|Lildballe, Dorte - AARHUS UNIV., DENMARK|
|Hardlei, Tore - AARHUS UNIV., DENMARK|
|Nexo, Ebba - AARHUS UNIV., DENMARK|
Submitted to: Clinical Chemistry and Laboratory Medicine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 21, 2008
Publication Date: February 4, 2009
Citation: Lildballe, D.L., Hardlei, T.F., Allen, L.H., Nexo, E. 2009. High concentrations of haptocorrin interfere with routine measurement of cobalamins in human serum and milk. A problem and its solution. Clin. Chem. Lab Med., 47:182-187. Interpretive Summary: Measurement of cobalamins (Cbl, vitamin B12) in serum is important in the initial screening for vitamin B12 deficiency in humans, but is also important for evaluating the supply of the vitamin from human milk to the infant. Today, most routine laboratories employ commercial methods available on semiautomated platforms developed for measurement in human serum. The methods are based on liberation of Cbl from its serum binding proteins transcobalamin (TC) and haptocorrin (HC) and subsequent measurement of the Cbl employing a competitive protein binding assay with intrinsic factor (IF) as the binding protein, as IF specifically binds intact forms of Cbl. Because of the design of the assay for Cbl, it is obviously important that the Cbl binding capacity of the sample is destroyed prior to addition of IF. Normal serum contains <1 nM HC (1) and 0.5-1.5 nM TC (2). Currently, little is known about the validity of measuring Cbl in samples containing a high concentration of unsaturated HC (apoHC). Human milk is reported to contain 100-fold more HC than serum, mostly in the form of apoHC (3). In addition, occasional serum samples, notably from patients with various malignancies, have an increased concentration of apoHC, and up to a 10,000 fold increase in apoHC concentration has been described in serum samples from patients with hepatoma (4). Here, we report that a high concentration of apoHC interferes with routine measurement of Cbl and, depending on the method employed, gives rise to spuriously high or low values. We show that removal of excess apoHC solves the problem, and we use this strategy to measure the amount of Cbl in human milk.
Technical Abstract: Background: Human milk and occasional serum samples contain high concentrations of unsaturated haptocorrin (apoHC), which may influence measurement of cobalamins (Cbl). Methods: Cbl in serum samples spiked with increasing amounts of apoHC were measured employing the Centaur, Cobas and Architect analysers. Cobinamide-coated EAH sepharose (Cbi-Seph) was employed for pretreatment of the samples. Human milk samples were collected from 24 healthy mothers. HC was measured by ELISA. Results: The measured concentration of Cbl either increased (Centaur Analyzer) or decreased (Architect, Cobas Analyzers) significantly for HC >10nM, and was 220, 52 or 45% of the expected values in a serum sample containing 50 nM HC. Following pre-treatment with Cbi-Seph, the expected Cbl concentration was obtained (Centaur). The milk samples contained 4.5-180 nM HC. In samples containing >10 nM HC (n=19), the median concentration of Cbl decreased from 1.3 nM to 0.67 nM after pre-treatment with Cbi-Seph. Conclusions: HC in concentrations above 10 nM influence measurement of Cbl giving rise to falsely elevated or decreased results. Removal of apoHC by pre-treatment with Cbi-Seph solves the problem.