THE VITAMIN B12 AND FOLATE PATHOLOGY INVESTIGATION
THE INVESTIGATION OF ERRORS IN PATHOLOGY TESTS
FOR VITAMIN B12 AND FOLATE DEFICIENCY
BY MEANS OF MEDICAL EXPERIMENTS
B12 AND FOLATE INFORMATION
Interpreting Results
Reference Intervals

Index

This page contains the following sections:

You can go to a section by selecting the link.

Introduction

Here I consider the importance of two sets of figures that appear in pathology reports:

  • Reference Intervals
  • Limits, or Cut-offs

I shall use the three analytes of particular interest to me, for the diagnosis of vitamin B12 deficiency, as examples of the use of incorrect limits:

  • Vitamin B12
  • Methylmalonic Acid
  • Total Homocysteine

Quoting incorrect reference intervals or limits can significantly affect the interpretation of results. In my case, investigating vitamin B12 deficiency, the use of incorrect limits caused an actual deficiency to be reported as a normal condition.

Reference Intervals

A reference interval is the range of values of a test result for a defined population. The range is usually specified as the central 95% of the values, from the lower 2.5% to the upper 97.5% of the population. In a normal distribution, 50% of the population will have a result below the mean, and 50 % will have a result above the mean, as shown in the following diagram:

What the Reference Interval Does Show

The reference interval shows:

  • How broad the range of results is for a specific lab, for a given test, for a given population
  • How the result for a patient compares with the rest of the population

What the Reference Interval Does Not Show

  • What result is likely to indicate disease
  • What range of results is indeterminate for indication of disease
  • What range is unlikely to indicate disease

So, the reference interval tells us how our result compares to the rest of the population, but does not tell us if the result is a healthy one.

For example, my weight and blood pressure are very much lower than the average Australian male of my age. With 60% of men overweight or obese, is quite possible that I am in the lower 2.5% of the population for both of these measures, but this, by itself,says nothing about my state of health.

What we need to know is what is a healthy range; this is why limits, or cut-offs, are important.

There are some excellent articles and slide shows on the subject of reference intervals, including Reference Intervals for New Methods, reference AB24, and Statistical Techniques for Reference Intervals, reference AB25

Limits

A limit, or cut-off is a value that indicates a change from a result that is not indicative of disease to one that is indicative of disease. The important points about limits are:

  • Their level is determined by research into the relationship between the analyte and the disease
  • They are independent of the reference interval
  • They are absolute values and are independent of the lab or the method of analysis
  • They are only meaningful if the analytical method is sufficiently accurate

For some analytes, for example methylmalonic acid and homocysteine, there is a single cut-off point for a given disease, although the actual level might not be universally agreed to:

For other analytes, for example vitamin B12, there is a range of indeterminate values:

Because limits are taken from research, and compare the patient's result to absolute values, accuracy is critically important. This is explained well in the ERNDIM article, reference AB01:

Not only is accuracy of the result important; so also is the use of the correct cut-off in the pathology report. Where inappropriate limits are quoted, the interpretation of the result can be very significantly affected.

The labs cited here are not alone in quoting incorrect cut-offs; they also appear in the RCPA Manual.

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Vitamin B12

A serum B12 level below 295 pmol/L should be considered suspicious of B12 deficiency in the presence of symptoms. This is clear in the flowchart from Oh and Brown, reference DA01:

Oh and Brown

It is also clearly stated in the Abbott AxSym B12 Package Insert, reference AD01:

A serum B12 cut-of level of 200 pmol/L, for defining B12 deficiency, is recommended by Clarke et al, reference DA04.

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Methylmalonic Acid

Most reputable laboratories quote limits between 0.34 and 0.40 umol/L, the lowest being 0.28 umol/L; several are listed in reference AF03, AF10, AF11 and AF13. This table lists some of these limits, in umol/L:

Mayo
Cleveland
Sundhed
RPH
CHW
QHPS
0.40
0.376
0.28
0.35
0.34
0.40

Holleland et al, reference DA07, reported these findings:

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Total Homocysteine

Many labs incorrectly quote a limit of 15.0 umol/L for total homocysteine (so also does the RCPA Manual, reference AF05).

A homocysteine level of 15.0 umol/L is not considered safe by most experts, including authors of reference AE11 and AE12. Both of these expert groups recommend a maximum level of 12.0 umol/L. Here is the diagnostic flowchart for homocysteine, as shown in reference AE12:D.A.C.H. - Liga Homocystein:

As you can see, a level of > 12 umol/L is considered high for a healthy population; a level > 10 umol/L is high for those patients at risk for cardiovascular disease.

Another expert report by Rasmussen et al, reference AF01, provides a range of age and gender-specific reference intervals. For a male aged 30 to 59 years, the recommended limit is 11.2 umol/L.

Reputable laboratories use much lower levels than the 15.0 umol/L; several are listed in reference AF08 to AF22. This table lists some of these limits, in umol/L:

-
Mayo
Cleveland
Calgary
Cedars-Sinai
Sundhed
Male
<13
13.7
13.7
12.0
11.2 - 12.0
Female
<13
12.9
9.9 - 12.8
10.0
11.2 - 12.0

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