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Vitamin B12 Deficiency Cascade

CPT: 82607
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Synonyms

  • Pernicious Anemia Cascade

Test Includes

Vitamin B12 testing is performed on all samples.

If Vitamin B12 is <200 pg/mL: Intrinsic Factor Blocking Antibodies and Antiparietal Cell Antibody (APCA) will be performed at an additional charge.

If Vitamin B12 is between 200 and 400 pg/mL: Methylmalonic Acid (MMA) will be performed at an additional charge.

If Methylmalonic Acid (MMA) is >378 nmol/L: Intrinsic Factor Blocking Antibodies and Antiparietal Cell Antibody (APCA) will be performed at an additional charge.


Special Instructions

This test may exhibit interference when sample is collected from a person who is consuming a supplement with a high dose of biotin (also termed as vitamin B7 or B8, vitamin H, or coenzyme R). It is recommended to ask all patients who may be indicated for this test about biotin supplementation. Patients should be cautioned to stop biotin consumption at least 72 hours prior to the collection of a sample.



Related Documents

For more information, please view the literature below.

Vitamin B12 Deficiency Cascade: Understanding Nutritional Deficit


Specimen Requirements


Specimen

Serum


Volume

3 mL


Minimum Volume

2 mL (Note: This volume does not allow for repeat testing).


Container

Red-top tube or gel-barrier tube


Collection

If a red-top tube is used, transfer separated serum to a plastic transport tube.


Storage Instructions

Store specimen at room temperature


Stability Requirements

Temperature

Period

Room temperature

7 days

Refrigerated

7 days

Frozen

14 days

Freeze/thaw cycles

Stable x3


Test Details


Use

Diagnosis of Vitamin B12 Deficiency. Although often used as the first-line screening test for B12 deficiency, serum B12 measurement used in isolation has a generally poor sensitivity and specificity for detection of B12 deficiency.1,6,19,32 The National Health and Nutrition Examination Survey (NHANES) opted to use the combination of serum total vitamin B12 and methylmalonic acid (MMA) to monitor B12 status in the United States population.35 In the interest of economy, a number of groups have suggested the use of a sequential selection algorithm for the detection of B12 deficiency.5,14,33,34 In this approach, a second-line assay (in this case MMA) is performed only when the outcome of the first-line assay (vitamin B12 level) falls in an "equivocal" range.1,7 It has been suggested that borderline B12 levels (200-400 ng/L) should be followed up with measuring MMA levels.1 MMA levels below the upper limit of the reference interval (0-378 nmol/L) are strongly suggestive of normal B12 status.


Limitations

Ninety per cent of patients with pernicious anemia have gastric parietal cell antibodies, but specificity of this test is poor since they are also found in 15% of elderly subjects.

If IFA results are negative but suspicion for pernicious anemia remains, an elevated serum gastrin level is consistent with the diagnosis.7

Mutations in the gene encoding intrinsic factor, can also lead to an inherited form of B12 malabsorption and deficiency, which resembles pernicious anemia, but without autoantibody involvement.36

In the presence of discordance between laboratory test result and strong clinical features of B12 deficiency, it remains important to proceed with treatment to avoid neurological impairment.14

MMA can increase (300-700 nmol/L) in renal failure and its refractory to B12 administration.1

Some patients with gastric atrophy and diminished parietal cell function are not positive for IFA or PCA. Diminished acid secretion caused by gastric atrophy regardless of the etiology can cause increased secretion of gastrin. Elevated gastrin levels can support the diagnosis of PA in antibody negative patients.24,29 It is important to diagnose hypergastrinaemia arising from loss gastric parietal cells drives development of antral enterochromaffin cell hyperplasia that can further develop into neoplasia and carcinoid syndrome.1,3,24,30,31


