Activated Partial Thromboplastin Time and Minor Coagulopathies WILLIAM E. HATHAWAY, M.D., SHERRY L ASSMUS, B.S., ROBERT R. MONTGOMERY, M.D., AND A. STEPHEN DUBANSKY, M.D.
Hathaway, William E., Assmus, Sherry L., Montgomery, Robert R., and Dubansky, A. Stephen: The activated partial thromboplastin time and minor coagulopathies. Am J Clin Pathol 71: 22-25, 1979. Five commercially available activated partial thromboplastin time (APTT) test systems were compared with the kaolin partial thromboplastin time (KPTT) method to determine sensitivity in detecting minor coagulation defects. All reagent systems detected severe factor VIII-, IX-, and Xl-deflcient hemophilia. Homozygous states of factor XII deficiency, Fletcher factor deficiency, and high-molecularweight kininogen deficiency (Fitzgerald trait) also showed abnormally long APTTs by all systems. Of 19 samples from patients with deficiencies of factors XII, VIII, IX, XI, and II ranging from 2.5 to 52%, eight had deficiencies that were not detected by reagent A (ellagic acid); two, by reagent B (ellagic acid); two, by reagent C (kaolin); one, by reagent D (silica); one, by the KPTT method. All deficiencies were detected by reagent E (celite). Heparin effect on plasma was less well detected by reagent A (ellagic acid) than with the other test systems. APTT test systems can vary greatly in their abilities to detect minor coagulation abnormalities. (Key words: Activated partial thromboplastin time; Minor coagulopathies; Hemophilia; Heparin monitoring.)
Department of Pediatrics, University of Colorado Medical Center, Denver, Colorado
method of Proctor and Rapaport, 6 which has been the standard method in our laboratory since 1962. Methods
THE ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) test is widely used as a plasma-clotting test to determine abnormalities due to defects in procoagulants or to assess anticoagulants such as heparin or the "lupus anticoagulant." Various reagents are commercially available for performance of this test. While these reagents are usually provided with positive controls to demonstrate efficacy in detection of severe coagulation defects like hemophilia due to deficiencies of factors VIII and IX, little information about the sensitivities of these reagents in the determination of minor and moderately severe coagulation defects is available. Recent experience in our laboratory has suggested that some of these APTT methods are not as sensitive as others in the detection of mild hemophilia. We report the results of a comparison of some of the commercially available methods for APTT testing with the kaolin partial thromboplastin time (KPTT)
The normal subjects were ten women and ten men who were members of a group of adult subjects used to obtain pooled normal plasma for all coagulation assays in this laboratory. Twenty-six patients with a variety of coagulation abnormalities (Table 2) were selected after prior complete coagulation analysis had established their diagnoses. In addition, each patient had the appropriate specific factor assay repeated on the same plasma sample used for the APTT testing. Methods for the specific factor assays have been described, 2,3 except for the Fitzgerald factor assay, which was performed with the use of Fitzgerald-deficient plasma and the KPTT system. The APTT tests were performed according to the instructions provided by the manufacturers (see Table 1) with an automatic timing device;t batches of reagents to complete the entire study were purchased in JulyAugust 1975; details of reagents and incubation times are shown in Table 1. The KPTT was prepared with the use of the method of Proctor and Rapaport and
Received October 20, 1977; received revised manuscript and accepted for publication January 3, 1978. Supported in part by U. S. Public Health Service Grant ROl HL 18507. Address reprint requests to Dr. Hathaway: Department of Pediatrics, University of Colorado, Denver, Colorado 80262.
* Falcon, Oxnard, California. t Fibrometer, Becton-Dickinson & Co., Cockeysville, Maryland.
0002-9173/79/0100/0022 $00.70 © American Society of Clinical Pathologists
22
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
All subjects' blood samples were collected by a careful two-syringe technic in plastic syringes containing a citrate-citric-acid anticoagulant (three parts 0.1 M sodium citrate plus two parts 0.1 M citric acid) in a proportion of one part anticoagulant to nine parts whole blood. Plasma was prepared by centrifugation by 3,500 x g for 20 minutes in a refrigerated centrifuge (4 C) and stored in capped plastic tubes* until used. The plasma was prepared and stored without contact with glass. Samples from patients and normal subjects were tested fresh or within a week of storage at - 7 0 C. Plasma to be frozen was recentrifuged at 35,000 x g (4 C) to remove all platelets. Plasma to be tested fresh was stored at 4 C for no longer than 1-2 hours.
