Coagulation and Transfusion Medicine / A “PERCENT CORRECTION” FORMULA FOR EVALUATION OF MIXING STUDIES
A “Percent Correction” Formula for Evaluation
of Mixing Studies
Sheng-hsiung Chang, MD, Veronica Tillema, MT(ASCP), and Doris Scherr, MT(ASCP)
Key Words: Prothrombin time; Activated partial thromboplastin time; Patient plasma; Citrated normal plasma; Mixing studies
Abstract
The study examined a “percent correction” formula
for evaluating mixing study results comparing a 1:1 mix
with a new 4:1 mix of patient plasma with citrated
normal plasma for a prolonged activated partial
thromboplastin time (aPTT) and/or prothrombin time
(PT). The study also examined 3 suggested definitions
of correction for evaluating mixing study results for
comparison. Applicability of percent correction for
evaluating the aPTT 4:1 mix testing with and without
incubation also was studied.
Our results showed that percent correction of the
aPTT or PT 4:1 mix had an overall good sensitivity and
specificity for detecting anticoagulant and factor
deficiency and was better than that of the aPTT or PT
1:1 mix. The 3 suggested definitions of correction all
had a poor sensitivity for detecting anticoagulant.
The percent correction of the aPTT 4:1 mix testing
after incubation had better sensitivity and specificity
that that of testing immediately. Nevertheless, these
procedures were complementary to each other. The
percent correction using the aPTT or PT 4:1 mix
seemed to offer a simple, objective, and effective
criterion for evaluating mixing study results.
62
Am J Clin Pathol 2002;117:62-73
In 1994, our laboratory began to use a “percent correction” of a prolonged prothrombin time (PT) or activated
partial thromboplastin time (aPTT) in a 1:1 mix of patient
plasma (PP) with citrated normal plasma (CNP) for evaluation of mixing study results after a retrospective study.1 The
percent correction is expressed in the following formula:
Percent Correction =
PP PT (or aPTT) – 1:1 Mix PT (or aPTT)
× 100
PP PT (or aPTT) – CNP PT (or aPTT)
For aPTT mixing studies, more than 70% correction
suggested the presence of a factor deficiency, less than 58%
correction suggested the presence of a circulating anticoagulant,
and a 58% to 70% correction was considered a borderline
result. For PT mixing studies, more than 75% correction
suggested the presence of a factor deficiency, less than 70%
correction suggested the presence of a circulating anticoagulant,
and 70% to 75% correction was considered a borderline result.
Based on the percent corrections, we prioritized follow-up
testing to workup prolonged PT, aPTT, or both with fairly
successful results. However, with reagent changes and a change
in instrumentation, the classification and testing scheme did not
seem to perform as well. We had noticed the 1:1 mix of PP with
CNP frequently seemed to “overcorrect” (false high percent
correction); samples with a lupus anticoagulant were misclassified as a factor deficiency. We undertook a reevaluation of
the procedure correlated with follow-up testing results.
Because it has been suggested a 4:1 mix of PP with CNP is
more sensitive for the detection of a lupus anticoagulant,2 we
incorporated a 4:1 mix of PP with CNP into the study.
We also studied 3 previously suggested definitions of
correction for evaluating mixing study results in the aPTT 1:1
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
mix of PP with CNP for comparision.3 In addition, applicability of percent correction for evaluating the aPTT 4:1 mix
testing after incubation also was studied to ascertain the
described time-dependent inhibitor effect of some anticoagulants in mixing studies.3
Materials and Methods
Instrument Used in the Study
We used the AMAX CS-190 coagulation analyzer (Sigma
Diagnostics, St Louis, MO). The following reagents were used:
(1) for PT, ThromboMax (Sigma Diagnostics; International
Sensitivity Index [ISI], 1.89 [mechanical] except for cases 1830 for which a new reagent lot was used, ISI, 1.78); (2) for
aPTT, Sigma Diagnostics APTT reagent; (3) for CNP,
CryoCheck (Precision Biologic, Dartmouth, Nova Scotia); (4)
factor-deficient plasma (factors II, V, VII, VIII, IX, X, XI, XII;
Sigma Diagnostics); (5) Accuclot Reference Plasma, Normal
(for preparation of standard curve for factor assay; Sigma Diagnostics); (6) Accuclot Reference Plasma, Abnormal (for preparation of mild factor-deficient plasma with the aforementioned
factor-deficient plasma; Sigma Diagnostics); (7) DRVVT
(dilute Russell viper venom time; for lupus anticoagulant), LAScreen and LA-Confirm (Gradipore, North Ryde, Australia);
and (8) PTT-LA (for lupus anticoagulant; StaClot LA, Diagnostica Stago, Asnieres-Surseine, France).
Coagulation Testing
Prothrombin Time
The test was performed using ThromboMax and the
automated AMAX CS-190 coagulation analyzer in the
mechanical mode. The reference range was 11.0 to 13.0
seconds (except for cases 18-30 for which a new reagent lot
was used; reference range, 11.7-14.1 seconds).
Activated Partial Thromboplastin Time
The test was performed using Sigma Diagnostic APTT
reagent and the automated AMAX CS-190 coagulation
analyzer in the mechanical mode. The reference range was
23.3 to 29.7 seconds.
Dilute Russell Viper Venom Time
The test was performed initially using LA-Screen
reagent and the automated AMAX CS-190 coagulation
analyzer in the mechanical mode. The reference range was
32.4 to 41.4 seconds. If the result was within reference
range, no further testing was performed. The sample was
considered negative for lupus anticoagulant. If the result was
higher than the reference range, the test was performed with
© American Society for Clinical Pathology
LA-Confirm reagent. The final result was expressed as a
ratio of LA-Screen/LA-Confirm clotting time. The ratio of
more than 1.3 was considered positive for lupus anticoagulant, 1.3 was borderline, and less than 1.3 was negative.