Additional Information

B12 is essential for certain enzymatic reactions that are required for numerous physiologic functions including erythropoiesis and myelin synthesis.1,2 Impaired DNA synthesis caused by B12 deficiency impacts nuclear maturation of rapidly dividing cells. This affects hematopoiesis and results in the presence of immature and ineffective red cells that are larger than normal (megaloblasts) in a context of severe anemia and pancytopenia. This megaloblastic anemia is characterized by the hypersegmented neutrophils that can be seen on peripheral smears and giant bands in bone marrow. Other rapidly dividing cells of the small-bowel epithelium can be affected resulting in malabsorption and diarrhea.3 Glossitis is a frequent hallmark of megaloblastic anemia, with the patient experiencing a painful, smooth, red tongue. Ineffective erythropoiesis and associated increased red cell turnover can result in elevation in bilirubin levels, manifesting as jaundice.3

B12 deficiency can also produce neurological manifestations including sensory and motor disturbances (symmetric paresthesias, numbness and gait problems), ataxia, cognitive decline leading to dementia and psychiatric disorders. These neurological symptoms often predominate and can frequently occur in the absence of hematological complications.3,7 In fact, the majority of patients with suspected B12 deficiency do not have anemia.5-8

Emerging evidence indicates that low (though not necessarily deficient) B12 is associated with increased risk of various chronic diseases of ageing including cognitive dysfunction, cardiovascular disease and osteoporosis.5,6 Dietary vitamin B12 is normally bound to proteins in food and requires release by gastric acid and pepsin in the stomach.7 In the small intestine, vitamin B12 binds to intrinsic factor (IF) produced by gastric parietal cells. In the ileum, the B12-IF complex binds to specific receptors, which facilitates absorption into the blood. Large amounts of absorbed vitamin B12 are stored in the liver such that any reduction in vitamin B12 intake/absorption may take many years to manifest clinically.8 Low B12 status, especially in older adults, is rarely attributable to dietary insufficiency9 and is more typically the result of malabsorption related to atrophic gastritis, inflammatory bowel disease or use of proton pump inhibitors or other gastric acid suppressant drugs.2,6,7,10-13

The diagnosis of vitamin B12 deficiency requires consideration of both the clinical state of the patient and the results of laboratory tests. Screening average-risk adults for vitamin B12 deficiency is not recommended.2 However, testing should be considered in patients with risk factors and/or clinical blood count and serum vitamin B12 level.2,5,7,14,15 The World Health Organization16 and the British Committee for Standards in Haematology14 suggested using 200 pg/mL as a cut-off to define B12 deficiency. In practice, detectable disturbances in metabolic networks consistent with possible deficiency occur at B12 levels as high as 400 pg/mL.17

A significant number of B12-deficient patients may be overlooked when serum B12 measurement is used in isolation.5,17 Further investigation using a second-line test can be useful for serum B12 results that fall within the indeterminate range. The enzyme, methylmalonyl-CoA mutase requires vitamin B12 as a cofactor for the conversion of methylmalonyl-CoA to succinyl-CoA.5 In vitamin B12 depletion, reduced activity of this enzyme leads to an accumulation of methylmalonyl-CoA which is, in turn, hydrolyzed to methylmalonic acid. Measurement of serum methylmalonic acid provides biochemical evidence of metabolic abnormalities consistent with B12 insufficiency.2,5,7,10,14,18,19

In the United States and the United Kingdom, the prevalence of vitamin B12 deficiency has been estimated to be approximately 6% of persons younger than 60 years, and nearly 20% in those older than 60 years.10 B12 status in the United States has been assessed in the National Health and Nutrition Examination Survey (NHANES).20 Using NHANES data from 1999 to 2004, the prevalence of B12 status defined as low was estimated to be 2.9%, 10.6% or 25.7% based on serum B12 cut-off values of 200, 300 and 400 pg/mL, respectively.20 Using these cut-off values, the prevalence of low B12 status increased with age from young adults (19-39 years of age) to older adults (greater than or equal to 60 years of age), and was generally higher in women than than in men (prevalence of 3.3% versus 2.4% with a serum B12 level of <200 pg/mL, respectively).20 Using increased levels of MMA as a functional indicator of B12 status, the prevalence of low B12 status was 2.3% or 5.8% based on cut-off values of >376 and >271 nmol/L, respectively.20 The prevalence of increased levels of MMA increased with age and was not different between men and women.20 Notably, only 50-75% of participants in NHANES with low levels of serum B12 had increased levels of MMA.20 It should also be noted that modest increases occur with renal failure.7

