Endocrine Scheme Guide · Filename: SP-QL1-ENDOGUIDEV2.7 Approved by:M.A.Thomas Version 2.7 Date of...

© Copyright WEQAS 2003. All rights reserved. No part of this document may be reproduced or utilised in any form without permission from WEQAS

Endocrine

Scheme Guide

Filename: SP-QL1-ENDOGUIDEV2.7 Approved by:M.A.Thomas Version 2.7 Date of revision: 23/09/2015 Page 3 of 16

Contents

1. Scheme details and repertoire 1.1. Source material and integrity

1.2. List of analytes and frequency of distribution

1.3. Instructions for use

2. Statistical Analysis 2.1. Performance criteria

2.2. Instrument Performance

3. Reference Methods 3.1 Progesterone Study

3.2 Cortisol Study

4. Macroprolactin scheme 4.1 Typical Instruction sheet

4.2 Typical Report 4.3 Typical summary sheet 5. Stability


1. Scheme details and repertoire

1.1 Source Material and Serum Integrity The material is human serum, tested negative for HIV and Hepatitis B and C at donor level. The donor serum is initially selected according to age and gender and analysed for testosterone, LH, FSH, E2 cortisol, progesterone and thyroid profile. Individual donor material is the sample of choice, however pooled units of similar concentrations may also be used. Certain pools are spiked with an exogenous source of hormones to enhance the analytical range. Only one of the pools distributed each month will contain analytes from an exogenous source. The pools are filtered aseptically down to a 0.2µm, and gentamicin added to maintain sterility. Preservatives such as sodium azide are not added as these are known to inhibit certain immuno enzymatic methods. Great care is taken to ensure that aseptic techniques are used throughout all procedures to maintain sterility. The serum is dispensed aseptically into 1.8ml aliquots and stored at -20°C until dispatched. The samples are dispatched by first class mail as frozen samples packaged in containers conforming to current Post Office guidelines. 1.2 List of Analytes and Frequency of Distribution Five samples covering an appropriate physiological range are distributed every month. 12 analytes are included in each distribution.

Analyte Approx. Range Covered

Cortisol 60 - 1400 nmol/L

Progesterone 1.0 - 100 nmol/L Oestradiol 37 - 1300 pmol/L Testosterone 0.1 - 40 nmol/L T4 64 - 280 nmol/L

T3 1.5 - 12 nmol/L FT4 8 - 50 pmol/L FT3 3.0 - 40 pmol/L TSH 0.1 - 21 mU/L

FSH 0.8- 100 IU/L LH 2 - 80 IU/L Prolactin 48 - 800 mU/L 1.3 Instructions for use

The samples are dispatched frozen, and will thaw in transit. Please ensure that the samples are well mixed before analysis. Please analyse on the day of arrival. If there is a delay in analysis please store at 4°C and assay within 5 days.

Although every effort is made to ensure that the material is free from any known infectious agent, the samples should be handled as for clinical specimens.


2. Statistical analysis Please refer to the accompanying Participants Manual for full details on statistical analysis and interpretation of results.

2.1 Performance Criteria

Precision profiles have been established for all the analytes which reflect the current “state of the art” of immunoassay methods. These criteria are used to calculate the WEQAS SD for each analyte at each level.

A plot of the standard deviation (SD) against concentration is given for each analyte over the distributions. The data reflects the interlaboratory variation and the bias between the methods currently in use.

Oestradiol 0-1500 sd y = 0.000014x2 + 0.071094x + 35.891047R2 = 0.766264

y = 0.000001x2 + 0.052390x + 28.927567R2 = 0.621906

y = 0.09391x + 12.81971R2 = 0.84783

y = 3E-05x2 + 0.0303x + 14.946R2 = 0.9695

0

20

40

60

80

100

120

140

160

180

200

0 300 600 900 1200 1500

Mean

SD

97

1203-0804

1004-0906

1006-1206

2007 - 2010

Biologic goals

S160-S184

S185 - S194

Linear (S160-S184)Poly. (S185 -S194)

TE = 27.2%

line in use

Cortisol sd

y = 0.1017x + 1.5466R2 = 0.9267

y = 0.0839x + 6.1764R2 = 0.7766

0

20

40

60

80

100

120

140

160

180

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

Mean

Sd

971203-08041004-09060106-12062007-2010Biologic goalsS160 - S184S185 - S194Linear (S185 - S194)