23
APTT AND MINOR COAGULOPATHIES
Vol. 71 . No. I
Table I. Reagent Specification for Activated Partial Thromboplastin Time
Reagent Description
Incubation Time (Min)
A. Rabbit brain cephalin plus ellagic acid B. Bovine-brain cephalin plus ellagic acid C. Rabbit-brain cephalin and kaolin D. Rabbit-brain cephalin plus micronized silica E. Rabbit-brain cephalin plus cetite KPTT (Proctor and Rapaport) rabbit-brain cephalin plus kaolin
3 3 3 5 1 3
Mean ± 2 SD (Sec)
Distribution Range (Sec)
Suggested Normal Range (Sec)*
29.5 (23.0-36.0) 28.0(23.5-33.0) 40.0 (32.0-45.5) 35.5 (30.0-40.5) 37.0(32.0-41.5) 42.0(33.0-51.5)
25.0-36.5 23.5-33.0 32.5-44.0 31.5-40.0 32.0-42.0 37.5-51.0
23.6-31.6 0.25-40.0 29.0-43.5 27.0-39.0 33.0-40.0 37.0-50.0
* The suggested norma] range refers to package inserts, although each manufacturer recommends that each user establish its own normal range.
performed exactly as described in the original publication,6 with the Bell and Alton chloroform extract of rabbit-brain thromboplastin; the end point in seconds was determined by tilt-tube method with the use of new glass tubes. The APTT tests were performed by
the same technician, while the KPTT tests were performed by the technician usually doing routine KPTT testing in this laboratory. All tests were done in duplicate or triplicate; the results were averaged expressed to the nearest 0.5 seconds.
Table 2. Subject Activated Partial Thromboplastin Times (Seconds) for Each Reagent
Diagnosis Normal range (±2 SD) Factor VHI-deficient Hemophilia 1 2 3 4 5 6 7 8 9t 10 lit 12 13* Factor IX-deficient Hemophilia 14 15 16 17 Factor Xl-deficient Hemophilia 18 19 20 Prothrombin deficiency Hageman (factor XII) homozygote Factor XII heterozygote Fletcher homozygote Fitzgerald homozygote LK (lupus anticoagulant)
Factor Assay Percentage
A Ellagic Acid
B Ellagic Acid
C Kaolin
D Silica
E Celite
KPTT Kaolin
23.0-36.0
23.5-33.0
32.0-45.5
30.0-40.5
32.0-41.5
33.0-51.5
0.0 4.5 4.8 14.0 15.0 22.5 27.0 29.0 40.0 48.0 49.0 50.0 48.0
78 76.5 76.5 62.5 54.0 54.5 34.0* 48.5 40.5 32.5* 38.0 36.0* 32.0*
58.5 51.5 51.5 47.0 42.5 77.0 35.0 59.0 33.5 33.5 32.0* 30.0* 33.5
111.0 109.0 108.0 84.5 70.0 75.0 48.0 69.5 42.0* 52.0 45.0* 49.0 46.0
93.5 106.5 89.0 81.0 64.5 80.5 45.0 58.5 46.0 45.0 53.0 39.5* 41.0
75.0 85.0 78.5 66.0 60.5 67.0 46.0 62.0 45.0 44.0 42.5 42.5 45.0
160.5 119.0 127.0 101.5 87.5 84.0 63.0 73.5 59.5 51.5* 52.5 57.0 52.5
0.0 3.0 16.0 13.0
81.5 81.5 32.5* 33.0*
58.0 62.0 34.0 35.0
115.5 85.5 46.5 54.5
136.5 106.0 45.0 47.5
78.0 83.0 44.5 43.0
157.0 91.0 61.5 65.0
0.0 2.5 52.0 9.5
87.5 99.5 33.5* 36.5
104.5 93.5 34.5 36.5
123.0 110.5 54.5 50.0
86.5 107.5 45.0 67.0
87.5 92.0 45.0 45.5
154.0 136.0 62.0 65.0
0.0 33.0 0.0 0.0
148 34.5* 62.0 130.0
— 38.5 — —
— 52.5 — —
220.0 45.0 186.0 202.0
179.0 44.5 192.0 133.0
337.0 56.5 247.0 215.0
—
66.5
45.0
60.0
80.0
45.0
103.0
* Individuals whose AFTTs fell within normal limits. t Hemophilia A carriers. t von Willebrand's disease.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Manufacturers: A, Dade Division, American Hospital Supply; B, Ortho Diagnostics, Inc.; C, Hyland Division Travenol Laboratories, Inc.; D, General Diagnostics, Division Warner-Lambert Co.; E. BBL, Division, Becton-Dickinson and Co.
