A fully automated method for clot based coagulation panels using an MCA Multistat Centrifugal Analyzer

THROMBOSIS RESEARCH 39; 485-499, 1985 0049-3848/85 $3.00 + .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

A FULLY AUTOMATEDMETHOD FOR CLOT BASED COAGULATION PANEIS USING AN MCA MULTISTAT CENTRIFUGALANALYZER

D.R. Hoak, E.F.

Mammen, S.K. Banerjee and G. Kaldor VA Medical Center, Allen Park, MI 48101 and Departments of Pathology, Physiology and Surgery, Wayne State University School of Medicine, Detroit, MI 48201 U.S.A.

(Received 2.1.1985; Accepted in revised form 17.4.1985 by Editor J.G. White) (Received in final form by Executive Editorial Office 7.6.1985) ABSTRACT Small modifications of the commercially available software of the I.L. Multistat Centrifugal Analyzer (MCA) enabled us to evaluate clot based prothrombin times (PT), activated partial thromboplastin times (APTT) This imparted and thrombin times (TT) simultaneously within one run. a great deal of flexibility to this procedure in that batches and/or panels of clot based and other types of turbidimetric tests can be performed concurrently in any combination. The PT and TT by this turbidimetric procedure correlated very well with those of the Fibrometer in normal specimens as well as in a wide variety of coagulation The turbldimetric APTT procedure, however, of ten produced defects. The clotting times longer than those achieved with the Fibrometer. discrepancy between the Fibrometer and turbidimetric APTT procedures was shown to be the consequence of greater sensitivity of the latter elevated fibrinogen split to detect low concentrations of heparin, products and mild factor deficiencies. The available clot based procedures are suitable for use in detecting specific factor deficiencies and the presence of coagulation inhibitors.

INTRODUCTION Prothrombin times, activated partial thromboplastin times and thrombin times as clot based tests are widely used to evaluate the integrity of the coagulation system. In the majority of diagnostic laboratories, fully automated or semiautomated dedicated instruments, equipped with electromechanical or optical detectors, are utilized to perform these tests. The flexibility of these dedicated instruments is obviously limited. In recent years several investigators have used centrifugal analyzers to perform certain coagulation tests successfully (l-4). We have explored the potential of the centrifugal analyzer in coagulation testing relative to flexibility, accuracy, precision and sensitivity. The fully computerized Multistat III Analyzer, equipped with nephelometric and turbidimetric capabilities, was used throughout this work.

Key words:

Centrifugal

Analyzer,

computer

485

directed

coagulation

panels.

486

METHOD FOR CLOT BASED COAGULATION

It was our intent to develop methods equally which could be performed in any combination account of this research is given below.

Vo1.39,

No.4

useful in batches and panels during a single run. A brief

METHODS Most of this work was carried out using the I.L. Multistat III Centrifugal Analyzer and loader system (Instrumentation Laboratories, Lexington, MA) and with the BBL Fibrometer (Becton, Dickinson and Co., Cockeysville, MD). Some preliminary experiments were performed on a Hitachi 557 dual wavelength, dual beam spectrophotometer (Hitachi, LTD., Tokyo, Japan). The MCA III is equipped with an 8K RAM programmable microcomputer. The programming language is FOCAL 8 (Digital Equipment Corporation, Maynard, MA). The “clot” tape was generously provided by Instrumentation Laboratories, Spokane, WA. The original programs of this tape were designed by scientists of Instrumentation Laboratories, to accI.lmulate data points at three different intervals. The relative number of data The time between points available for all three intervals was limited to 55. data points at any given interval was left to the discretion of the operator. We chose 40 data points at 1 second each, 10 at 2 seconds each and 5 at 4 seconds each, giving a total reaction time of 80 seconds for each test. Thus, we were able to accommodate not only the normal range of PT, APTT and TT, but a The original tape directed the analyzer to use perreasonable abnormal range. cent transmittance in the calculation of clot reaction times. Our software modification directed the analyzer to read the instantaneous absorbance change This approach minimized potential (delta A), rather than the actual absorbance. inaccuracies due to lipemia, turbidity and bilirubinemia. The next modif ication of the software was aimed at determining the reaction time. As seen in Figure 1, turbidity increases at a point where the sigmoid curve begins to ascend. In order to insure that the signal is indeed the beginning of a positive slope and not just noise, the curve has to first achieve at least 10% of the maximal delta A over two consecutive readings before it is recognized as the TI Tll TIYI (sac) reaction time. The equation listed in Figure 1 and its FIG. 1 heading achieves this goal. Schematic illustration of the reaction time AI and AI1 reflect the for the automated APTT, PT and TT tests. change in optical density The computer is instructed to record early over two consecutive readchanges in optical density rather than the ings of at least 10%. TI is of fibrin formation, i.e. terminal phase the initial time relative the formation of a solid clot. to AI while TII is the time recorded. AII-AI [TII-TI] + TI + TD Reaction time = [z]