Pernicious Anemia (PA) caused by autoimmune destruction of gastric parietal cells and atrophy of the gastric mucosa is the most common cause of vitamin B12 deficiency.3,6,7,21 Asymptomatic autoimmune gastritis, a chronic inflammatory disease of the gastric mucosa, precedes the onset of mucosal atrophy by 10-20 years.22

With disease progression, an increasing number of the parietal cells that produce hydrochloric acid and intrinisic factor are destroyed.22 This may present initially as iron deficiency anemia due to loss of gastric acid, which is required for iron absorption.1,23 Ultimately, diminished production of intrinsic factor together with development of neutralizing antibody against intrinsic factor itself leads to B12 malabsorption.2,3,10,24 The autoimmune nature of PA is reflected by the presence of autoantibodies against the parietal cell proton pump protein (H/K ATPase) and to intrinsic factor.3,20,24,25 This condition frequently coexists with other autoimmune disorders including Hashimoto's thyroiditis and type 1 diabetes mellitus.3,24,29

Parietal Cell Antibodies (PCA) are present at a high frequency in PA (80%-90%), especially in early stages of the disease and are considered a predictive marker of subsequent gastric mucosa atrophy and its hematologic manifestations.3,24 In the later stages of the disease, the incidence of PCA decreases due to the progression of autoimmune gastritis and a loss of gastric parietal cell mass, as a result of the decrease in antigenic rate.26 PCA can precede the clinical symptoms of the gastric disease by several years.3 PCA are found in 90% of patients with PA, but have low specificity and are seen in various autoimmune disorders.1 Intrinsic Factor Antibodies (IFA) are less sensitive, but are considered highly specific for PA.3 Studies have reported positivity for IFA in 40%-60% of patients with PA, which rises to 60%-80% with increasing duration of disease.3,27 The combined assessment of both PCA and IFA increases diagnostic performance, with 73% sensitivity and 100% specificity.28

Diminished acid secretion caused by gastric atrophy resulting from autoimmune disease or some other etiology elevates secretion of gastrin. Elevated gastrin levels can support the diagnosis of PA.24,29 Hypergastrinaemia arising from loss gastric parietal cells drives development of antral enterochromaffin cell hyperplasia that can further develop into neoplasia and carcinoid syndrome.1,3,24,30,31