Te 29.8%

line in use

Progesterone sd 0 - 110

y = 0.0002x2 + 0.0782x + 0.4535R2 = 0.9521

y = -0.0001x2 + 0.1306x + 0.2573R2 = 0.9312

y = 0.0005x2 + 0.0573x + 0.3787R2 = 0.9059

y = 0.0004x2 + 0.0502x + 0.2126R2 = 0.9459

0

2

4

6

8

10

12

14

16

0 10 20 30 40 50 60 70 80 90 100 110

Mean

SD

971203-08041004-09061006-12062007 -2010S160 - S184S185 - S194Poly. (S160 - S184)Poly. (S185 - S194)

line in use

Testosterone sd y = 0.091910x + 0.296990R2 = 0.861308

y = 0.1026x + 0.0296R2 = 0.8324

y = 0.0022x2 + 0.0463x + 0.1249R2 = 0.8539

0

1

2

3

4

5

6

0 5 10 15 20 25 30 35 40 45

Mean

SD

97

1203-0804

1004-0906

1006-1206

2007 - 2010

Biologic goals

S160 - S184

S185 - S194

Linear (S160 -S184)Poly. (S185 -S194)

TE = 13.1%

line in use

FREE T4 sd y = 0.003007x2 - 0.002498x + 0.956388R2 = 0.910460

y = 0.006x2 - 0.0928x + 1.7782R2 = 0.8741

y = -0.003x2 + 0.2309x - 0.9095R2 = 0.7043

0

2

4

6

8

10

12

14

0 10 20 30 40 50 60 70

Mean

SD

9720041004 - 09061006-12062007 - 2010Biologic goalsS160 - S184S185 - S194Poly. (S160 - S184)Poly. (S185 - S194)

TE = 8%

line in use Ft3 sd 0-45 y = 0.002295x

2 + 0.153751x - 0.013645R2 = 0.900116

y = 0.0012x2 + 0.1762x - 0.2789R2 = 0.9514

y = -0.0006x2 + 0.2187x - 0.6178R2 = 0.9776

y = 0.0638x1.2245

R2 = 0.9018

0

2

4

6

8

10

12

0 5 10 15 20 25 30 35 40 45

Mean

Sd

Series1

1203 - 0804

0904-0906

1006-1206

2007 -2010

Biologic goals

S160-S184

S185 - S194

Poly. (S160-S184)Power (S185 -S194)

TE = 11.3%

line in use


TSH sd y = 0.008229x2 + 0.051965x + 0.042059R2 = 0.972839

y = 0.0009x2 + 0.0505x + 0.0792R2 = 0.9049

y = -0.0007x2 + 0.1265x - 0.0601R2 = 0.8244

y = -0.0027x2 + 0.1293x - 0.0076R2 = 0.9177

y = -0.0012x2 + 0.0702x + 0.066R2 = 0.8282

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 5 10 15 20 25 30

Mean

SD

4/96 to 7/9912/03 to 8/041004-09061006-12062007 -2010Biologic goalsS160-S184S185 - S194Poly. (S160-S184)Poly. (S185 - S194)

current biol goals line 0.123x (24.6%) - new biol goals - TE=23.7% - no change required

Prolactin sd 0-800

y = 0.00008x2 + 0.06949x + 6.33051R2 = 0.84655

y = -8E-05x2 + 0.182x - 5.4803R2 = 0.8069

0

20

40

60

80

100

120

140

0 100 200 300 400 500 600 700 800

Mean

Sd

Series1

1203 -0804

1004 - 0906

0106-1206

2007 -2010

Old BiologicgoalsS160-S184

S185 - S194

New biol goals

Old TE = 21.1%

line in use

New TE = 29.4%

FSH sd 0-100 y = 0.096847x - 0.011800R2 = 0.885716

y = 0.08093x + 0.30192R2 = 0.76894

y = 0.0005x2 + 0.0382x + 0.2476R2 = 0.9575

0

2

4

6

8

10

0 10 20 30 40 50 60 70 80 90 100

Mean

sd

971203-08041004-0906106-12062007 -2010S160-S184S185 - S194biologic goalsPoly. (S185 - S194)Linear (biologic goals)

TE = 17.1%

line in use

LH sd 0 - 80y = 0.000689x2 + 0.146666x - 0.106780

R2 = 0.924040

y = 0.0015x2 + 0.3396x - 1.6326R2 = 0.9754

y = -0.0012x2 + 0.1303x + 0.032R2 = 0.7724

y = 0.1646x0.8615

R2 = 0.9544

0

10

20

30

0 20 40 60 80

Mean

Sd

Series11203-08041004-09061006-12062007 - 2010Biologic goalsS160-S184S185 - S194Power (S160-S184)

