WHO/BS/2014.2244 ENGLISH ONLY

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WHO/BS/2014.2244 ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 13 to 17 October 2014

COLLABORATIVE STUDY ON THE 1st INTERNATIONAL REFERENCE PANEL (PLASMA) FOR LUPUS ANTICOAGULANT

Helen Wilmot and Elaine Gray¹

Haemostasis Section,

National Institute for Biological Standards and Control Potters Bar, Hertfordshire, EN6 3QG, UK

1Principal Investigator

NOTE:

This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on Biological Standardization (ECBS). Comments MUST be received by 4 October 2014 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:

Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible Officer: Dr David Wood at email: [email protected].

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Summary

Nineteen laboratories from 11 different countries participated in a collaborative study to evaluate 3 plasma samples as the 1st International Reference Panel (Plasma) Lupus anticoagulant, 13/172.

The candidates were prepared by titration of lupus negative and lupus positive patient plasmas to obtain a negative (12/148), a moderate positive (12/150) and a strong positive (12/152) lupus anticoagulant samples. Another lupus positive plasma sample, 96/560, was also included in the study.

Nineteen laboratories returned 22 sets of data for dilute Russell Viper’s venom time (dRVVT), 13 laboratories returned 21 sets of data for activated partial thromboplastin time (APTT), 4 laboratories returned results for silica clotting time (SCT), 2 laboratories returned data for dilute Prothrombin time (dPT), while one set of result was returned for kaolin clotting time (KCT), activated seven lupus anticoagulant assay (ASLA) and Taipan snake venom time (TSVT). The laboratories performed screening tests and, where appropriate, confirmation and mixing studies for each method.

The intra-laboratory variability was low. The majority of laboratories obtained ratios that had intra-laboratory coefficients of variation (CV) of less than 5% when the test samples were calculated relative to the local pooled normal plasma (PNP) indicating that the participants performed assays for lupus anticoagulant reproducibly and with high precision.

Inter-laboratory variability was low for dRVVT, but higher for the other assay methods. Both intra- and inter-laboratory variation were reduced when the ratios were recalculated relative to sample A, 12/148, the candidate lupus negative plasma.

All methods employed in this study found 12/148 to be lupus negative and both 12/150 and 12/152 to be lupus positive. The other lupus positive plasma, 96/560 was also confirmed as lupus positive. The ranking order of lupus positivity for the 3 lupus positive samples in all the assays were C>B>Z.

The results of this study confirmed the lupus status of the 3 candidates and it is therefore recommended that all 3 plasmas be established as:

The 1st International Reference Panel (Plasma) for Lupus Anticoagulant, 13/172, with the following components:

12/148 Lupus negative 12/150 Lupus moderate positive 12/152 Lupus strong positive

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Introduction

The presence of antiphospholipid antibodies characterises Lupus Anticoagulant. These immunoglobulins are heterogeneous and bind to phospholipid, disrupting the clotting process and leading to a prolonged clotting time in vitro tests. However, the clinical manifestations are occurrence and recurrence of thrombotic events rather than bleeding issues. Lupus

anticoagulant positivity also carries increased risk of recurrent spontaneous miscarriages, foetal death and foetal retardation. Patients require long-term anticoagulant therapy and clotting assay based laboratory tests are employed to help clinical decision on diagnosis and treatment regimen.

Diagnosis is difficult as patients with a clotting factor deficiency also have prolonged clotting times. Although mixing studies are performed to identify if the clotting time is restored by normal plasma (indicating factor deficiency) or by washed platelets (phospholipid), indicating the presence of antiphospholipid antibodies, there is no specific test for lupus and therefore misdiagnosis is common as full correction is not always observed. There are national and international guidelines on laboratory testing for lupus anticoagulants (1, 2). Numerous studies, including quality assessment schemes illustrate large numbers of false negative and positive diagnoses are still an issue (3, 4, 5, 6, 7, 8). The use of appropriate reference materials have been recommended by lupus testing guidelines as means of improving the quality of testing. The 1^st British Reference Panel(Plasma) for Lupus Anticoagulant was established by NIBSC in 1999 and proven to be valuable in helping laboratories to improve assay performance of lupus anticoagulant tests. The stock of this panel was deleted within 5 years of its establishment and there is no replacement available. Currently there is no international reference material for Lupus Anticoagulant. This study serves to evaluate the proposed 1st International Reference Panel(Plasma) for Lupus Anticoagulant.

Participants

Nineteen laboratories from 11 different countries (1 Australia, 1 Austria, 1 Belgium, 1 Canada, 1 France, 1 Germany, 2 Italy, 1 Japan, 1 The Netherlands, 5 UK, 4 USA) agreed to participate in the study, all of which returned data in time for the statistical analysis.

The participants included 6 clinical laboratories, 8 diagnostics manufacturers, 4 academic

laboratories and 1 regulatory authority. A list of participants is given in Appendix 1 at the end of this report. Each laboratory is referred to in this report by an arbitrarily assigned number, not necessarily representing the order of listing in the Appendix.

Samples

The following coded samples were sent to each participant:

A Proposed 1st International Reference Panel (Plasma) for Lupus Anticoagulant, Negative plasma (12/148)

B Proposed 1st International Reference Panel(Plasma) for Lupus Anticoagulant, Moderate positive plasma (12/150)

C Proposed 1st International Reference Panel (Plasma) for Lupus Anticoagulant, Strong positive plasma (12/152)

Z 1st British Reference Plasma Panel for Lupus Anticoagulant, Weak positive Plasma (96/560)

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The Candidates, 12/148 (lupus negative), 12/150 (lupus moderate) and 12/152 (lupus positive)

Samples A, B and C were freshly prepared at NIBSC from frozen platelet poor normal plasma and platelet poor positive plasma. All donations had been tested and found to be virology negative for HIV1&2, Hepatitis B surface antigen and Hepatitis C. The negative pool was made using plasma from a total of 10 donors and the positive pool was made using plasma from a total of 17 donors. Each pool had HEPES added to a total of 0.8% w/v. The positive pool was diluted 1:1 in negative plasma to generate sample C (strong positive pool) and 1:3.3 to generate sample B (moderate positive). The remaining negative plasma served as sample A (lupus negative). All 3 preparations were filled and freeze-dried into siliconised ampoules at NIBSC and stored at - 20°C. Stocks were sufficient to generate approximately 2500 ampoules of each preparation.

Product characteristics are shown in the tables below:

NIBSC Code 12/148

Presentation Sealed, siliconised glass 3 ml DIN

ampoules

Filling date 21st June 2012

Number of Ampoules available 2933

Liquid filling weight (g) (n=132, measurements taken

from all 3 pumps throughout the duration of the fill) 1.0087

CV of fill mass (%) 0.259

Homogeneity of the fill by activity: 3 ampoules selected from the beginning, middle and end of the fill were assayed by DRVVT method (duplicate

measurements for each). Effect of fill position was assessed by ANOVA.

