Testing for Pernicious Anaemia

As soon as the Pernicious Anaemia Society was formed it became obvious that some, though not all patients faced a long journey in being diagnosed quickly and accurately. And the results of the members’ survey showed that one-third had experienced symptoms for up to a year before diagnosis.
Nearly 22% had to wait 2 years, 19% for 5 years and 4% for 10 years. Regrettably, 14% of individuals experienced symptoms for more than 10 years before arriving at their diagnosis¹. For many years we have wanted to know why many of our members had to wait so long for an accurate and quick diagnosis. And it is only recently that research papers have been published that show that one of the reasons is because the current tests used to diagnose vitamin B12 deficiency in general and whether that deficiency is caused by Pernicious Anaemia are seriously flawed.

There are a number of different tests that are used to determine the patient’s B12 status and whether that deficiency is caused by Pernicious Anaemia. Below is a brief explanation of each of these tests but it is useful to note:

“The clinical picture is the most important factor in assessing the significance of test results assessing cobalamin status since there is no ‘gold standard’ test to define deficiency”.²
Despite studies consistently demonstrating that no single biomarker of B12 status exhibits the performance characteristics necessary to definitively define status in all patients, the majority of diagnostic laboratories rely solely on serum B12.⁷

Tests to determine patient’s B12 status

Serum B12 Test

There are two main problems with the current test used to ascertain how much B12 the patient has in his or her blood.
Firstly the current threshold used to diagnose a deficiency may be far too low. Although the threshold figures will vary from laboratory to laboratory because different laboratories use analytical machines made by different manufacturers the typical cut-off value used to define B12 deficiency is around 148 pmol/L (200 pg/mL) and for a growing number of scientists this is far too low and, if patients are showing symptoms of B12 deficiency yet their blood result shows their B12 to be above 148 pmol/L the patient should be treated. They classify patients who have a range of B12 between 148 pmol/L and 300 pmol/L as being Subclinical Cobalamin Deficient.
Secondly the machines that are currently used to evaluate B12 status of patients have been shown to be inaccurate and are giving false high readings in 22%, 26% and 35% of patients⁵ (the different percentages are due to the different machines used).

Definitive cut-off points to define clinical and subclinical deficiency states are not possible, given the variety of methodologies used and technical issues, and local reference ranges should be established.²

The NICE Guideline states:
Less than 180 ng/l (133 pmol/l) : Confirmed vitamin B12 deficiency
Between 180 and 350 ng/l (133 and 258 pmol/l) : Indeterminate test result – possible vitamin B12 deficiency. Consider further testing in patients with symptoms.

Be aware that people of Black ethnicity may have a higher reference range for serum vitamin B12 concentrations than people of White or Asian ethnicity.

Serum Holotranscobalamin (HoloTC) – The ‘Active’ B12 Test

Serum (Total) B12 measures the total of B12 in blood. HoloTC in serum tests the abundance of B12 available for transportation into cells, the ‘active B12’ (B12 bound to the transport-protein transcobalamin), as opposed to the ‘inactive B12’ (HoloHC, B12 bound to haptocorrin). There are a few hospitals in the UK where this test is available but results are sometimes inconclusive though in other cases there has been a substantial difference between the patient’s total serum B12 results and the HoloTC result. HoloTC seems to be a little better compared to serum B12, but a large indeterminate area exists. HoloTC <25 pmol/L indicates deficiency. A result of 25 to 70 pmol/L is considered to be indeterminate and a second line test can be used to clarify B12 status (MMA/homocysteine).^8,9
During pregnancy this test should be used instead of serum B12.^8,9

Methylmalonic Acid (MMA)

This test is not routinely carried out though it is a better indicator of B12 deficiency than serum B12 is. When a patient has low B12 levels then their MMA will almost always be raised. If their level of MMA is extremely high then it is inevitable that the patient will have low B12. However, high MMA serum values are also found in people with impaired kidney function, impaired thyroid function, inherited methylmalonic aciduria and small-bowel overgrowth with bacteria. In these cases MMA levels cannot be used to diagnose B12 deficiency though a B12 deficiency might simultaneously exist.
A significant in MMA concentration after treatment with B12 confirms a previous existing B12-deficiency (even with low-normal values of serum B12).
More on MMA

Plasma Total Homocysteine (tHcy)

This is another test that is a reliable indicator of low B12 as it rises quite significantly early on in a deficiency. However, tHcy is also raised in folate deficiency, B6 and B2 deficiency and a few other causes. Although seldomly done, age- and sex-specific reference values should be used as Hcty is lower in children and pregnant women and higher in the elderly. It is also slightly higher in men than women. Although Hcty has it’s limitations, it can still be helpful in the diagnosis.

The Full Blood Count (or FBC)

This is the first-line test and provides doctors with a general picture of the patient’s blood. It includes an assessment of the size of the patient’s red blood cells which can be an indicator of a deficiency in vitamin B12. Doctors refer to enlarged red blood cells as megaloblasts and the patient as having macrocytosis (his or her blood cells will be enlarged). The test to establish whether there is any evidence of macrocytosis is the Mean Corpuscular Volume (MCV). It is often wrongly assumed that red blood cells will always be enlarged if there is a B12 Deficiency. However, only around 60% of patients with a B12 deficiency will have enlarged red blood cells (macrocytosis)^{3 4}. Another problem with relying on the MCV to indicate a B12 Deficiency is that if the patient also has an iron deficiency that deficiency will lead to a reduction in the MCV – an iron deficiency and a B12 deficiency will in effect cancel out each deficiency’s effect on the size of the red blood cell. We would like for the patient’s B12 status to be made part of the full blood count but, as more and more doctors become aware that B12 deficiency is more common than first thought, it seems that most doctors now ask for the patients B12 to be investigated at the same time as the FBC is performed. There is, however, a problem with the B12 test – it’s not accurate.

