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
Laboratory Performance
Sullivan Nicolaides
Interpretation Errors
Immunoassays

Index

This page contains the following sections:

You can go to a section by selecting the link.

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Introduction

Here you will find my specific claims about what I consider to be the poor performance by Sullivan Nicolaides Pathology, Brisbane (SNP).

For each issue raised, I explain:

  • What Went Wrong
  • Why it Matters

Notes for Charts and Tables

Where a chart is shown, you can view a larger chart in a new window by selecting the link shown above the chart.

Where there is a link to a table, above a chart, you can view the complete Excel table in a new window.

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, available on this site from Evidence.

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SNP B12 - Use of Incorrect "Normal" Level

Summary of what went wrong

Sullivan Nicolaides Pathology incorrectly quotes a "normal" level of >130 pmol/L for serum vitamin B12:

A level of 130 pmol/L is not considered normal by experts, reference DA01 to DA10, and a 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, supplied with the instrument used by Sullivan Nicolaides Pathology:

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

Why it Matters

Sullivan Nicolaides Pathology failed to report the onset of an actual vitamin B12 deficiency that should have been interpretable from their serum B12 results...

As my serum B12 level fell, after I ceased taking the oral B12, my MMA levels increased during 2006. Here is a chart showing the relationship between my serum B12 measured by SNP, and MMA measured by Westmead:

Chart C24 Table C8

My MMA level exceeded the Westmead (CHW) cut-off, of 0.34 umol/L, between 24 February (Day 114) and 5 June 2006 (Day 215). This means that I was becoming vitamin B12 deficient, by the very latest, after 5 June 2006.

According to experts, methylmalonic acid is a sensitive and selective marker of vitamin B12 deficiency. Some excellent articles on this can be found by selecting reference DA0 to DA10.

After ceasing taking the oral B12, my serum B12 level rapidly fell to 323 pmol/L, in mid-March 2006, then fell much more slowly to a minimum of 140 pmol/L in October 2006. The anomalous peak on 9 October will be considered separately in the next section.

During 2006, my B12 level did not fall below the "normal" level of >130 pmol/L quoted by Sullivan Nicolaides in their reports, so they did not report a vitamin B12 deficiency..

There is clearly a problem when the Westmead MMA results unambiguously indicate the onset of vitamin B12 deficiency, whereas the SNP serum B12 results remain "normal" and do not even enter their quoted "borderline range".

For a detailed investigation of the reference Intervals and limits, select Reference Intervals and Limits under Interpreting Results.

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SNP B12 - Misleading Comments in Reports

Summary of what went wrong

The report SNP reports use the heading Anaemia Profile for the B12 results, and include the comment "but unexplained low levels do occur in some patients without B12 deficiency".

Why it Matters

The heading Anaemia Profile is misleading because anaemia is not present in 30% of cases of vitamin B12 deficiency. This is very well documented in many of the references cited here; an excellent example is the article by Lindenbaum et al, reference CA12.

The comment "but unexplained low levels do occur in some patients without B12 deficiency", is true but very misleading because the quoted SNP "normal" and "borderline" levels are far too low. B12 deficiency can also occur in patients with high levels of serum B12, but this is not commented on in the reports.

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SNP B12 - Anomaly in Results

Summary of what went wrong

On 30 October 2006, we received an anomalous result for my serum vitamin B12 test from Sullivan Nicolaides Pathology.

According to the SNP result for 30 October, my B12 level increased from 169 pmol/L to 250 pmol/L in one week. As I was not taking any supplements, and am on a very strictly controlled vegetarian diet, there was nothing to drive my serum B12 level higher.

Here is a chart showing the anomaly:

Chart C11 Table C8

You will see that it appears that my levels fell gradually to a low of 140 pmol/L on 16 October, then climbed to a peak of 250 pmol/L on 30 October, then fell again to a low of 143 pmol/L. There is no reasonable explanation for this other than very significant analytical error. The rapid increase in B12 level after 13 November was caused by me re-starting B12 therapy.

With a claimed SD (Standard Deviation) of 18 pmol/L, an error of 110 pmol/L represents about 5 SD, placing it outside the 99.9999% confidence interval. The spike in B12 level cannot be explained as being within normal error limits; something is seriously wrong.