PTT-LA
The test was performed initially using StaClot LA
reagent with reagent 1 (buffer) and the automated AMAX
CS-190 coagulation analyzer in the mechanical mode. The
reference range was 40.1 to 50.4 seconds. If the result was
within reference range, no further testing was performed.
The sample was considered negative for lupus anticoagulant.
If the result was higher than the reference range, the test was
performed with StaClot LA reagent with reagent 2 (hexagonal phase phospholipid). The final result was expressed as
the difference between the clotting time result with reagent 1
and the result with reagent 2. A difference of more than 3.2
seconds was considered positive for lupus anticoagulant, a
difference of less than 3.2 seconds was considered negative,
and a difference of 3.2 seconds was considered borderline.
Factor Assays
Factor assays were performed using the automated
AMAX CS-190 coagulation analyzer. Assays for factors II, V,
VII, and X used a PT-based assay, while assays for factors VIII,
IX, XI, and XII used an aPTT-based assay. For each assay, a
standard curve was prepared with Accuclot normal reference
plasma and factor-deficient plasma. The clotting times of dilutions of the plasma to be tested were obtained, and the factor
activity of each dilution was determined from the standard
curve. The reference range was 50% to 150% (0.50-1.50).
Plasma Samples
We used 3 sources of plasma samples: (1) patient plasma
samples with an abnormal PT and/or aPTT submitted for
routine 1:1 mixing studies and eventually worked-up for a factor
deficiency or circulating anticoagulant; (2) selected plasma
samples from patients receiving oral anticoagulant therapy; and
(3) factor-deficient plasma samples routinely used to perform
factor assays were used as severe factor deficiency plasma
samples; 1:1 mixtures of a specific factor-deficient plasma
sample with the abnormal factor-deficient control plasma
(Accuclot Reference Plasma, Abnormal) were used as mild
factor-deficient plasma samples. The mixtures had specific
factor levels of approximately 25% to 37% (0.25-0.37).
Plasma samples (from sources 1 and 2) were obtained
via atraumatic venipuncture and collected into a 3.2%
buffered sodium citrate solution with a ratio of 9 parts
whole blood to 1 part anticoagulant. The citrated whole
blood was centrifuged at no less than 2,500g for 15 minutes
to obtain platelet-poor plasma with a platelet count of less
than 10 × 103/µL (10 × 109/L).
Am J Clin Pathol 2002;117:62-73
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Chang et al / A “PERCENT CORRECTION” FORMULA FOR EVALUATION OF MIXING STUDIES
❚Table 1❚
Summary of Results for Plasma Samples (Part 1 Study)*
Case No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
PT
aPTT
Group
N
N
N
N
20.1
14.6
13.4
13.5
17.2
N
N
N
39.2
24.7
N
N
N
18.8
19.9
18
22.2
N
N
17.4
16.8
15.8
15
14.4
14.5
N
24.7
21.1
17.9
20.2
17.7
N
N
N
N
N
N
27.4
15.7
68.8
14.9
59.1
15.4
60
14.3
N
N
33.9
33.7
36.1
33.2
40.7
35.1
50.9
N
33
37.9
30
29.9
44.1
54.2
31.8
30.7
33.2
N
N
N
56.8
35.2
33.8
72.2
35.2
32
32.9
32.4
N
31.6
44.4
53
32.4
42.8
35.5
67.7
36.7
63.9
34
122
30.1
N
N
153.9
30.7
120.1
34.4
87.8
34.3
80.8
36.6
3
2
3
3
1
3
3
1
3
3
3
2
1
1
3
3
2
1
1
1
1
3
3
1
1
3
3
2
1
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Pertinent Laboratory Data
DRVVT+
Indeterminate
PTT-LA+
PTT-LA+
Factor VII, 14% (0.14); factor V, 34% (0.34); factor X, 54% (0.54)
DRVVT+; PTT-LA+
DRVVT+
Factor VII, 39% (0.39)
PTT-LA+
PTT-LA+
PTT-LA+
Indeterminate
Factor VII, 1% (0.01); factor X, 11% (0.11); factor V, 96% (0.96)
Factor VII, 5% (0.05); factor X, 19% (0.19); factor V 120% (1.20)
DRVVT+; PTT-LA+
PTT-LA+
Indeterminate
Factor VII, 14% (0.14)
Factor VII, 48% (0.48)
Factor VII, 21% (0.21)
Factor VII, 6% (0.06); factor X, 47% (0.47); factor V, 101% (10.1)
PTT-LA+
DRVVT+; PTT-LA+
Factor VII, 32% (0.32); factor X, 43% (0.43); factor V, 75% (0.75)
Factor V, 40% (0.40); factor VII, 65% (0.65); factor X, 65% (0.65)
DRVVT+; PTT-LA+
DRVVT+
Indeterminate
Factor VII, 32% (0.32)
PTT-LA+
INR, 4.0
INR, 2.8
INR, 2.2
INR, 2.7
INR, 2.1
Factor VIII, <1% (<0.01)
Factor VIII, approximately 26% (0.26)
Factor IX, <1% (<0.01)
Factor IX, approximately 32% (0.32)
Factor XII, <1% (<0.01)
Factor XII, approximately 35% (0.35)
Factor VII, <1% (<0.01)
Factor VII, approximately 32% (0.32)
Factor II, <1% (0.01)
Factor II, approximately 30% (0.30)
Factor V, <1% (<0.01)
Factor V, approximately 37% (0.37)
Factor X, <1% (<0.01)
Factor X, approximately 33% (0.33)
Factor XI, <1% (<0.01)
Factor XI, approximately 25% (0.25)
+, positive for lupus anticoagulant; aPTT, activated partial thromboplastin time; DRVVT, dilute Russell viper venom time; INR, international normalized ratio; N, normal; PT,
prothrombin time; PTT-LA, PTT-LA (for lupus anticoagulant): StaClot LA, Diagnostica Stago, Asnieres-Surseine, France.
