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 HOMOCYSTEINE INVESTIGATION
HCY ERRORS
Summary
HCY Lab Errors
Laboratory Performance
Interpretation Errors
Immunoassays

Index

This page contains the following sections:

You can go to a section by selecting the link.

Introduction

This investigation was performed to investigate very significant differences in serum total homocysteine results reported by several laboratories during the The Serum B12 Experiment.

These are the laboratories:

Series 1:

  • Sullivan Nicolaides Pathology (SNP)
  • Queensland Health Pathology Service (QHPS)
  • NSW Biochemical Genetics Service at The Children's Hospital Westmead (CHW)
  • Pacific Laboratory Medicine Service (PALMS)
  • Royal Prince Alfred Hospital Pathology (RPAH

Series 1b:

  • QML Pathology (QML)
  • Sullivan Nicolaides Pathology (SNP)
  • NSW Biochemical Genetics Service at The Children's Hospital Westmead (CHW)

Serum samples were collected on 20 sample dates for series 1, and on 6 samples dates for series 3.

Notes for Charts and Tables

Results 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 chart.

Most of the 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 from Evidence.

These results can be verified from the scanned original pathology reports, which may be downloaded from Evidence.

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SNP HCY Compared to CHW HCY

The important points to note, about homocysteine levels, from the SNP Sample A and Westmead Sample A results are:

  • The maximum difference between the labs was 8.71 SD.
  • All 15 results were outside the 95% Confidence Interval.
  • 13 of those results were also outside the 99.0% Confidence Interval.
  • All 13 of those results were also outside the 99.9% Confidence Interval.
Chart E12 Table B18

The important points to note, about homocysteine levels, from the SNP Sample B and Westmead Sample B results are:

  • The maximum difference between the labs was 10.30 SD.
  • 15 out of 16 results were outside the 95% Confidence Interval.
  • All 15 of those results were also outside the 99.0% Confidence Interval.
  • All 15 of those results were also outside the 99.9% Confidence Interval.
Chart E13 Table B18

The Westmead results for homocysteine were significantly more consistent with the relationship between homocysteine and serum B12, as reported by Refsum et al, reference AE11 Figure 7, than were the SNP results.

The important points to note are:

  • All of the SNP homocysteine results were significantly lower than would be expected from the findings of Refsum.
  • The Westmead homocysteine results are generally very close to those predicted by Refsum.
  • The Westmead results that are significantly higher than the Refsum curve, shown as points B, were recorded at a time of rapid change in cellular deficiency, as defined by the rapidly increasing MMA levels. During a time of such rapid change, because of differences in analyte response times, the relationship between them is temporarily affected.
  • The result at point A is the B12 anomaly of 9 October 2006.
Chart C31 Table C9

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PALMS HCY Compared to CHW HCY

The important points to note, about homocysteine levels, from the PaLMS Sample A and Westmead Sample A results are:

  • The maximum difference between the labs was 9.62 SD.
  • All 13 results were outside the 95% Confidence Interval.
  • All 13 of those results were also outside the 99.0% Confidence Interval.
  • 12 of those results were also outside the 99.9% Confidence Interval.
Chart E26 Table B18

The important points to note, about homocysteine levels, from the PaLMS Sample B and Westmead Sample B results are:

  • The maximum difference between the labs was 14.18 SD.
  • 15 out of 16 results were outside the 95% Confidence Interval.
  • 14 of those results were also outside the 99.0% Confidence Interval.
  • All 14 of those results were also outside the 99.9% Confidence Interval.
Chart E27 Table B18

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Conclusion

None of the three labs to measure my homocysteine, SNP, Westmead and PaLMS, gave results that were entirely consistent within all pairs of identical samples. This indicates the possibility of either sample mishandling or antibody interference. The "loss" of all 29 samples by RPAH has prevented any further investigation of these anomalies.

The Westmead homocysteine results were the only ones to indicate the onset of cellular vitamin B12 deficiency, consistent with the Westmead MMA results. These results were also the only ones consistent with the known relationship between serum vitamin B12 and homocysteine levels.

The homocysteine results from SNP and PaLMS were very significantly lower than those from Westmead, never indicating a vitamin B12 deficiency.

The SNP homocysteine results also included an anomaly, which cannot be explained by my biological variation, indicating a serious problem with quality control of the immunoassay.

The errors were, in my opinion, contributed to by failure in quality control. The allowed control ranges, for some immunoassays, are so wide that results obtained using these methods cannot, in my opinion, be relied on for diagnosis or monitoring of vitamin B12 deficiency.

Sullivan Nicolaides Pathology, acknowledges that it relies on the controls and standards supplied by the kit manufacturer, for the measurement of homocysteine, and has no knowledge of the existence of the primary standard for homocysteine, NIST SRM 1955.

In addition to very significant analytical error, both Sullivan Nicolaides Pathology and PaLMS quote a homocystein cut-off that is significantly above the level recommended by experts. This could cause some cases of vitamin B12 deficiency to be missed.

Having regard to the results of my investigation, set out in this web site, in my opinion, Sullivan Nicolaides Pathology and PaLMS homocysteine results, and quoted cut-off, cannot be relied on for the diagnosis or monitoring of vitamin B12 deficiency.

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