0 0.0 8.0 0.0 481.5 L6 6.5 0.7 0.0 5.0 0.0 0.0 1006.6 L7 3.5 1.2 7.5 0.0 0.0 0.0 233.5 L8 4.0 2.0 6.0 0.0 0.0 0.0 405.5 L9 4.5 1.6 6.0 0.0 0.0 0.0 432.5 L10 12.0 0.0 0.0 0.0 0.0 0.5 215.5

(9) TABLE-US-00002 TABLE 2 Composition Of The Nonlipemic Samples Composition Native Lipid- Plasma with a Final Sam- sample containing deficiency of Heparin triglyceride ple amount supernatant FV FVII PC solution concentration No. mL mL mL mL mL mL mg/dL NL1 12.0 0.0 0.0 0.0 0.0 148.0 NL2 12.0 0.0 0.0 0.0 0.0 120.5 NL3 12.0 0.0 0.0 0.0 0.0 73.5 NL4 12.0 0.0 0.0 0.0 0.0 52.6 NL5 6.0 6.0 0.0 0.0 0.0 143.5 NL6 12.0 0.0 0.0 0.0 0.0 83.0 NL7 1.2 0.0 10.8 0.0 0.0 100.0 NL8 3.0 0.0 9.0 0.0 0.0 99.5 NL9 7.0 5.0 0.0 0.0 0.0 62.5 NL10 6.0 6.0 0.0 0.0 0.0 105.5 NL11 6.6 5.4 0.0 0.0 0.0 107.0 NL12 12.0 0.0 0.0 0.0 0.2 92.2 NL13 11.5 0.0 0.0 0.0 0.6 53.8 NL14 12.0 0.0 0.0 0.0 0.7 73.8

(10) 1b) Determination of the Lipid Interference in a Method for Determining the APTT Using Dade Actin Reagent

(11) In the case of the activated partial thromboplastin time (APTT), a plasma sample is mixed with reagents containing phospholipids and a surface activator (Dade Actin reagent, Siemens Healthcare, Marburg, Germany). After an incubation time, coagulation is initiated by the addition of CaCl.sub.2. In the example described here, the APTT was automatically processed on the Sysmex CS-5100 analyzer (Siemens Healthcare, Marburg, Germany). The coagulation reaction is measured photometrically as an increase in absorbance. The time until a particular increase in absorbance is the coagulation time in seconds, which represents the result of the APTT assay.

(12) From all of the samples prepared under 1a), a subamount was used for the measurement of the APTT (assay result 1). A second subamount of each of the samples prepared under 1a) was centrifuged at 133 000g for 1 hour and the lipid-depleted phase was used as sample for the APTT measurement (assay result 2). If the centrifugation has no influence, the lipid-depleted phase of the centrifuged aliquot should contain the same analyte amount as the noncentrifuged sample. The two assay results (coagulation times in seconds) of each sample were compared with one another, and the relative difference in % was calculated: 100(assay result 1-assay result 2)/assay result 2.

(13) From the relative differences of the assay results for the nonlipemic samples, the mean was calculated (in this case: 0.6). Since the nonlipemic samples exhibit no interference due to lipemia, this mean difference is attributed to a nonspecific influence factor of the method (e.g., the centrifugation). For the correction of this effect, a corrected difference was ascertained for each sample by subtracting said mean from the relative difference. The corrected difference is a measure of the interference caused by lipids.

(14) Table 3 shows for each sample the assay results 1 and 2, the ascertained relative difference and the corrected difference. The difference values are rounded values.

(15) TABLE-US-00003 TABLE 3 APTT Assay Results And Deviations Lipid-depleted phase of a Noncentrifuged centrifuged subamount of the subamount of the Triglyceride sample sample Relative Corrected Sample concentration 1st assay result 2nd assay result difference difference No. mg/dL APTT [s] APTT [s] % % NL1 Nonlipemic 148.0 26.4 26.2 0.6 0.0 NL2 120.5 27.8 26.6 4.5 3.9 NL3 73.5 28.5 27.1 5.2 4.6 NL4 52.6 23.0 23.1 0.2 0.8 NL5 143.5 34.6 34.5 0.3 0.3 NL6 83.0 32.7 31.4 4.3 3.7 NL7 100.0 30.1 31.0 2.9 3.5 NL8 99.5 28.4 29.9 5.0 5.6 NL9 62.5 39.8 40.8 2.3 2.9 NL10 105.5 38.2 38.8 1.5 2.1 NL11 107.0 37.6 38.1 1.3 1.9 NL12 92.2 65.0 61.1 6.5 5.9 NL13 53.8 115.8 115.5 0.3 0.3 NL14 73.8 100.1 100.3 0.2 0.8 Mean: 0.6 L1 Lipemic 237.5 27.1 26.7 1.5 0.9 L2 399.5 22.8 22.8 0.0 0.6 L3 731.0 22.2 20.7 7.5 6.9 L4 316.5 28.7 29.4 2.2 2.8 L5 481.5 30.6 29.0 5.5 4.9 L6 1006.6 25.2 23.8 6.1 5.5 L7 233.5 42.0 42.1 0.2 0.8 L8 405.5 36.2 36.3 0.3 0.9 L9 432.5 40.7 40.0 1.6 1.0 L10 215.5 68.9 70.6 2.5 3.1