24
HATHAWAY ET AL.
Table 3. Effect of Heparin on the Activated Partial Thromboplastin Time for Each Reagent* APTT Reagent and Normal Range (±2 SD)
APTT (Sec)
1 25 1 15
33.0 41.5 35.5 39.5
1 15 1 15
47.0 54.0 45.5 50.5
1 15 05 1
47.5 56.0 53.0 62.0
A (23.0-36.0) B (23.5-33.0) C (32.0-45.5) D (30.0-40.5) E (32.0-41.5) KPTT (33.0-51.5)
* The amount of heparin that prolongs the APTT beyond the normal range is shown.
Heparin^ was mixed w|th pooled normal plasma in final concentrations ranging from 0.05 to 0.25 u/ml plasma and the APTT and KPTT tests were done on each sample with the use of the above-described methods. Results Based on recent (past two years) stpdies of control subjects in our laboratory, the range for the KPTT for 70 normal adults was 37-50 seconds (mean = 44 sec). These values agree well with the values found for the KPTT for the 20 control subjects of this study (see Table 1). Table 1 also shows the range of values for each of the commercial reagents obtained in the testing of the same normal controls. The upper limits of normal for the clotting times for each reagent were set at a value equal to the mean plus two standard deviations (SD). This value also agrees well with the upper limit of the distribution range. However, the suggested normal range is frequently wider than those determined here. Table 2 shows the data obtained when each patient's blood was tested with each reagent. As can be seen, all reagents were equally satisfactory in determining severe deficiencies of factors VIII, IX, XI, and XII and the homozygous states of Fletcher (prekallikrein) and Fitzgerald (high-molecular-weight kininogen) traits. However, the ellagic acid reagents (A and B) missed ten minor factor deficiencies. The commercial kaolin (C) reagent missed two, the silica reagent (D) missed one, the KPTT reagent missed one, and the celite reagent missed none. The magnitudes of the de% Panheparin 1,000 u/ml, Abbott North Chicago, Illinois.
ficiencies not detected ranged from 13 to 50%. In addition, strict adherence to the upper limits of normal was necessary in order to detect the minor deficiencies shown in Table 2. All reagents detected the moderately severe anticoagulant effect in the sample from the lupus patient. In Table 3 the effect of heparin on the APTT for each reagent is shown. Again, the ellagic acid reagent (A) was less sensitive to the anticoagulant effect of heparin than were the other reagents. That is, reagent A did not detect in-vitro heparin until the concentration was 0.25 units/ml plasma, while the other reagents detected 0.1 unit/ml. The KPTT, with its higher upper limits, detected heparin at the 0.05 unit/ml concentration. Discussion This study was stimulated by the clinical observation of three patients with mild hemophilia, who had been referred to our laboratory for diagnosis after two other hospitals had determined that their APTTs were normal.5 Data for two of the patients, with 13% and 16% factor IX deficiencies, are shown in Table 2 (Patients 16 and 17). The third patient had mild factor Vlll-deficient hemophilia (22%). A shown by Proctor and Rapaport6 in their classic paper in 1961, the activated partial thromboplastin time test should be able to detect deficiencies of 30% or less in order to be a clinically useful screening test. They also demonstrated that the sensitivity of their test was related to the type of phospholipid material in their reagents; i.e., there was more sensitivity with a chloroform extract of rabbit-brain thromboplastin than with human-brain cephalin. Table 1 suggests that the differences in sensitivities of the commercial tests are related to the activating agents used rather than the sources of phospholipid, since all manufacturers used rabbit-brain cephalin except the manufacturer of reagent B, which used bovine-brain cephalin. No correlation with incubation times was observed. However, some other variable could be responsible for the lesser sensitivity of the ellagic-acid-activating reagents (A and B). A previous study by Sibley and others9 compared APTT reagents by recording the sensitivities of the reagents used in specific factor assays. They found the ellagic acid reagent (A) to be relatively insensitive to factor IX; however, another reagent (D) was even more insensitive to factor IX. Reagent D did detect the minor factor IX deficiencies in our studies. Abildgaard and Harrison1 showed that the ellagic acid reagent (A) would not detect the homozygous state of Fletcherfactor deficiency at an incubation time of 4 minutes; Fletcher-factor deficiency was detected by reagent A with 3 minutes' incubation in our study.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Heparin (Units/ml)