* Reference ranges: PT, 11.0-13.0 seconds; for cases 18-30, 11.7-14.1 seconds (new reagent lot); aPTT, 23.3-29.7 seconds. Group 1, factor deficiency; group 2, indeterminate;
group 3, anticoagulant.
The plasma samples were classified into 3 groups representing a factor deficiency, circulating anticoagulant, or indeterminate, based on the follow-up results and available information about the samples. Cases with any factor assay result
of less than 50% (<0.50) were classified in the factor-deficiency group (group 1). Cases with a positive DRVVT (LAScreen and LA-Confirm) and/or positive PTT-LA (StaClot
LA) were classified in the circulating anticoagulant group
(group 3). One case with factor VIII inhibitor was classified
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Am J Clin Pathol 2002;117:62-73
in the anticoagulant group. Cases were classified in the indeterminate group (group 2) if available factor assay results
showed more than 50% (>0.50) and were negative for circulating anticoagulants with the DRVVT and PTT-LA tests.
The Part 1 Study
The study involved 51 samples ❚Table 1❚. Included were
30 patient samples received for mixing studies (cases 1-30),
5 samples from patients receiving oral anticoagulant therapy
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
❚Table 2❚
Summary of Results for Plasma Samples (Part 2 Study)*
Case No.
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
PT
15.8
15
14.4
14.2
16.5
N
N
23.8
29.3
22.4
23.1
21.5
62
16.2
N
N
N
N
N
N
N
18.6
N
15.1
aPTT
32
32.9
32.4
31.2
40.4
30.1
31.5
34.2
34.7
38.3
48.8
33.8
150.7
36.3
62.9
36.5
61.2
33.8
92.8
34
32.5
54.3
33.2
49.7
Group
Pertinent Laboratory Data
3
3
2
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
3
3
DRVVT+; PTT-LA+
DRVVT+
Indeterminate
DRVVT+
DRVVT+; factor VII, 34% (0.34); factor V, 106% (1.06); factor X, 106% (1.06)
DRVVT+; PTT-LA+
Factor XII, 43% (0.43); factor VIII, 105% (1.05); factor IX, 86% (0.86); factor XI, 97% (0.97)
INR, 3.3
INR, 3.6
INR, 2.7
INR, 2.8
INR, 2.5
Factor V, <1% (<0.01)
Factor V, approximately 37% (0.37)
Factor VIII, <1% (<0.01)
Factor VIII, approximately 26% (0.26)
Factor IX, <1% (<0.01)
Factor IX, approximately 32% (0.32)
Factor VIII, <1% (<0.01); factor VIII inhibitor, 12.4 Bethesda U
DRVVT+
PTT-LA+
PTT-LA+
DRVVT+; PTT-LA+
DRVVT+; PTT-LA+
+, positive for lupus anticoagulant; aPTT, activated partial thromboplastin time; DRVVT, dilute Russell viper venom time; INR, international normalized ratio; N, normal; PT,
prothrombin time; PTT-LA, PTT-LA (for lupus anticoagulant): StaClot LA, Diagnostica Stago, Asnieres-Surseine, France.
* Reference ranges: PT, 11.0-13.0 seconds; aPTT, 23.3-29.7 seconds. For a description of the groups, see Table 1.
with international normalized ratios ranging from 2.1 to 4.0
(cases 31-35), and 16 artificially prepared factor-deficient
samples that included severe and mild deficiencies of factors
II, V, VII, VIII, IX, X, XI, and XII (cases 36-51).
An initial PT and aPTT were performed on all samples.
Mixing studies were performed on samples with a prolonged
PT and/or aPTT result using a 1:1 mixture and a 4:1 mixture
of PP/CNP. A percent correction was calculated based on the
original formula and the following formula, respectively:
The 3 definitions of correction suggested by Brandt et
al3 were as follows: (1) aPTT 1:1 mix result less than or
equal to the upper limit of normal, (2) aPTT 1:1 mix result
less than or equal to the CNP aPTT plus 5 seconds, and (3) a
Rosner index of 15 or less. The Rosner index was calculated
from the following formula:
PP PT (or aPTT) – 4:1 Mix PT (or aPTT)
× 100
PP PT (or aPTT) – CNP PT (or aPTT)
For definitions 1 and 2, the actual aPTT 1:1 mix results
and the CNP aPTT results were used for evaluation. The
Rosner index for each case was calculated from the
preceding formula. Sensitivity and specificity for each category then were calculated for comparison.
Percent Correction =
The aPTT or PT 1:1 and 4:1 mix results, CNP aPTT or
PT results, their respective percent correction, and group
classification (as defined in “Plasma Samples”) were
compiled.
For the aPTT or PT 1:1 mix, the original criteria1 of
percent correction cutoff values were used for evaluation.
Sensitivity and specificity for detecting anticoagulant and
factor deficiency were calculated based on the cutoff values.