at AII. mixed.

487

METHOD FOR CLOT BASED COAGULATION

Vo1.39, No.4

TD is the time delay beginning after reaction constituents have AA max is the maximal optical density change during the reaction.

been

The second term The first term of the equation is the delay time (TD). is the time of the beginning of the first interval (TI) during which the O.D. The third term of the equation is an interreached 10% of the maximum. MI-AI) I. Since the analyzer measures the O.D. polation factor [(TII-TI) * AA max theoretically there is one interof each sample at the end of each interval, val (TII - TI) margin of uncertainty between the “true time” at which the required O.D. (10% of maximal) was first reached and the time at which the actual measurement was performed. The numerical value of the interpolation Therefore, including this term is always a fraction of that of the interval. term to the equation reduces the margin of uncertainty regarding the PT, APTT and TT to a fraction of the interval between two measurements. Finally, a program alteration flags reactions conditions such as a minimal acceptable clot density. Instrumentation: components : the

The cuvette

that

fail

is composed instrumentation system rotor, the loader and the analyzer.

to

of

meet

three

specific

distinct

The rotor consists of 20 cuvettes. Each cuvette has two distinct chambers, an inner sample chamber and an outer reagent chamber, separated by a dam. In most cases the first cuvette is used as a reference, containing water, and the remaining 19 for the unknowns and control specimens. We used both known normal and known abnormal controls at each 20 patient interval or at least three times in an eight hour period. The loader is fully automated. It fills the cuvette rotor with samples and reagents. The volumes of the syringes which load the reagents and samples are 250 and 100 ~1, respectively. A total volume switch allows the addition of diluent or wash solution, in addition to sample or reagent. The term primary reagent refers to a solution which, when mixed with the plasma, invariably initiates the reaction. The term secondary reagent refers to a solution which is a necessary component of the reaction, but which will not initiate it. As the data in Table 1 indicate, the loader delivered 30 ul plasma plus 45 ul distilled water in the sample compartment of the cuvette-rotor in case of the PT. For the same test the 75 pl thromboplastin were delivered into the reagent compartment of the cuvette-rotor without additional distilled water wash, The data regarding APTT and TT test are also specified in Table 1. The so-called wash volume served the purpose to ensure a fixed final volume for each test rather than a wash out of the pipette. To insure that the difference between the sample aspirated and the sample delivered did not introduce an analytical error, we compared mixtures in which the final concentration of the plasma coagulation factors was kept constant but the plasma versus wash volume ratio varied between 1: 1 and 1:3. These experiments (not shown here) failed to reveal any difference in the PT, APTT or TT results. Table 1 illustrates the loader settings used in our experiments.