A.J.C.P. • January 1979
Vol. 71 . No. 1
APTT AND MINOR COAGULOPATHIES
of the systems. Each laboratory should know the sensitivity of the APTT system used in relation to detection of minor coagulopathies. When it becomes necessary to change to a new reagent system, steps to ensure sensitivity for detection for minor defects should be taken. Also, each clinician should understand the variability of the different APTT systems in monitoring heparin therapy and interpret published and personally obtained data accordingly. Acknowledgment. Dr. Robert Waldman supplied the Fitzgeralddeficient plasma.
References 1. Abildgaard CF, Harrison J: Fletcher factor deficiency: Family study and detection. Blood 43:641-644, 1974 2. Hathaway WE, Solomons CC, Ott JE: Platelet function and pyrophosphates in osteogenesis imperfecta. Blood 39:500509, 1972 3. Hathaway WE, Belhasen LP, Hathaway HS: Evidence fora new plasma thromboplastin factor. I. Case report coagulation studies, and physical physiochemical properties. Blood 26: 521-532, 1965 4. Markay AZ, Waterbury L: The activated partial thromboplastin time as a monitor of heparin therapy: A warning. John Hopkins Med J 140:311-315, 1977 5. Montgomery RR, Dubansky AS, Hathaway WE: Failure to detect mild hemophilia using the activated partial thromboplastin time (APTT). Clin Res 24:178A, 1976 6. Proctor RR, Rapaport SI: The partial thromboplastin time with kaolin. A simple screening test for first stage plasma clotting factor deficiencies. Am J Clin Pathol 36:212-219, 1961 7. Salzman EW, Deykin D, Shapiro RM, et al: Management of heparin therapy. Controlled prospective study. N Eng J Med 292:1046-1050, 1975 8. Shapiro GA, Huntzinger SW, Wilson JE: Variation among commercial activated partial thromboplastin time reagents in response to heparin. Am J Clin Pathol 67:477-480, 1977 9. Sibley C, Singer JW, Wood RJ: Comparison of activated partial thromboplastin reagents. Am J Clin Pathol 59:581-586, 1973
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Because both commercial reagents and reagents made in the laboratory may vary from batch to batch and year to year, certain precautions should be taken by each laboratory that uses the APTT to screen for bleeding diatheses, including mild hemophilia. Each laboratory should establish the normal ranges for their reagents and APTT by study of blood from healthy subjects. The upper limit for healthy subjects should be compared with values obtained for patients with minor defects of factors VIII, IX, and XI in the 2040% range. New batches of reagents should be standardized by measurement of values for pooled normal control plasma, upper- and lower-limit normal-subject plasmas, or plasmas with known minor deficiencies. In this manner the reproducibility and validity of the APTT in detection of minor coagulopathies can be maintained. The APTT has also been used to monitor heparin therapy.78 Recent studies by Shapiro and others8 have compared various APTT systems in the detection of the heparin effect on plasma. Reagent systems that detected heparin in a dose-related linear fashion were comparable to our study's B, D, and KPTT (by tilt tube) methods. Less satisfactory were reagents comparable to our A, C, and E. The relative insensitivity of the ellagic acid reagent (A) was confirmed by the results in Table 3. Comparisons of the APTT with whole-blood clotting times in samples from patients receiving heparin therapy showed poor correlation related to characteristics of the APTT system used.4 The results reported here should not be construed as advocating one APTT system rather than another. Emphasis is placed on the differences in sensitivities
25