Screen/confirm ratio (%CV) p DRVVT 1.09 (4.02) 0.903

Mean dry weight (g) (n=6) 0.0830 (CV 0.62%)

Mean head space oxygen (%) (n=12) 0.48 (CV 57.80%)

Residual moisture (%) (n=12) 0.52 (CV 19.74%)

pH of plasma Pre-fill: 6.9, freeze-dried 7.8

Storage temperature -20°C

Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK

NIBSC Code 12/150

ampoules

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Mean dry weight (g) (n=6) 0.0832 (CV 0.37%) Mean head space oxygen (%) (n=12) 0.48 (CV 53.61%)

Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK

NIBSC Code 12/152

ampoules

Mean dry weight (g) (n=6) 0.0837 (CV 0.51%)

Mean head space oxygen (%) (n=12) 0.54 (CV 20.34%)

Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK A clotting factor screen was carried out on each freeze-dried preparation, where possible using methods insensitive to the presence of lupus anticoagulant. The results are shown in Table 1.

Table 1: Clotting factor screen of 12/148, 12/150 and 12/152

Clotting factor IU/ml (95% confidence intervals)

12/148 12/150 12/152

FII 0.93

(0.87-1.00)

0.84 (0.78-0.90)

0.74 (0.69-0.79)

FV 0.78

(0.74-0.82)

0.79 (0.75-0.83)

0.87 (0.83-0.92)

FVII 0.90

(0.82-0.99)

0.80 (0.73-0.88)

0.75 (0.68-0.83)

FVIII 1.06

(1.02-1.11)

1.07 (1.03-1.12)

1.02 (0.98-1.06)

FIX 0.89

(0.82-0.97)

0.80 (0.73-0.86)

0.74 (0.69-0.81)

FX 0.96

(0.92-1.01)

0.85 (0.82-0.89)

0.73 (0.70-0.77) Antithrombin Activity 0.97

(0.94-1.02)

0.96 (0.92-1.00)

0.94 (0.90-0.97)

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Antithrombin Antigen 1.02 (0.99-1.05)

0.92 (0.90-0.95)

0.94 (0.91-0.97)

Protein C 1.06

(1.02-1.10)

0.98 (0.95-1.01)

0.90 (0.87-0.93) Free Protein S Antigen 0.93

(0.90-0.96)

0.86 (0.83-0.89)

0.79 (0.77-0.82) Protein S functional activity 0.94

(0.89-1.00)

0.82 (0.78-0.87)

0.75 (0.71-0.80)

Assays for FII, V, VII and X were performed using tissue factor-based assays; FVIII and FIX assays were performed using chromogenic kits, antithrombin assays were chromogenic (activity) and latex bead-based (antigen); protein C assays were chromogenic, and protein S antigen was latex bead-based, with protein S activity APTT-based. Although the protein S activity assay was APTT-based, the assay involves dilution of the test sample in normal plasma and involves activation of protein S, thus the contribution of lupus anticoagulant to the result is likely to be small.

Study design and assay methods

Participants were requested to carry out three independent assays of each chosen method(s) using freshly reconstituted ampoules of samples A, B, C, and Z. Each set of assays was to be carried out on separate days.

Each participant performed their routine in-house dilute Russell’s Viper Venom Time (dRVVT) method(s) for Lupus Anticoagulant, plus any additional routine in-house tests (e.g. Activated Partial Thromboplastin Time, Silica Clotting Time, Kaolin Clotting Time etc.). A list of

reagents, methods and instruments used by the participants is given in Appendix 2, together with details of positive and negative controls used by each laboratory.

All 19 laboratories performed dRVVT, 13 performed Activation Partial Thromboplastin Time (APTT), 4 performed Silica Clotting Time (SCT), 2 performed dilute Prothrombin Time (dPT), 1 lab performed Kaolin Clotting Time (KCT), 1 lab performed Activated Seven Lupus

Anticoagulant assay (ASLA), 1 performed the Taipan Snake Venom Time (TSVT), with Ecarin time as confirmation test, and one performed an anti-cardiolipin (aCL) enzyme linked

immunosorbent assay (ELISA). Mixing study regimes for all the clot-based assays when performed were as indicated in Appendix 2.

Raw assay data were returned to NIBSC together with calculated ratios where appropriate (e.g.

dRVVT screen/confirm ratios) for each participant. NIBSC calculated ratios for each individual laboratory (where possible) are shown in Appendix 3. Where it was not possible to analyse results centrally at NIBSC (e.g. where mix only results were returned), then the laboratory’s reported data are shown. The collaborative study protocol is shown in Appendix 4.

Analysis of data

An independent calculation of ratios from raw data and statistical analysis of ratios were performed at NIBSC. Screen, confirm and mix ratios were calculated relative to local pooled normal plasma (PNP) and the candidate Lupus negative plasma, Sample A (12/148). The formulae used for calculation of the ratios are as shown in Table 2. For each sample, duplicate clotting times from each individual assay were used to calculate the mean clotting times which were then used to calculate ratios for each individual assay. Laboratory mean ratios for each

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independent assay were combined to give overall laboratory mean ratios and intra-laboratory variability was expressed as % coefficient of variation (CV). Where there were sufficient numbers of data sets for each method, overall mean ratios for each method and inter-laboratory variation (%CV) were calculated from overall individual laboratory mean and this was carried out for dRVVT and APTT.

Table 2: Calculation of ratios for different assay methods dRVVT, SCT, dPT, ASLA, TSVT

Screen ratio test screen clotting time/PNP screen clotting time or sample A screen clotting time

Confirm ratio test confirm clotting time/PNP confirm clotting time or sample A confirm clotting time

Screen/ Confirm

ratio test screen clotting time/test confirm clotting time Normalised

ratio (NR) screen ratio/confirm ratio

% correction of

ratio ((Screen ratio – confirm ratio)/Screen ratio)x100

Mix ratio test mix clotting time/PNP screen clotting time or sample A screen clotting time

APTT

KCT

Rosner Ratio= (KCT-mix - KCT-PNP) / KCT-sample.

Results

Some laboratories performed more than one type of assay for each method and the data from each type of assay were treated as separate sets of results and, for example, referred to Lab 18a and Lab 18b.

Nineteen laboratories returned 22 sets of data for dRVVT, with Lab 8 returning 3 sets of results and Lab 18 returning 2 sets of results employing different dRVVT methods.

Thirteen laboratories returned 21 sets of data for APTT, with Lab 8 returning 6 sets of data, each set using different APTT reagents and instruments, and Lab 11, 15 and 19 returning 2 sets of results using different APTT reagents.

Four laboratories returned results for SCT, 2 laboratories returned data for dPT, while one set of results each were returned for KCT, ASLA, TSVT and aCL.

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With the exception of the following, all the laboratories returned full set of results with duplicates clotting times for each sample:

DRVVT:

 Lab 1: Day 3; no results for sample C confirm.

 Lab 3: No local positive control.

 Lab 4: Mix results only; data cannot be interpreted in the usual manner.

 Lab 6: No mix results reported for days 1 or 2. Day 3 mix results reported for B, C and Z only.

