BlueCross and BlueShield of Montana Medical Policy/Codes
Serum Holotranscobalamin (holo-TC) as a Marker of Vitamin B12 (i.e., Cobalamin)
Chapter: Medicine: Tests
Current Effective Date: April 18, 2013
Original Effective Date: April 18, 2013
Publish Date: January 18, 2013
Revised Dates: This policy is no longer scheduled for routine literature review and update.

Vitamin B12 (i.e., cobalamin) is an essential vitamin that is required for one-carbon metabolism and cell division.  Cobalamin deficiency can result from nutritional and/or dietary deficiencies (e.g., vegetarians, elderly), malabsorption of vitamin B12 (e.g., pernicious anemia, after gastrectomy), or other relatively uncommon gastrointestinal conditions (e.g., Whipple’s disease). Clinical signs and symptoms include megaloblastic anemia, paresthesia and neuropathy, and psychiatric symptoms such as irritability, dementia, depression, or psychosis.  While the hematologic abnormalities disappear promptly after treatment, neurologic disorders may become permanent if left untreated.

The diagnosis of cobalamin deficiency has traditionally been based on low total serum cobalamin levels, typically less than 200 pg/ml in conjunction with clinical evidence of disease.  However, this laboratory test has been found to be poorly sensitive and poorly specific.  Therefore, attention has turned to measuring metabolites of cobalamin a surrogate marker.  For example, in humans only two enzymatic reactions are known to be dependent on cobalamin—the conversion of methylmalonic acid (MMA) to succinyl-CoA, and the conversion of homocysteine and folate to methionine.  Therefore, in the setting of cobalamin deficiency, serum levels of MMA and homocysteine are elevated, and have been investigated as surrogate markers.

There has also been interest in the direct measurement of the subset of biologically active cobalamin.  Cobalamin in serum is bound to two proteins, transcobalamin and haptocorrin.  Transcobalamin-cobalamin complex (called holo-transcobalamin, or holo-TC) functions to transport cobalamin from its site of absorption in the ileum to specific receptors throughout the body.  Less than 25% of the total serum cobalamin exists as holo-TC, but this is considered the clinically relevant biologically active form.  Serum levels of holo-TC can be measured using a radioimmunoassay (RIA).  The Axis-Shield Holo TC™ RIA is an example of a radioimmunoassay for holo-TC that was cleared for marketing by the United States Federal Food and Drug Administration (FDA) in 2004, with the following labeled indication for use:  “The Axis-Shield Holo TC™ RIA is an in-vitro diagnostic assay for quantitative measurement of the fraction of cobalamin (vitamin B12) bound to the carrier protein transcobalamin in the human serum or plasma.  Measurements obtained by this device are used in the diagnosis and treatment of vitamin B12 deficiency.”



Blue Cross and Blue Shield of Montana (BCBSMT) considers measurement of serum holo-transcobalamin (holo-TC) experimental, investigational and unproven in the diagnosis and management of vitamin B12 deficiency.

Federal Mandate

Federal mandate prohibits denial of any drug, device or biological product fully approved by the FDA as investigational for the Federal Employee Program (FEP). In these instances coverage of these FDA-approved technologies are reviewed on the basis of medical necessity alone.

Rationale for Benefit Administration

This medical policy was developed through consideration of peer reviewed medical literature, FDA approval status, accepted standards of medical practice in Montana, Technology Evaluation Center evaluations, and the concept of medical necessity. BCBSMT reserves the right to make exceptions to policy that benefit the member when advances in technology or new medical information become available.

The purpose of medical policy is to guide coverage decisions and is not intended to influence treatment decisions. Providers are expected to make treatment decisions based on their medical judgment. BCBSMT recognizes the rapidly changing nature of technological development and welcomes provider feedback on all medical policies.

When using this policy to determine whether a service, supply or device will be covered, please note that member contract language will take precedence over medical policy when there is a conflict.


Validation of the clinical use of any diagnostic test focuses on three main principles:

  • Technical feasibility of the test;
  • Diagnostic performance of the test, such as sensitivity, specificity, and positive and negative predictive value both in different populations of patients and compared to the gold standard;
  • Clinical utility of the test, i.e., how the results of the diagnostic test will be used to improve management of the patient.

Technical Feasibility

The serum measurements of holo-TC involve the use of standard laboratory immunoassay techniques.  In the first step, holo-TC in the serum sample is separated by magnetic microspheres coated with monoclonal anti-human transcobalamin antibodies.  The cobalamin bound to the holo-TC is then released and measured by a competitive binding radioimmunoassay.