METHOD FOR CLOT BASED COAGULATION

488

Vo1.39, No.4

After the rotor has been filled, it is placed in the analyzer and subjected to a centrifugal field capable of propelling the sample over the separating dam into the reagent chamber. After a dramatic stop, to insure proper mixing, the ensuing reaction is read through the polished window at the terminal end of the cuvette's reagent compartment while spinning at 1000 RPM. Actual readings begin after 4 seconds, using a wave length of 405 nM at 37O C. TABLE 1 Cuvette Rotor Filling Data

Assay

Sample Volume

PT

30 nl plasma

APTT

50 nl plasma

TT

5Oul plasma

Secondary Reagent None

75 nl Thrombofax

None

Total Volume

Primary Reagent Volume

Total

30 nl plasma +45 ~1 wash

75 nl thromboplastin (30% of 250)

75 ~1 (no wash used)

50 pl plasma +45 ~1 Thrombofax +3 ~1 wash

50 ~1 CaCl (20% of 250)

50 ul (no wash used)

50 ~1 plasma +5 ul wash

50 ~1 thrombin (20% of 250)

50 ~1 thrombin +50 ~1 wash (40% of 250)

In the majority of experiments, we used brain thromboplastin for PT, Thrombofax for APTT and Fibrindex for TT, all of which were obtained from Ortho Diagnostics (Raritan, NJ). In addition to the commercial reagents mentioned, we have prepared a number of specialized coagulation reagents using well established methods. Citrated Plasma: Venous blood was drawn into a 5 ml blue top tube containing Plasma was obtained by centrifuging the samples at 0.11 M sodium citrate. 2000 x g for 5 minutes at 25O C. We used both fresh and frozen plasma for the screening studies. of a Adsorbed Plasma: Ten ml of normal titrated plasma were mixed with 100 1.11 6M BaC12 slurry at 25O C, stirred for 15 minutes and centrifuged at 1000 x g for 10 minutes. After decanting the solution, the pH was adjusted to 7.5 with 1.0 N NaOH. 1000 units per ml of USP porcine Na heparin were obtained from Heparin: Organon Inc. (West Orange, NJ) and diluted I:10 with deionized water, giving 100 U/ml. Aged Serum: for 3 days.

Serum from normal blood was allowed to stand at room temperature

Lyophilized reference "NORMAL" and "ABNORMAL" controls were Controls: obtained from Ortho Diagnostics and reconstituted with deionized water. The

Vo1.39, No.4

METHOD FOR CLOT BASED COAGULATION

489

controls were assayed by the manufacturer to yield the following: FIBRINOGEN 280-300 mg/dl, TT NORMAL 7-10 seconds, PT NORMAL lo-14 seconds, PT ABNORMAL 2-2.5 x NORMAL, APTT NORMAL 22-36 seconds, APTT ABNORMAL 2-2.5 x NORMAL. In some experiments, we attempted to differentiate between Plasma Mixtures: factor deficiencies and inhibitors. Therefore, we repeated the PT and/or APTT with a mixture (1:l v/v> of the patient's plasma and standard normal plasma. The volumes of these plasma mixtures were the same as the sample volumes in Table 1. Protamine Sulfate Titration: Protamine sulfate reagent (0.20%) was obtained The from American Dade and reconstituted per manufacturer's instructions. protamine reagent was mixed with heparinized plasma in concentrations of 0.01 to 10.0 U/ml. Fibrinogen Degradation Products (FDP): One ml of 10 NIH U/ml thrombin in 0.10 M CaC12 was added to 9 ml of normal plasma. The plasma was allowed to clot at 37V. The plasminogen in the clot was converted to plasmin with 10,000 IU of streptokinase. The Thrombo-Wellcotest was used to assay the FDP concentration. The average concentration of these mixtures was 1200 &ml. In some experiments normal plasma was diluted with increasing amounts of FDP ranging from 10 to 300 pg/ml. The effect of dilution was assessed by diluting with 50 millimolar Tris-HCL, pH 7.2. Controls and samples were then assayed for PT, APTT and TT. Prothrombin Time (PT): Lyophilized brain thromboplastin was obtained from Ortho Diagnostics (Raritan, NJ) and reconstituted with deionized water per manufacturer's recommendations. Thirty pl of control or patient plasma sample volume were added to 75 ~1 of thromboplastin primary reagent along with 45 pl of deionized water (wash volume) (Table 1). Activated Partial Thromboplastin Time (APTT): The APTT reagent, Thrombofax, was obtained from Ortho. CaC12 reagent was prepared by adding 11.1 g CaC12 to 1 1 deionized water, giving a concentration of 0.10 M CaC12, and then further diluted to give 0.05 M/l. Fifty ~1 of control or sample plasma (sample volume) were mixed with 45 ul of Thrombofax (secondary reagent) and incubated at 37'C for 3 minutes. After incubation, 50 ~1 CaC12 were added when the analyzer initiated its centrlfugation sequence (Table 1). Thrombin Time (TT) and Fibrinogen Concentration: Lyophilized thrombin containing 100 NIH U/ml was obtained from American Dade (Miami, FL). It was reconstituted with 1 ml of deionized water per manufacturer's recommendations. A further dilution was prepared by adding 9 ml of 0.1 M CaC12 to the reconstituted thrombin, yielding a final concentration of 10 NIH U/ml. TT and fibrinogen concentration were determined by mixing 50 ~1 of thrombin reagent with 50 pl of control plasma (sample volume) and 50 pl of deionized water (wash volume) (Table 1). Between 0.05 and 0.70 absorbance the maximum absorbance change at the end of the reaction (80 seconds) was directly proportional to the fibrinogen concentration. When the maximal absorbance change exceeded 0.70, the plasma was diluted 1:l with physiological saline solution and the test repeated.