Footnotes

1. Green R, Datta Mitra A. Megaloblastic Anemias: Nutritional and Other Causes. Med Clin North Am. 2017 Mar;101(2):297-317.28189172
2. Langan RC, Goodbred AJ. Vitamin B12 Deficiency: Recognition and Management. Am FamPhysician. 2017 Sep 15;96(6):384-389.28925645
3. Bizzaro N, Antico A. Diagnosis and classification of pernicious anemia. AutoimmunRev. 2014 Apr-May;13(4-5):565-568.24424200
4. Green R. Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood. 2017 May 11;129(19):2603-2611.28360040
5. Harrington DJ. Laboratory assessment of vitamin B12 status. J Clin Pathol. 2017 Feb;70(2):168-173.27169753
6. Hughes CF, McNulty H. Assessing biomarker status of vitamin B12 in the laboratory: no simple solution. Ann Clin Biochem. 2018 Mar;55(2):188-189.29169259
7. Stabler SP. Vitamin B12 deficiency. N Engl J Med. 2013 May 23;368(21):2041-2042.23697526
8. Shipton MJ, Thachil J. Vitamin B12 deficiency - A 21st century perspective. Clin Med(Lond). 2015 Apr;15(2):145-150.25824066
9. Hughes CF, Ward M, Hoey L, McNulty H. Vitamin B12 and ageing: current issues and interaction with folate. Ann Clin Biochem. 2013 Jul;50(Pt 4):315-329.23592803
10. Hunt A, Harrington D, Robinson S. Vitamin B12 deficiency. BMJ. 2014 Sep 4;349:g5226.25189324
11. Toh BH. Pathophysiology and laboratory diagnosis of pernicious anemia. ImmunolRes. 2017 Feb;65(1):326-330.27538411
12. de Jager J, Kooy A, Lehert P, et al. Long term treatment with metformin in patients with type 2 diabetes and risk of vitamin B-12 deficiency: randomised placebo controlled trial. BMJ. 2010 May 20;340:c2181.20488910
13. Lam JR, Schneider JL, Zhao W, Corley DA. Proton pump inhibitor and histamine 2 receptor antagonist use and vitamin B12 deficiency. JAMA. 2013 Dec 11;310(22):2435-2442.24327038
14. Devalia V, Hamilton MS, Molloy AM; British Committee for Standards in Haematology. Guidelines for the diagnosis and treatment of cobalamin and folate disorders. Br J Haematol. 2014 Aug;166(4):496-513.24942828
15. Kaferle J, Strzoda CE. Evaluation of macrocytosis. Am Fam Physician. 2009 Feb 1;79(3):203-208.19202968
16. de Benoist B. Conclusions of a WHO Technical Consultation on folate and vitamin B12 deficiencies. Food Nutr Bull. 2008 Jun;29(2 Suppl):S238-244.18709899
17. Herrmann W, Obeid R, Schorr H, Geisel J. The usefulness of holotranscobalamin in predicting vitamin B12 status in different clinical settings. Curr Drug Metab. 2005 Feb;6(1):47-53.15720207
18. Smith AD, Refsum H. Do we need to reconsider the desirable blood level of vitamin B12? J InternMed. 2012 Feb;271(2):179-182.22092891
19. Carmel R. Biomarkers of cobalamin (vitamin B-12) status in the epidemiologic setting: a critical overview of context, applications, and performance characteristics of cobalamin, methylmalonic acid, and holotranscobalamin II. Am J Clin Nutr. 2011 Jul;94(1):348S-358S.21593511
20. Bailey RL, Carmel R, Green R, et al. Monitoring of vitamin B-12 nutritional status in the United States by using plasma methylmalonic acid and serum vitamin B-12. Am J Clin Nutr. 2011 Aug;94(2):552-561.21677051
21. Green R. Folate, cobalamin and megaloblastic anemias. In :Kaushansky K, Lichtman MA, Prchal JT, Levi MM, Press OW, Burns LJ, Caligiuri M, eds. Williams Hematology. 9th ed. New York, NY:MacGraw-Hill; 2016.
22. Toh BH, Sentry JW, Alderuccio F. The causative H+/K+ ATPase antigen in the pathogenesis of autoimmune gastritis. Immunol Today. 2000 Jul;21(7):348-354.10871877
23. Hershko C, Ronson A, Souroujon M, Maschler I, Hevd J, Patz J. Variable hematologic presentation of autoimmune gastritis: age-related progression from iron deficiency to cobalamin depletion. Blood. 2006 Feb 15;107(4):1673-1679.16239424
24. Toh BH. Diagnosis and classification of autoimmune gastritis. Autoimmun Rev. 2014 Apr-May;13(4-5):459-462.24424193
25. Rusak E, Chobot A, Krzywicka A, Wenzlau J. Anti-parietal cell antibodies – diagnostic significance. Adv Med Sci. 2016 Sep;61(2):175-179.26918709
26. Toh BH, Alderuccio F. Pernicious anaemia. Autoimmunity. 2004 Jun;37(4):357-361.15518059
27. Carmel R. How I treat cobalamin (vitamin B12) deficiency. Blood. 2008 Sep 15;112(6):2214-2221.18606874
28. Lahner E, Norman GL, Severi C, et al. Reassessment of intrinsic factor and parietal cell autoantibodies in atrophic gastritis with respect to cobalamin deficiency. Am J Gastroenterol. 2009 Aug;104(8):2071-2079.19491828
29. Antico A, Tampoia M, Villalta D, Tonutti E, Tozzoli R, Bizzaro N. Clinical usefulness of the serological gastric biopsy for the diagnosis of chronic autoimmune gastritis. Clin Dev Immunol. 2012;2012:520970.23251219
30. Campana D, Ravizza D, Ferolla P, et al. Risk factors of type 1 gastric neuroendocrine neoplasia in patients with chronic atrophic gastritis. A retrospective, multicentre study. Endocrine. 2017 Jun;56(3):633-638.27592118
31. Vanoli A, La Rosa S, Luinetti O, et al. Histologic changes in type A chronic atrophic gastritis indicating increased risk of neuroendocrine tumor development: the predictive role of dysplastic and severely hyperplastic enterochromaffin-like cell lesions. Hum Pathol. 2013 Sep;44(9):1827-1837.23642738
32. Hannibal L, Lysne V, Bjorke-Monsen AL, et al. Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency. Front Mol Biosci. 2016 Jun 27;3:27.27446930
33. Berg RL, Shaw GR. Laboratory evaluation for vitamin B12 deficiency: the case for cascade testing. Clin Med Res. 2013 Feb;11(1):7-15.23262189
34. Green R, Kinsella LJ. Current concepts in the diagnosis of cobalamin deficiency. Neurology. 1995 Aug;45(8):1435-1440.7644036
35. Yetley EA, Pfeiffer CM, Phinney KW, et al. Biomarkers of vitamin B-12 status in NHANES: a roundtable summary. Am J Clin Nutr. 2011 Jul;94(1):313S–321S.21593512
36. Nielsen MJ, Rasmussen MR, Andersen CB, Nexo E, Moestrup SK. Vitamin B12 transport from food to the body's cells--a sophisticated, multistep pathway. Nat Rev Gastroenterol Hepatol. 2012 May 1;9(6):345-354.22547309