Diagnostic Performance

The diagnostic performance must be compared to the established gold standard for measuring cobalamin deficiency.  This is particularly problematic, since there is currently no established gold standard.  As noted in the Description section, serum levels of total cobalamin are considered poorly sensitive and specific, and there have been several reports of the serum measurements proposing serum measures of methylmalonic acid (MMA) and homocysteine as an alternative gold standard.  Two general categories of patients may be considered; for example, those with symptoms suggestive of cobalamin deficiency, for whom the diagnosis may be confirmed by a treatment response to cobalamin supplementation.  One possible strategy would be to develop diagnostic parameters for holo-TC (i.e., the establishment of cut-off points for normal versus low values) based on this known population, followed by remeasuring holo-TC after treatment.  In a second step, the established diagnostic parameters could be applied to an independent population (representative of the United States’ population and diet) with suggestive symptoms.  One population of interest is composed of asymptomatic patients who are considered at risk for cobalamin deficiency, such as those with high-risk nutritional factors (i.e., elderly patients or those with restrictive diets), or those with a predisposing disease or condition, such as gastrectomy or autoimmune disease.  It is thought that identification of subclinical disease can prompt early treatment such that clinical symptoms do not develop.  Given the absence of a definitive gold standard, confirmation of a diagnosis of subclinical disease is problematic. 

According to the FDA decision summary, the cut-off values for holo-TC were based on a normal population instead of a population of those with known cobalamin deficiency.  For example, the low value of holo-TC, 37 pmol/L, was based on a study of 303 normal Finnish individuals.  This study has also been published in the peer-reviewed literature.  Participants included 226 normal elderly subjects and 80 normal, non-elderly adult subjects.  Patients were excluded from the trial if they had hyperhomocysteinemia, evidence of a possible cobalamin deficiency.  In addition, patients in the lowest one third of holo-TC results underwent additional testing with MMA; those with elevated MMA levels were also excluded.  In the normal reference population, the holo-TC range was 25–254 pmol/L with a 95% central reference interval of 37–171 pmol/L.  Therefore, the cut-off value for a low result was established at 37 pmol/L.  This cut-off value was then applied to the results of 107 patients with presumed cobalamin deficiency, as evidenced by different combinations of an increased plasma homocysteine or MMA level, or a low total serum cobalamin level, defining patients with potential, possible, or probable cobalamin deficiency.  A total of 48% of those with presumed deficiency had a holo-TC below 37 pmol/L.  The frequencies of low holo-TC levels increased with increasing pretest probability of cobalamin deficiency.  For example, among the 16 patients thought to have the highest pretest probability of cobalamin deficiency, based on elevated levels of homocysteine and MMA, 100% had low levels of holo-TC.  Therefore, this study used levels of homocysteine and MMA as the gold standard.  Based on this standard, the sensitivity of the test was only 48% among those with potential, possible, or probable cobalamin deficiency.  The authors conclude that further studies are needed to confirm the clinical utility and specificity of holo-TC in diagnosis of subclinical cobalamin deficiency.

Hvas and Nexo reported on a study of 143 subjects who were divided into four groups, those with a confirmed diagnosis of cobalamin deficiency based on a decreased total serum cobalamin (<200 pmol/L) and increased MMA (>0.70 ųmol/L), a second group thought to be normal based on normal values of total serum cobalamin and MMA, and finally two additional groups with an uncertain diagnosis due to conflicting values of total cobalamin and MMA.  Although these authors used the reference interval established in the above study (i.e., 24–157 pmol/L), the cut-off for a low result was set at 50 pmol/L.  Using this cut-off point, measurements of holo-TC had a sensitivity of 1.00 and specificity of 0.89 in classifying patients very likely to be, or not be, cobalamin deficient.  Among the 73 patients with conflicting levels of MMA and total cobalamin, 39 had low holo-TC levels.  Without a gold standard, it is difficult to interpret the results in this group with an uncertain diagnosis.  As noted by the authors, it is not possible to determine whether or not holo-TC correctly classified the individual as deficient or not.

Hermann and colleagues reported on another series of patients using the same 37 pmol/L cut-off established by Loikas.  This study included 93 omnivorous German controls, and several other groups of patients considered at risk for cobalamin deficiency: 111 German and Dutch vegetarians, 122 apparently health Syrians, 127 elderly Germans, and 92 patients with renal failure.  In addition to holo-TC, MMA, total serum cobalamin, and homocysteine were measured. A total of 72%, 50%, and 21% of vegetarians, Syrians, and the elderly had holo-TC levels of less than 35 pmol/L.  Similar to the study above, these low levels of holo-TC were associated with either normal or high levels of MMA.  Conversely, high levels of MMA were associated with normal holo-TC levels in other patients.  Again, it is difficult to interpret the clinical significance of these conflicting laboratory values.