METHOD FOR CLOT BASED COAGULATION

490

Vo1.39, No.4

The absorbance of the unknown was calculated from a calibration The calibration curve was prepared with normal plasma containing 40-400 fibrinogen (FBG). A calibration curve was prepared once a day.

tests

In the screening tests were done concurrently.

compiled

in

Tables

3 and

4

the

PT,

curve. mg/dl

APTT and

TT

RESULTS At the beginning of this work we accepted the results obtained with the Fibrometer as the “standard”. Initial experiments were carried out using the however, these proportions did same plasma/reagent ratios as the Fibrometer. not always provide comparable results when applied to the MCA. Therefore, we had to change the proportions of plasma and/or reagents. As the results in Figure 2 show, using normal plasmas in the MCA, a mixture of 30 pl of plasma, 45 ul of wash and 75 pl of reagent gave a reaction time between 11 and 13 seconds for the PT. At this plasma/reagent ratio we achieved excellent agreement between the two PT methods. Since the original plasma/reagent proport ions the were altered , we invariably changed the calcium concentration and necessary to investigate ionic strength of these mixtures. It was, therefore, the influence of Ca2+, ionic strength, pH and temperature on the PT, APTT and TT. Ca2+ concentrations between 50 and 100 pmol/ml did not alter significantly the PT, APTT and TT. of less than 5?u~~~/~~e~~~~~ PT, APTT and TT were prolonged (Figure 3).

. \

. \

OOIb

Somplsvolume (pl)

FIG. 2 MCA prothrombin times relative to sample volume. PT of a markedly prolonged (o), moderately prolonged (A) and normal (0) plasma relative to sample volumes used on the MCA. The Fibrometer comparison revealed PTs of 35, 17 and 12 set, respectively. Reagent mixture see Table 1.

Increasing the ionic strength of the incubation mixture invariably increased the time prior to the turbidity and depressed the extent of the maximal absorbance change of all three tests studied (Figure 4). In both respects the APTT was sensitive most and the TT least sensitive to the change in ionic strength. The pH optimum for the PT, APTT, in terms of the and TT, defined shortest delay prior to the onset of turbidity and the maximum absorbance achieved, was between pH 7.5 and pH 8.5.

Vo1.39, No.4

METHOD FOR CLOT BASED COAGULATION

491

‘---_p---*-s-s

0

. 0

60-

./* 1 .

3 2 ; F

_

/

30-

/

,/’

0’.

