Lesson of the week

Diagnosing vitamin B-12 deficiency on the basis of serum B-12 assay

BMJ 2006; 333 doi: http://dx.doi.org/10.1136/bmj.333.7564.385 (Published 17 August 2006)
Cite this as: BMJ 2006;333:385

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We too continue to find low b12 levels and after a FY2 doctor attached to our practice did some basic research now offer patients the choice of buying o.t.c. Vitamin b12 form a health food (about £7 for 100 tablets of 1000 micrograms) shop or have injections at the surgery. This has been summarised and published in GP magazine.

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john sharvill, General Practitioner

Deal CT14 7EQ

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Dr. Gibbs, have a look at my eResponse above. The easy and cheap solution is the Internet. This is the link to a $36/year + mailing cost multivitamin with 100 mcg B12: http://www.vitacost.com/Twinlab-Daily-One-Without-Iron Also, since vitamin deficiency rarely happens for a single vitamin alone, such multi prevents any other common deficiency, and would deal with patients such as you describe, alcoholic, or other.

Multivitamins should be taken at the end of a meal to 'look' like food and slow absorption. I do not have a conflict with any vitamin maker and have paid full price myself for the above mentioned supplement for about 10 years, and there are many other sources.

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Eddie Vos, maintains health-heart.org

Sutton (Qc) Canada J0E 2K0

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I have been looking for information on this, as we are finding an increasing number of patients with significant deficiency which would appear to relate to diet (teenagers) or other conditions (alcoholism, pregnancy) which respond readily to oral supplements (the only one I am aware of that is easily available is Forceval - only 3mcg cyanocobalamin daily though, so it would not be enough where malabsorption was the problem). Yet hospital doctors and other GPs institute what is often lifetime treatment with injections on the basis of only slight reduction in B12, without consideration of whether this is a deficiency due to malabsorption or not. I strongly suspect that there is a tendency to look no further than injection treatment as it is safe, cheap and easy: however, it distresses patients (it appears more painful than most other injections) and ignores other causes.

Forceval is certainly more expensive on a drug cost basis, but when the cost of a professional actually giving a B12 injection every three months is taken into account, I suspect it would be no more expensive, and may well be less. It is more appropriate when malnutrition is a cause. Clearly it is important to check on response, but that is easy enough. I have just had a result back on an alcoholic patient, with poor diet, started on Forceval three months ago when his B12 level was 86ng/L: this week it was 209 ng/L. MCV is not raised, so I am not concerned about missing a true B12 deficiency.

I note the reference in one of the responses to "physiological" deficiency in pregnancy - I would be interested to know how low can be considered "physiological". It seems impossible locally to get midwives to check folate and B12 when treating anaemia, but we often find low levels of both. I have recently found a lady who had severe hyperemesis early on, whose ferritin is low normal, but her B12 is only 66ng/L (and her folate is low too). I treat pregnant women with B12 below 100ng/L with one or two injections of B12, but then follow them up to make sure their levels remain normal. This is based purely on what seems safe to me. I cannot find any help locally - the haematology department here never agree to Schilling tests if asked, and simply tell us to give B12 injections.

I note that DTB (Vol 47, No 2) recommends high dose B12 tablets (1mg) instead of injections, which are simply not available here, but this high dose is not really necessary for most younger people I think. They refer to oral treatment with 50mcg-150mcg daily, but I am not sure what this is based on - I do not know of any such tablets.

I would be interested in any response to this, even though I am writing three years late.

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Mary G Gibbs, Genral Practitioner

City Road Surgery M15 4EA

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Congratulations on Authoring and Publishing such an excellent Article regarding the inaccuracies of Vitamine B12 serum levels. Dr William Osler, the Father of Internal Medicine, would roll over in his grave if he knew these sorts of things were occurring. 'Dr Osler was a man of immense personal charm, he epitomized Peabody’s saying that "the secret of patient care is caring for patients." '

The Art of Medicine perspective fortunately allowed Dr Devalia to arrive at the correct choice for treating the Patients' anemia with a Vitamine B-12 Supplement, when their clinical conditions didn't seem to be as life threatening as the "normal" B12 levels indicated.

It is unfortunate that some individuals, on this side of the Pond, measure solely a B-12 level when attempting to evaluate the entire Nutritional Status of a Patient. In my opinion, if you want to measure a Thiamine or Niacin serum level, or their metabolites, then that is the entity that has to be sent to the Lab, not a B-12 measurement which is then used to erroneously relate, or extrapolate, to other Vitamine levels. This is how, in my opinion, other medical misadventures could be initiated.