For the aPTT or PT 4:1 mix, a dot plot was made for
initial correlation of percent correction with the groups.4 An
optimal cutoff then was selected for differentiation of factor
deficiency and anticoagulant. Sensitivity and specificity for
detecting factor deficiency and anticoagulant were calculated
based on the selected cutoff for evaluation.5
© American Society for Clinical Pathology
Index =
1:1 Mix aPTT – CNP aPTT
× 100
PP aPTT
The Part 2 Study
The study involved 24 samples ❚Table 2❚. Included were
7 samples received for mixing studies (cases 52-58); 5
samples from patients receiving oral anticoagulant therapy
with international normalized ratios ranging from 2.5 to 3.6
(cases 59-63); 6 artificially prepared factor-deficient samples
that included severe and mild deficiencies of factors V, VIII,
and IX (cases 64-69); and 6 patient samples with circulating
anticoagulant, including 1 with a factor VIII inhibitor (case
70) and 5 samples with a documented lupus anticoagulant
(cases 71-75).
Am J Clin Pathol 2002;117:62-73
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Chang et al / A “PERCENT CORRECTION” FORMULA FOR EVALUATION OF MIXING STUDIES
❚Table 3❚
Results for aPTT 1:1 and 4:1 Mixes and Percent Corrections and Group Classification*
Case No.
aPTT
CNP
1
2
3
4
5
6
7
9
10
11
12
13
14
15
16
17
21
22
23
24
25
26
27
28
30
31
32
33
34
35
36
37
38
39
40
41
44
45
46
47
48
49
50
51
33.9
33.7
36.1
33.2
40.7
35.1
50.9
33
37.9
30
29.9
44.1
54.2
31.8
30.7
33.2
56.8
35.2
33.8
77.2
35.2
32
32.9
32.4
31.6
44.4
53
32.4
42.8
35.5
67.7
36.7
63.9
34
122
30.1
153.9
30.7
120.1
34.4
87.8
34.3
80.8
36.6
25.9
26.9
26.5
28.1
26.7
27.2
26.5
28.1
26
26.1
25.5
26.1
24.9
24.9
25.2
25.2
25.4
26.1
25.6
25.6
26.4
26.2
25.2
24.9
25.6
25.5
25.5
25.5
25.5
25.5
24.7
24.1
24.7
24.1
24.7
24.1
24.7
24.1
24.7
24.1
24.7
24.1
25.4
25.1
1:1 Mix
28.2
27.9
30.7
28
30.1
28.4
44.7
27.8
28.6
28.3
26.8
26.2
26.4
26.6
29
27
33.1
28.9
28
32
27.8
28.3
26.5
27.5
27.4
27.4
27.9
25.4
27.1
27.3
28.3
27.5
27
26.9
24.7
25.8
25
25.8
27.1
27.3
27.6
27.4
29.6
28.9
% Correction
4:1 Mix
% Correction
Group
71
85
56
102
76
85
25
106
78
44
70
99
95
75
31
78
75
69
71
87
84
64
83
65
70
90
91
101
91
81
92
73
94
72
100
72
99
74
96
69
95
68
93
67
30.4
30.8
33.5
30.8
33.6
32.2
43.5
30.8
32.6
29.6
28
31.4
35.7
28.8
29
29.4
43.1
32.7
30.1
52.1
31.8
30.3
30.7
29.1
32.4
30.9
36.7
27.7
32.3
29.3
33.5
29.8
30.7
28.7
26.9
26.1
27.3
26.5
30.9
28.5
31.8
28.8
34.8
30.8
44
43
27
47
51
37
30
45
44
10
43
71
63
43
30
48
44
27
46
49
38
30
29
44
–13
71
59
68
61
62
79
54
84
54
97
67
98
64
94
57
89
54
83
50
3
2
3
3
1
3
3
3
3
3
2
1
1
3
3
2
1
3
3
1
1
3
3
2
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
aPTT, activated partial thromboplastin time; CNP, citrated normal plasma.
* All data given in seconds except % correction. aPTT reference range, 23.3-29.7 seconds. For a description of the groups, see Table 1.
For this part of the study, mixing studies were performed
only on samples with a prolonged aPTT. The mixing studies
used a 4:1 mixture of PP/CNP and were tested immediately
and after 60 minutes of incubation in a 37°C water bath. The
CNP was incubated in the same water bath used for incubating the test mixture. The percent correction for the immediate testing and percent correction after incubation then
were calculated using the same percent correction formula as
for the aPTT 4:1 mix.
The aPTT 4:1 mix results and the CNP aPTT results for
testing immediately and testing after incubation, the immediate
percent correction and the incubated percent correction, and group
classification (as defined in “Plasma Samples”) were compiled.
For the aPTT 4:1 mix tested immediately, the same percent
correction cutoff of 50% or more in the part 1 study of the aPTT
66
Am J Clin Pathol 2002;117:62-73
4:1 mix was used for evaluation. For the aPTT 4:1 mix tested
after incubation, a dot plot was made for initial correlation
and selection of an optimal cutoff value. Sensitivity and specificity for detecting factor deficiency and anticoagulant were
calculated based on the selected cutoff value for evaluation.
Results
The Part 1 Study
In the part 1 study, 44 samples had a prolonged aPTT
and 30 samples had prolonged PT. The results of the aPTT
1:1 and 4:1 mixes, percent correction, and group classification are shown in ❚Table 3❚.
© American Society for Clinical Pathology
120
100
100
80
80
60
Percent Correction
Percent Correction
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
70
60 58
50
40
40
20
20
0
–20
0
1
2
1
3
2
3
Group
Group
❚Figure 1❚ Results for activated partial thromboplastin time
1:1 mix, correlation of percent correction with groups,
cutoffs at >70% and <58% (dotted lines, original criteria).
Group 1, factor deficiency (n = 25); group 2, indeterminate
(n = 4); group 3, anticoagulant (n = 15).
