TITLE: FOLATE (CH205)

I. PRINCIPLE:

Principles of the Procedure

The Access Folate assay is a competitive binding receptor assay. For the assay of folate in serum or plasma (heparin), no pre-treatment is required.

A serum, plasma (heparin) sample is treated to release folate from endogenous binding proteins. Folate binding protein, mouse anti-folate binding protein, folic acid-alkaline phosphatase conjugate, and goat anti-mouse capture antibody coupled to paramagnetic particles are added to the reaction vessel. Folate in the sample competes with the folic acid-alkaline phosphatase conjugate for binding sites on a limited amount of folate binding protein. Resulting complexes bind to the solid phase via mouse anti-folate binding protein. After incubation in a reaction vessel, materials bound to the solid phase are held in a magnetic field while unbound materials are washed away. Then, the chemiluminescent substrate Lumi-Phos* 530 is added to the vessel and light generated by the reaction is measured with a luminometer. The light production is inversely proportional to the concentration of folate in the sample. The amount of analyte in the sample is determined from a stored, multi-point calibration curve.

Summary and Explanation

Folate is an essential vitamin vital to normal cell growth and DNA synthesis. It is present in a wide variety of foods such as dark, leafy vegetables, citrus fruits, yeast, beans, eggs, and milk. It is absorbed by the small intestine and stored in the liver. A folate deficiency can lead to megaloblastic anemia and ultimately to severe neurological problems.(2,3)

Folate deficiency can be caused by insufficient dietary intake, malabsorption or excessive folate utilization. Excessive utilization occurs very commonly during pregnancy. Alcoholism, hepatitis, or other liver-damaging diseases can also cause excessive folate utilization.(3,4) Folate levels in both serum and red blood cells are used to assess folate status. The serum folate level is an indicator of recent folate intake. Red blood cell (RBC) folate is the best indicator of long term folate stores. A low RBC folate value can indicate a prolonged folate deficiency.

Folate and vitamin B12 are linked by the reaction pathway for methionine synthesis. A deficiency in either leads to a disruption of this pathway and to similar clinical symptoms.(2,3) Another consequence of this common metabolic pathway is that a B12 deficiency disrupts the uptake of folate into red blood cells. This leads to a low RBC folate value even with adequate folate intake. For the above reasons, it is often necessary to measure both vitamins in a clinical workup. The treatment depends on which vitamin is deficient.

II. SPECIMEN: A. Serum and lithium heparin plasma from fasting individuals are the recommended samples. If the assay will not be completed immediately, refrigerate the sample at 2 to 8°C. If the assay will not be completed within eight hours, or for shipment of samples, freeze at -20°C. (5,6)

B. Observe the following recommendations for handling, processing, and storing blood samples:(7) 1. Collect all blood samples observing routine precautions for venipuncture; see Phlebotomy Standard PE1100. 2. Allow serum samples to clot completely before centrifugation. 3. Criteria for rejection: see GL016 Unacceptable Specimen Policy. 4. Beckman Coulter, Inc. recommends that frozen specimens can be stored up to six months before testing. 5. Thaw samples only once. After thawing, samples should be centrifuged again prior to analysis.

C. Use the following guidelines when preparing specimens: 1. Ensure residual fibrin and cellular matter has been removed prior to analysis. 6. Follow blood collection tube manufacturer’s recommendations for centrifugation.

D. Each laboratory should determine the acceptability of its own blood collection tubes and serum separation products. Variations in these products may exist between manufacturers and, at times, from lot-to-lot.

E. Do not use hemolyzed samples. The folate level in the red cells is much greater than that of the serum or plasma (heparin), leading to spuriously high results.

F. Sample size is 55 uL for each determination in addition to the sample container and system dead volume.

III. MATERIALS: A. Beckman Coulter Access 2 Immunoassay System and supplies

B. R1: Access Folate Reagent Pack Cat. No. A14208: 100 determinations, 2 packs, 50 tests/pack

Provided ready to use. Store upright and refrigerate at 2 to 10°C. Refrigerate at 2 to 10°C for a minimum of two hours before use on the instrument. Stable until the expiration date stated on the label when stored at 2 to 10°C. Stable at 2 to 10°C for 14 days after initial use. Signs of possible deterioration are a broken elastomeric layer on the pack or control values out of range. If the reagent pack is damaged (i.e., broken elastomer), discard the pack. All antisera are polyclonal unless otherwise indicated.