Your Article is excellent food for thought, keep up the good work.

Cordially,

Joseph W Arabasz MD PC Past Division Chairman, Anesthesiology, Cook County Hospital, Chicago, Illinois Past Chairman, Respiratory Therapy, Cook County Hospital, Chicago, Illinois Diplomate ABA Mensa Sigma Xi, The Professional International Science Research Society PO Box 6939 Denver, CO 80206 303-316-1740 http://www.topica.com/lists/josepharabasz@topica.com

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Joseph W Arabasz MD, Physician

Denver, Colorado 80206 USA

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Dear Sir,

We have read with interest the paper entitled “Diagnosing vitamin B12 deficiency on the basis of serum B12 assay” from the last BMJ issue (1).

We have been aware that the paper has stirred some anxiety among British colleges. To our opinion, we feel that "dysmyelopoietic appearance" might be a greater problem than neurological lesions nowadays, especially in countries in which homocysteine is not a routine test. In fact there is a possible risk to confuse “atypical forms” of vitamin B12 (cobalamin) deficiency with dysmyelopoetic stages, especially in elderly patients. In our experience (of more than 300 patients with documented cobalamin deficiency), this risk exists in at least 20% of the patient (2,3). Thus to exclude this possibility of confusion, we usually propose a "traitement d'épreuve" (exclusion therapy) with oral cobalamin therapy (1000 µg per day of oral cyanocobalamin for at least 7 days). In fact, in several studies, we have reported that one week of oral vitamin B12 therapy may be effective to improve or cure cobalamin deficiency (4-6). The readers of the BMJ may find several informations in a practical review of cobalamin deficiency management we have recently published in the CAMJ (7).

Professor Emmanuel ANDRES
For the study group of cobalamin deficiency of the University Hospital of Strasbourg, Department of Internal Medicine, University Hospital of Strasbourg, France, emmanuel andres@chru-strasbourg.fr

1. Devalia V. Diagnosing vitamin B-12 deficiency on the basis of serum B12 assay. BMJ 2006; 333: 385-386.

2. Andrès E, Affenberger S, Vinzio S, Kurtz JE, Noel E, Kaltenbach G et al. Food-cobalamin malabsorption in elderly patients: clinical manifestations and treatment. Am J Med 2005; 118: 1154-1159.

3. Andrès E, Affenberger S, Zimmer J, Vinzio S, Grosu D, Pistol G et al. Current hematological findings in cobalamin deficiency. A study of 201 consecutive patients with documented cobalamin deficiency. Clin Lab Haem 2006; 28: 50-56.

4. Kaltenbach G, Noblet-Dick M, Andrès E, Barnier-Figue G, Noel E, Vogel T et al. Réponse précoce au traitement oral par vitamine B12 chez des sujets âgés hypovitaminiques. Ann Med Interne (Paris) 2003; 154: 91-95.

5. Andrès E, Kaltenbach G, Noblet-Dick M, Noel E, Perrin AE, Vinzio S et al. Hematological response to short-term oral cyanocobalamin therapy for the treatment of cobalamin deficiencies in elderly patients. J Nutr Health Aging 2006; 10: 3-6.

6. Andrès E, Kaltenbach G, Noel E, Noblet-Dick M, Perrin AE, Vogel T et al. Efficacy of short-term oral cobalamin therapy for the treatment of cobalamin deficiencies related to food-cobalamin malabsorption. A study of 30 patients. Clin Lab Haem 2003; 25: 161-166.

7. Andrès E, Loukili NH, Noel E, Kaltenbach G, Ben Abdelgheni M, Perrin AE et al. Vitamin B12 (cobalamin) deficiency in elderly patients. CAMJ 2004; 171: 251-260.

Competing interests: None declared

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Emmanuel ANDRES, Professor of Internal Medicine

Department of Internal Medicine B, University Hospital, 1 place de l'Hôpital, 67091 STRASBOURG, F

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1 September 2006

Hamilton et al further highlight the fact that the problem of a ‘false-normal’ serum B12 assay truly exists, and suggest that this may be due to the presence of a high titre anti-intrinsic factor antibody interfering with the assay. Further studies would help in confirming this possibility. However, it remains unclear whether the magnitude of the problem is ‘rare’, given the fact that literally hundreds of thousands of assays are done globally and clinical information on patients with ‘normal’ B12 levels is unavailable. Laboratories providing serum B12 assays need to be aware of possible technical problems resulting in ‘false -normal’ results, and data collected centrally (i.e. MHRA) of such cases will help to clarify the magnitude any reasons for it.