❚Figure 2❚ Results for activated partial thromboplastin time
4:1 mix, correlation of percent correction with groups, cutoff
at 50% or more (dotted line, new criterion). For a description
of the groups, see Figure 1.
The correlation of the aPTT 1:1 mix percent correction with group classification is shown in ❚Figure 1❚. The
original criteria1 of percent correction cutoff at more than
70% and at less than 58% were used in the evaluation.
The sensitivity and specificity for detecting factor deficiency and anticoagulant based on the criteria are shown
in ❚Table 4❚ . There was a slight discrepancy in case
number between cases correctly classified and sensitivity
because there were 2 cutoff values in the original criteria.
The cases with percent correction values between 70%
and 58% were considered borderline and were excluded
in the calculation of sensitivity and specificity. All indeterminate cases also were excluded in all calculations of
sensitivity and specificity.
The correlation of the aPTT 4:1 mix percent correction
with group classification is shown in ❚Figure 2❚. The percent
❚Table 4❚
Sensitivity and Specificity for Detecting Factor Deficiency and Anticoagulant of aPTT 1:1 and 4:1 Mixes at Defined Percent
Correction Cutoffs
aPTT Mix
1.1
% Correction cutoff at >70% and at <58%
(original criteria)
4:1
% Correction cutoff at 50% or more
Group
Cases Correctly Classified
Sensitivity
Specificity (%)
Factor deficiency
Anticoagulant
22/25 (88%)
4/15 (27%)
22/22 (100%)
4/12 (33%)
33
100
Factor deficiency
Anticoagulant
22/25
15/15
88%
100%
100
88
aPTT, activated partial thromboplastin time.
* Percent correction cutoff >70% positive for factor deficiency; <58% positive for anticoagulant; <70% and >58% considered borderline, not included in calculation of
sensitivity and specificity. As a result there is a slight discrepancy in case number between cases correctly classified and sensitivity.
© American Society for Clinical Pathology
Am J Clin Pathol 2002;117:62-73
67
Chang et al / A “PERCENT CORRECTION” FORMULA FOR EVALUATION OF MIXING STUDIES
❚Table 5❚
Results for PT 1:1 and 4:1 Mixes and Percent Correction and Group Classification*
Case No.
5
6
7
8
9
13
14
18
19
20
21
24
25
26
27
28
29
31
32
33
34
35
42
43
44
45
46
47
48
49
PT
CNP
1:1 Mix
% Correction
4:1 Mix
% Correction
Group
20.1
14.6
13.4
13.5
17.2
39.2
24.7
18.8
19.9
18
22.2
17.4
16.8
15.8
15
14.4
14.5
24.7
21.1
17.9
20.2
17.7
27.4
15.7
68.8
14.9
59.1
15.4
60
14.3
12.4
12.8
12.4
12.2
12.5
12.3
12.2
13.7
13.5
13.7
13.2
13.5
13.4
13.8
13.9
13.7
12.7
12.2
12.2
12.2
12.2
12.2
12.5
13.3
12.5
12.2
12.5
12.2
12.5
12.2
13.7
13.1
13.3
12.4
14
13.5
13.2
15
13.9
14.6
14.2
14.3
14.3
14.3
14.1
13.9
13
12.6
12.9
13
13
12.7
12.8
13.7
12.8
12.8
13.6
13.2
12.7
12.6
83
83
10
85
68
96
92
75
94
79
89
79
74
75
82
71
83
97
92
86
90
91
98
83
99
78
98
69
99
81
15.7
14
13.2
12.8
15.5
16.6
16.3
13.9
14.9
15
16
15.7
15.5
15
14.7
14.2
13.6
14.7
15.1
14.5
15.2
14.1
14.3
14.7
14.7
13
16.1
13.6
14.3
13
57
33
20
53
36
84
67
96
78
70
69
43
38
40
27
29
50
80
67
60
63
66
87
42
96
70
92
56
96
62
1
3
3
1
3
1
1
1
1
1
1
1
1
3
3
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
CNP, citrated normal plasma; PT, prothrombin time.
* All data given in seconds except % correction. PT reference range: 11.0–13.0 seconds; cases 18-30, 11.7-14.1 seconds (new reagent lot). For a description of the groups, see Table 1.
correction of 50% or more was the selected cutoff value. The
sensitivity and specificity for detecting factor deficiency and
anticoagulant based on the cutoff of 50% or more also are
shown in Table 4.
The results of the PT 1:1 and 4:1 mixes, percent correction, and group classification are shown in ❚Table 5❚.
The correlation of the PT 1:1 mix percent correction
with the group classification is shown in ❚Figure 3❚. The
original criteria1 of percent correction cutoff at more than
75% and at less than 70% were used in the evaluation. The
sensitivity and specificity for detecting factor deficiency and
anticoagulant based on the criteria are shown in ❚Table 6❚.
There was a slight discrepancy in case number between cases
correctly classified and sensitivity for the same reason
described for the aPTT 1:1 mix.
The correlation of the PT 4:1 mix percent correction
with the group classification is shown in ❚Figure 4❚. The
percent correction of more than 40% was the selected cutoff
value. The sensitivity and specificity for detecting factor
deficiency and anticoagulant based on the cutoff of more
than 40% also are shown in Table 6.
The sensitivity and specificity using the 3 suggested
definitions for correction for evaluating the aPTT 1:1 mix
68
Am J Clin Pathol 2002;117:62-73
based on patient aPTT, CNP aPTT, and 1:1 mix aPTT results
in Table 3 are shown in ❚Table 7❚.
The Part 2 Study
The part 2 study involved 24 samples with a
prolonged aPTT. The results of the aPTT 4:1 mix with
immediate testing or testing after a 60-minute incubation,
percent correction, and group classification are shown in
❚Table 8❚.
