vitamin B12/folate measurement
Reference interval
Table
| analyte | value |
|---|---|
| Vitamin B-12: | 210-920 pg/mL (< 100 pg/mL is diagnostic) |
| Serum Folate: | 2.0-14.0 ng/mL |
| Red Cell Folate: | 110-700 ng/mL |
Principle
The Quantaphase B-12/Folate is performed by combining a serum or plasma sample with Vitamin B-12 (57Co) &/or folate (125I)in a solution containing dithiothreitol (DTT) & cyanide. The mixture is boiled to inactivate endogenous binding proteins & to convert the various forms of Vitamin B-12 to cyanocobalamin. The reduced folate & its analog are stabilized by DTT during the heating. The mixture is cooled & then combined with immobilized affinity- purified porcine intrinsic factor & folate binding proteins. This addition adjusts & buffers the pH of the reaction mixture to 9.2. The reaction is then incubated for one hour at room temperature.
During incubation, the endogenous & labeled vitamin compete for the limited number of binding sites on the basis of their relative concentrations. The reaction mixtures are then centrifuged & decanted. Labeled & unlabeled vitamins binding to the immobilized binding proteins are concentrated at the bottom of the tube in the form of a pellet. The unbound vitamins on the supernatant are discarded & the radioactivity associated with the pellet is counted. Standard curves are prepared using precalibrated Vitamin B-12/Folate standards in a human serum albumin base. The concentrations of the Vitamin B-12 & folate in the patient serum are determined from the standard curves.
In the erythrocyte folate procedure, the sample is first diluted 1:1 with ascorbic acid in water & incubated for 90 minutes. The ascorbic acid stabilizes the folate liberated from erythrocytes. The 90 minute incubation is necessary for hemolysis of erythrocytes & allows the endogenous folate conjugases to hydrolyze the conjugated pteroylpolyglutamates to pteroylmonoglutamates prior to assay. The sample is further diluted 1:2 with protein diluent. The concentration of protein in this diluent is 2 times that found in the standards. The resulting diluted hemolysate thus contains a protein concentration & matrix similar to that used in the standards.
Clinical significance
Serum folate, serum Vitamin B-12 & red blood cell folate are among the most common tests ordered in the investigation of anemia. Since deficiency of either vitamin may be a cause of megaloblastic anemia, it is essential to determine the levels of both Vitamin B-12 & folic acid to establish the etiology of the anemia. Vitamin B-12 is an essential cofactor in intermediary metabolism & is required for the biosynthesis of DNA. Untreated vitamin B-12 deficiency may lead to severe anemia & potentially irreversible central nervous system degeneration. Folic acid is required in cellular metabolism & hematopoiesis, & prolonged folic acid deficiency may lead directly to megaloblastic anemia.
Red cell folate concentration shows a much better correlation with the presence of megaloblastic changes than serum levels & is unaffected by short term dietary changes. However erythrocyte folate levels may be normal in megaloblastic state due to folate lack when anemia is slight or absent. Also, low erythrocyte folate levels have been observed in approximately 1/2 of patients with primary cobalamin deficiency. Therefore it is necessary to measure the serum Vitamin B-12 level along with the red cell folate level.
Specimen
The determination of B-12FOLATE should be performed on serum or plasma. AVOID HEMOLYSIS. Collect blood sample in a red-top or plasma (EDTA) Centrifuge the sample & separate from cells.
When measurement of red cell folate is required, sample should be collected in EDTA blood collection tubes. Determine & record the hematocrit. If the specimen is to be analyzed within 3-4 hours after collection, it may be stored at 2-8 *C. Otherwise, specimens should be stored as follows & frozen until just prior to analysis.
Whole Blood Hemolysate: Add 100 uL of well-suspended blood to 1 mL of 0.4% ascorbic acid solution. Vortex gently If the sample is to be assayed at a later date, the hemolysate should be frozen immediately. The hemolysate is stable for 10 days when stored at -20*C. On the day of the assay, allow the hemolysate to thaw. Add 100 uL of the hemolysate to an appropriately labeled reaction tube. Add 100 uL of red cell folate diluent to the same reaction tube. Proceed with the assay without delay.
If assay is performed within 24 hours after collection, the specimen should be stored in the refrigerator at 2-8 C. If the testing will be delayed more than 24 hours, the specimen should be frozen. Mix thoroughly after thawing to ensure consistency in the results. Avoid repeated freezing & thawing.
Specimens showing particulate matter, erythrocytes, or turbidity should be centrifuged before testing.
More general terms
More specific terms
- folate in blood
- folate in erythrocytes
- folate in serum/plasma
- folate measurement
- vitamin B12 in body fluid
- vitamin B12 in serum/plasma
Additional terms
References
- ↑ Quantaphase B-12/Folate Radioassay. Clinical Division, Product information published by Bio-Rad, Hercules, California, April, 1989.
- ↑ Guidelines for Evaluating a B-12 (Cobalamin) Assay. National Committee for Clinical Laboratory Standards, March 1980.
- ↑ Gutcho, S. & Mansback, L., Simultaneous radiassay of serum vitamin B-12 & folic acid. New Eng., 23: 1609-1614, 1977.
- ↑ Herbert, Vit B-12 & folate deficiency. Nuclear Medicine, edited by B. Rothfeld, Lippincott, Philadelphia, pp. 69-84, 1974.
- ↑ Green R. Screening for vitamin B12 deficiency: caveat emptor. Ann Intern Med. 1996 Mar 1;124(5):509-11. PMID: https://www.ncbi.nlm.nih.gov/pubmed/8602711