LOINC® Map

Order Code Order Code Name Order Loinc Result Code Result Code Name UofM Result LOINC
141503 Vitamin B12 Deficiency Cascade 001503 Vitamin B12 pg/mL 2132-9
Reflex Table for Vitamin B12
Order Code Order Name Result Code Result Name UofM Result LOINC
Reflex 1 010424 APCA+IF Ab 010422 Intrinsic Factor Abs, Serum AU/mL 31443-5
Reflex Table for Vitamin B12
Order Code Order Name Result Code Result Name UofM Result LOINC
Reflex 1 010424 APCA+IF Ab 006522 Antiparietal Cell Antibody Units 8087-9
Reflex Table for Vitamin B12
Order Code Order Name Result Code Result Name UofM Result LOINC
Reflex 1 010424 APCA+IF Ab 010435 Interpretation n/a
Reflex Table for Vitamin B12
Order Code Order Name Result Code Result Name UofM Result LOINC
Reflex 1 706990 Methylmalonic Acid, Serum 706798 Methylmalonic Acid, Serum nmol/L 13964-2
Reflex 2 010427 Interpretation 010427 Interpretation n/a
Reflex Table for Vitamin B12
Order Code Order Name Result Code Result Name UofM Result LOINC
Reflex 1 706990 Methylmalonic Acid, Serum 706798 Methylmalonic Acid, Serum nmol/L 13964-2
Reflex 2 010423 APCA+IF Ab 010422 Intrinsic Factor Abs, Serum AU/mL 31443-5
Reflex Table for Vitamin B12
Order Code Order Name Result Code Result Name UofM Result LOINC
Reflex 1 706990 Methylmalonic Acid, Serum 706798 Methylmalonic Acid, Serum nmol/L 13964-2
Reflex 2 010423 APCA+IF Ab 006522 Antiparietal Cell Antibody Units 8087-9
Reflex Table for Vitamin B12
Order Code Order Name Result Code Result Name UofM Result LOINC
Reflex 1 706990 Methylmalonic Acid, Serum 706798 Methylmalonic Acid, Serum nmol/L 13964-2
Reflex 2 010423 APCA+IF Ab 010434 Interpretation n/a
Reflex Table for Vitamin B12
Order Code Order Name Result Code Result Name UofM Result LOINC
Reflex 1 010426 Interpretation 010426 Interpretation n/a

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