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).
As the BCSH puts it:
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.

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

Following on from the above, there is now a test that is being introduced gradually in many countries that doesn’t measure the total amount of B12 in the patient’s blood but distinguishes between the ‘active B12’ (holotranscobalamin) and the ‘inactive B12’ (holohaptocorrin). 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. It seems that this new test offers the potential to replace the current, inaccurate test but there does need to be more investigation into its effectiveness. Here’s what the new guidelines say: “Serum holotranscobalamin has the potential as a first line test, but an indeterminate ‘grey area’ may still exist”.

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. Indeed until recently the UK’s National Institute for Health and Care Excellence (NICE) guidance on this issue specifically stated that investigating the patient’s B12 status should only be carried out if there was evidence of enlarged red blood cells. Thankfully that advice was removed after we pointed out the error. 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.

Red Blood Cell Folate (RBC folate) and Serum Folate

RBC measures the amount of Folic Acid in the patient’s red blood cell. Serum Folate is an evaluation of the patient’s folic acid in the blood (rather than the red blood cells) and the latest guidelines suggest that Serum Folate is as effective as Red Blood Cell Folate as a method of evaluating the patient’s folic acid. There are three things worth mentioning about Folic Acid and B12. The first is that if the patient has been supplementing using folic acid tablets (as pregnant women are advised to do) then the increase in folic acid will prevent the red blood cells from becoming enlarged (megaloblastic) as discussed above. Therefore high folic acid levels means that one of the indicators of low B12 (enlarged red blood cells) will not be present when doctors interpret the results of any full blood count. Secondly, low level of folic acid has the 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. And finally, a healthy level of folic acid is needed to enable the patient to convert any cyanocobalamin (a form of injectable B12) to convert to Hydroxocobalamin and then to the biologically ‘active’ forms of B12 – Methylcobalamin and Adenosylcobalamin. The current test does not distinguish between the active and inactive forms of B12 and so if a patient is deficient in folic acid he or she will not be converting the inactive forms of B12 into the active forms. The result will be that the patient’s B12 status will show normal or high, but very little of this B12 will be in the active form.

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.

This is what the new guidelines say: “Serum folate offers equivalent diagnostic capability to red cell folate and is the first line test of choice to assess folate status”.

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. Low iron can be the result of haemorrhaging following an accident or from heavy menstruation among other causes.

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 ‘classic’ or ‘full-blown’ 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 guidelines say:
“…and the finding of a negative intrinsic factor antibody assay does not therefore rule out 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 anemia 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

Other tests carried out as part of the Full Blood Count include the following:
• Mean Corpuscular Haemoglobin (MCH) This measures the amount of Haemoglobin per red blood cell. A high concentration is indicative of B12 Deficiency.
• MCHC (mean corpuscular haemoglobin concentration) This measures the average concentration of haemoglobin in the cells.
• Red blood cell distribution width (RDW) A measure of the volume of the red blood cells.

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
  1. 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.
  2. 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.
  3. Beck WS. Neuropsychiatric consequences of cobalamin deficiency. Adv Intern Med. 1991;36:33-56. PMID: 2024586.
  4. 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.
  5. 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.
  6. 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
  7. 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.
  8. Nexo E, Parkner T. Vitamin B12-Related Biomarkers. Food and Nutrition Bulletin. 2024;45(1_suppl):S28-S33. doi:10.1177/03795721241227114

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