Hathaway, William E., Assmus, Sherry L., Montgomery, Robert R., and Dubansky, A. Stephen: The activated partial thromboplastin time and minor coagulopathies. Am J Clin Pathol 71: 22-25, 1979. Five commercially available activated partial thromboplastin time (APTT) test systems were compared with the kaolin partial thromboplastin time (KPTT) method to determine sensitivity in detecting minor coagulation defects. All reagent systems detected severe factor VIII-, IX-, and Xl-deflcient hemophilia. Homozygous states of factor XII deficiency, Fletcher factor deficiency, and high-molecularweight kininogen deficiency (Fitzgerald trait) also showed abnormally long APTTs by all systems. Of 19 samples from patients with deficiencies of factors XII, VIII, IX, XI, and II ranging from 2.5 to 52%, eight had deficiencies that were not detected by reagent A (ellagic acid); two, by reagent B (ellagic acid); two, by reagent C (kaolin); one, by reagent D (silica); one, by the KPTT method. All deficiencies were detected by reagent E (celite). Heparin effect on plasma was less well detected by reagent A (ellagic acid) than with the other test systems. APTT test systems can vary greatly in their abilities to detect minor coagulation abnormalities. (Key words: Activated partial thromboplastin time; Minor coagulopathies; Hemophilia; Heparin monitoring.)
Department of Pediatrics, University of Colorado Medical Center, Denver, Colorado
method of Proctor and Rapaport, 6 which has been the standard method in our laboratory since 1962. Methods
THE ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) test is widely used as a plasma-clotting test to determine abnormalities due to defects in procoagulants or to assess anticoagulants such as heparin or the "lupus anticoagulant." Various reagents are commercially available for performance of this test. While these reagents are usually provided with positive controls to demonstrate efficacy in detection of severe coagulation defects like hemophilia due to deficiencies of factors VIII and IX, little information about the sensitivities of these reagents in the determination of minor and moderately severe coagulation defects is available. Recent experience in our laboratory has suggested that some of these APTT methods are not as sensitive as others in the detection of mild hemophilia. We report the results of a comparison of some of the commercially available methods for APTT testing with the kaolin partial thromboplastin time (KPTT)
The normal subjects were ten women and ten men who were members of a group of adult subjects used to obtain pooled normal plasma for all coagulation assays in this laboratory. Twenty-six patients with a variety of coagulation abnormalities (Table 2) were selected after prior complete coagulation analysis had established their diagnoses. In addition, each patient had the appropriate specific factor assay repeated on the same plasma sample used for the APTT testing. Methods for the specific factor assays have been described, 2,3 except for the Fitzgerald factor assay, which was performed with the use of Fitzgerald-deficient plasma and the KPTT system. The APTT tests were performed according to the instructions provided by the manufacturers (see Table 1) with an automatic timing device;t batches of reagents to complete the entire study were purchased in JulyAugust 1975; details of reagents and incubation times are shown in Table 1. The KPTT was prepared with the use of the method of Proctor and Rapaport and
Received October 20, 1977; received revised manuscript and accepted for publication January 3, 1978. Supported in part by U. S. Public Health Service Grant ROl HL 18507. Address reprint requests to Dr. Hathaway: Department of Pediatrics, University of Colorado, Denver, Colorado 80262.
* Falcon, Oxnard, California. t Fibrometer, Becton-Dickinson & Co., Cockeysville, Maryland.
0002-9173/79/0100/0022 $00.70 © American Society of Clinical Pathologists
22
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
All subjects' blood samples were collected by a careful two-syringe technic in plastic syringes containing a citrate-citric-acid anticoagulant (three parts 0.1 M sodium citrate plus two parts 0.1 M citric acid) in a proportion of one part anticoagulant to nine parts whole blood. Plasma was prepared by centrifugation by 3,500 x g for 20 minutes in a refrigerated centrifuge (4 C) and stored in capped plastic tubes* until used. The plasma was prepared and stored without contact with glass. Samples from patients and normal subjects were tested fresh or within a week of storage at - 7 0 C. Plasma to be frozen was recentrifuged at 35,000 x g (4 C) to remove all platelets. Plasma to be tested fresh was stored at 4 C for no longer than 1-2 hours.