1. R1a: Mouse monoclonal anti-folate binding protein, paramagnetic particles coated with goat anti-mouse IgG, buffer, human serum albumin (HSA). 2. R1b: 1.0M Ascorbate, 0.05N HCl, pH 5.5. 3. R1c: Milk folate binding protein (bovine) in buffer, HSA. 4. R1d: Folic acid alkaline phosphatase (bovine) conjugate in buffer, HSA. 5. R1e: 0.6M K3PO4.

Mix contents of new (unpunctured) reagent packs by gently inverting pack several times before loading on instrument. Do not invert open (punctured) packs.

C. Access Folate Calibrators Cat. No. A14207: S0-S5, 4.0 mL/vial

Provided ready to use. Store at -20°C. Thaw only once. Mix contents by gently inverting before use. Avoid bubble formation. Stable until the expiration date stated on the label when stored at -20°C. Stable for three months when stored at 2–10°C. Signs of possible deterioration are control values out of range. Refer to calibration card for exact concentrations.

1. S0: Buffered matrix with human serum albumin (HSA), surfactant, < 0.1% sodium azide, and 0.25% ProClin** 300. Contains 0.0 ng/mL (nmol/L) folate. 7. S1–S5: Folate (pteroylglutamic acid) in buffered matrix at levels of approximately 1.0, 2.5, 5.0, 10.0, and 20.0 ng/mL (2.3, 5.7, 11.3, 22.7, and 45.3 nmol/L), respectively, with HSA, surfactant, < 0.1% sodium azide, and 0.25% ProClin 300. 8. Calibration Card: 1.

D. Access Substrate Cat. No. 81906: 4 x 130 mL

Provided ready to use. Refer to the following chart for storage conditions and stability. An increase in substrate background measurements may indicate instability. Do not pool bottles of substrate.

|Condition |Storage |Stability |
|Unopened |2 to 8°C |Until expiration date stated on the |
| | |label |
|Equilibration prior to use (unopened) |15 to 30°C (room temperature) |Minimum 18 hours |
| | |Maximum 14 days |
|In use (opened) |Internal substrate supply position |Maximum 5 days |
| | | |
|In use (opened) |External fluids tray substrate |Maximum 14 days |
| |position | |

R2 Substrate: Lumi-Phos 530 (buffered solution containing dioxetane Lumigen* PPD, fluorescer, and surfactant).

Refer to the appropriate system manuals and/or Help system for detailed instructions.

E. Access Wash Buffer II, Cat. No. A16792

Provided ready to use. Stable until the expiration date stated on the label when stored at room temperature (15 to 30°C). An increase in substrate background measurements or increased relative light units for the zero calibrators in “sandwich”-type assays may indicate instability.

R3 Wash Buffer: TRIS buffered saline, surfactant, < 0.1 sodium azide, and 0.1% ProClin 300.

Refer to the appropriate system manuals and/or Help system for detailed instructions.

F. Quality Control (QC) materials.

IV. CALIBRATION: An active calibration curve is required for all tests. For the Access Folate assay, calibration is required every 28 days or when quality control or instrument maintenance dictate. Refer to the appropriate system manual and/or Help system for information on calibration theory, configuring calibrators, calibrator test request entry, and reviewing calibration data. Also see CH217 Beckman Maintenance and Calibration.

The Access Folate Calibrators are provided at six levels - zero and approximately 1.0, 2.5, 5.0, 10.0, and 20.0 ng/mL – prepared gravimetrically from purified folic acid and buffered HSA-based matrix. Assay calibration data are valid up to 28 days; current in-use lots are recalibrated accordingly.

Calibrators run in duplicate.