Sharvill raises the question of how long should one treat a patient empirically if the anti-intrinsic actor antibody is negative. My pragmatic practice is to give an initial treatment of five injections (1mg/ml) of hydroxycobalamin and then to measure serum B12 at six monthly intervals, without any further treatment, for two years. If the serum B12 remains well within the reference range without any clinical problems, I suggest a further check in a year’s time and then no further monitoring or treatment if stable. It will be interesting to see what recommendations are proposed by Raghunath and colleagues.

Morton describes other scenarios where a low serum B12 level may have uncertain clinical significance, which again emphasises the importance of a very thorough clinical evaluation in the context of the laboratory result.

Vos’s suggestion of empirical oral B12 supplementation has to be taken with caution since it may not contain adequate amounts of B12 for an individual with pernicious anaemia 1.

Reference

1 Solomon LR. Oral vitamin B12 therapy: a cautionary note. Blood 2004; 103: 2863.

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Vinod Devalia, Consultant Haematologist

Princess of Wales Hospital, Coity Road, Bridgend CF31 1RQ

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Sir,

One of the cases in the report by Devalia is a timely reminder that current “no boil” automated cobalamin immunoassays may produce normal results in the presence of severe cobalamin deficiency. Since 2004 the external quality assessment scheme for cobalamin assays UKNEQAS Haematinics has become aware of a further eight cases of sera from patients with severe megaloblastic anaemia or subacute combined degeneration of the cord in whom current commercial assays have failed to detect cobalamin deficiency. In all eight cases intrinsic factor antibody levels were strongly positive, suggesting that intrinsic factor antibody interference may be the cause of assay failure. These cases are not just a difficulty in the selection of a cut off point for the reference range but are “false normal results” with levels well within the manufacturers normal reference range and yet severe clinical deficiency. Manufacturers’ product literature stresses that diagnosis of cobalamin deficiency should not be based on the results of a single assay and that all clinical features and other laboratory tests including intrinsic factor antibody levels should be considered.

Devalia suggests heterophilic antibody interference may be relevant to the mechanism but the cases reported are more likely to be due to high titre anti-intrinsic factor antibody levels.

In two patients both with megaloblastic anaemia, which subsequently responded to cobalamin treatment, the initial cobalamin assay results were normal, but the clinical features aroused suspicion that the laboratory result was incorrect. Pre treatment sera were available in both cases for further study by UKNEQAS haematinics. Both sera had high titre anti-intrinsic factor, heterophilic antibodies were not detected in one case, and the pre-treatment sera were investigated further in a round robin exercise of cobalamin assays.

Table 1. Pretreatment serum cobalamin in two cases with false normal cobalamin results with current commercial assays.

 

Manufacturer Abbott Architect Bayer  AdviaCentaur Beckman Coulter Access Diagnostic products CorporationImmulite2000 Roche Elecsys E170 E2010 Intrinsic factor antibody Launch Diagnostics
Reference range 189-883(pg/ml) 211-911(pg/ml) 180-914(pg/ml) 193-982(pg/ml) 191-663(pg/ml) 0-5.9 (u/ml)
Reference range  (S.I.units) 139-651(pmol/l) 156-672(pmol/l) 133-674(pmol/l) 143-725(pmol/l) 141-489(pmol/l)  
Case 1 (pg/ml) <60   145   356   310   81 244 u/ml
Case 2 (pg/ml) <60 217 317 360 70 >100 u/ml

The results demonstrate that only two of five methods found extremely low levels of cobalamin consistent with the clinical presentation of severe cobalamin deficiency in both cases. The Bayer centaur assay detected one of the two patient samples as low. The mechanism of assay failure is not yet clear, nor the frequency with which this may occur. The current commercial assays are competitive binding immunoassays which are no boil, and rely on alkaline hydrolysis and dithiothreitol or monothioglycerol treatment to release cobalamin from transcobalamin and other binders, and denature intrinsic factor antibody, prior to competitive binding against labelled cobalamin to assay intrinsic factor. We postulate that intrinsic factor antibody present in the patient serum at high titre fails to be denatured by the alkaline hydrolysis and dithiothreitol treatment and persists into the binding stage, resulting in assay failure. UKNEQAS Haematinics has alerted all manufacturers to this issue.

Ideally assays should be tested pre-commercial release with sera which contain high titre anti-intrinsic factor antibody to ascertain whether the assays will detect low cobalamin levels in this circumstance. It is not yet clear whether some assays are more vulnerable to assay interference that others.