I believe that this episode is an example of problems with calibration, and control ranges. For more details select Control Ranges - Open to Abuse under Immunoassays.

Why it Matters

There is a totally different interpretation for the result of 250 pmol/L compared to a result of 140 pmol/L. This potentially has very serious implications for other patients, if their results contain similar errors.

In the absence of any symptoms, a result of 250 pmol/L would not require further investigation, according to the criteria given in the Abbott AxSym B12 Package Insert, reference AD0C1, whereas a result of 140 pmol/L would be considered abnormal.

A result of 140 pmol./L satisfies the criteria for vitamin B12 deficiency, as defined by Clarke et al, reference DA04.

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SNP Homocysteine - Errors

Summary of What Went Wrong

There were very significant differences between Sullivan Nicolaides Pathology and Westmead results for the same samples.

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.

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.

I am confident that the Westmead results are correct because:

  • Westmead uses Stable Isotope Dilution Tandem Mass Spectrometry, the reference method also used by Mayo Medical Laboratories.
  • Westmead proved their reliability as a very high quality lab by producing excellent MMA results
  • Westmead is involved in an international, external quality control program (ERNDIM) for homocysteine
  • Westmead results were consistent with the findings of Refsum et al, reference AE11; see note below.

I am confident that the SNP results are incorrect because:

  • The Immunoassay method used by SNP is subject to interferences
  • Immunoassay control ranges are open to abuse, allowing for very significant errors
  • SNP results were very significantly inconsistent with the findings of Refsum et al, reference AE11; see note below.

The Relationship Between Homocysteine and B12

The following chart from Refsum et al, reference AE11 Figure 7, shows the relationship between serum vitamin B12 and total homocysteine:

The Westmead results for homocysteine were significantly more consistent with the relationship between homocysteine and serum B12, as reported by Refsum, than were the SNP results. This may be seen in the following chart:

Chart C31 Table C9

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, described in detail in the previous section.

Why it Matters

Sullivan Nicolaides Pathology failed to report the onset of vitamin B12 deficiency.

According to experts, homocysteine is a sensitive marker of vitamin B12 deficiency. Some excellent articles on this can be found in reference DA01 to DA6.

Here is a chart showing my homocysteine measured by SNP, compared to my homocysteine measured by Westmead:

Chart B22 Table B18

As you can see, at no time did the SNP homocysteine level exceed their quoted maximum level of 15 umol/L, whereas the Westmead results are frequently above, or very close to, their quoted maximum of 13.7 umol/L.

The differences between the labs produce totally different interpretations of my vitamin B12 status. According to the SNP results, my homocysteine was always low and did not indicate vitamin B12 deficiency. According to the Westmead results, my homocysteine level increased dangerously and clearly indicated vitamin B12 deficiency.

In addition to the offset errors, there was was a spike in the level reported by SNP for 9 October 2006. The homocysteine level had apparently increased from 10.0 to 12.6 umol/L in one week, an increase of 26%. The within-person biological variation reported by experts is 4.5%; select reference AB15, AE10 to AE12 and AF02 to view expert reports.

The need for both accuracy and precision is clearly explained in an ERNDIM Report, reference AB01:

There are many excellent expert reports on the subject of homocysteine measurement accuracy and precision, including reference AE14 to AE19.

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SNP Homocysteine - Use of Incorrect Cut-off

Summary of What Went Wrong

Sullivan Nicolaides Pathology incorrectly quotes a limit of 15.0 umol/L for total homocysteine:

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. The following table from Refsum et al, reference AE11 Table 6, shows the recommended maximum homocysteine levels for different patient groups:

In Australia, many processed foods, including breakfast cereals, are folate enriched, so the folate supplemented figure of 12 umol/L should be used for people aged 15-65 years..

Here is the diagnostic flowchart for homocysteine, as shown by D.A.C.H. - Liga Homocystein in reference AE12:

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.

The following chart from Refsum et al, reference AE11 Figure 4, shows the normal total homocysteine level compared to age:

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 quoted by SNP; 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

Sullivan Nicolaides Pathology is not alone in quoting an incorrect homocysteine cut-off; it also appears in the RCPA manual, reference AF05.

