Presented at the XX Congress of The International Society on Thrombosis and
Haemostasis, August 6-12, 2005, Sydney, Australia
Characterization and Performance of the HemosIL™ Silica
Clotting Time (SCT) Test for Detection of Lupus Anticoagulants (LA).
R. Bottenus, C. Cueto, M. Triscott
Instrumentation Laboratory, Orangeburg, NY, USA.
Introduction
The Silica Clotting Time (SCT) is an APTT-based assay
first described by Chantarangkul, et.al., Thrombosis
Research 67: 355-365, 1992. The current version
utilizes synthetic phospholipid for the cephalin
component of the reagents, instead of the original
bovine cephalin. The kit contains a low concentration
phospholipid screening reagent and a high concentration
phospholipid confirmatory reagent. A calcium chloride
activator is also provided that contains polybrene to
minimize heparin interference. It is recommended that
the results of the screening and confirmatory assays are
calculated using a normalized ratio, to help increase the
specificity of the assay. These studies evaluated the
performance of the HemosIL SCT assay, relative to the
HemosIL LAC DRVVT assay.
Materials
• HemosIL Silica Clotting Time (SCT) kit contains vials
of three liquid reagents:
- SCT Screen Reagent
- SCT Confirm Reagent
- SCT Calcium Chloride
• HemosIL LAC Screen and LAC Confirm kits
• HemosIL Normal Calibrator
• Staclot LA (Diagnostica Stago)
Methods and Results
SCT Ratio - Procedures and Calculations
The following procedure should be used to calculate the
SCT Screen and SCT Confirm ratios:
1. For each new lot of SCT Screen and SCT Confirm
kit, determine new Normal Range according to NCCLS
Document H21-A4, Vol. 23, No. 35.
2. Determine the Mean of each Normal Range in
seconds.
3. Use the mean of each normal range as a constant
denominator in the calculations of ratios.
SCT Screen Ratio = Patient Screen results (in seconds)
Mean of Screen Normal Range
(in seconds)
SCT Confirm Ratio = Patient Confirm results (in seconds)
Mean of Confirm Normal Range
(in seconds)
Normalized SCT Ratio = Screen Ratio
Confirm Ratio
Interpretation
The final result should be expressed as the Normalized
SCT Ratio.
If ratio is > 1.24 (ACL Futura/ACL Advance) or > 1.20
(ACL 8000/9000/10000), then LA is present
If ratio ≤ 1.24 (ACL Futura/ACL Advance) or ≤ 1.20
(ACL 8000/9000/10000) and SCT Screen and SCT
Confirm clotting times are prolonged, then mixing
studies should be performed to investigate factor
deficiencies or inhibitors. If the mixing test is still
prolonged, some anticoagulant other than LA may be
present in the test plasma.
Sample Type
SCT
Screen
SCT
Confirm
LAC
Screen
LAC
Confirm
Normalized Ratio
SCT
LAC
Sec
Sec
Sec
Sec
Normal
35.9
33.4
31.6
28.2
1.06
0.98
OAT
64.1
63.2
67.3
52.1
1.03
1.12
DIC
33.4
40.9
34.9
35.7
0.83
0.85
UFH
32.7
34.8
35.2
35
0.95
0.87
LMWH
32.6
28.6
30.5
26.5
1.16
1.00
Factor VIII Def.
46.9
49.1
28.1
28.2
0.97
0.86
Factor IX Def.
73.6
88.1
37.9
31.7
0.85
1.04
Factor XI Def.
54.5
56.3
39.9
36.6
0.98
0.94
Factor XII Def.
43.6
49.1
45.4
39.5
0.9
1.00
LA-ACA
101.3
38.8
100.2
39.1
2.65
2.22
LA-APA
84.2
50.1
124.6
44.3
1.7
2.44
A-ß2GPI
57.6
37.3
55.6
29.6
1.57
1.63
Table 2
Normal Range Study
One hundred twenty fresh, citrated plasma samples (60
female and 60 male) were obtained from apparently
healthy donors and tested on an ACL Advance using a
single lot of HemosIL Silica Clotting Time (SCT Screen
and SCT Confirm) reagents. The distribution of the
normalized ratios is parametric as shown in the
frequency distribution plot below.
Abnormal Samples
OAT
DIC
LA-APA
A-ß2GPI
LA-ACA
UFH
LMWH
Factor VIII Def.