The incidence of these”false normal” sera are rare but in our own clinical laboratory we detected four similar cases over a two year period from 12,000 cobalamin assay requests. Laboratories should not report cobalamin results in isolation from intrinsic factor antibody results or full blood count data. Clinicians are urged to be vigilant for these potentially dangerous “false normal” cobalamin results, and once suspected laboratories should store pre-treatment sera where available for repeat analysis on an alternative assay. Elevated homocysteine and methylmalonic acid levels which correct on B12 treatment, and positive anti-intrinsic factor antibody will confirm that the megaloblastic anaemia or neuropathy is indeed due to cobalamin deficiency.

These cases of assay failure should be reported to the in vitro diagnostics section of Medicines and health care related products agency (MHRA) as adverse events via http://www.mhra.gov.uk. UKNEQAS Haematinics would be pleased to assist laboratories to investigate any suspected false normal sera ( email: ukneqas.haematinics.org.uk) and collect data on any further cases.

A trial of therapy in suspected cobalamin therapy once serum has been stored will prevent delay in treatment and adverse clinical consequences.

Malcolm.S.Hamilton
Director UKNEQAS Haematinics Good Hope NHS Trust Rectory Rd. Sutton Coldfield B77 7RR

Sheena Blackmore
Deputy director UKNEQAS Haematinics

Anne Lee
UKNEQAS Haematinics Scheme scientist

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Malcolm S Hamilton, Director UKNEQAS Haematinics

Sheena Blackmore, Anne Lee

Dept Haematology Good Hope NHS Trust, Rectory Rd, Sutton Coldfield, B75 7RR

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Approximately 20 % of women show a physiological fall in serum vitamin B12 during pregnancy without signs of megaloblastic anaemia or a rise in homocysteine concentrations. Treatment with metformin may be associated with a fall in serum vitamin B12 and increases in homocysteine both in the short term (after 16 weeks) and up to 30 % of those on long term therapy. While individuals have been described with megaloblastic anaemia, subacute combined degeneration of the cord and memory loss in the setting of vitamin B12 deficiency and metformin therapy, with improvement following vitamin B12 replacement, the clinical significance of vitamin B12 deficiency with metformin therapy is uncertain. In about half of individuals vitamin B12 deficiency will resolve with withdrawal of the medication or calcium supplementation.

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Adam P Morton, Physician

Mater Hospital South Brisbane Australia 4101

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Based on a price of US$660 per kg of vitamin B12 [watsonii.com product V013], 1 gram or $0.66 provides about 1 million daily required doses of 1 mcg and would be sufficient for 34 eighty year long life-times, with 1 gram arguably representing the only non-toxic true megadose of any nutrient.

Conversely, $0.02 is the life-time cost for vitamin B12 for a human. Even assuming worst case scenarios or when elderly and that only 1/100th is absorbed, this likely represents a fraction of the cost of the blood analysis of the 4.25% of the patient population measured yearly, and found to contain 18% individuals 'below normal' for B12.

Considering the current debate about folate fortification and its theoretical harm from masking vitamin B12 deficiency, this is a golden opportunity to fortify grain products with both vitamins with, for example, targeting B12 at 10 mcg/d. A zero cost remedy to known deficiency problems that simultaneously solves the majority of the diagnosis issues raised by Devalia.

While scientists debate and before politicians act regarding food fortification, my U.S. mail-ordered 'high potency' multivitamin / mineral capsule contains 100 mcg B12 [and 800 mcg folic acid plus 6 other B- vitamins] and I have yet to see science that this amount of B12 does not eventually replete most worst case scenario patients at virtually no cost, risk or effort. The vitamin bottle has a six month supply at $0.12/day and comes without a child-proof cap; it's considered that safe! vos{at}health-heart.org

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Eddie Vos, maintains health-heart.org

Sutton (Qc) Canada J0E 2K0

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We have through the offices of our local research network (WoReN)have just completed data collection in over 500 patients on Injectable B12 from a cross-section of UK general pratices in the North-East. We have undertaken both quantitative and qualitative research. We have only just started analysing our results. Our preliminary impressions are that intiation and long-term maintenance of parenteral B12 therapy in a significant number of patients are made on "doubtful" clincial and biochemical results. This approach appears to exist across both primary and secondary care. Qulitative data from health professionals appear to suggest significant lack of consenus and confusion around interpretation of results, intitation of therapy and route of administration (oral vs parenteral). Patients similarly and not suprisingly appear to have little understanding of their B12 therapy. We are in the process of writing up this paper which will include guidelines on an integrated approach to diagnosis and management of patients with possible B12 deficiency.

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Anan Raghunath, General Practitioner

Kathleen Howlett, Val Featherstone

Hull HU143 3PA

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