23
APTT AND MINOR COAGULOPATHIES
Vol. 71 . No. I
Table I. Reagent Specification for Activated Partial Thromboplastin Time
Reagent Description
Incubation Time (Min)
A. Rabbit brain cephalin plus ellagic acid B. Bovine-brain cephalin plus ellagic acid C. Rabbit-brain cephalin and kaolin D. Rabbit-brain cephalin plus micronized silica E. Rabbit-brain cephalin plus cetite KPTT (Proctor and Rapaport) rabbit-brain cephalin plus kaolin
3 3 3 5 1 3
Mean ± 2 SD (Sec)
Distribution Range (Sec)
Suggested Normal Range (Sec)*
29.5 (23.0-36.0) 28.0(23.5-33.0) 40.0 (32.0-45.5) 35.5 (30.0-40.5) 37.0(32.0-41.5) 42.0(33.0-51.5)
25.0-36.5 23.5-33.0 32.5-44.0 31.5-40.0 32.0-42.0 37.5-51.0
23.6-31.6 0.25-40.0 29.0-43.5 27.0-39.0 33.0-40.0 37.0-50.0
* The suggested norma] range refers to package inserts, although each manufacturer recommends that each user establish its own normal range.
performed exactly as described in the original publication,6 with the Bell and Alton chloroform extract of rabbit-brain thromboplastin; the end point in seconds was determined by tilt-tube method with the use of new glass tubes. The APTT tests were performed by
the same technician, while the KPTT tests were performed by the technician usually doing routine KPTT testing in this laboratory. All tests were done in duplicate or triplicate; the results were averaged expressed to the nearest 0.5 seconds.
Table 2. Subject Activated Partial Thromboplastin Times (Seconds) for Each Reagent
Diagnosis Normal range (±2 SD) Factor VHI-deficient Hemophilia 1 2 3 4 5 6 7 8 9t 10 lit 12 13* Factor IX-deficient Hemophilia 14 15 16 17 Factor Xl-deficient Hemophilia 18 19 20 Prothrombin deficiency Hageman (factor XII) homozygote Factor XII heterozygote Fletcher homozygote Fitzgerald homozygote LK (lupus anticoagulant)
Factor Assay Percentage
A Ellagic Acid
B Ellagic Acid
C Kaolin
D Silica
E Celite
KPTT Kaolin
23.0-36.0
23.5-33.0
32.0-45.5
30.0-40.5
32.0-41.5
33.0-51.5
0.0 4.5 4.8 14.0 15.0 22.5 27.0 29.0 40.0 48.0 49.0 50.0 48.0
78 76.5 76.5 62.5 54.0 54.5 34.0* 48.5 40.5 32.5* 38.0 36.0* 32.0*
58.5 51.5 51.5 47.0 42.5 77.0 35.0 59.0 33.5 33.5 32.0* 30.0* 33.5
111.0 109.0 108.0 84.5 70.0 75.0 48.0 69.5 42.0* 52.0 45.0* 49.0 46.0
93.5 106.5 89.0 81.0 64.5 80.5 45.0 58.5 46.0 45.0 53.0 39.5* 41.0
75.0 85.0 78.5 66.0 60.5 67.0 46.0 62.0 45.0 44.0 42.5 42.5 45.0
160.5 119.0 127.0 101.5 87.5 84.0 63.0 73.5 59.5 51.5* 52.5 57.0 52.5
0.0 3.0 16.0 13.0
81.5 81.5 32.5* 33.0*
58.0 62.0 34.0 35.0
115.5 85.5 46.5 54.5
136.5 106.0 45.0 47.5
78.0 83.0 44.5 43.0
157.0 91.0 61.5 65.0
0.0 2.5 52.0 9.5
87.5 99.5 33.5* 36.5
104.5 93.5 34.5 36.5
123.0 110.5 54.5 50.0
86.5 107.5 45.0 67.0
87.5 92.0 45.0 45.5
154.0 136.0 62.0 65.0
0.0 33.0 0.0 0.0
148 34.5* 62.0 130.0
— 38.5 — —
— 52.5 — —
220.0 45.0 186.0 202.0
179.0 44.5 192.0 133.0
337.0 56.5 247.0 215.0
—
66.5
45.0
60.0
80.0
45.0
103.0
* Individuals whose AFTTs fell within normal limits. t Hemophilia A carriers. t von Willebrand's disease.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Manufacturers: A, Dade Division, American Hospital Supply; B, Ortho Diagnostics, Inc.; C, Hyland Division Travenol Laboratories, Inc.; D, General Diagnostics, Division Warner-Lambert Co.; E. BBL, Division, Becton-Dickinson and Co.