There are inadequate data to establish holo-TC testing as an alternative to either total serum cobalamin or levels of MMA or homocysteine.  Cut-off points for low levels of cobalamin were based on a study of a homogeneous population of 303 Finnish subjects.  These values for a homogeneous population of Finnish subjects with a diet high in fish might not be able to be extrapolated to the heterogeneous American population and diet.  Furthermore, these cut-off points require confirmation in a larger population of patients whose cobalamin status is unknown.

Clinical Utility

Advocates of holo-TC testing posit that this laboratory test can identify early subclinical stages of cobalamin deficiency, permitting prompt initiation of treatment, specifically supplementary cobalamin dietary supplementation.  This hypothesis was not directly tested in any of the identified published literature.  In the absence of a gold standard, the clinical significance of subclinical cobalamin deficiency must be further studied by understanding the natural history of this condition.  Does subclinical deficiency inevitably progress to clinical deficiency?  Does cobalamin supplementation normalize the values?  How variable are cobalamin levels within patients?  These clinical issues have not been well addressed in the literature.  Finally, for all patients at risk, i.e., vegetarians, the elderly, and post-gastrectomy patients, the recommended treatment of subclinical disease is further dietary supplementation of cobalamin.  This recommendation is appropriate, regardless of the level of measured cobalamin.

A MEDLINE database literature search through July 2007 did not identify any new published literature that would prompt reconsideration of the policy statement, which remains unchanged.

Serum holo-transcobalamin as a marker of vitamin B12 deficiency is not supported by evidence in the peer-reviewed medical literature that:

  • permits conclusions on the effect on health outcomes;
  • demonstrates an improvement in net health outcome;
  • demonstrates that use of this test is as beneficial as established alternatives.


Disclaimer for coding information on Medical Policies

Procedure and diagnosis codes on Medical Policy documents are included only as a general reference tool for each policy. They may not be all-inclusive.

The presence or absence of procedure, service, supply, device or diagnosis codes in a Medical Policy document has no relevance for determination of benefit coverage for members or reimbursement for providers. Only the written coverage position in a medical policy should be used for such determinations.

Benefit coverage determinations based on written Medical Policy coverage positions must include review of the member’s benefit contract or Summary Plan Description (SPD) for defined coverage vs. non-coverage, benefit exclusions, and benefit limitations such as dollar or duration caps. 

ICD-9 Codes

266.2, 266.9, 281.0-281.9, V12.1, V12.2

ICD-10 Codes
Investigational for all codes.
Procedural Codes: 0103T
  1. Summer, A.E., Chin M.M., et al.  Elevated methylmalonic acid and total homocysteine levels show high prevalence of vitamin B12 deficiency after gastric surgery.  Annals of Internal Medicine (1996) 124:469-76.
  2. Elin, R.J., and W.E. Winter.  Methylmalonic acid: a test whose time has come?  Archives of Pathology and Laboratory Medicine (2001) 125:824-27.
  3. Oh, R.C., and D.L. Brown.  Vitamin B12 deficiency.  American Family Physician (2003): 67:979-86.
  4. Loikas, S., Lopponen, M., et al.  RIA for serum holo-transcobalamin: method evaluation in the clinical laboratory and reference interval.  Clinical Chemistry (2003) 49:455-62.
  5. Hvas, A.M., and E. Nexo.  Holotranscobalamin as a predictor of vitamin B12 status.  Clinical Chemistry and Laboratory Medicine (2003) 41:1489-92.
  6. Herrmann, W., Obeid, R., et al.  Functional vitamin B12 deficiency and determination of holotranscobalamin in populations at risk.  Clinical Chemistry and Laboratory Medicine (2003) 41:1478-88.
  7. Serum Holo-Transcobalamin as a Marker of Vitamin B12 (i.e., Cobalamin) Status.  Chicago, Illinois: Blue Cross Blue Shield Association Medical Policy Reference Manual (2006 April) Medicine 2.04.39.
  8. Clarke, R., Sherliker, P., et al.  Detection of vitamin B12 deficiency in older people by measuring vitamin B12 or the active fraction of vitamin B12, holotranscobalamin.  Clinical Chemistry (2007 May) 53(5):963-70.
January 2013  New 2013 BCBSMT medical policy.  Considered investigational.
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Serum Holotranscobalamin (holo-TC) as a Marker of Vitamin B12 (i.e., Cobalamin)