The correlation of the aPTT 4:1 mix percent correction
for immediate testing with group classification is shown in
❚Figure 5❚. The same percent correction cutoff of 50% or
more as in the part 1 study of the aPTT 4:1 mix was used in
the evaluation. The sensitivity and specificity for detecting
factor deficiency and anticoagulant based on the cutoff of
50% or more are shown in ❚Table 9❚.
The correlation of the aPTT 4:1 mix percent correction
for testing after incubation with group classification is
shown in ❚Figure 6❚. The percent correction of more than
10% was the selected cutoff value. The sensitivity and specificity for detecting factor deficiency and anticoagulant based
on the percent correction cutoff of more than 10% are also
shown in Table 9.
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
100
100
80
80
75
Percent Correction
Percent Correction
70
60
40
60
40 40
20
20
0
0
1
2
1
3
2
3
Group
Group
❚Figure 3❚ Results for prothrombin time 1:1 mix, correlation
of percent correction with groups, cutoffs at >75% and
<70% (dotted lines, original criteria). Group 1, factor
deficiency (n = 24); group 2, indeterminate (n = 1); group 3,
anticoagulant (n = 5).
❚Figure 4❚ Results for prothrombin time 4:1 mix, correlation
of percent correction with groups, cutoff at >40% (dotted
line, new criterion). For a description of the groups, see
Figure 3.
Discussion
uniform agreement as to what criteria should be used to
judge correction.3 The normal range usually is used as the
guide for correction. However, it often is inadequate because
a circulating anticoagulant with a minimally prolonged PT or
aPTT may be corrected to normal in a 1:1 mix of PP with
CNP. 8 In contrast, factor deficiencies with a markedly
prolonged PT or aPTT may not be corrected to normal in a
1:1 mix of PP with CNP.3
As to the mixing study procedure itself, 1:1 mix of PP
with CNP often is used.2,3,6-8 A 4:1 mix of PP with CNP also
Mixing studies have long been used for the evaluation of
an unexplained prolongation of PT and aPTT. If a prolonged
PT or aPTT of patient plasma is “corrected” to normal when
mixed with CNP, a factor deficiency is indicated.6,7 If a
prolonged PT or aPTT is not corrected to normal when
mixed with CNP, a circulating anticoagulant is indicated.
The principle of the mixing study seems simple, but
results often are difficult to interpret in practice.8 There is no
❚Table 6❚
Sensitivity and Specificity for Detecting Factor Deficiency and Anticoagulant of PT 1:1 and 4:1 Mixes at Defined Percent
Correction Cutoffs
PT Mix
1.1
% Correction cutoff at >75% and at <70%
(original criteria)
4:1
% Correction cutoff at >40%
Group
Cases Correctly Classified
Factor deficiency
Anticoagulant
21/24 (88%)
2/5 (40%)
Factor deficiency
Anticoagulant
23/24
5/5
Sensitivity
Specificity (%)
21/22 (95%)
2/4 (50%)
50
95
96%
100%
100
96
PT, prothrombin time.
* Percent correction cutoff >75% positive for factor deficiency; <70% positive for anticoagulant; <75% and >70% considered borderline, not included in calculation of sensitivity
and specificity. As a result there is a slight discrepancy in case number between cases correctly classified and sensitivity.
© American Society for Clinical Pathology
Am J Clin Pathol 2002;117:62-73
69
Chang et al / A “PERCENT CORRECTION” FORMULA FOR EVALUATION OF MIXING STUDIES
❚Table 7❚
Sensitivity and Specificity for Detecting Factor Deficiency and Anticoagulant of aPTT 1:1 Mix Using Three Suggested
Definitions3 of Correction*
aPTT 1:1 Mix
Group
Upper limit of normal
Cases Correctly Classified
Factor deficiency
Anticoagulant
Factor deficiency
Anticoagulant
Factor deficiency
Anticoagulant
CNP aPTT + 5 seconds
Rosner index† 15 or less
22/25
2/15
23/25
1/15
25/25
1/15
Sensitivity (%)
Specificity (%)
88
13
92
7
100
7
13
88
7
92
7
100
aPTT, activated partial thromboplastin time; CNP, citrated normal plasma.
* Based on data in Table 3.
† See the text.
❚Table 8❚
Results for aPTT 4:1 Mix, Immediate or Incubated, and Percent Correction and Group Classification*
Case No.
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
aPTT
32
32.9
32.4
31.2
40.4
30.1
31.5
34.2
34.7
38.3
48.8
33.8
150.7
36.3
62.9
36.5
61.2
33.8
92.8
34
32.5
54.3
33.2
49.7
CNP
4:1 Mix
% Correction
CNP†
4:1 Mix†
% Correction†
26.2
25.2
24.9
25.4
26.5
25.4
26.2
25.2
25.1
24.3
24.3
24.3
25
25
24.3
24.3
24.3
24.3
25.1
25.4
25.4
25.4
25.2
25.2
30.3
30.7
29.1
29.2
35.1
27.9
28.8
29.1
28.5
29.4
33.6
28.4
33.2
31.6
33.4
31.1
30.7
30.2
52.1
31.5
30.3
46.3
30.8
43
29
29
44
34
38
47
51
57
65
64
62
57
93
42
76
44
83
38
60
29
31
28
30
27
25.1
25.5
25.4
25.3
26
25.1
25.8
25
26.1
24.9
24.9
24.9
26.7
26.3
24.9
24.9
24.9
24.9
26.1
25.5
25.5
25.5
25
25
34.5
33.7
34.3
32.7
38.9
31.4
30.9
30.8
32.4
31.6
42.3
31.2
33.2
31.1
34
31.9
31.1
30.1
87.3
34.2
34.4
64.5
36.8
47.3
–36
–11
–27
–25
10
–26
11
37
27
50
27
29
95
52
76
40
83
42
8
–2
–27
–35
–44
10
Group
3
3
2
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
3
3
aPTT, activated partial thromboplastin time; CNP, citrated normal plasma.