 Lab 8b: Day 1 – no duplicates reported. Day 2 – no duplicates for sample A screen or confirm.

 Lab 10: No duplicates for PNP or local positive screen or confirm (days 1, 2 or 3).

 Lab 12: Uses Vipera lebetina venom instead of Russell’s viper venom.

 Lab 13: No results for PNP confirm or sample A mix confirm (days 1, 2 or 3)

 Lab 19: no results for sample A confirm days 2 and 3; no results for Z confirm day 3 APTT:

 Lab 4: Mix results only; data cannot be interpreted in the usual manner.

 Lab 5: No duplicates for mix results for A, B, C or Z on days 1 and 3. On day 3, no duplicates for mix results for B, C or Z and no mix results for sample A.

 Lab 6: Day 1 – no mix results reported. Days 2 and 3, mix results reported for samples B, C and Z only.

 Lab 8a: No confirm or mix for Actin FSL (8a1) or Pathromtin (8a2).

 Lab 8b: No confirm or mix for Actin FSL (8a1) or Pathromtin (8a2).

 Lab 8c: No confirm or mix for Actin FSL (8a1) or Pathromtin (8a2).

 Lab 9: Mixes at 50:50 and 80:520 ratios for samples B, C and Z only.

 Lab 11b: Mix results only for STAclot LA.

 Lab 15a and b: No mix results reported.

 Lab 17: Uses LCA index for APTT results so cannot be analysed alongside the other APTT results.

 Lab 19: Mix results only. Cannot analyse results against sample A as no screen results reported for sample A.

SCT:

 Lab 2: no mix results.

 Lab 5: No mix results for sample A.

 Lab 13: No PNP confirm, no confirm or mix for sample A, no duplicate confirm for sample Z.

 Lab 14: No duplicate for local positive confirm on day 2.

dPT:

 Lab 18: Day 1– no duplicate for sample B confirm. 50:50 mix carried out for screen reagent on positive samples only each day.

ASLA:

 Lab 6: Mix carried out for samples A, B, C and Z on day 1 only.

TSVT:

 Lab 6: Days 1 & 2 – no confirm test carried out for samples A or Z

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Ratios calculated for each independent assay, carried out by individual laboratories, using different assay methods are presented in Appendix 3. Where analysis was not possible (e.g. mix results only reported), then the results as reported by the originating laboratory are shown.

DRVVT

The revised ISTH guidelines for lupus anticoagulant detection (Pengo et al., 2009) provide information on how to obtain local cut-off values, but does not give “common” cut-off values for any lupus anticoagulant assays. The BCSH guidelines (Greaves et al., 2000) for dRVVT stated that the upper limit of normal ratio should be between 1.1 –1.2. DRVVT ratios of >1.1 should be retested using the Platelet Correction Procedure (PCP) and a significant shortening (10%) of the DRVVT is suggestive of the presence of lupus anticoagulant. A 50% normal to 50% test mixture giving a ratio of more than 1.1 with DRVVT is also suggestive of the presence of lupus anticoagulant.

Screen ratios, confirm ratios, normalised ratios, % correction of ratio and mix and screen ratios were calculated relative to local pooled normal plasma (PNP) for Samples A, B, C and Z and these are presented in Tables 3, 4, 5 and 6 respectively. When compared against the local PNP, with the exception of Lab 12, sample A (12/148), the candidate negative plasma was found to be negative by all the participants, with all screen ratios being close to 1 (overall mean ratio: 1.00, range: 0.90 – 1.29). The screen ratio for Lab 12 was 1.29 and was slightly higher than the limit of 1.2 as stated by the BCSH guidelines. However, the laboratory’s own cut off value was reported to be 1.3 and therefore sample A would deem to be negative. For sample B (12/150), the candidate moderate positive plasma and sample C (12/152), the strong positive plasma, screen ratios were all greater than 1.2, with mean screen ratios of 1.51 (range: 1.31 – 1.93) and 1.86 (range 1.51 – 2.31) for samples B and C respectively. The results for confirm ratios and normalised ratios, together with greater than 30% correction of ratio in majority of cases indicate that both samples B and C are lupus positive, with sample C stronger than sample B. The mix ratios for both samples B and C were also greater than 1.1 thus supporting lupus positivity status of these materials. Sample Z is a weak positive sample and is not part of the proposed reference panel for Lupus anticoagulant. The screen (mean 1.36, range 1.10 -1.79), confirm (mean 0.99, range 0.93 – 1.17) and normalised (mean 1.38, range 1.14 – 1.70) ratios verified sample Z as lupus positive. All laboratories obtained the same order of positivity for samples B, C, Z (C>B>Z), indicating that all the test methods were able to correctly distinguish the activity of these lupus positive samples. The intra-laboratory variability for dRVVT ratios relative to PNP are shown in Tables 7, 8, 9 and 10 which present the %CV for screen, confirm and normalised ratios for all 4 samples. It is clear that the majority of the participants were able to perform dRVVT with good precision as the majority of the CVs was less than 5%.

Tables 4, 5 and 6 also present the calculated ratios for samples, B, C and Z relative to sample A and it is encouraging that the ratios were similar to those obtained relative to the local PNP.

Table 11 shows direct comparison of the ratios and the %CVs for samples B, C and Z relative to PNP or sample A and there is a clear improvement to inter-laboratory agreement for all the ratios calculated when sample A was used.

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Table 3: dRVVT Screen ratios, confirm ratios, normalised ratios, % correction of ratio and mix and screen ratios calculated relative to local pooled normal plasma (PNP) for sample A

Against Laboratory PNP Lab Screen ratio Confirm

ratio NR

% correction

of ratio

Mix/Screen Screen/

confirm ratio

1 0.94 0.88 1.06 5.91 NA 1.02

2 0.94 0.94 1.00 -4.21 NA 1.02

3 0.99 0.93 1.06 5.75 NA 1.08

4 NA NA NA NA NA

5 1.02 1.04 0.98 -2.40 NA 1.04

6 1.04 0.96 1.08 7.17 NA 1.14

7 0.91 0.85 1.08 7.17 0.85 1.04

8a 0.97 0.96 1.02 1.77 NA 1.07

8b 1.00 0.98 1.02 2.09 NA 0.95

8c 1.01 0.98 1.02 2.22 NA 1.09

9 1.05 1.05 1.00 -0.19 1.01 1.06

10 0.98 0.97 1.01 0.52 0.97 1.10

11 1.09 1.03 1.06 5.66 1.05 1.09

12 1.29 1.09 1.18 15.10 NA 1.38

13 0.98 NA NA NA NA NA

14 1.02 1.02 1.00 0.24 0.99 1.08

15 0.91 0.90 1.01 1.42 - 0.97

16 0.93 0.98 0.95 -4.86 0.99 1.10

17 1.00 0.83 1.21 16.94 - 1.21

18a 1.07 1.03 1.05 4.04 1.03 0.93

18b 0.90 0.95 0.95 -4.85 0.93 1.05

19 1.02 1.02 1.03 3.23 NA 1.03

Mean 1.00 0.97 1.04 3.14 0.98 1.07

Range 0.90-1.29 0.85-1.09 0.95-1.21 -4.86-16.94 0.85-1.05 0.93-1.38

CV 8.29 6.98 6.27 184.15 6.42 9.01

NA: Not applicable

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Table 4: dRVVT Screen ratios, confirm ratios, normalised ratios, % correction of ratio and mix and screen ratios calculated relative to local pooled normal plasma (PNP) or relative to sample A for sample B