(16) The corrected difference, i.e., the deviation of the assay results of the noncentrifuged samples from the assay results of the centrifuged samples (decreased by the mean of the difference of the nonlipemic samples), was plotted against the triglyceride concentration of the noncentrifuged samples (FIG. 1, line 1). A polynomial fitting method was applied and the associated confidence interval calculated (FIG. 1, lines 2). The point of intersection of the limit of the confidence interval with the criterion of 10% relative deviation identifies the triglyceride concentration from which a lipid interference is to be expected in the tested APTT assay method (676.5 mg/dL).

Example 2

Determination of the Lipid Interference in a Method for Determining Protein C

(17) 2a) Inventive Preparation of Lipemic Plasma Samples for the Determination of the Lipid Interference in a Method Fort Determining Protein C Using the Berichrom Protein C Assay

(18) Use was made of 12 native lipemic citrate plasma samples (triglyceride level >150 mg/dL) from 12 donors, and the procedure as described under 1a) was carried out. The triglyceride level of the thus prepared samples was measured again and was between 199 mg/dL and 1007 mg/dL.

(19) Table 4 contains the final triglyceride concentration of the lipemic sample Nos. L1-L12 prepared according to the invention and of a further native lipemic sample (sample No. L13) and also the mixing ratio of the amount of native (noncentrifuged, untreated) plasma sample from the donor, lipid-containing supernatant, deficiency plasma and a heparin solution. The mixing with deficiency plasma was done to expand the protein C measurement range.

(20) Table 5 contains the composition and the final triglyceride concentration of the nonlipemic sample Nos. NL1-NL12, which were likewise used in the interference study.

(21) TABLE-US-00004 TABLE 4 Composition Of The Lipemic Samples Composition Native Lipid- Plasma with a Final Sam- sample containing deficiency of Heparin triglyceride ple amount supernatant FV FVII PC solution concentration No. mL mL mL mL mL mL mg/dL L1 3.0 3.0 0.0 0.0 6.0 0.0 330.5 L2 3.7 2.1 0.0 0.0 5.7 0.0 351.0 L3 3.0 2.5 0.0 0.0 8.0 0.0 481.5 L4 2.8 1.7 0.0 0.0 7.5 0.0 566.5 L5 3.5 1.2 7.5 0.0 0.0 0.0 233.5 L6 5.5 2.3 0.0 0.0 3.8 0.0 545.5 L7 6.3 1.2 0.0 0.0 4.5 0.0 967.5 L8 4.3 0.9 6.8 0.0 0.0 0.0 199.0 L9 3.0 2.7 0.0 6.3 0.0 0.0 369.5 L10 4.5 1.6 6.0 0.0 0.0 0.0 432.5 L11 11.1 0.9 0.0 0.0 0.0 0.0 731.0 L12 6.5 0.7 0.0 5.0 0.0 0.0 1006.6 L13 12.0 0.0 0.0 0.0 0.0 0.0 237.5

(22) TABLE-US-00005 TABLE 5 Composition Of The Nonlipemic Samples Composition Native Lipid- Plasma with a Final Sam- sample containing deficiency of Heparin triglyceride ple amount supernatant FV FVII PC solution concentration No. mL mL mL mL mL mL mg/dL NL1 3.0 0.0 0.0 9.0 0.0 91.0 NL2 4.0 0.0 0.0 8.0 0.0 95.5 NL3 4.0 0.0 0.0 8.0 0.0 103.0 NL4 4.0 0.0 0.0 8.0 0.0 87.5 NL5 7.0 5.0 0.0 0.0 0.0 62.5 NL6 11.5 0.0 0.0 0.0 0.6 53.8 NL7 6.0 0.0 0.0 6.0 0.0 88.0 NL8 7.0 0.0 0.0 5.0 0.0 92.0 NL9 6.0 6.0 0.0 0.0 0.0 105.5 NL10 12.0 0.0 0.0 0.0 0.0 83.0 NL11 12.0 0.0 0.0 0.0 0.0 120.5 NL12 12.0 0.0 0.0 0.0 0.0 73.5