&_d----A-----&

Oi

0

OD5 CoCl~ (moles/l)

0.10

02

0.3 KC1 hdes/l)

a4

05

0.6

The

(Figure 5A and 5B respectively). The temperature optimum of al.1 three tests was 37’C. APTT and PT, unlike TT, were significantly prolonged as the temperof the reaction mixtures was ature lowered from 35’C to 15“C (Figure 6).

60‘3

I=

0.1

FIG. 4 effect oE ionic strength on the APTT, PT and TT of normal plasma. Figure 4A shows the effect of increasing ionic strength on absorbance; Figure 4B shows the effect on clot time. Sample ?? =APTT, o=PT, A-TT. volumes are identical to those in Figure 3.

FIG. 3 The effect of Ca2+ on PT (o), APTT (0) and TT (A) using the react ion mixture MCA. Each contained Ca2+ as indicated.

J E

]A_ 0

.-----

-a

_

The data produced by these experiments established the methodological criteria for all subsequent testing.

30-

0-L , 5

,

I

6

)

,

7

PH

,

,

6

,

,

,

9

FIG. 5 The effect of pH using the MCA on PT (o), APTT (0) and TT (A) relative to A absorbance and B clot time.

r

10

Figures 7 through 9 are scatterograms obtained by comparing the PT, TT and APTT results of the MCA method with those of the Fibrometer. The results shown in Figures 7 and 8 demonstrate that there is excellent agreement between the MCA and the Fibrometer methods in a wide range

METHOD FOR CLOT BASED COAGULATION

492

. 'i;603 : F_

\

30-

%,

?? \. \ .\ .-----• O\

A---A-~-b oJ-0 , I 20

O\ -A-A I

0-O

of normal, moderately and significantly prolonged PTs (Figure 7) and TTs (Figure 8). Table 2 contains the results of the statistical evaluation of these experiments. It may be seen that the S(y,x) of the PT was 1.7297 and that of the TT indicating 1.8788 that the intermethod variability between the MCA and the fibrometer is low. It is obvious that, with regard to the numerical values, the PT and TT results of either method can be interchanged without ambiguity.

I

1

30 Temperature

Vo1.39, No.4

40

(‘Cd

FIG. 6 The effect of temperature using the MCA on PT (o), APTT (0) and TT (A), relative to the reaction time.

TABLE 2 Statistical Evaluation of the Comparison of Coagulation Tests Performed Using Fibrometer and MCA Coagulation Test

Number

Type of Patients Tested

PT 100 Normal + TT 100 Normal + APTTk 50 Normal APTT* 50 Abnormal APTT'k 100 Normal + * The calculations were done on the first separately and then combined.

Statistical Parameters Slope Intercept S(y,x)

Abnormal Abnormal

0.8252 2.3851 1.7297 0.9168 0.8253 1.8788 0.8550 4.6118 1.9169 1.0952 14.0020 10.2950 Abnormal 1.3499 5.3004 11.8216 same normal and abnormal patient population,

The APTT results compared favorably in the lower half of the normal range However, in the upper half of the "generally accepted" normal (Figure 9). range (24-34 seconds) the fibrometer gave significantly shorter APTTs than the MCA. This discrepancy between the two methods was always unidirectional and, therefore, significant because while the MCA reflected increased APTT values, the Fibrometer indicated normal. The statistical evaluation of the APTT comparison gave also low intermethod variability between the MCA and the fibrometer on a patient population whose APTT was in the lower half of the normal APTT range, S(y,x) = 1.9169. On the other hand the S(y,x) of the APTT method was 10.2950 on a patient population whose APTT was in the upper half of the normal APTT range or was abnormal.

voi.39,

METHOD FOR CLOT BASED COAGULATION

No.4

493

4OJ

.

3o-

.

c 8 t

/ ??

I

I

/

??

. d@@ .

.:

.

20 -

2

IO -

/

1

IO

.

/

?? 0. .

??

/

.

,*

.

00 0

40

$0 20 FibromslsrPT kc)

IO

20 Fibrometer

30 TT (set)

40

FIG. 8 Scatterogram of TTs on 100 plasma samples correlating the MCA with the Fibrometer.