Against Laboratory PNP Against Sample A

Screen/

confirm ratio Lab Screen

ratio

Confirm

ratio NR % correction of ratio

Mix/

Screen ratio

Confirm

ratio NR

% correction

of ratio

Mix/

1 1.64 0.93 1.77 43.37 NA 1.76 1.06 1.66 39.82 NA 1.70

2 1.35 0.97 1.39 51.20 NA 1.44 1.04 1.39 72.22 NA 1.42

3 1.40 0.99 1.43 29.65 NA 1.41 1.05 1.34 25.42 NA 1.44

4 NA NA NA NA NA NA NA NA NA NA NA

5 1.49 1.07 1.39 27.86 NA 1.47 1.03 1.42 29.53 NA 1.48

6 1.82 1.01 1.81 44.77 NA 1.76 1.05 1.68 40.50 NA 1.92

7 1.31 0.90 1.45 30.88 1.05 1.43 1.06 1.34 25.53 NA 1.40

8a 1.46 0.98 1.48 32.49 NA 1.50 1.03 1.45 31.27 NA 1.55

8b 1.56 1.00 1.56 35.99 NA 1.56 1.02 1.53 34.63 NA 1.45

8c 1.59 1.02 1.55 35.60 NA 1.58 1.04 1.52 34.13 NA 1.65

9 1.47 1.11 1.33 24.58 1.28 1.40 1.06 1.33 24.70 1.22 1.41

10 1.67 1.01 1.65 39.54 1.34 1.71 1.04 1.65 39.23 1.38 1.81

11 1.63 1.08 1.51 33.88 1.31 1.49 1.05 1.43 29.91 1.20 1.56

12 1.93 1.14 1.69 40.77 NA 1.50 1.05 1.43 30.23 NA 1.98

13 1.45 NA NA NA 1.24 1.48 NA NA NA 1.26 1.51

14 1.40 1.04 1.34 25.52 1.21 1.38 1.03 1.34 25.34 1.19 1.45

15 1.35 0.95 1.43 30.13 NA 1.48 1.05 1.41 29.13 NA 1.37

16 1.50 1.03 1.47 31.77 0.81 1.61 1.05 1.54 34.93 NA 1.69

17 1.42 0.87 1.64 38.27 NA 1.41 1.05 1.35 25.78 NA 1.64

18a 1.36 1.12 1.22 17.77 1.13 1.27 1.09 1.17 14.35 NA 1.10

18b 1.40 0.98 1.43 30.15 1.25 1.55 1.04 1.50 33.38 NA 1.58

19 1.40 1.03 1.36 26.20 NA 1.38 NA NA NA NA 1.34

Mean 1.51 1.01 1.49 33.52 1.18 1.50 1.05 1.45 32.63 1.25 1.54

Range 1.31-1.93 0.87-1.14 1.22-1.81 17.77-51.20 0.81-1.34 1.27-1.76 1.02-1.09 1.17-1.68 24.70-72.22 1.19-1.38 1.10-1.98

CV 10.70 6.92 10.30 23.56 13.89 8.42 1.48 8.98 35.19 6.17 13.17

NA: Not applicable

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Table 5: dRVVT Screen ratios, confirm ratios, normalised ratios, % correction of ratio and mix and screen ratios calculated relative to local pooled normal plasma (PNP) or relative to sample A for sample C

Screen/

ratio

Confirm

ratio NR

% correction

of ratio

Mix/

Confirm

ratio NR

% correction

of ratio

Mix/

1 2.17 NA NA NA NA 2.32 NA NA NA NA NA

2 1.66 1.02 1.63 82.58 NA 1.77 1.08 1.64 115.85 NA 1.67

3 1.71 1.06 1.64 38.59 NA 1.73 1.13 1.54 34.90 NA 1.66

5 1.83 1.11 1.65 39.47 NA 1.80 1.07 1.69 40.87 NA 1.76

6 2.24 1.08 2.08 51.89 NA 2.16 1.12 1.93 48.18 NA 2.20

7 1.60 0.99 1.62 38.17 1.22 1.75 1.16 1.50 33.40 1.34 1.56

8a 1.84 1.04 1.77 43.47 NA 1.89 1.09 1.74 42.45 NA 1.85

8b 1.98 1.07 1.85 46.02 NA 1.98 1.09 1.81 44.87 NA 1.72

8c 1.99 1.09 1.82 45.12 NA 1.97 1.11 1.78 43.87 NA 1.93

9 1.67 1.16 1.44 30.48 1.44 1.60 1.11 1.44 30.61 1.38 1.53

10 2.08 1.06 1.95 48.73 1.64 2.12 1.09 1.94 48.47 1.69 2.13

11 2.07 1.15 1.79 44.26 1.59 1.90 1.12 1.69 40.92 1.46 1.85

12 2.31 1.20 1.92 47.88 NA 1.80 1.10 1.63 38.62 NA 2.25

13 1.77 NA NA NA 1.46 1.81 NA NA NA 1.49 1.77

14 1.73 1.09 1.58 36.71 1.45 1.69 1.07 1.58 36.56 1.42 1.70

15 1.71 1.00 1.71 41.53 NA 1.87 1.11 1.69 40.69 NA 1.64

16 1.90 1.08 1.76 43.13 0.78 2.03 1.10 1.84 45.76 1.59 2.02

17 1.75 0.94 1.87 45.98 NA 1.74 1.13 1.54 35.03 NA 1.87

18a 1.51 1.21 1.25 19.18 1.05 1.41 1.18 1.19 15.86 1.22 1.13

18b 1.77 1.03 1.72 41.81 1.54 1.97 1.09 1.80 44.50 1.70 1.90

19 1.72 1.06 1.63 38.47 NA 1.69 NA NA NA NA 1.60

Mean 1.86 1.08 1.72 43.34 1.35 1.86 1.11 1.67 43.41 1.48 1.79

Range 1.51-2.31 0.94-1.21 1.25-2.08 19.18-82.58 0.78-1.64 1.41-2.32 1.07-.18 1.19-1.94 15.86-115.85 1.22-1.70 1.13-2.25

CV 11.68 6.53 10.99 27.55 20.83 10.98 2.63 11.11 45.25 10.99 14.44

NA: Not applicable

(13)

Table 6: dRVVT Screen ratios, confirm ratios, normalised ratios, % correction of ratio and mix and screen ratios calculated relative to local pooled normal plasma (PNP) or relative to sample A for sample Z