Quantitative assay calibration is the process by which samples with known analyte concentrations (i.e. assay calibrators) are tested like patient samples to measure the response. The mathematical relationship between the measured responses and the known analyte concentrations establishes the calibration curve. This mathematical relationship, or calibration curve, is used to convert RLU (Relative Light Unit) measurements of patient samples to specific quantitative analyte concentrations.

VI. QUALITY CONTROL: Quality Control materials simulate the characteristics of patient samples and are essential for monitoring the system performance of immunochemical assays. The control material is BioRad Liquichek Immunoassay Plus, levels 1 and 3, run once every 24 hours. Follow manufacturer's instructions for reconstitution and storage. Follow laboratory internal QC procedures if the results obtained are outside acceptable limits.

VII. SAFETY: 1. For in vitro diagnostic use. 9. Patient samples and blood-derived products may be routinely processed with minimum risk using the procedure described. However, handle these products as potentially infectious according to universal precautions and good clinical laboratory practices, regardless of their origin, treatment, or prior certification. Use an appropriate disinfectant for decontamination. Store and dispose of these materials and their containers in accordance with local regulations and guidelines. 10. Human source material used in the preparation of the reagent has been tested and found negative or non-reactive for Hepatitis B, Hepatitis C (HCV), and Human Immunodeficiency Virus (HIV-1 and HIV-2). Because no known test method can offer complete assurance that infectious agents are absent, handle reagents and patient samples as if capable of transmitting infectious disease.(8) 11. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal of liquids, flush with a large volume of water to prevent azide build-up.(9) 12. Xi. Irritant: 0.6M K3PO4. R 36/38: Irritating to eyes and skin. S 26-37: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable gloves. 13. Xi. Irritant: 0.25% ProClin 300. R 43: May cause sensitization by skin contact. S 28-37: After contact with skin, wash immediately with plenty of soap and water. Wear suitable gloves. 14. The Material Safety Data Sheet (MSDS) is available upon request from Beckman Coulter.

VIII. PROCEDURE: Refer to the appropriate system manuals and/or Help system for information on managing samples, configuring tests, requesting tests, reviewing test results, and system operation.

IX. CALCULATIONS: Patient test results are determined automatically by the system software using a weighted four parameter logistic curve (4PLC) math model. The amount of analyte in the sample is determined from the measured light production by means of the stored calibration data. Patient test results can be reviewed using the appropriate screen. Refer to the appropriate system manual and/or Help system for complete instructions on reviewing sample results.

X. REPORTING OF RESULTS: Reference Range: Normal: >3.0 ng/mL Indeterminate: 2.5-3.0 ng/mL Deficient: 20 ng/mL. • Samples are not diluted.

B. For assays employing antibodies, the possibility exists for interference by heterophile antibodies in the patient sample. Patients who have been regularly exposed to animals or have received immunotherapy or diganostic procedures utilizing immunoglobulins ot immunoglobulin fragments may produce antibodies, e.g. HAMA, that interfere with immunoassays. Additionally, other heterophile antibodies such as human anti-goat antibodies may be present in patient samples.(16,17)

Such interfering antibodies may cause erroneous results. Carefully evaluate the results of patients suspected of having these antibodies.

C. The Access Folate results should be interpreted in light of the total clinical presentation of the patient, including: symptoms, clinical history, data from additional tests, and other appropriate information.

D. Samples containing up to 10 mg/dL (171 µmol/L) bilirubin and lipemic samples containing the equivalent of 1800 mg/dL (20.32 mmol/L) triglycerides do not affect the concentration of folate assayed. In addition, samples with 5 g/dL (50 g/L) human albumin added to the endogenous albumin in the samples do not affect the concentration of folate assayed.

E. The following table describes the cross-reactivity of the assay with substances which are similar in structure to folate. The analytes were spiked into serum samples.

|Substance |Analyte Added |Cross-Reactivity |
| | |(%) |
|Aminopterin |500 ng/mL |0.2 |
|Phenytoin |100 µg/mL |< 0.1 |
|Methotrexate |100 ng/mL |< 0.2 |
|Folinic Acid |100 ng/mL |< 1.1 |

F. The lowest detectable level of folic acid distinguishable from zero (Access Folate Calibrator S0) with 95% confidence is 0.5 ng/mL (1.1 nmol/L).