FIG. 7 Scatterogram of PTs on 100 plasma samples correlating the MCA reaction time with the Fibrometer readings.

TABLE 3 Coagulation Profile for Plasmas with Prolonged APTT Using the MCA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

APTT(MCA)

APTT

Sample Number

APTT(FIB)

(Set)

(Se4

FIB

MCA

27 31 25 25 24 29 28 34 31 40 33 27 32 34 23 24 28 30 37 22

43 50 39 40 38 46 50 47 51 58 41 46 42 54 52 37 39 43 50 34

Sample

16 19 14 15 14 17 22 13 20 18 8 19 10 20 19 13 11 13 13 12

TT

FEG

Other MCA

(Set>

Sample + Normal Plasma

MCA

MCA

4 11 2 5 5 1 20 9 15 2 1 3 1 14 19 5 5 1 11 6

7

450

7 8 7 6 14 19 7 7 7 6 5 22 17 8 7 7 6 7

340 355 410 320

FDP=>40pg/ml

520 500 475 380 375

Protamine=5 set FDP=>40ug/ml with serum=24 set with serum=26 set

FDP=>40pg/ml Protaminez7 set 360 480 570 280 325

METHOD FOR CLOT BASED COAGULATION

494

.

60 4

This discrepancy concerning the two methods was followed up by scrutinizing 20 randomly selected samples that gave different APTTs by performing a few additional coagulation tests. These tests included protamine titration, to detect the presence of heparin, FDP assays and mixing experiments using reference plasmas and aged sera. Table 3 shows the results of these experiments. In two cases (#7,15) protamine titration indicated the presence of heparin and three samples (f/2,8,14) contained FDPs. In each instance of heparin or FDP the MCA gave prolonged APTT values, while the Fibrometer reflected no The remainder of the abnormalities. patient samples were normalized with the addition of reference plasma or serum suggesting a factor deficiency or the presence of an anticoagulant.

.

I

I

60

30 Fibromhr

APT1

Vo1.39, No.4

ircc)

FIG. 9 Scatterogram of APTTs on 100 plasma samples correlating the MCA with the Fibrometer. Note the discrepancies in the higher ranges.

TABLE 4 Comparative Methodological Diagnostic Evaluation

Diagnosis

Number of Cases

APTT (Set) MCA FIB

PT (Set) MCA FIB

TT (Set) MCA FIB

t of Pts in Normal Range MCA FIB

Liver Disease

20

AVG

53

38

18

17

8

9

0

11

Oral Anticoagulants Heparin

30

AVG

51

39

24

22

7

10

0

18

50

AVG

76

57

18

16

12

16

0

7

10

AVG

63

53

41

36

29

38

4

10

Thrombolytic Therapy MI

3

58

41

24

23

32

37

-

Leukemia (Myeloblastic) Sepsis

2

72

48

16

14

7

8

-

2

52

48

23

19

7

6

-

Pneumonia

3

33

31

12

11

7

7

Upper G.I.

