The assay is traceable to an internal standard manufactured using qualified materials and measurement procedures.

Analytical Sensitivity: 0.5 µmol/L

Precision: Samples were repeatedly assayed in quadruplicate over the course of several days, for a total of 20 runs and 80 replicates. (See "Precision" table.)

Linearity: Samples were assayed under various dilutions. (See "Linearity" table for representative data.)

Recovery: Samples spiked 1 to 19 with three homocysteine solutions (125, 250, 500 µmol/L), were assayed. (See "Recovery" table for representative data.)

Specificity: The antibody is highly specific for homocysteine. (See "Specificity" table.)

Bilirubin: Presence of conjugated and unconjugated bilirubin in concentrations up to 200 mg/L has no effect on results, within the precision of the assay.

Hemolysis: Presence of hemoglobin in concentrations up to 512 mg/dL has no effect on results, within the precision of the assay.

Lipemia: Presence of triglycerides in concentrations up to 3,000 mg/dL has no effect on results, within the precision of the assay.

Alternate Sample Type: To determine whether serum can be used in the IMMULITE 2000 Homocysteine procedure, blood was collected on ice from 34 laboratory volunteers into heparinized, EDTA and plain vacutainer tubes. Samples were separated from the cells, and matched samples were spiked with homocysteine and then assayed by the IMMULITE 2000 Homocysteine procedure, with the following results.

(EDTA) = 0.98 (Heparin) + 0.85 µmol/L r = 0.954

(Serum) = 1.02 (Heparin) – 0.53 µmol/L r = 0.975

Means:

20.2µmol/L (Heparin)

20.7µmol/L (EDTA)

20.1µmol/L (Serum)

To assess the effect of storage temperature, heparinized, EDTA and plain vacutainer tubes were collected from five volunteers for each tube type. Some tubes were stored at room temperature, while other tubes were kept on ice for various

IMMULITE 2000 Homocysteine (PIL2KHO-14, 2007-11-20)

time periods prior to separation. The graphs below show the effect of storage time and temperature for heparin, EDTA and serum. (See graphs 1-3).

Method Comparison 1: The assay was compared to a commercially available manual enzyme immunoassay (Kit A) on 168 plasma samples (Concentration range: approximately 4 to 43 µmol/L. See “Method Comparison 1” graph.) By linear regression:

(IML 2000) = 1.0 (Kit A) + 0.24 µmol/L r = 0.966

Means:

12.9µmol/L (IMMULITE 2000)

12.7µmol/L (Kit A)

Method Comparison 2: The assay was also compared to an in-house HPLC method in use at a reference laboratory in the United States on 95 heparinized plasma samples (Concentration range: approximately 4 to 44 µmol/L. See “Method Comparison 2” graph.) By linear regression:

(IML 2000) = 0.97 (HPLC) + 0.71 µmol/L r = 0.974

Means:

13.4µmol/L (IMMULITE 2000)

13.2µmol/L (HPLC)

Method Comparison 3: The assay was compared to another commercially available immunoassay (Kit B) on 113 plasma samples (Concentration range: approximately 4 to 23 µmol/L. See “Method Comparison 3” graph.) By linear regression:

(IML 2000) = 0.90 (Kit B) – 0.02 µmol/L r = 0.925

Means:

8.7µmol/L (IMMULITE 2000)

9.6µmol/L (Kit B)

References

1)Selhub J, Miller JW. The pathogenesis of homocysteinemia: interruption of the coordinate regulation by s-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Am J Clin Nutr 1992;55:131-8. 2) Jacques PF, Bostom AG, Williams RR, et al. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase and plasma homocysteine concentrations. Circulation 1996;93:7-9. 3) Malinow MR. Plasma homocysteine and arterial occlusive diseases: A mini review. Clin Chem. 1995;40:173-76. 4) Clarke R, Daly L, Robinson K, Naughten E, et al. Hyperhomocysteinemia: An independent risk

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