XII. REFERENCES AND MANUFACTURER LITERATURE: Beckman Coulter, Inc. Access Folate product insert, Fullerton, CA 92835, WB A36254. Beckman Coulter, Inc. Access Substrate product insert, Fullerton, CA 92835, 386966. Beckman Coulter, Inc. Access Wash Buffer product insert, Fullerton, CA 92835, A16534 (Access), A16543 (UniCel).

1. Hoffbrand AV, Newcombe BFA and Mollin DL. Method of assay of red cell activity and the value of the assay as a test for folate deficiency, J Clin Pathol, 1966; 19: 17-28. 2. Hoffbrand AV. Vitamin B12 and folate metabolism: the megaloblastic anæmias and other nutritional anæmias. In Blood and its disorders, 1982; 199-263. Edited by Hardistry RM and Weatherall D. Philadelphia, PA: Blackwell Scientific Publications. 3. Chanarin I. Megaloblastic anæmia, cobalamin and folate, J Clin Pathol, 1987; 40: 978-984. 4. Herbert V. In Modern nutrition in health and disease, 1973; 221-244. Edited by Goodhart RS and Shils ME. Philadelphia, PA: Lea and Febiger.

5. Mastropaolo W, Wilson MA. Effect of light on serum B12 and folate stability. Clinical Chemistry, 1993; 5: 913. 6. Burtis CA, Ashwood ER. Tietz textbook of clinical chemistry, 2nd edition, 1994; W.B. Saunders Co: 2056. 7. Approved Guideline – Procedures for the handling and processing of blood specimens, H18-A2. 1999. National Committee for Clinical Laboratory Standards. 8. HHS Publication, 4th ed., May 1999. Biosafety in Microbiological and Biomedical Laboratories. Available http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm 9. DHHS (NIOSH) Publication No. 78-127, August 1976. Current Intelligence Bulletin 13 - Explosive Azide Hazard. Available http://www.cdc.gov/niosh. 10. Cembrowski GS, Carey RN. Laboratory Quality Management: QC & QA. ASCP Press, Chicago, IL, 1989. 11. CDC, Folate status in women of childbearing age, by race/ethnicity – United States, 1999-2000. MMWR 2002 Sep 13; 51 (36): 808-810. 12. Jacques PF, Selhub J, Bostom AG, Wilson PW, Rosenberg IH. The effect of folic acid fortification on plasma folate and total hemocysteine concentrations. New England Journal of Medicine 1999; 340 (19): 1499-1454. 13. Choumenkovitch SF, Jacques PF, Nadeau MR, Wilson PW, Rosenberg, Selhub J. Folic acid fortification increases red blood cell folate concentrations in Framingham study. Journal of Nutrition 2001; 131 (12): 3277-3280. 14. Cuskelly GJ, McNutty H, Scott JM. Fortification with low amounts of folic acid makes significant difference in folate status in young women: implications for prevention of neural tube defects. American Journal Clinical Nutrition 1999; 70 (2): 234-239. 15. Wiltshire EJ, Couper JJ. Improved folate status in children and adolescents during voluntary fortification of food with folate. Journal Pediatric Child Health 2004; 40 (1-2): 44-47. 16. Kricka, L. Interferences in immunoassays – still a threat. Clin Chem 2000; 46: 1037. 17. Bjerner J, et al. Immunometric assay interference: incidence and prevention. Clin Chem 2002; 48: 613–621. 18. Pfeiffer CM, Caudill SP, Gunter EW, Osterloh J, Sampson EJ. Biochemical indicators of B vitamin status in the US population after folic acid fortification: results from the National Health and Nutrition Examination Survey 1999-2000. Am J Clin Nutr 2005; 82: 442-50. 19. Approved Guideline – Evaluation of precision performance of clinical chemistry devices, EP5-A. 1999; 19:2 Villanova, PA: National Committee for Clinical Laboratory Standards. 20. In-house Heartland Regional Medical Center.