Screen/

ratio

Confirm

ratio NR

% correction

of ratio

Mix/

Confirm

ratio NR

% correction

of ratio

Mix/

1 1.29 0.99 1.31 23.85 NA 1.38 1.12 1.24 19.07 NA 1.26

2 1.24 0.97 1.28 37.62 NA 1.32 1.04 1.28 54.17 NA 1.31

3 1.46 0.93 1.57 36.08 NA 1.47 1.00 1.47 32.19 NA 1.59

5 1.37 1.06 1.30 23.07 NA 1.35 1.02 1.33 24.86 NA 1.39

6 1.51 0.95 1.59 37.07 NA 1.46 0.99 1.48 32.20 NA 1.68

7 1.16 0.86 1.35 25.83 1.03 1.27 1.01 1.25 20.10 1.13 1.30

8a 1.39 0.97 1.43 30.03 NA 1.43 1.02 1.40 28.76 NA 1.50

8b 1.45 1.00 1.46 31.45 NA 1.45 1.01 1.43 29.99 NA 1.35

8c 1.47 1.00 1.47 32.03 NA 1.46 1.02 1.44 30.49 NA 1.56

9 1.28 1.11 1.15 13.35 1.14 1.23 1.06 1.16 13.51 1.09 1.23

10 1.42 0.98 1.45 30.74 1.20 1.46 1.01 1.44 30.39 1.23 1.58

11 1.57 1.02 1.53 34.84 1.23 1.44 1.00 1.45 30.93 1.13 1.58

12 1.79 1.17 1.52 34.41 NA 1.39 1.07 1.29 22.72 NA 1.79

13 1.30 NA NA NA 1.14 1.33 NA NA NA 1.17 1.35

14 1.35 1.00 1.35 25.79 1.17 1.32 0.98 1.34 25.61 1.15 1.45

15 1.10 0.95 1.16 13.72 NA 1.21 1.06 1.14 12.47 NA 1.11

16 1.23 1.04 1.18 15.39 0.91 1.31 1.06 1.24 19.31 1.20 1.36

17 1.40 0.84 1.70 39.41 NA 1.40 1.01 1.39 27.53 NA 1.70

18a 1.24 1.09 1.14 11.46 0.85 1.15 1.06 1.09 7.82 1.05 1.02

18b 1.29 0.94 1.38 27.70 1.14 1.44 0.99 1.45 31.05 1.27 1.53

19 1.27 1.00 1.26 20.52 NA 1.24 NA NA NA NA 1.25

Mean 1.36 0.99 1.38 27.22 1.09 1.36 1.03 1.33 25.96 1.16 1.42

Range 1.10-1.79 0.93-1.17 1.14-1.70 11.46-39.41 0.85-1.23 1.15-1.47 0.98-1.12 1.09-1.48 7.82-54.17 1.05-1.27 1.02-1.79

CV 11.27 7.85 11.56 32.03 12.05 7.06 3.49 9.10 38.47 5.89 13.93

NA: Not applicable

(14)

Table 7: %CV for screen, confirm and normalised ratios for sample A Against Laboratory PNP

Screen ratio Confirm ratio NR

Lab Mean %CV Mean %CV Mean %CV

1 0.94 0.84 0.88 1.37 1.06 0.58

2 0.94 0.01 0.94 0.01 1.00 0.01

3 0.99 6.45 0.93 10.98 1.06 5.38

4 NA NA NA NA NA NA

5 1.02 1.61 1.04 1.72 0.98 2.93

6 1.04 0.74 0.96 1.44 1.08 2.10

7 0.91 3.47 0.85 1.80 1.08 2.01

8a 1.97 1.11 0.96 0.52 1.02 1.17

8b 1.00 0.27 0.98 0.52 1.02 0.74

8c 1.01 0.82 0.98 1.43 1.02 0.71

9 1.05 0.97 1.05 1.78 1.00 1.65

10 0.98 3.39 0.97 1.24 1.01 2.15

11 1.09 1.14 1.03 1.57 1.06 0.46

12 1.29 1.48 1.09 0.23 1.18 1.26

13 0.98 1.07 NA NA NA NA

14 1.02 0.64 1.02 0.31 1.00 0.43

15 0.91 1.71 0.90 1.52 1.01 0.19

16 0.93 3.16 0.98 2.73 0.95 0.71

17 1.00 3.90 0.83 11.85 1.21 11.79

18a 1.07 5.91 1.03 0.93 1.05 6.77

18b 0.90 1.66 0.95 1.39 0.95 0.51

19 1.02 5.24 1.02* NA 1.03* NA

Range 0.90-1.29 0.01-6.45 0.83-1.09 0.01-11.85 0.95-1.21 0.01-11.79

*Result from one day only; NA: Not applicable

(15)

Table 8: %CV for screen, confirm and normalised ratios for sample B

1 1.64 1.06 0.93 0.73 1.77 0.36

2 1.35 0.002 0.97 0.01 1.39 0.01

3 1.40 6.66 0.99 12.58 1.43 7.91

4 NA NA NA NA NA NA

5 1.49 1.92 1.07 0.74 1.39 1.72

6 1.82 1.23 1.01 1.41 1.81 2.46

7 1.31 2.85 0.90 0.89 1.45 1.96

8a 1.46 0.81 0.98 0.73 1.48 0.21

8b 1.56 1.10 1.00 2.00 1.56 1.33

8c 1.59 1.03 1.02 0.60 1.55 0.43

9 1.47 1.92 1.11 0.68 1.33 2.27

10 1.67 4.02 1.01 1.19 1.65 2.84

11 1.63 0.53 1.08 0.67 1.51 0.93

12 1.93 0.70 1.14 1.23 1.69 1.80

13 1.45 1.73 NA NA NA NA

14 1.40 2.58 1.05 0.99 1.34 3.58

15 1.35 0.94 0.95 1.16 1.43 1.00

16 1.50 4.45 1.03 3.88 1.47 3.08

17 1.42 6.31 0.87 11.68 1.64 13.88

18a 1.36 8.61 1.12 0.64 1.22 9.14

18b 1.40 1.38 0.98 1.72 1.43 0.97

19 1.40 0.91 1.03 0.99 1.36 1.75

Range 1.31-1.93 0.002-8.61 0.90-1.14 0.01-12.58 1.22-1.81 0.01-9.14 NA: Not applicable

(16)

Table 9: %CV for screen, confirm and normalised ratios for sample C

1 2.17 2.21 NA NA NA NA

2 1.66 0.003 1.02 0.02 1.63 0.02

3 1.71 5.91 1.06 13.42 1.64 8.45

4 NA NA NA NA NA NA

5 1.83 2.06 1.11 1.00 1.65 2.52

6 2.24 1.52 1.08 1.58 2.08 2.64

7 1.60 5.06 0.99 1.61 1.62 3.70

8a 1.84 1.54 1.04 0.82 1.77 0.90

8b 1.98 1.26 1.07 1.21 1.85 0.06

8c 1.99 1.07 1.09 0.40 1.82 1.40

9 1.67 1.79 1.16 1.30 1.44 2.52

10 2.08 4.17 1.06 1.21 1.95 3.54

11 2.07 1.59 1.15 1.35 1.79 2.13

12 2.31 1.45 1.20 1.45 1.92 1.85

13 1.77 1.55 NA NA NA NA

14 1.73 2.77 1.09 1.24 1.58 3.37

15 1.71 0.50 1.00 0.88 1.71 0.62

16 1.90 3.27 1.08 2.16 1.76 1.24

17 1.75 5.39 0.94 13.54 1.87 13.74

18a 1.51 11.05 1.21 0.95 1.25 11.41

18b 1.77 1.73 1.03 1.50 1.72 0.72

19 1.72 0.91 1.06 0.20 1.63 0.75

Range 1.51-2.24 0.003-

11.05 0.94-1.20 0.02-13.54 1.25-2.08 0.02-11.41 NA: Not applicable

(17)