(23) 2b) Determination of the Lipid Interference in a Method for Determining Protein C Using the Berichrom Protein C Assay

(24) In the assay mix, a plasma sample is incubated with a snake venom activator, resulting in the activation of protein C. Furthermore, a chromogenic peptide substrate which is cleaved by activated protein C is added. This reaction achieves an increase in absorbance that is measured at 405 nm. The increase in absorbance is converted to the protein C result (% of the norm) on the basis of a calibration curve. In the example described here, the protein C assay was automatically processed on the Sysmex CS-5100 analyzer (Siemens Healthcare, Marburg, Germany).

(25) From all of the samples prepared under 2a), a subamount was used for the measurement of protein C (assay result 1). A second subamount of each of the samples prepared under 2a) was centrifuged at 133 000g for 1 hour, and the lipid-depleted phase was used as sample for the protein C measurement (assay result 2). If the centrifugation has no influence, the lipid-depleted phase of the centrifuged aliquot should contain the same analyte amount as the noncentrifuged sample. The two assay results (% of the norm) of each sample were compared with one another, and the relative difference in % was calculated: 100(assay result 1-assay result 2)/assay result 2.

(26) From the relative differences of the assay results for the nonlipemic samples, the mean was calculated (in this case: 2.4). Since the nonlipemic samples exhibit no interference due to lipemia, this mean difference is attributed to a nonspecific influence factor of the method (e.g., owing to the centrifugation). For the correction of this effect, a corrected difference was ascertained for each sample by subtracting said mean from the relative difference. The corrected difference is a measure of the interference caused by lipids.

(27) Table 6 shows for each sample the assay results 1 and 2, the ascertained relative difference and the corrected difference. The difference values are rounded values.

(28) TABLE-US-00006 TABLE 6 Protein C Assay Results And Deviations Lipid-depleted phase of a Noncentrifuged centrifuged subamount of the subamount of the Triglyceride sample sample Relative Corrected Sample concentration 1st assay result 2nd assay result difference difference No. mg/dL PC [% of the norm] PC [% of the norm] % % NL1 Nonlipemic 91.0 28.7 29.3 2.0 0.3 NL2 95.5 30.4 31.1 2.3 0.1 NL3 103.0 40.8 42.7 4.4 2.1 NL4 87.5 30.6 31.3 2.2 0.1 NL5 62.5 76.3 76.5 0.3 2.1 NL6 53.8 74.1 73.8 0.4 2.8 NL7 88.0 56.1 62.1 9.6 7.2 NL8 92.0 53.5 54.9 2.6 0.2 NL9 105.5 91.5 89.2 2.5 4.9 NL10 83.0 96.2 98.3 2.1 0.2 NL11 120.5 110.4 114.0 3.2 0.8 NL12 73.5 113.9 116.7 2.4 0.1 Mean: 2.4 L1 Lipemic 330.5 48.7 50.9 4.2 1.9 L2 351.0 44.7 46.4 3.7 1.3 L3 481.5 50.4 52.1 3.3 0.9 L4 566.5 39.5 43.1 8.4 6.0 L5 233.5 81.3 85.3 4.7 2.3 L6 545.5 96.7 99.3 2.6 0.2 L7 967.5 62.7 75.4 16.8 14.4 L8 199.0 83.5 85.7 2.5 0.2 L9 369.5 94.1 97.3 3.2 0.9 L10 432.5 83.0 87.1 4.7 2.3 L11 731.0 100.3 109.9 8.8 6.4 L12 1006.6 97.8 106.5 8.2 5.8 L13 237.5 105.5 110.3 4.4 2.0

(29) The corrected difference, i.e., the deviation of the assay results of the noncentrifuged samples from the assay results of the centrifuged samples (decreased by the mean of the difference of the nonlipemic samples), was plotted against the triglyceride concentration of the noncentrifuged samples (FIG. 2, line 1). A polynomial fitting method was applied and the associated confidence interval calculated (FIG. 2, lines 2). The point of intersection of the limit of the confidence interval with the criterion of 10% relative deviation identifies the triglyceride concentration from which a lipid interference is to be expected in the tested protein C assay method (867.5 mg/dL).