24
HATHAWAY ET AL.
Table 3. Effect of Heparin on the Activated Partial Thromboplastin Time for Each Reagent* APTT Reagent and Normal Range (±2 SD)
APTT (Sec)
1 25 1 15
33.0 41.5 35.5 39.5
1 15 1 15
47.0 54.0 45.5 50.5
1 15 05 1
47.5 56.0 53.0 62.0
A (23.0-36.0) B (23.5-33.0) C (32.0-45.5) D (30.0-40.5) E (32.0-41.5) KPTT (33.0-51.5)
* The amount of heparin that prolongs the APTT beyond the normal range is shown.
Heparin^ was mixed w|th pooled normal plasma in final concentrations ranging from 0.05 to 0.25 u/ml plasma and the APTT and KPTT tests were done on each sample with the use of the above-described methods. Results Based on recent (past two years) stpdies of control subjects in our laboratory, the range for the KPTT for 70 normal adults was 37-50 seconds (mean = 44 sec). These values agree well with the values found for the KPTT for the 20 control subjects of this study (see Table 1). Table 1 also shows the range of values for each of the commercial reagents obtained in the testing of the same normal controls. The upper limits of normal for the clotting times for each reagent were set at a value equal to the mean plus two standard deviations (SD). This value also agrees well with the upper limit of the distribution range. However, the suggested normal range is frequently wider than those determined here. Table 2 shows the data obtained when each patient's blood was tested with each reagent. As can be seen, all reagents were equally satisfactory in determining severe deficiencies of factors VIII, IX, XI, and XII and the homozygous states of Fletcher (prekallikrein) and Fitzgerald (high-molecular-weight kininogen) traits. However, the ellagic acid reagents (A and B) missed ten minor factor deficiencies. The commercial kaolin (C) reagent missed two, the silica reagent (D) missed one, the KPTT reagent missed one, and the celite reagent missed none. The magnitudes of the de% Panheparin 1,000 u/ml, Abbott North Chicago, Illinois.
ficiencies not detected ranged from 13 to 50%. In addition, strict adherence to the upper limits of normal was necessary in order to detect the minor deficiencies shown in Table 2. All reagents detected the moderately severe anticoagulant effect in the sample from the lupus patient. In Table 3 the effect of heparin on the APTT for each reagent is shown. Again, the ellagic acid reagent (A) was less sensitive to the anticoagulant effect of heparin than were the other reagents. That is, reagent A did not detect in-vitro heparin until the concentration was 0.25 units/ml plasma, while the other reagents detected 0.1 unit/ml. The KPTT, with its higher upper limits, detected heparin at the 0.05 unit/ml concentration. Discussion This study was stimulated by the clinical observation of three patients with mild hemophilia, who had been referred to our laboratory for diagnosis after two other hospitals had determined that their APTTs were normal.5 Data for two of the patients, with 13% and 16% factor IX deficiencies, are shown in Table 2 (Patients 16 and 17). The third patient had mild factor Vlll-deficient hemophilia (22%). A shown by Proctor and Rapaport6 in their classic paper in 1961, the activated partial thromboplastin time test should be able to detect deficiencies of 30% or less in order to be a clinically useful screening test. They also demonstrated that the sensitivity of their test was related to the type of phospholipid material in their reagents; i.e., there was more sensitivity with a chloroform extract of rabbit-brain thromboplastin than with human-brain cephalin. Table 1 suggests that the differences in sensitivities of the commercial tests are related to the activating agents used rather than the sources of phospholipid, since all manufacturers used rabbit-brain cephalin except the manufacturer of reagent B, which used bovine-brain cephalin. No correlation with incubation times was observed. However, some other variable could be responsible for the lesser sensitivity of the ellagic-acid-activating reagents (A and B). A previous study by Sibley and others9 compared APTT reagents by recording the sensitivities of the reagents used in specific factor assays. They found the ellagic acid reagent (A) to be relatively insensitive to factor IX; however, another reagent (D) was even more insensitive to factor IX. Reagent D did detect the minor factor IX deficiencies in our studies. Abildgaard and Harrison1 showed that the ellagic acid reagent (A) would not detect the homozygous state of Fletcherfactor deficiency at an incubation time of 4 minutes; Fletcher-factor deficiency was detected by reagent A with 3 minutes' incubation in our study.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Heparin (Units/ml)