TE = 19.8%

line in use

Total T3 y = 0.075745x + 0.084043R2 = 0.613248

y = 0.005x2 + 0.0021x + 0.1848R2 = 0.3765

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Mean

Sd

Total T4 y = 0.000263x2 + 0.014829x + 2.432797

R2 = 0.546143

y = 0.0001x2 - 0.0207x + 5.0857R2 = 0.1889

0

5

10

15

20

25

30

35

40

45

50

50 100 150 200 250 300 350

Mean

Sd c


2.2 Instrument Performance

A comparison of instrument performance is included with each distribution. This is given in tabular form, as the mean and standard deviation. Analytical performance of methods, instruments and kits are available to participants for all Schemes on request.

Testosterone (nmol/l)

Method Instrument 1 2 3 4 5

Overall Mean 6.48 0.59 5.41 11.34 0.60

Overall SD 0.52 0.21 0.39 1.60 0.13

Est. Uncertainty of Consensus 0.113 0.045 0.086 0.340 0.030

Overall Number 21 22 21 22 20

Reference Value

Reference Value NS

Access/DxI 800 Method Mean 6.22 0.80 5.27 8.94 0.65

Method SD 0.56 0.12 0.26 0.27 0.14


Number 6 6 6 5 6

Access Instrument Mean 6.57 0.83 5.40 9.53 0.67

Instrument SD 0.57 0.09 0.29 0.77 0.09

Number 3 3 3 3 3

DX Instrument Mean 5.87 0.77 5.13 8.90 0.63

Instrument SD 0.21 0.12 0.09 0.29 0.17

Number 3 3 3 3 3

Tandem MS Method Mean 8.70 0.90 7.10 12.20 No Returns

Method SD 0.00 0.00 0.00 0.00 No Returns

Est. Uncertainty of Consensus 0.000 0.000 0.000 0.000 No Returns

Number 1 1 1 1 No Returns

Miscellaneous Instrument Mean 8.70 0.90 7.10 12.20 No Returns

Instrument SD 0.00 0.00 0.00 0.00 No Returns

Number 1 1 1 1 No Returns

Advia Centaur Method Mean 7.03 0.96 5.82 10.93 0.98

Method SD 0.00 0.00 0.00 0.00 0.00


Number 1 1 1 1 1


Advia Centaur Instrument Mean 7.03 0.96 5.82 10.93 0.98

Instrument SD 0.00 0.00 0.00 0.00 0.00

Number 1 1 1 1 1

Elecsys Testo II Method Mean 6.95 0.40 5.70 12.95 0.53

Method SD 0.13 0.06 0.16 0.29 0.04


Number 7 8 8 8 8

Elecsys E170 Instrument Mean 7.00 0.30 5.50 12.60 0.50

Instrument SD 0.00 0.00 0.00 0.00 0.00

Number 1 1 1 1 1

Cobas E Module Instrument Mean 6.94 0.42 5.73 13.00 0.54

Instrument SD 0.14 0.05 0.15 0.27 0.04

Number 6 7 7 7 7

Tosoh AIA Method Mean 6.14 0.52 5.11 11.24 0.65

Method SD 0.33 0.10 0.43 0.80 0.16


Number 6 6 6 6 6

AIA Instrument Mean 6.14 0.52 5.11 11.24 0.65

Instrument SD 0.33 0.10 0.43 0.80 0.16

Number 6 6 6 6 6


3 Reference Methods - Assessment of Analyser bias

Reference methods using Isotope Dilution Gas Chromatography-Mass Spectrometry (ID-GCMS) have been developed for Progesterone, Cortisol and Testosterone. The Oestradiol method is awaiting validation. The following studies (pages 9-11) highlight the usefulness of reference targeted data as an accuracy target in EQA Schemes. WEQAS is committed to provide traceable reference method target values for the majority of analytes. ID GC-MS values will be assigned to all steroid pools allowing a comparison of results to the reference method for each distribution. For these studies, uncertainty measurements were estimated according to GUM, with traceability established by use of certified reference material. On the following graphs, the deviations from the ‘true’ result (the reference method) for the main analyser groups are illustrated in the form of Bias plots (Bland –Altman). The pools were distributed on several occasions over the time period. The x-axis represents the reference value and for each pool the scatter on the y-axis represents the deviation of the analyser method mean from this value. The ‘---‘ on the graph represents the 2 * WEQAS SD limits of acceptance. From the linear regression analysis, the slope provides a measure of the systematic proportional error (calibration) and the intercept the systematic constant error (specificity).