* All data given in seconds except % correction. aPTT reference range, 23.3-29.7 seconds. For a description of the groups, see Table 1.
† 60-minute incubation.
has been described.2,3,7,8 However, it was not clear from the
publications which of the 2 procedures was preferred.
Kaczor et al2 suggested that the 4:1 mix of PP with CNP
might be more sensitive for detecting a lupus anticoagulant,
while Brandt et al3 found that the 4:1 mix was ineffective for
correcting the aPTT of the mixture and that it might misclassify samples with factor deficiency as anticoagulant.
Our results for the aPTT 1:1 mix with percent correction
cutoff at more than 70% and at less than 58% (original
criteria) (Figure 1 and Table 4) showed a very poor sensitivity (33%) for detecting anticoagulant, thus confirming our
impression of overcorrection in the mixing studies resulting
in frequent misclassification of samples with lupus anticoagulant as samples with factor deficiency.
70
Am J Clin Pathol 2002;117:62-73
Results of the aPTT 4:1 mix with percent correction
cutoff at 50% or more (Figure 2 and Table 4) showed
markedly improved sensitivity (100%) for detecting anticoagulant while maintaining a good specificity (88%). In addition, there was a better separation of percent correction of
anticoagulant from that of factor deficiency in the 4:1 mix
procedure, ie, 1 cutoff value was reasonably sufficient in
classifying the anticoagulant and factor deficient groups,
while the 1:1 mix procedure required 2 cutoff values owing
to more overlapping borderline percent correction values
between the anticoagulant group and the factor deficiency
group in the original study in 1994.1
As shown in Table 4, at a defined percent correction
cutoff value, the sensitivity for detecting factor deficiency
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
100
100
80
80
Percent Correction
Percent Correction
60
60
50
40
40
20
10
0
–20
20
–40
0
–60
1
2
1
3
2
3
Group
Group
❚Figure 5❚ Results for activated partial thromboplastin time
4:1 mix, immediate (part 2 study), correlation of percent
correction with groups, cutoff at 50% or more (dotted line,
new criterion). Group 1, factor deficiency (n = 12); group 2,
indeterminate (n = 1); group 3, anticoagulant (n = 11).
❚Figure 6❚ Results for activated partial thromboplastin time
4:1 mix, incubated (part 2 study), correlation of percent
correction with groups, cutoff at >10% (dotted line, new
criterion). For a description of the groups, see Figure 5.
coincided with the specificity for detecting anticoagulant and
vice versa. Therefore, no further discussion of sensitivity and
specificity for detecting factor deficiency at the same cutoff
value is necessary.
There were 2 interesting observations: (1) Plasma
samples with more severe factor deficiency tended to have
higher percent corrections. They usually had percent corrections much higher than the cutoff of 50%. Samples with mild
factor deficiency (eg, cases 5, 37, 39, 49 in Table 3, aPTT 4:1
mix) tended to have borderline percent correction. Occasional
cases with mild factor deficiency (eg, cases 24 and 25 in
Table 3, aPTT 4:1 mix) had percent correction below the
cutoff of 50%. (2) One sample with a lupus anticoagulant
(case 30) had a percent correction of –13% resulting from a
more prolonged aPTT on the mixture than on the patient
plasma alone. This phenomenon has been termed the lupus
cofactor effect.8,9
Results of the PT 1:1 mix with percent correction cutoffs
at more than 75% and at less than 70% (original criteria)
(Figure 3 and Table 6) also showed a poor sensitivity (50%)
❚Table 9❚
Sensitivity and Specificity for Detecting Factor Deficiency and Anticoagulant of aPTT 4:1 Mix, Immediate and Incubated, at
Defined Percent Correction Cutoff
aPTT 4:1 Mix
Immediate
% Correction cutoff at 50% or more
Incubated
% Correction cutoff at >10%
Group
Cases Correctly Classified
Sensitivity (%)
Specificity (%)
Factor deficiency
Anticoagulant
9/12
10/11
75
91
91
75
Factor deficiency
Anticoagulant
12/12
11/11
100
100
100
100
aPTT, activated partial thromboplastin time
© American Society for Clinical Pathology
Am J Clin Pathol 2002;117:62-73
71
Chang et al / A “PERCENT CORRECTION” FORMULA FOR EVALUATION OF MIXING STUDIES
for detecting anticoagulant. As in the aPTT 4:1 mix, the
results for the PT 4:1 mix with a percent correction cutoff at
more than 40% showed markedly improved sensitivity
(100%) for detecting anticoagulant while maintaining very
good specificity (96%).
In addition, as in the aPTT 4:1 mix, the PT 4:1 mix had
a better separation of samples with factor deficiency from
samples with anticoagulant. Only 1 cutoff value was needed
in the PT 4:1 mix compared with 2 cutoff values in the PT
1:1 mix procedure for the same reason described for the
aPTT 4:1 mix. Like the aPTT 4:1 mix, the samples with
more severe factor deficiency tended to have higher percent
correction.
As shown in Table 7, all 3 suggested definitions of correction for evaluating the aPTT 1:1 mix3 showed poor sensitivity
(13%, 7%, and 7%, respectively) for detecting anticoagulant.
Brandt et al,3 in their evaluation of these 3 definitions of
correction, also showed less than satisfactory results.