Serum Folate

There is a close relationship between folate and vitamin B12. In addition to vitamin B12, the body also needs folate (vitamin B9 (or B11)) for the growth and maintenance of the body, the health of the nervous system and the production of white and red blood cells. The effect of vitamin B12 is closely intertwined with that of folate, but these two vitamins are not interchangeable. Administering large amounts of folic acid can restart both vitamin B12 and folate-dependent reactions in the body, but this is only temporary. When anaemia caused by a vitamin B12 deficiency (temporarily) disappears after the administration of folic acid, the premature conclusion is sometimes drawn that there was (only) a folic acid deficiency. Administration of folic acid for a vitamin B12 deficiency does show an improvement in the blood values (Hb and MCV), and can therefore mask a vitamin B12 deficiency. Also, low level of folic acid could have a surprising effect of increasing the amount of B12 loss in urine. The lower the patient’s folic acid, the more B12 will be excreted via urine⁶.

If you have been supplementing your diet using folic acid tablets you should tell your doctor this before or after you have given blood to be analysed.

Ferritin (iron stores)

Iron plays an important part in the formation of healthy red blood cells and so it is useful to ascertain the patient’s iron stores. NICE guidelines recommend a ferritin level below 30 micrograms/litre (µg/L) as indicative of iron deficiency. Causes for a deficiency include dietary insufficiency, poor absorption, blood loss, or increased iron demands. Over 40% of people with Pernicious Anaemia reported to have also been diagnosed with iron deficiency.
More on iron

Tests for Pernicious Anaemia

When a patient is identified as having a deficiency in vitamin B12 the cause of that deficiency should be investigated. These are the tests used to determine whether the deficiency is caused by the patient having developed Pernicious Anaemia.

Anti-intrinsic Factor Antibody Test

This is the test used to find out if the patient has Pernicious Anaemia. The test is classified as highly specific (>95%): if tested positive the patient will be deemed as having auto-immune Pernicious Anaemia. However the test has a low sensitivity (50-70%) which means that a negative result does not rule out Pernicious Anaemia.

Here’s what the new guideline says:
“A negative test result does not rule out the presence of Pernicious Anaemia”.
Important: A vitamin B12 injection within 48 hours of testing and, in some cases, within one to two weeks of testing can interfere with test results, so this test should either be done before treatment or at least a week or more after an injection.

Parietal Cell Antibodies

This is another classic test for Pernicious Anaemia. About 90% of those with Pernicious Anaemia will have parietal cell antibodies. However, as up to 10% of healthy individuals will test positive for this test and the test can also be positive in people with other auto-immune diseases and people with gastritis – it is, therefore, a low specificity test.

Serum Gastrin

When a patients tests negative for antibodies, but Pernicious Anaemia is likely, this test can be used. 85-90% of patients with Pernicious Anaemia will have a high serum gastrine.

Other Tests

CobaSorb test to measure whether vitamin B12 can be absorbed
Gastroscopy with gastric body biopsy

IMPORTANT

If you are concerned about any part of your blood results you should discuss this with your doctor. However, as we have seen above, some of the tests are seriously flawed.

References

Hooper M, Hudson P, Porter F, McCaddon A. Patient journeys: diagnosis and treatment of pernicious anaemia. Br J Nurs. 2014 Apr 10-23;23(7):376-81. doi: 10.12968/bjon.2014.23.7.376 PMID: 24732991.
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. doi: 10.1111/bjh.12959. Epub 2014 Jun 18. PMID: 24942828.
Beck WS. Neuropsychiatric consequences of cobalamin deficiency. Adv Intern Med. 1991;36:33-56. PMID: 2024586.
Lindenbaum J, Healton EB, Savage DG, Brust JC, Garrett TJ, Podell ER, Marcell PD, Stabler SP, Allen RH. Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis. N Engl J Med. 1988 Jun 30;318(26):1720-8. doi: 10.1056/NEJM198806303182604. PMID: 3374544.
Carmel R, Agrawal YP. Failures of cobalamin assays in pernicious anemia. N Engl J Med. 2012 Jul 26;367(4):385-6. doi: 10.1056/NEJMc1204070. Erratum in: N Engl J Med. 2012 Sep 6;367(10):976. PMID: 22830482.
Eric Van Royen, Pierre Blockx, Fritz Molter; The influence of serum folate on urinary excretion of vitamin B12; EuropeanJournal of Nuclear Medicine, 1.7.1978, Volume 3, Issue 3, pp 175-178
Harrington DJ, Stevenson E, Sobczyńska-Malefora A. The application and interpretation of laboratory biomarkers for the evaluation of vitamin B12 status. Ann Clin Biochem. 2025 Jan;62(1):22-33. doi: 10.1177/00045632241292432. Epub 2024 Oct 27. PMID: 39367523; PMCID: PMC11707970.
Nexo E, Parkner T. Vitamin B12-Related Biomarkers. Food and Nutrition Bulletin. 2024;45(1_suppl):S28-S33. doi:10.1177/03795721241227114
NICE Guideline: Vitamin B12 deficiency in over 16s: diagnosis and management March 2024