3.1 Progesterone Study (Distributions 70 to 90)

6 Pools:were circulated over 18 months (Distributions 70 to 90), containing both spiked (n=4) and endogenous (n=2) progesterone.


The regression analysis suggests good agreement with the reference value for the Roche Elecys (E170). The Bayer Centaur, DPC Immulite, Immuno 1 and Beckman Access show a systematic mixed error, with positive bias of 1 to 3 nmmol/l at low concentration, near zero bias at 20nmol/l and a negative bias at high concentration. A negative bias is observed across the analytical range for the Bayer ACS 180. Future pool selection for reference value assignment will include samples with endogenous progesterone at more elevated levels. 3.2 Cortisol Study (Distributions 58-72). 15 Pools:were circulated over 18 months (Distributions 58-72); containing both spiked and endogenous cortisol.


Bayer Centaur - Spiked

y = 1.2423x + 48.571

-300

-200

-100

0

100

200

300

400

200 700 1200

Bia

s

The data suggests a predominant positive proportional bias for the DPC Immulite (22%) and Roche Elecsys (13%), which is also mirrored in the spiked samples. A mixed systematic bias is observed for the Tosoh AIA giving a +60nmol/l positive bias at 200 nmol/l, which reduces to zero at 1200nmol/l. The Bayer Centaur shows a large proportional bias of approximately 30% over the analytical range with an associated constant error which increases the bias to 40% at low concentration. All methods overrecovered for cortisol at low concentration. For pool 69 and 70, both DPC Immulite and Centaur gave higher than expected results, suggesting some cross-reacting interference. A similar effect was also observed for pool 64 on the Immulite.


4. Macroprolactin Scheme More challenging samples are distributed in the Immunoassay scheme on a regular basis with an aim of assessing both analytical performance and current laboratory practice. Samples with significant macroprolactin levels are distributed every six months to assess current practice in macroprolactin investigation. 4.1 Typical Macroprolactin Input sheet

Immunoassay Scheme - Prolactin Assay

Sample Code: Sent out on:

Please note that for this distribution there are 6 samples for Prolactin. Enter your results for Samples 1 to 5 on the standard return form. Please treat sample 6 as you would a patient sample and carry out any further investigation you feel may be appropriate. I would be grateful if you could supply us with your comments and the results of your findings. Thank you for your continued co-operation Lab: _______ (please complete) Section: _______ (please complete) Analyser: _______ (please complete)

Clinical details

Please provide additional information based on the Prolactin result of Sample 6:

What was the analyser result for Sample 6? Did you carry out any further investigation on this sample? Yes No Please give details of any further analytical procedures and results. _________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ What is your interpretation of this result? _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ Please send a copy of your report as if it were a patient sample.


4.2 Typical Macroprolactin Report

IMMUNOASSAY SCHEME DISTRIBUTION S180 (SAMPLE 6) Dear Colleague, Please find enclosed the Prolactin results for the above Distribution. Clinical Details The sample was from a healthy female donor, aged 30 years. Her endocrine profile showed a normal TFT with an oestradiol of 375 pmol/L, progesterone 6.9 nmol/L, testosterone 2.7 nmol/L, LH 8.9 IU/L, FSH 4.2 IU/L with an elevated prolactin. Gel filtration confirmed the presence of significant macroprolactin (77%) with a normal monomeric prolactin. This sample had previously been distributed on Distribution S62, S123 and S163. Result Summary A wide variation of results was observed for this sample, even within the same analyzer group. Unfortunately only nine laboratories returned a result for this sample. Four of the results were above the reference ranges for their analyser. Two out of the 4 would have carried out further investigation. One laboratory stated that they had carried out PEG precipitation and a monomeric result of 129 mU/L (male range 61-196) was obtained. They stated that they would not have carried out a PEG precipitation on a female as the result was within their reference range. The Abbott Architect continues to provide one of the highest results for macroprolactin at 836 mU/L, although in this distribution only 1 laboratory returned a result for this analyser. A result of 964 mU/L was observed from one laboratory using a Beckman Access , which is neither consistent with the other users in the group or previous results reported for this sample. The response to the current distribution indicates that a wide variation in reactivity for macroprolactin still exists; despite improvements in method specificity, the presence of macroprolactin should still be considered. Thank you for your continue co-operation Yours Sincerely

Annette Thomas (Scheme Organiser)

Endocrine Scheme – Macroprolactin Report

WEQAS Unit 6, Parc Tŷ Glas, Llanishen Cardiff, UK CF14 5DU Tel: +44 (0) 2920 314750 Fax: +44 (0) 2920 314760 E-mail: [email protected]


Method Lab Result Further investigation Interpretation

Beckman Access 2 AE 583 Yes

Increased prolactin - assuming normal thyroid and renal function suggest exclude stress, drugs (eg dopamine blockers or antagonists) and pregnancy

(is female) as cause. Suggest repeat to confirm. Marginally elevated prolactins are also seen in some PCOs patients. our reference range is up to 560 - we probably would'nt put a comment on that close to the RR. However

if asked for interpretation would give the above.