Why it Matters

Not only is a high level of homocysteine an indicator of vitamin B12 deficiency, it is also an indicator or risk factor for other diseases including:

  • depression
  • Alzheimer's disease
  • Cardiovascular disease

There are many expert reports on the association between homocysteine and disease, including reference AE11, AE12, CA21 to CA26 and CB01 to CB13. As an example, the following chart from Nygard al, reference CB04, Figure 2, shows the relationship between Mortality Ratio for patients with CAD, and total homocysteine:

Note the very significant increase in mortality ratio, as homocysteine increases from 10 umol/L to 15 umol/L.

In my case, Sullivan Nicolaides Pathology failed to report the onset of an actual vitamin B12 deficiency that should have been interpretable from their homocysteine results.

For a detailed investigation of the reference Intervals and limits, select Reference Intervals and Limits under Interpreting Results.

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SNP Homocysteine - Sent Out Samples Frozen in SST Tubes

Summary of What Went Wrong

For each of the three days, 5, 6 and 7 February 2007, at the request of Dr PM, Sullivan Nicolaides Pathology collected samples of my blood and tested them for homocysteine. The samples were then sent to Dr KC at Westmead, for homocysteine testing, along with a series of samples collected by QML Pathology.

Dr KC at Westmead reported that the three samples from SNP had been sent frozen in Serum Separator Tubes (SST tubes), unlike the 18 samples from QML that had been correctly sent frozen in transfer tubes. My Internet research found that freezing SST tubes is not acceptable by other labs. I contacted the tube manufacturer, BD Vacutainer, and received the following response in their Email of 15 February 2007:

Why it Matters

Although there was no significant difference between results, for the samples frozen in SST tubes and those in transfer tubes, the samples were mishandled.

Any homocysteine results obtained from the samples in frozen SST tubes must be considered invalid.

I believe that any laboratory receiving such samples would either comment on their condition, as Westmead did, or refuse to accept them.

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SNP - Loss of Two Blood Samples

Summary of What Went Wrong

On 17 July 2006, I had my first blood test for which Dr PM requested that my samples be sent to both QHPS and MP. There was no trouble from the collection centre; they took 4 tubes; sufficient for the requested tests.

I later discovered that QHPS never received their sample for 17 July or 14 August, although MP did receive their ones. QHPS did receive their sample for 31 July, and all ones after 14 August, so the instructions from Dr PM were understood. The instruction could not have been clearer:

Here is an extract from the Email that I received from Dr MH, the CEO of Sullivan Nicolaides Pathology, on 10 August, in response to my complaint about the loss of the first blood sample:

Here is how Dr MH responded to my question about the lost sample for 17 July:

Dr Dr MH did not respond to my message about the lost sample for 14 August.

Why it Matters

The loss, or destruction, of two blood samples by any pathology lab, before testing is completed is, in my opinion, totally unacceptable, and a sign of failure of quality control.

For me, the loss of these samples significantly interfered with monitoring of my vitamin B12 status, and investigation of the erroneous results from Mater Pathology.

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SNP - Sent Samples to MP for MMA Testing and Failed to Investigate Complaints

Summary of What Went Wrong

Because Sullivan Nicolaides Pathology does not perform MMA analyses in-house, they refer samples to Mater Pathology for this test.

I have no problem with Sullivan Nicolaides Pathology sending out my samples to another lab, if the other lab is capable of performing the analyses to the required standard of precision and accuracy required for the indicated condition.

I am aware that Mater pathology also tests patients with suspected methylmalonic aciduria, a condition in which the MMA levels are far higher than those expected for vitamin B12 deficiency, and which might require less precision. The indicated condition, vitamin B12 deficiency, was clearly stated under Clinical Notes on the request forms signed by Dr PM:

It is very clear, from the next few sections, that Mater Pathology was not able to achieve the required standard of precision and accuracy required for the diagnosis or monitoring of vitamin B12 deficiency.

Here is an extract from the Email that I received from Dr MH on 10 August, in response to my complaint about Mater Pathology:

Why it Matters

As shown in later sections, Mater Pathology failed to report the onset of an actual vitamin B12 deficiency.

In my opinion, any MMA results provided for other patients by Mater Pathology at the same time as mine, for B12 deficiency diagnosis or monitoring, are likely to also have been very significantly erroneous, potentially leading to incorrect interpretation of cellular vitamin B12 status.

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