How does Vitamin B12 deficiency get diagnosed?

How does Vitamin B12 deficiency get diagnosed?

There are several steps for doctors to take before they can diagnose vitamin B12 deficiency:

1. First, the doctor has to think of B12 deficiency. For me, this is usually a combination of a patient’s symptoms and risk factors.
2. Then, the doctor does blood tests to confirm B12 deficiency.
3. Once B12 deficiency is confirmed, the doctor will want to know why someone has B12 deficiency. This is important for their treatment.

What are the tests for B12 deficiency?

Vitamin B12 deficiency is diagnosed on the basis of blood tests. First, the doctor will have to suspect B12 deficiency as a possible cause of the patient’s symptoms, combined with their risk factors. The NICE guideline recommends doctors to do blood tests when a patient has at least one symptom and one risk factor. One or more blood tests can confirm it.

There is no gold standard test that gives a clear, unequivocal answer to the question ‘does this person have a vitamin B12 deficiency, yes or no’. However, there are four blood tests that can help with diagnosis.

The NICE guideline recommends that doctors first use one of the primary tests for B12. These tests measure how much B12 there is in the blood. If the result of the primary test is low, they should diagnose vitamin B12 deficiency. If the result of the primary test is in the indeterminate zone, they can request one of the secondary tests for B12. These tests indicate how well the B12 is used in the cells.

Two primary tests measure the amount of B12

The primary tests that measure how much B12 there is, are:

First, total (or serum) B12. This test measures all of the B12 in the blood. However, not all of this B12 is available to the cells of the body. Only about 5-20% of the total is available for the cells to absorb and use. Say that two people both have a total B12 result of 300 One of them, in whom 20% is available to cells, may be fine. The other, in whom only 5% is available to cells, is not. But you can’t see that from the result of the B12 test.

Two secondary tests measure B12 function

The secondary tests that measure how well B12 functions in the cells, are called functional tests. They measure how much B12 functions in the cells. If there is enough B12 at work in the cells, it keeps these two substances down, and the results of these tests will be low. If there is not enough B12 at work in the cells, these tests will be raised.

The two functional tests are:
Firstly, methylmalonic acid (MMA). MMA rises when there is not enough B12 in the mitochondria (the cell’s “energy factories”) to process fats and proteins so that they can be burnt as fuel for energy. However, MMA is not only influenced by the person’s B12 status.

Kidney function, genetic factors, and the amount of a fat (propionate) that the gut microbiome produces, also influence MMA.

Secondly, homocysteine. Homocysteine rises when there is not enough B12 in the cell fluid to support methylation, an important process where a methyl group is added to all sorts of molecules in the cell. Again, homocysteine is not only influenced by the person’s B12 status. It is also influenced by how much folate (the body’s form of folic acid) there is, kidney function, and genetic factors.

MMA is the most practical test. It can be done from the same blood sample taken for the primary test.

Homocysteine has a couple of drawbacks. Blood for this test needs to be drawn in hospital, because it needs to be put on ice and taken to the lab immediately. And it is only a reliable indicator of B12 status when the person’s folate is normal (above 4 ng/mL). But in people who use laughing gas (also called nitrous oxide, nos, whippets), homocysteine is the best test.

In practice, the functional tests are more expensive, and laboratories don’t have the budget to do a lot of these functional tests. So often the GP cannot request them. We hope these tests will become more available in the future.²

How do you know what is the cause of someone’s vitamin B12 deficiency?

Detecting why someone has B12 deficiency, is through a combination of taking the patient’s history and doing further tests.
First, I ask the patient, and check their records, for:
a. Diet: if it is low in animal-source foods (e.g. vegans, or restricted eating for any other reason, in people not supplementing with B12), this is a risk factor for B12 deficiency.
b. Personal or family history of auto-immune disease – for instance, thyroid disease, vitiligo, type 1 diabetes, Sjogren’s disease (dry mouth and other symptoms), Addison’s disease. In families of patients with PA (pernicious anaemia), ulcerative colitis, rheumatoid arthritis, polymyalgia, psoriasis, sarcoidosis, lupus were in one study more common than in those of people without PA. Any of these make an auto-immune cause of B12 deficiency (PA, also called autoimmune gastritis³) more likely.
c. Any issue that can affect the stomach, pancreas or the last 3-4 cm of the small bowel (called the terminal ileum):

1. Stomach: gastrectomy, gastric bypass
2. Pancreas: chronic pancreatitis, removal of the pancreas
3. Terminal ileum: Crohn’s disease, coeliac disease (an allergy to gluten requiring a very strict gluten-free diet), removal of terminal ileum, ileostomy, radiotherapy to the pelvis (e.g. for bowel, ovarian or prostate cancer).

d. Medications that reduce absorption of B12 – metformin (for diabetes) and antacids (that reduce the amount of stomach acid) are the main ones.

Second, we can do further tests:

1. Antibodies against intrinsic factor (IFAB): these are specific for PA and make the diagnosis. However, only about half of people with PA have these IFAB. And if the PA is so far advanced that there is no intrinsic factor left, the antibodies can disappear. Therefore, if someone does not have these IFAB antibodies, it does not rule out PA.
2. Other tests are possible, but not usually offered, to indicate a diagnosis of PA:
       a. Antibodies against parietal cells (the cells lining the stomach that make intrinsic factor and stomach acid). These can also be present in people who do not have PA. Therefore, many laboratories advise not to test for these.
       b. Gastrin (increased in PA)
       c. Cobasorb test (the patient is given a small dose of oral B12 and then tested to see if it increases the B12 in their blood)⁴
       d. Gastroscopy (a camera into the stomach)
3. Tests for coeliac disease (an auto-immune disease that makes people allergic to gluten and reduces their absorption of a lot of nutrients).
4. Looking for calprotectin in the stool can indicate inflammatory bowel disease, such as Crohn’s or ulcerative colitis.