A.J.C.P. • January 1979
Vol. 71 . No. 1
APTT AND MINOR COAGULOPATHIES
of the systems. Each laboratory should know the sensitivity of the APTT system used in relation to detection of minor coagulopathies. When it becomes necessary to change to a new reagent system, steps to ensure sensitivity for detection for minor defects should be taken. Also, each clinician should understand the variability of the different APTT systems in monitoring heparin therapy and interpret published and personally obtained data accordingly. Acknowledgment. Dr. Robert Waldman supplied the Fitzgeralddeficient plasma.
References 1. Abildgaard CF, Harrison J: Fletcher factor deficiency: Family study and detection. Blood 43:641-644, 1974 2. Hathaway WE, Solomons CC, Ott JE: Platelet function and pyrophosphates in osteogenesis imperfecta. Blood 39:500509, 1972 3. Hathaway WE, Belhasen LP, Hathaway HS: Evidence fora new plasma thromboplastin factor. I. Case report coagulation studies, and physical physiochemical properties. Blood 26: 521-532, 1965 4. Markay AZ, Waterbury L: The activated partial thromboplastin time as a monitor of heparin therapy: A warning. John Hopkins Med J 140:311-315, 1977 5. Montgomery RR, Dubansky AS, Hathaway WE: Failure to detect mild hemophilia using the activated partial thromboplastin time (APTT). Clin Res 24:178A, 1976 6. Proctor RR, Rapaport SI: The partial thromboplastin time with kaolin. A simple screening test for first stage plasma clotting factor deficiencies. Am J Clin Pathol 36:212-219, 1961 7. Salzman EW, Deykin D, Shapiro RM, et al: Management of heparin therapy. Controlled prospective study. N Eng J Med 292:1046-1050, 1975 8. Shapiro GA, Huntzinger SW, Wilson JE: Variation among commercial activated partial thromboplastin time reagents in response to heparin. Am J Clin Pathol 67:477-480, 1977 9. Sibley C, Singer JW, Wood RJ: Comparison of activated partial thromboplastin reagents. Am J Clin Pathol 59:581-586, 1973
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Because both commercial reagents and reagents made in the laboratory may vary from batch to batch and year to year, certain precautions should be taken by each laboratory that uses the APTT to screen for bleeding diatheses, including mild hemophilia. Each laboratory should establish the normal ranges for their reagents and APTT by study of blood from healthy subjects. The upper limit for healthy subjects should be compared with values obtained for patients with minor defects of factors VIII, IX, and XI in the 2040% range. New batches of reagents should be standardized by measurement of values for pooled normal control plasma, upper- and lower-limit normal-subject plasmas, or plasmas with known minor deficiencies. In this manner the reproducibility and validity of the APTT in detection of minor coagulopathies can be maintained. The APTT has also been used to monitor heparin therapy.78 Recent studies by Shapiro and others8 have compared various APTT systems in the detection of the heparin effect on plasma. Reagent systems that detected heparin in a dose-related linear fashion were comparable to our study's B, D, and KPTT (by tilt tube) methods. Less satisfactory were reagents comparable to our A, C, and E. The relative insensitivity of the ellagic acid reagent (A) was confirmed by the results in Table 3. Comparisons of the APTT with whole-blood clotting times in samples from patients receiving heparin therapy showed poor correlation related to characteristics of the APTT system used.4 The results reported here should not be construed as advocating one APTT system rather than another. Emphasis is placed on the differences in sensitivities
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