Advia Centaur 400 AF 598 Yes - PEG PrecipitationPost - PEG prolactin is normal for a male. Apparent hyperprolactinaemia is likely due to macroplactin (an ig-prolactin complex) which is not bilogically

active.

Roche Cobas 6000 HK 92.7 NO Normal result - no further analysis needed (ref ranges, Male: 102 - 496 mU/L, Female: 86 - 324 mU/L)

Normal Result

Beckman Access 2 QR 964 NO Abnormal

Roche Cobas 6000 GY 468 NO

None StatedAbbott Architect GU 836 NO

None Stated

None Stated

Beckman Access 2 FF 452.2 NO

Beckman Access Dxi CT 144 NO

Summary Sheet Distribution S180 Sample 6

None StatedBeckman Access Dxi AZ 451 NO

4.3 Typical Summary Sheet


Cortisol Stability

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

Dec-02 Mar-03 Jun-03 Oct-03 Jan-04 Apr-04 Aug-04 Nov-04

Date (months)

Ove

rall

mea

n (n

mol

/l)

Pool 60 (spiked)Pool 59 (Endogenous)Pool 49 (Spiked)

Testosterone Stability

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00


Date (months)

Ove

rall

mea

n (n

mol

/l)

Pool 60 (Endogenous)Pool 59 (Endogenous) Pool 49 (Spiked)

Progesterone Stability

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00


Date (months)

Ove

rall

mea

n (n

mol

/l)

Pool 60 (Spiked)Pool 59 (Endogenous)Pool 49 (Spiked)

Oestradiol Stability

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00


Date (months)

Ove

rall

mea

n (p

mol

/l)

Pool 60 (Spiked)Pool 59 (Endogenous)Pool 49 (Enogenous)

FT4 Stability

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00


Date (months)

Ove

rall

mea

n (p

mol

/l)

Pool 60 (Spiked)Pool 59 (Endogenous)Pool 49 (spiked)

FT3 Stability

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00


Date (months)

Ove

rall

mea

n (p

mol

/l)

Pool 60 (Spiked)Pool 59 (Endogenous)Pool 49 (spiked)

5. Stability Long term stability at--20 °C The 3 pools were distributed on at least 3 occasions over a 16 month period Pool 49 was spiked with Testosterone, Progesterone, T4, T3, LH, FSH and Cortisol, Pool 60 was spiked with Oestradiol, Progesterone, T4, T3, LH, FSH TSH and Cortisol and Pool 59 contained endogenous hormones only. The following graphs illustrate the change in analyte concentration (overall mean of all the results) for each of the pools distributed to all WEQAS participants over the 16 month period.


TSH Stability

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00


Date (months)

Ove

rall

mea

n (Im

U/l)

Pool 60 (spiked)Pool 59 (Endogenous)Pool 49 (Endogenous)

LH Stability

0.00

10.00

20.00

30.00

40.00

50.00

60.00


Date (months)

Ove

rall

mea

n (IU

/l)


FSH Stability

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00


Date (months)

Ove

rall

mea

n (IU

/l)


Prolactin Stability

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

180.00

200.00


Date (months)

Ove

rall

mea

n (Im

U/l)

Pool 60 (Endogenous)Pool 59 (Endogenous)Pool 49 (Endogenous)

Long term stability at--20 °C- continued


3. Reference Methods3.1 Progesterone Study4. Macroprolactin scheme4.1 Typical Instruction sheet4.2 Typical Report1. Scheme details and repertoire1.1 Source Material and Serum Integrity1.2 List of Analytes and Frequency of DistributionTestosterone (nmol/l)/

The regression analysis suggests good agreement with the reference value for the Roche Elecys (E170). The Bayer Centaur, DPC Immulite, Immuno 1 and Beckman Access show a systematic mixed error, with positive bias of 1 to 3 nmmol/l at low concentrati...//

4.1 Typical Macroprolactin Input sheet4.2 Typical Macroprolactin Report

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