Limitations of the tests for B12 Deficiency (and most other tests)

Virtually all tests have limitations. We know this from everyday life. We think we know a cat when we see one. But at night, it can be hard to tell a fox from a cat. Telling car models apart on the motorway can also be difficult. The tests for B12 are no different.

Here are some issues that can make tests difficult to interpret:

• There can be spurious results. Something could have gone wrong with the blood while it was in transport (too hot, too cold, delayed) before it got to the laboratory. If a blood sample is delayed in its transport to the hospital, some of the red blood cells can burst, spilling their B12 into the blood, which raises the B12 test. If the blood is visibly haemolysed (lots of burst blood cells) the lab will not do the B12 test for that reason. To repeat the test, it can be better to have the blood taken in hospital, so it gets to the lab faster.
• Something could have gone wrong with the test itself, though the lab usually detects it if all their tests get unusual results. Again, if there is an unexpected result, it is useful to repeat the test.
• Nearly all tests have false positives and false negative results. This is the most common reason a test does not give the correct result. The question you ask of a serum B12 test is, does this person have B12 deficiency? If the test result is low, the answer is yes, which we call a positive test. If the test result is high, the answer is no, which we call a negative test. The NICE guideline has now introduced a maybe (indeterminate) range for the B12 test. However, tests results can be false when they give a result that does not match the reality of the disease. For instance:
  ◦ False positive: the test result is positive, but the person does not have the disease. For instance, a small proportion of people have genetically low b12 values, while they do not have a deficiency.
  ◦ False negative: the test result is negative, but the person does have the disease. For instance, the NICE guideline says that with total B12 values of over 350 ng/L,0 deficiency is unlikely. However, some people with B12 levels over 350 still have a deficiency.
• Unhelpful reference ranges. Reference ranges are difficult to set. Any cut-off is a trade-off. If the cut-off for deficiency is set higher, you diagnose more people who have deficiency, but you also end up treating more people who don’t have a deficiency. If it is set lower, you diagnose less people with deficiency, and you have less people treated (or investigated further) who don’t have deficiency. It’s a matter of skippering between the two. It remains less than perfect. And we keep having to use our clinical judgment, rather than trusting blindly on test results ⁵.
• A further problem with reference ranges (also called the normal range, i.e. values that are considered normal and therefore not needing treatment) is that they are usually set to indicate people with obvious disease. That does not tell us what the optimum level is for a person’s long-term health. For instance, in the past decades we have seen the optimum level for vitamin D increase, as we have more evidence that lower levels of vitamin D are associated with disease in the long term (e.g. osteoporosis, but also mental health and generally feeling unwell). A new study just published shows that older people have slower processing speed in the brain at B12 levels in the lower half of the reference range. [ref. 1] This means older people need higher B12 levels, in the upper half of the reference range.
• For B12 testing, one of the big problems is that we measure the B12 in blood, but we need it in the cells, and most of all in the brain. A blood test is an indication for how much the brain might have access to B12, but if for instance the blood-brain barrier does not absorb enough B12, the brain will have too little B12, while the blood test is normal. That is one of the reasons, as the NICE guideline says, that we need to adjust treatment to the patient’s symptoms, not do a blood test or prescribe a one-size-fits-all dose.
• The context of a test result needs to be taken into account. For instance, homocysteine is one of the secondary tests for vitamin B12 deficiency. However, a high result can indicate either B12 deficiency, or folate deficiency, or something in the person’s genetic make-up.

So you can see from the above the challenges for doctors to consider so many factors to get to the correct diagnosis.

1. Active B12/Holo TC is not widely available in the UK. Our enquiries also established that there are no laboratories using this test in the USA. Some private blood test laboratories offer this test, but it still has limitations on sensitivity and specificity.
2. Members of the Pernicious Anaemia Society regularly report that their GPs in primary care and even some doctors in Secondary care tell them that they do not have access to Active B12 tests or MMA/Homocysteine or are not willing to order these tests. We are not aware of any movement to make these tests more available even though the NICE Guideline on B12 Deficiency recommended the use of these tests, together with presentation of symptoms, to diagnose B12 deficiency and Pernicious Anaemia.
3. Note by PAS: The Nice Guideline referred to Pernicious Anaemia as autoimmune gastritis. Most research papers, medical guidelines and documents call the condition Pernicious Anaemia and there is no strong argument, in our view, to change the name.
4. The CobaSorb test is mentioned in the NICE Guideline and as far as PAS is aware is only available in Denmark. In our experience from member comments, no primary care doctor in the UK has access to, or has heard of, this test.
5. Reference ranges mentioned here are based on adults. Reference ranges for children and adults of black family background are higher than these.

Ref. [1] Beaudry-Richard, A. et al. (2025) Vitamin B12 Levels Association with Functional and Structural Biomarkers of Central Nervous System Injury in Older Adults. Annals of neurology. [Online]