Factor IX Def.
Factor XI Def.
Factor XII Def.
Quanity
20
10
10
10
10
7
7
4
4
4
4
Normalized
Ratios
Method Comparison
N
Normal Range Study
Figure 1
The parametric reference interval (normal range) was
calculated according to the guidelines established by
NCCLS C28-A2, i.e. 95% reference interval with 90%
confidence intervals (2 SD) around the reference limits.
These data support the normal range for the normalized
ratio in the HemosIL Silica Clotting Time product insert
of the ACL Advance, n=120, Normalized SCT Ratio:
0.70 to 1.24.
Due to many variables that may affect results, each
laboratory should establish its own normal range.
Method Comparison Using SCT and LAC Reagents
with Patient Samples
A comparative performance study was conducted using
210 patient citrated plasma samples (120 normals and
90 abnormals) analyzed in singlet with HemosIL Silica
Clotting Time (SCT Screen and SCT Confirm) versus
the predicate HemosIL LAC Screen/LAC Confirm on
an ACL Advance.
SCT Normalized Ratio
4.00
3.00
y = 1.0989x - 0.086
r = 0.8738
n = 210
2.00
1.00
0.00
0.00
1.00
2.00
3.00
4.00
LAC Normalized Ratio
Figure 2
Sensitivity and Specificity
The sensitivity and specificity of HemosIL Silica
Clotting Time (SCT Screen/SCT Confirm) as compared
directly to the predicate HemosIL LAC Screen/LAC
Confirm were also calculated from this study:
Table 3
LAC Screen/Confirm
Normalized Ratio
SCT Screen/Confirm
Normalized Ratio
• Relative Sensitivity (31/33)
+
-
+
31
3
-
2
174
= 93.9%
(95% C.I. = 77.0 - 96.9)
• Relative Specificity (174/177) = 98.3%
(95% C.I. = 95.9 - 98.7)
• Overall Agreement (205/210) = 97.6%
Final Interpretation Calculation
Mixing studies and an additional qualitative test, Staclot
LA from Diagnostica Stago was used to resolve the five
discrepant results. The table below shows the final
interpretation of results.
other interferences. The ease of use of SCT on
automated instruments for screening and confirmation
of Lupus inhibitors makes this test a convenient
alternative for LA Screening.
Table 4
Final Interpretation
SCT Screen/Confirm
Normalized Ratio
+
-
+
31
3
-
0
176
• Relative Sensitivity (31/31)
= 100%
(95% C.I. = 89.0 - 100.0)
• Relative Specificity (176/179) = 98.3% (95% C.I. = 95.9 - 98.7)
• Overall Agreement (207/210)
= 98.6%
Table 5
Sample Type
SCT
LAC
Staclot
Mixing Study
Final
Cut-off > 1.24 Cut-off > 1.2 Cut-off > 8 sec N. Ratio > 1.24 Interpretation
Normal
1.34(+)
1.00(-)
2.6(-)
UF Heparin
OAT
Factor VIII
Factor XII
1.13(-)
Negative
1.28(+)
1.12(-)
1.01(-)
1.22(+)
4.5(-)
1.10(-)
Negative
2.5(-)
Not Done
Negative
1.44(+)
0.82(-)
0.98(-)
-1.4(-)
1.02(-)
Negative
1.21(+)
2.4(-)
1.04(-)
Negative
Conclusions
The HemosIL Silica Clotting Time assay is a convenient
automated test for detection of LA, that runs on ACL
instrument platforms. The SCT assay can be used with a
second screening test (e.g. DRVVT- based) to satisfy
current guidelines on the use of at least two different
LA assay principles for detection of LA.
The sensitivity and specificity of the SCT assay is
comparable to the DRVVT-based HemosIL LAC assay.
Analysis of 210 patient citrated plasma samples (120
normals and 90 abnormals) with SCT and LAC assays
gave a relative Sensitivity of 93.9% (versus LAC), a
relative Specificity of 98.3% and an overall agreement
of 97.6%. Both tests identified all the Lupus-positive
samples correctly. Five samples were found to be
discrepant between the two assays, but upon follow-up
mixing studies, the samples were correctly identified as
negative samples.
Using a normalized ratio to calculate the Screen and
Confirm assay results allows the SCT assay to be
insensitive to OAT patients, heparin patients, as well
SCTBOT.PS 08/05