Table 10: %CV for screen, confirm and normalised ratios for sample Z

1 1.29 1.41 0.99 0.80 1.31 1.46

2 1.24 0.0003 0.97 0.01 1.28 0.01

3 1.46 7.52 1.03 12.95 1.57 5.95

4 NA NA NA NA NA NA

5 1.37 1.56 1.06 0.49 1.30 1.99

6 1.51 1.04 0.95 1.25 1.59 0.50

7 1.16 3.85 0.86 1.50 1.35 2.36

8a 1.39 1.81 0.97 1.77 1.43 2.00

8b 1.45 1.16 1.00 0.50 1.46 1.49

8c 1.47 2.56 1.00 0.16 1.47 2.71

9 1.28 2.26 1.11 0.90 1.15 2.39

10 1.42 3.04 0.98 2.88 1.45 4.81

11 1.57 0.36 1.03 0.57 1.53 0.89

12 1.79 1.00 1.17 2.62 1.52 1.86

13 1.30 0.92 NA NA NA NA

14 1.35 1.72 1.00 0.64 1.35 1.30

15 1.10 0.54 0.95 0.70 1.16 0.44

16 1.23 1.78 1.04 1.93 1.18 0.38

17 1.40 11.12 0.84 9.40 1.70 21.53

18a 1.24 8.44 1.09 1.34 1.14 9.74

18b 1.29 4.30 0.94 1.68 1.38 2.70

19 1.27 2.38 1.00* 1.59* 1.26* 6.73*

Range 1.10-1.79 0.0003-

11.12 0.84-1.17 0.01-12.95 1.14-1.59 0.01-21.53

*Results from 2 days only; NA: Not applicable

(18)

Table 11: Comparison of dRVVT ratios against local pooled normal plasma (PNP) and Sample A: Mean and inter-laboratory %CV with p values obtained by two tailed paired t-test

Sample B

Screen ratio Confirm ratio NR % correction of ratio Mix/Screen

Vs PNP Vs A Vs PNP Vs A Vs PNP Vs A Vs PNP Vs A Vs PNP Vs A

mean 1.51 1.50 1.01 1.05 1.49 1.45 33.52 32.63 1.18 1.25

range 1.31-1.93 1.27-1.76 0.87-1.14 1.02-1.09 1.22-1.81 1.17-1.68 17.77- 51.20

24.70-

72.22 0.81-1.34 1.19-1.38

%CV 10.70 8.42 6.92 1.48 10.30 8.98 23.56 35.19 13.89 6.17

Sample C

mean 1.86 1.86 1.08 1.11 1.72 1.67 43.34 43.41 1.35 1.49

range 1.51-2.31 1.41-2.32 0.94-1.21 1.07-1.18 1.25-2.08 1.19-1.94 19.18- 82.58

15.86-

115.85 0.78-1.64 1.22-1.70

%CV 11.68 10.98 6.53 2.63 10.99 11.11 27.55 45.25 20.83 8.06

Sample Z

mean 1.36 1.36 0.99 1.03 1.38 1.33 27.22 25.96 1.09 1.15

Range 1.10-1.79 1.15-1.47 0.93-1.17 0.98-1.12 1.14-1.70 1.09-1.48 11.46-

39.41 7.82-54.17 0.85-1.23 1.05-1.27

%CV 11.27 7.06 7.85 3.49 11.56 9.10 32.03 38.47 12.05 4.45

(19)

APTT

APTT is recommended by the BCSH guidelines as a screening test, with mixing tests

recommended if APTT clotting time is prolonged. If the samples were clotting factor deficient rather than positive for lupus anticoagulant, mixing with normal pooled plasma should correct the prolonged clotting time. The revised ISTH guidelines also recommend a sensitive APTT (low phospholipids and silica as activator) as one of the two tests that should be carried out, the first choice being dRVVT. Sixteen sets of data were analysed for screen ratios. Ten laboratories returned results for mixing studies, with 2 labs returning data for mixing studies only. Table 8 shows the individual laboratories’ mean screen ratio and mix ratio for sample A. The overall laboratories’ mean screen ratio was found to be 1.08 (range 0.95 – 1.19) indicating that sample A is lupus negative. Tables 9, 10 and 11 give summary of individual laboratory APTT results for samples B, C and Z relative to PNP and sample A (where possible). The overall mean screen ratios for samples B, C and Z are 1.40 (range 1.01 – 1.92), 1.68 (range 1.07 – 2.52) and 1.27 (range 1.06 – 1.56) respectively. For samples B and C, the prolongation of clotting times were not corrected in the mixing studies confirming that these samples are lupus positive rather than factor deficient. For sample Z, the mixing studies results suggest correction of some of the screen clotting times, however, this could be due to the “over” dilution of a very weak lupus anticoagulant. With the exception of Labs 8a2, 8b2, 8c2 and 15a who obtained screen ratio results that suggest sample B was weaker than sample Z, the individual laboratory mean and overall mean screen ratio showed the same order of lupus positivity as those found with dRVVT:

C>B>Z. The differences in the order of positivity were probably due to the APTT reagents used since 8a1, 8b1, 8c1 obtained the correct order of lupus positivity and they have used the same instruments as Lab 8a2, 8b2, 8c2. The intra-laboratory variation for APTT was similar to that found for dRVVT, with %CV ranges of 0.08-5.70, 0.18-9.96, 0.02-5.75, 0.02-5.75 for samples A, B, C, Z, respectively (see Appendix 3).

The screen and mix ratios relative to sample A are also shown in Tables 9, 10 and 11 and a summary of comparison of ratios against PNP and sample A, together with inter-

laboratory %CV is given in Table 12. In general, both screen and mix ratios were lower relative to sample A than against local PNP. With the exception of screen ratio for Sample C, the

agreement between laboratories was improved when the ratios were calculated relative to sample A.

