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
THE SERUM VITAMIN B12 INVESTIGATION
SERUM B12 ERRORS
Summary
Laboratory Performance
Interpretation Errors
Immunoassays
Abuse of Control Ranges

Index

This page contains the following sections:

You can go to a section by selecting the link.

Introduction

I considered the possibility of abuse of control ranges, used to monitor analytical precision, for the following reasons:

  • Unexplained spike in Sullivan Nicolaides Pathology (SNP) serum B12 result for 30 October 2006
  • Unexplained spike in SNP total homocysteine result for 9 October 2006
  • Very significant differences between Westmead, SNP and PaLMS total homocysteine results

I added holotranscobalamin to the list in September 2007, after adding Active B12 to my test program, not as a result of any suspicion about the performance of the testing laboratory.

My investigation of immunoassay control ranges targeted the following analytes:

  • Total Serum Vitamin B12
  • Holotranscobalamin (Active B12)
  • Total homocysteine
  • Red-cell folate

The data for each analyte was taken from the Abbott AxSym package insert provided with each kit. These package inserts can be found at reference AD01 to AD04.

The results of my investigation are presented as tables, charts and a brief list of the significant findings for each analyte.

Summary of Findings

This table compares the maximum imprecision (CVA) required for desirable performance, with the potential maximum imprecision of the Abbott AxSym immunoassay method:

Analyte
Max. Desirable CVA
Max. Potential Error
Vitamin B12
7.5%
34% = 4.5 SD
Holotranscobalamin
7.5%
57% = 7.6 SD
Homocysteine
4.5%
25% = 5.0 SD
Red-cell Folate
6.0%
44% = 7.3 SD

This table lists the allowed range of readings for examples of actual analyte values:

Analyte
Actual Value of Sample
AAllowed Reading Range
Vitamin B12
313 pmol/L
207 - 419 pmol/L
Holotranscobalamin
35 pmol/L
18 - 52 pmol/L
Homocysteine
12.5 umol/L
10.0 - 15.0 umol/L
Red-cell Folate
700 nmol/L
450 - 950 nmol/L

This table lists the possible actual analyte values for examples of readings:

Analyte
Reading
Allowed Range of Actual Values
Vitamin B12
200 pmol/L
150 - 305 pmol/L
Holotranscobalamin
35 pmol/L
22 - 62 pmol/L
Homocysteine
12.0 umol/L
9.9 - 15.0 umol/L
Red-cell Folate
700 nmol/L
500 - 1000 nmol/L

Notes for calculation of Maximum CVA

Westgard, reference AB15, lists the the day-to-day intra-individual variation (CVW) for B12, homocysteine and red-cell folate.

According to Fraser et al, reference AB31, cited by Pfeiffer et al, reference AE13, analytical imprecision (CVA) should be less than 0.5 times the within-subject biological variation (CVW) for desirable performance, or less than 0.75 times the within-subject biological variation (CVW) for minimum performance.

From the Westgard list, I obtained the following values for maximum CVA for desirable performance.:

  • B12, 7.5%
  • Homocysteine, 4.5%
  • Red-cell folate, 6.0%

As Westgard does not list Holotranscobalamin, I have used 7.5%. This is the mean of the values for the LOW PANEL and HIGH PANEL data listed in the Abbott AxSym Package Insert. This is also consistent with the finding of an intra individual variation of 13% by Bor et al, reference ED01.

Notes for Charts and Tables

Control ranges are presented as charts and tables. You can view a larger chart in a new window by selecting the link shown above the chart. You can view the complete Excel table in a new window by selecting the link shown above the table.

Tables will initially appear at low resolution in the new window. To enlarge the table to full size, move the cursor from the white space below the table to inside the lower right corner of the table; an icon will then appear in the lower right corner of the table. Click on the icon to view the table full-size. Use the horizontal scroll-bar, in the new window, to view the entire table.

The chart and table numbers used here correspond to the sheet numbers in the Excel file, Series 1 - Serum B12 Investigation, which may be downloaded by selecting Access Documents under Evidence.

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Total Serum Vitamin B12 Immunoassays

The significant points to note, from the tables, about the total serum vitamin B12 control ranges are:

  • The allowed control range represents a potential error of +/- 34%
  • The maximum CVA for vitamin B12, for satisfactory performance is 7.5%
  • The allowed control range therefore represents a potential error of +/- 4.5 SD for all three ranges
  • The allowed control range therefore permits results to be accepted if they are outside the 99.999% CI

The significant points to note, from the charts, about the vitamin B12 control ranges are:

  • A sample with an actual value of 313 pmol/L could give an accepted result between 207 and 419 pmol/L
  • An accepted result of 200 pmol/L could be for a sample with an actual value between 150 and 305 pmol/L
Table F1
Chart F2
Chart F3

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