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
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 =
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
63
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
+, 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
64
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
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
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.
Percent Correction =
Index =
65
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
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
70
60 58
50
40
40
20
20
20
0
1
Group
Group
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
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
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
67
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
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
100
80
80
75
Percent Correction
Percent Correction
70
60
40
60
40 40
20
20
0
1
Group
Group
Discussion
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
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
69
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
Factor deficiency
Anticoagulant
Factor deficiency
Anticoagulant
Factor deficiency
Anticoagulant
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
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
100
100
80
80
Percent Correction
Percent Correction
60
60
50
40
40
20
10
0
20
20
40
60
1
Group
Group
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
Sensitivity (%)
Specificity (%)
Factor deficiency
Anticoagulant
9/12
10/11
75
91
91
75
Factor deficiency
Anticoagulant
12/12
11/11
100
100
100
100
71
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
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.
References
1. Chang S, Tillema V. Mixing study procedure changes.
Laboratory News Update Marshfield Laboratories. March 8,
1994;17(3):1-7.
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