Table 8: Summary of laboratory APTT results for sample A Against Laboratory PNP

Lab Screen ratio Mix/Screen ratio

1 1.12 1.02

5 1.04 1.04

6 1.16 NA

7 1.19 1.09

8a1 1.09 NA

8a2 1.09 NA

8b1 1.12 NA

8b2 1.11 NA

8c1 1.12 NA

8c2 1.11 NA

9 1.03 1.00

10 0.97 0.89

11a 1.16 1.06

(20)

15a 0.95 NA

15b 1.02 NA

18 1.00 0.97

19a NA 1.07

19b NA 1.08

Mean 1.08 1.02

Range 0.95-1.19 0.89-1.09

%CV 6.53 6.44

NA: Not applicable

Table 9: Summary of laboratory APTT results for sample B

Against Laboratory PNP Against Sample A

Lab Screen ratio Mix/Screen ratio Screen ratio Mix/Screen ratio

1 1.92 1.55 1.71 1.39

5 1.46 1.10 1.40 1.06

6 1.63 1.71 1.41 1.46

7 1.64 1.30 1.38 1.09

8a1 1.36 NA 1.24 NA

8a2 1.21 NA 1.12 NA

8b1 1.33 NA 1.19 NA

8b2 1.24 NA 1.11 NA

8c1 1.35 NA 1.20 NA

8c2 1.24 NA 1.11 NA

9 1.56 1.11 1.51 1.08

10 1.26 1.25 1.30 1.29

11a 1.68 1.47 1.46 1.27

15 a 1.01 NA 1.07 NA

15b 1.25 NA 1.22 NA

18 1.20 1.09 1.20 1.09

19a NA 1.10 NA NA

19b NA 1.49 NA NA

Mean 1.40 1.30 1.29 1.22

Range 1.01-1.92 1.09-1.71 1.07-1.71 1.06-1.39

%CV 16.70 17.72 13.66 13.10

NA: Not applicable

(21)

Table 10: Summary of laboratory APTT results for sample C

1 2.52 1.72 2.26 1.54

5 1.70 1.32 1.63 1.27

6 2.10 1.71 1.81 1.46

7 2.16 1.54 1.81 1.29

8a1 1.62 NA 1.49 NA

8a2 1.33 NA 1.22 NA

8b1 1.51 NA 1.35 NA

8b2 1.37 NA 1.23 NA

8c1 1.55 NA 1.37 NA

8c2 1.38 NA 1.24 NA

9 1.89 1.55 1.83 1.49

10 1.58 1.24 1.63 1.28

11a 2.10 1.61 1.81 1.40

15 a 1.07 NA 1.13 NA

15b 1.55 NA 1.52 NA

18 1.40 1.20 1.40 1.20

19a NA 1.12 NA NA

19b NA 1.63 NA NA

Mean 1.68 1.44 1.55 1.37

Range 1.07-2.52 1.12-1.72 1.13-2.26 1.20-1.54

%CV 22.65 15.78 19.58 9.04

NA: Not applicable

(22)

Table 11: Summary of laboratory APTT results for sample Z

1 1.33 1.08 1.19 0.96

5 1.25 1.02 1.20 0.98

6 1.56 1.31 1.35 1.12

7 1.28 1.14 1.08 0.95

8a1 1.22 NA 1.12 NA

8a2 1.28 NA 1.18 NA

8b1 1.19 NA 1.07 NA

8b2 1.34 NA 1.21 NA

8c1 1.21 NA 1.08 NA

8c2 1.35 NA 1.21 NA

9 1.23 1.07 1.19 1.03

10 1.20 1.01 1.24 1.04

11a 1.48 1.23 1.28 1.07

15 a 1.06 NA 1.12 NA

15b 1.24 NA 1.21 NA

18 1.12 1.07 1.12 1.07

19a NA 1.11 NA NA

19b NA 1.24 NA NA

Mean 1.27 1.11 1.18 1.03

Range 1.06-1.56 1.01-1.31 1.08-1.35 0.95-1.12

%CV 9.64 8.97 6.54 5.70

NA: Not applicable

(23)

Table 2: Comparison of APTT ratios against local pooled normal plasma (PNP) and Sample A:

Mean and inter-laboratory %CV test

Sample B

Screen ratio Confirm ratio

Vs PNP Vs A Vs PNP Vs A

mean 1.40 1.29 1.30 1.22

range 1.01-1.92 1.07-1.71 1.09-1.71 1.06-1.39

%CV 16.70 13.66 17.72 13.10

Sample C

mean 1.68 1.55 1.44 1.55

range 1.07-2.52 1.13-2.26 1.12-1.72 1.13-2.26

%CV 22.65 19.58 15.78 19.58

Sample Z

mean 1.27 1.18 1.11 1.03

range 1.06-1.56 1.08-1.35 1.01-1.31 0.95-1.12

%CV 9.64 6.54 8.97 5.70

SCT

Four sets of data were analysed for SCT. Tables 13, 14, 15 and 16 show SCT results for samples A, B, C and Z. The screen and normalised ratio indicated that sample A is lupus negative with mean screen ratio of 0.97 (range 0.89 – 1.03) and mean normalised ratio of 0.93 (range 0.89 - 0.98). The screen, confirm and normalised ratios for samples B, C and Z relative to PNP indicate these samples are all lupus positive with mean screen ratio of 1.82 (range 1.58 – 2.03), 2.31 (range 2.02 – 2.59) and 1.57 (range 1.45 – 1.70) for samples B, C and Z respectively.

The >30% correction of ratio and that incomplete neutralisation of activity in mixing study also support the lupus positivity identity of these materials. Overall, when the ratios were calculated relative to sample A, the inter-laboratory agreement as expressed by %CV was improved. The ranking order of positivity was the same as those found for dRVVT and APTT: C>B>Z.

(24)

Table 13: SCT results for sample A

Against Laboratory PNP

Screen/

Confirm ratio Lab Screen

ratio

Confirm

ratio NR

% correction

of ratio

Mix ratio

2 0.89 0.91 0.98 -1.79 NA 0.95

5 1.03 1.16 0.89 -12.35 NA 0.85

13 1.00 NA NA NA NA NA

14 0.94 1.01 0.92 -8.21 0.95 0.89

Mean 0.97 1.03 0.93 -10.27 0.95 0.90

Range 0.89-1.03 0.91-1.16 0.89-0.98 -8.21-12.35 NA 0.85-0.95

%CV 6.49 12.22 5.04 -20.18 NA 5.76

NA: not applicable

(25)

Table 14: SCT results for sample B

Screen/

ratio

Confirm

ratio NR

%

correction of ratio

Mix ratio

Screen ratio

Confirm

ratio NR % correction

of ratio

Mix ratio

2 1.58 0.93 1.71 41.32 NA 1.80 1.05 1.72 41.92 NA 1.60

5 2.03 1.23 1.66 39.54 NA 1.97 1.06 1.86 46.19 NA 1.58

13 1.95 NA NA NA 1.52 1.95 NA NA NA 1.52 1.90

14 1.70 1.06 1.61 37.81 1.52 1.82 1.04 1.74 42.53 1.62 1.55

Mean 1.82 1.07 1.66 39.56 1.52 1.52 1.05 1.77 43.55 1.57 1.66

Range 1.58-2.03 0.93-1.23 1.6-1.71 37.81-41.32 NA 1.80-1.97 1.04-1.06 1.74-1.86 41.92-46.19 NA 1.55-1.90

