How reliable is the determination method of reference ranges for blood tests?

Question

A reference range is usually defined as the set of values 95 percent of the normal population falls within (that is, 95% prediction interval). It is determined by collecting data from vast numbers of laboratory tests. ¹

When it comes to a "normal" range of a Vitamin B12 blood test, I find several ranges that are so widely defined that they almost contradict each other. The "normal" ranges I found vary between 200 pg/mL (150 pmol/L) to 1200 pg/mL (900 pmol/L).

According to this publication ² the first signs of a B12 deficiency at cell level sometimes occur at serum B12 concentrations up to 400 pg/mL (300 pmol/L).

Why does the lower limit in some standards differ 200 pg/mL (150 mpol/L) from other standards? Also, the upper limit seems to create an even wider range. I found examples where the upper limit differs from others with 500 pg/mL (400 pmol/L).

Moreover, what is a "normal population"? Is it global or national? Are environmental factors considered? For example, I can imagine that Nordic countries tend to have more cases of vitamin D shortage because of a lack of sun compared to countries close to the equator. What if the whole country has "a shortage" of something and that values are used to determine "normal values"? Therefore my question: How reliable is the determination method of reference ranges for blood tests?

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^{1 https://en.wikipedia.org/wiki/Reference_ranges_for_blood_tests
2 https://onlinelibrary.wiley.com/doi/abs/10.1002/ajh.2830340205}

Answer 1

Question: How reliable is the determination method of reference ranges for blood tests?

Short answer: The tests for which you can find different reference ranges may not be unreliable because of different ranges but because they are unreliable as such. For example, all reference ranges for vitamin B12 in the blood are unreliable, because the blood levels of B12 only poorly reflect the actual B12 status in the body. It is often only a combination of tests that can reliably reflect a certain situation.

In general, the reference ranges of the blood tests have been determined on the basis of decades of studies from around the world that have shown which ranges have been associated with minimal incidence of symptoms and diseases. Different health authorities may judge the background evidence differently, so this is why reference ranges can be different.

There is a suggestion that every country should determine their own reference ranges:

International guidelines recommend that every country must establish reference intervals for healthy individuals belonging to a group of homogeneous population. (Indian Journal of Clinical Biochemistry, 2009)

...and some countries have and others haven't determined them. Sometimes, reference ranges can also differ among hospitals and labs, and they can change with time.

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Here are few examples how the blood tests need to be judged regardless of reference ranges:

1) NORMAL BLOOD VALUES MAY NOT REFLECT DEFICIENCY - Calcium

Blood calcium levels can remain normal for years even in a person with severe calcium deficiency, because calcium leached from the bones maintains normal blood levels.

What serum calcium can tell us and what it can't (Nephrology, Dialysis, Transplantation, 2005):

When dietary calcium intake is inadequate (<600 mg/day in young adults) and/or intestinal calcium absorption abnormal, the serum calcium level can be kept stable only at the cost of a gradual depletion of bone calcium stores.

So, the bone mineral density can more accurately show the calcium status.

2) PERSONAL DIFFERENCES - Vitamin B12

Symptoms of vitamin B12 deficiency can occur at very different B12 levels in different individuals:

Subclinical vitamin B12 deficiency (usually defined as a total serum B12 of <200 pmol/L) presents asymptomatically or with rather subtle generic symptoms that oftentimes are mistakenly ascribed to unrelated disorders. Numerous studies have now established that serum vitamin B12 has limited diagnostic value as a stand-alone marker. Low serum levels of vitamin B12 not always represent deficiency, and likewise, severe functional deficiency of the micronutrient has been documented in the presence of normal and even high levels of serum vitamin B12. (Frontiers in Biomolecular Sciences, 2016)

Methylmalonic acid (MMA) levels may more accurately reflect the B12 status, because they indicate a metabolic change that is highly specific to B12 deficiency (Office of Dietary Supplements).

3) ETHNIC FACTORS - blood cells

Normal reference ranges can differ among ethnic groups:

This current study included 3,077 participants aged 18–65 years who reported their health status as “Excellent,” “Very good,” or “Good,” with known race/ethnicity as white, black, Hispanic, or Asian. Quantile regression analyses adjusted for sex were conducted to evaluate racial/ethnic differences in the normal ranges of 38 laboratory tests. Significant racial/ethnic differences were found in almost all laboratory tests. Compared to whites, the normal range for Asians significantly shifted to higher values in globulin and total protein and to lower values in creatinine, hematocrit, hemoglobin, mean cell hemoglobin, mean cell hemoglobin concentration, and mean platelet volume. (Hawai'i Journal of Medicine and Public Health, 2015)

4) ENVIRONMENTAL FACTORS - Vitamin D

Nordic populations have low sun exposure but have one of the highest milk consumption per capita on the world (milk is high in vitamin D) and they have one of the highest blood vitamin D levels in average (a map). So, the environmental factors can influence actual blood levels but they do not need to influence what reference ranges should be. For example:

The large majority of adults living close to the Arctic Circle in Sweden have adequate D3 levels even during the second half of the dark winter.

Levels used for definitions were...adequate, D3≥50 nmol/l (20 ng/ml). (International Journal of Circumpolar Health)

So, the "adequate" D3 level in the above study in Sweden was considered ≥50 nmol/l (20 ng/ml), which is the same as "sufficient" level in the United States (CDC.gov). Anyway, to properly evaluate vitamin D levels, one should also check calcium levels and other tests.