The aPTT 4:1 mix tested immediately in the part 2 study
was the same procedure as used for the aPTT 4:1 mix in the
part 1 study. We applied the same percent correction cutoff
of 50% or more established in the part 1 study to the second
set of samples of the part 2 study for evaluation (Figure 5
and Table 9).
The sensitivity and specificity of the aPTT 4:1 mix
tested immediately using the percent correction cutoff of
50% or more for detecting anticoagulant were 91% and 75%,
respectively, compared with 100% and 88% in the part 1
study (Table 4). They had fairly comparable sensitivity and
specificity values. Furthermore, review of Figures 2 and 5
showed a similar pattern of percent correction distribution.
Both the part 1 study and the part 2 study showed a few
cases of factor deficiency with false-negative results (below
the cutoff of 50% or more). Those cases generally had mild
factor deficiency, such as cases 24 and 25 in the part 1 study
and cases 65, 67, and 69 in the part 2 study.
One case stood out in the anticoagulant group in the part
2 study of the aPTT 4:1 mix with immediate testing (Figure
5 and Table 8). Case 70 with factor VIII inhibitor had a
percent correction of more than 50% and was classified as
factor deficiency on immediate testing.
For aPTT 4:1 mix testing after incubation, the selected
percent correction cutoff of 10% was much lower than that
for 4:1 mix immediate testing. However, the cutoff had a
good separation of the anticoagulant group from the factordeficient group (Figure 6 and Table 9). The sensitivity and
specificity of the aPTT 4:1 mix tested after incubation with
the percent correction cutoff at more than 10% were each
100% for detecting anticoagulant compared with 91% and
75% for the aPTT 4:1 mix tested immediately with the
percent correction cutoff of 50% or more. This was a
substantial improvement.
72
Am J Clin Pathol 2002;117:62-73
❚Table 10❚
Interpretation of Activated Partial Thromboplastin Time
4:1 Mixing Studies
Immediate %
Correction
Incubated %
Correction
50% or more
<50%
50% or more
<50%
>10%
>10%
10% or less
10% or less
% Correction
Results Suggest
Factor deficiency
Mild factor deficiency
Factor inhibitor
Lupus anticoagulant
Three cases (cases 65, 67, and 69) with mild factor deficiency were misclassified as anticoagulant by immediate
testing (immediate percent correction <50%). They were
classified correctly as factor deficiency on testing after incubation (incubated percent correction >10%). Case 70 with
factor VIII inhibitor, initially classified as factor deficiency
according to the immediate percent correction (percent
correction >50%) was classified correctly as circulating anticoagulant on testing after incubation (percent correction
<10%). A similar incubation effect in an aPTT mixing study
of factor VIII inhibitor has been described.3,10
Although the percent correction of the aPTT 4:1 mix
testing after incubation seemed to have better sensitivity and
specificity for detecting anticoagulant and factor deficiency
than that of the aPTT 4:1 mix with immediate testing, both
incubated and immediate testing procedures were complementary to each other for interpretation of mixing study
results ❚Table 10❚.
An additional interesting observation was that in the
part 2 study, 8 of 10 cases (cases 52, 53, 55, 57, 71, 72, 73,
and 74) with lupus anticoagulant showed the lupus cofactor
effect after incubation, while none of those cases and only
1 case (case 30) of 15 cases with lupus anticoagulant in the
part 1 study showed the lupus cofactor effect on immediate
testing. Incubation of the mixture seemed to enhance the
lupus cofactor effect and made samples with a lupus anticoagulant more distinct from samples with a factor deficiency.
In addition to good sensitivity and good specificity, the
percent correction formula in combination of aPTT or PT 4:1
mixing procedure offers an additional advantage in that it
gives an objective and quantitative measure for differentiating samples with anticoagulant from those with factor deficiency, even samples with minimally or markedly prolonged
aPTT or PT results as demonstrated in our study cases
(Tables 3, 5, and 8).
Whether the percent correction procedure is applicable to
other combinations of instruments and reagents is an open
question. Our experience of adapting the procedure to our
new instrument and new reagents suggested that the procedure could be applied to other instruments and reagent
© American Society for Clinical Pathology
Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
combinations as well. However, like most laboratory
procedures, each laboratory must establish its own reference cutoff values to derive meaningful interpretations.
Twenty to 30 samples each of anticoagulant and factor
deficiency cases probably suffice for study to establish
cutoff values.
Conclusion
The study showed that the percent correction for the
aPTT or PT 4:1 mix had an overall good sensitivity and
specificity for detecting anticoagulant and factor deficiency. The percent correction for the aPTT or PT 4:1 mix
had better sensitivity and specificity than that for the aPTT
or PT 1:1 mix.
The 3 definitions of correction suggested by Brandt et
al3 showed a poor sensitivity for detecting anticoagulant in
our study. The percent correction of the aPTT 4:1 mix tested
after incubation showed an improvement in sensitivity and
specificity for detecting anticoagulant and factor deficiency
compared with the percent correction of the aPTT 4:1 mix
tested immediately. Nevertheless, these procedures were
complementary for interpretation of mixing studies. Therefore, both the immediate and the incubated percent corrections are useful. The interpretation of percent correction
results of the aPTT 4:1 mix are summarized in Table 10. No
incubation study of PT was performed.
The percent correction of the aPTT or PT 4:1 mix of PP
with CNP testing immediately and testing after incubation
(aPTT only) seemed to offer a simple, objective, and effective criterion for evaluating mixing study results.
© American Society for Clinical Pathology
From the Hematology Laboratory, Marshfield Clinic, Marshfield, WI.
Address reprint requests to Dr Chang: Dept of Pathology,
Marshfield Clinic, 1000 N Oak Ave, Marshfield, WI 54449.
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