%CV 11.56 14.01 3.01 4.44 NA 4.64 0.83 4.19 5.31 NA 9.69

NA: not applicable

Table 15: SCT results for sample C

Screen/Confir m ratio Lab Screen

ratio

Confirm

of ratio Mix ratio Screen ratio

Confirm ratio NR

%

correction of ratio

Mix ratio

2 2.02 1.00 2.02 50.54 NA 2.30 1.12 2.04 51.05 NA 1.90

5 2.59 1.29 2.01 50.30 NA 2.51 1.11 2.26 55.78 NA 1.93

13 2.43 NA NA NA 1.81 2.42 NA NA NA 1.81 2.21

14 2.20 1.15 1.92 47.41 1.84 2.35 1.14 2.07 51.43 1.97 1.84

Mean 2.31 1.15 1.98 49.42 1.83 2.40 1.12 2.13 52.75 1.89 1.97

Range 2.02- 2.59

1.00-

1.29 1.92-2.02 47.41-50.54 NA 2.30- 2.51

1.11- 1.14

2.04-

2.26 51.05-55.78 NA 1.84-2.21

%CV 10.88 12.58 3.02 3.52 NA 3.90 1.21 5.61 4.98 NA 8.31

NA: not applicable

(26)

Table 16: SCT results for sample Z

Against Laboratory PNP Against Sample A Screen/

ratio

Confirm

of ratio

Mix ratio

Screen ratio

Confirm

of ratio Mix ratio

2 1.45 0.96 1.50 33.41 NA 1.65 1.09 1.52 34.08 NA 1.41

5 1.70 1.24 1.37 26.78 NA 1.65 1.07 1.53 34.84 NA 1.31

13 1.64 NA NA NA 1.23 1.64 NA NA NA 1.23 1.59

14 1.49 1.08 1.39 27.84 1.23 1.59 1.06 1.50 33.32 1.32 1.33

Mean 1.57 1.09 1.42 44.23 1.23 1.63 1.07 1.52 55.63 1.27 1.41

Range 1.45-1.70 0.96-1.24 1.37-1.50 26.78-78.06 NA 1.59-1.65 1.06-1.09 1.5-1.53 33.32-98.75 NA 1.31-1.59

%CV 7.59 12.80 5.21 66.26 NA 1.60 1.11 1.16 67.12 NA 8.94

NA: not applicable

(27)

dPT

Two laboratories carried out dPT and the results for samples A, B, C and Z relative to PNP or sample A are shown in Tables 17, 18, 19 and 20 respectively. Although inter-laboratory variation cannot be assessed, the ratios calculated for each laboratory were in close agreement and indicate sample A is lupus negative with mean screen ratio of 0.98, sample B and C are lupus positive with respective mean screen ratios of 1.24 and 1.70. However, the mean screen ratio for sample Z was 0.97 indicating that the weak Lupus Anticoagulant in sample Z is not detected by dPT. The ranking order of positivity was C>B>Z.

Table 17: dPT results for sample A relative to PNP Against Laboratory PNP

Screen/Confirm ratio Lab Screen

ratio

Confirm

ratio NR

% correction

of ratio

Mix/Screen ratio

16 1.01 0.97 1.04 3.52 0.97 0.94

18 0.96 0.88 1.11 7.88 NA 0.94

Mean 0.98 0.93 1.07 5.70 NA 0.94

NA: not applicable

(28)

Table 18: dPT results for sample B relative to PNP or sample A

ratio

Confirm

ratio NR

% correction

of ratio

Mix/Screen ratio

Screen ratio

Confirm

ratio NR

% correction

of ratio

Mix/Screen ratio

16 1.26 1.10 1.15 12.99 0.85 1.25 1.13 1.11 9.86 NA 1.05

18 1.22 1.07 1.16 11.38 1.04 1.27 1.22 1.04 4.05 1.08 0.98

Mean 1.24 1.09 1.15 12.18 0.94 1.26 1.17 1.08 6.96 NA 1.02

Table 19: dPT results for sample C relative to PNP or sample A

ratio

Confirm

ratio NR

% correction

of ratio

Mix/Screen ratio

Screen ratio

Confirm

ratio NR

% correction

of ratio

Mix/Screen ratio

16 1.74 1.20 1.44 30.10 0.72 1.72 1.24 1.39 27.64 NA 1.31

18 1.66 1.19 1.42 28.22 1.15 1.73 1.35 1.28 22.03 1.20 1.21

Mean 1.70 1.20 1.43 29.16 0.93 1.72 1.29 1.33 24.84 NA 1.26

Table 20: dPT results for sample Z relative to PNP or sample A

ratio

Confirm

ratio NR

% correction

of ratio

Mix/Screen ratio

Screen ratio

Confirm

ratio NR

% correction

of ratio

Mix/Screen ratio

16 1.02 0.96 1.06 5.24 0.97 1.01 0.99 1.02 1.79 NA 0.96

18 0.93 0.82 1.16 11.94 NA 0.97 0.93 1.05 4.40 NA 0.99

Mean 0.97 0.89 1.11 8.59 NA 0.99 0.96 1.03 3.09 NA 0.97

NA: not applicable

(29)

KCT, ASLA, TSVT

Only one set of data was submitted for KCT, ASLA and TSVT and the summaries of calculated ratios are shown in Tables 21, 22 and 23 for KCT, ASLA and TSVT respectively. Both NIBSC and local calculated ratios from all three test methods found sample A to be lupus negative, while samples B and C were lupus positive. KCT and ASLA found sample Z lupus positive, while it was negative/ borderline by TSVT.

aCL

Lab 10 returned ELISA results for anticardiolipin antibodies measurement. As shown in Table 24, Sample A was found to contain low concentrations of both IgG and IgM, while substantially higher concentrations of IgG were detected in samples B, C and Z. Interestingly, lower amount of IgM by comparison with IgG were found in these samples. The IgG results confirm that samples B, C and Z contain anticardiolipin antibodies and fit well with ranking order of these samples by clot-based assays.

(30)

Table 21: Summary of calculated ratios for KCT

Sample

Screen Ratio

Intra- lab %

CV

Mix Ratio

Intra- lab %

CV

Rosner Ratio

Intra- lab %

CV

Screen Ratio

Intra- lab %

CV

Mix Ratio

Intra- lab %

CV

Roser Ratio

Intra- lab %

CV

A 1.11 1.92 1.00 1.66 0.00 627.67 NA NA NA NA NA NA

B 2.27 1.02 1.81 0.28 0.36 0.73 2.05 2.89 1.82 1.80 0.31 2.68

C 2.85 1.37 2.28 1.35 0.45 3.60 2.58 2.75 2.29 0.58 0.41 1.98

Z 2.03 1.10 1.53 1.37 0.27 2.63 1.84 1.31 1.54 0.58 0.21 2.60

Local

positive 4.31 5.62 2.95 1.33 0.46 5.20 3.90 5.91 2.97 2.64 0.43 5.39

Rosner Ratio= (KCT-mix - KCT-PNP) / KCT-sample. Cutoff is 0.16 NA: Not applicable