1

44

41

19

18

7

7

-

Massive Trauma

1

43

41

18

18

7

7

-

Yo1.39, No.4

METHOD FOR CLOT BASED COAGULATION

495

To explore the discrepancy further, we studied PTs, APTTs and TTs with both the MCA and the Fibrometer in plasma of 100 patients known to have minor or major coagulation abnormalities. The results compiled in Table 4 show that in 20 patients with significant liver function damage, with or without jaundice, the MCA always showed longer APTTs. The same pattern was true in the case of patients who received oral anticoagulants or heparin therapy. Of 30 patients who had received thrombolytic therapy four had normal APTTs, when the MCA was used. With the Fibrometer, 10 patients had normal APTTs. No significant differences were noted in the PTs and TTs. Since the discrepancies observed between the Fibrometer APTTs and the MCA APTTs could be due to factor deficiencies, we investigated the time related deterioration of the labile plasma factors, factors V and VIII. One aliquot of plasma from young healthy donors was incubated at 25'C. A second aliquot of the same plasma was kept at 4“C. The APTT was determined at 0 time and in successive one hour time intervals over six hours with both procedures. While the APTT of the plasma kept at 4'C did not change over a six hour period with both procedures, the plasma incubated at 25'C gave with the MCA assay, unlike the Fibrometer, longer APTTs as the incubation progressed (Figure 10). It is suspected that decreases in the labile factor concentrations explain the progressively may increasing APTTs in the MCA, and that the MCA based APTT is more sensitive to changes in factor concentrations. Under comparable conditions, over a 6 hour incubation period with both methods, the PTs and TTs remained the same. In order to compare the effect of factor deficiencies on the APTT of both methods further, we performed these tests with various factor deficient plasmas. The results shown in Table 5 demonstrates that the APTTs increased with the MCA assay more significantly than with the Fibrometer when the factor level was decreased. It thus seems that the MCA APTT is the more sensitive test.

40?? -• ./

20

, 0

I I

I 2 lncutmtii

I 3 time h8rs)

I 4

, 5

I 6

FIG. 10 The effect of storage time on the APTT of normal plasma as measured by the MCA (0) and the Fibrometer (A). ?? = 25' C MCA; A= 25'C Fibrometer; o = 4'C MCA; A= 4'C Fibrometer.

Next, we studied the effect of fibrin(ogen) degradatlon products on the APTT, PT and TT of normal plasma. Increasing amounts of FDP were added to plasma. As can be seen in Figure 11, there was no significant difference in the PTs and TTs with both methods, but a difference was noted with the APTTs at higher FDP concentrations. Again, the MCA APTT seemed to give longer clotting times, suggesting greater sensitivity.

Vo1.39, No.4

METHOD FOR CLOT BASED COAGULATION

496

TABLE 5 APTTs: Factor XI

X

VIII

The Influence of Factor Deficient Plasma on Both Procedures

% Deficient

MCA

Fibrometer

0

--I31 33 40 45 84

32 30 34 43 60

V

25 50 75 100 0 25 50 75 100

33 37 40 46 84

32 33 34 36 64

I

0

33 34 35 41 62

32 27 32 35 56

25 50 75 100

Factor

% Deficient MCA 32 37 34 41 84

30 33 31 36 90

- 31 31 30 31 84

30 32 31 30 100

0 25 50 75 100

0 25 50 75 100

Fibrometer

Finally, we investigated the effect of heparin on the PTs, TTs and APTTs of both methods. Heparin was added in increasing amounts to normal plasma. While no differences were noted in the PTs and TTs, the MCA APTTs were again longer than the Fibrometer APTTs, as can be seen in Figure 12. Figure 13 illustrates the precision of the APTTs determined with the MCA and the Fibrometer in one normal plasma and three patient plasmas that gave prolonged APTTs. It can be seen that the MCA APTTs have far less of a scatter than the Fibrometer measured APTTs. statistical The results of the 6. analysis are presented in Table They indicate that the average coeffirepeated cient of variation of the measurements was 14.34% in the fibrometer and 6.1575% in the MCA.

0)

0

,

,

,

,

,

1

50 FDP lpghll

I

,

I

,

loo

FIG. 11 The effect of Fibrin(ogen) degradation products on APTT (o,e), PT (0 ,I > and TT (A,A) using the MCA and Fibrometer respectively.

METHOD FOR CLOT BASED COAGULATION

Vo1.39, No.4

497

TABLE 6 Statistical Fibrometer and MCA Fibrometer ST. DEV. APTT Mean

d of of Tests 1 2 3 4

20 20 20 20

APTT Mean

COEFFICIENT OF VARIATION

42.2 t8.7661 20.77 66.1 k8.8625 13.41 42.7 27.04 16.49 29.55 k1.9783 6.69 Avg. coeff. var. = 14.34%

COEFFICIENT OF VARIATION

48.3 3.1287 68.2 4.2895 42.4 3.8355 30.9 0.8756 Avg. coeff. var. =

WA

90-

MCA ST. DEV.

FIB

WA

FIB

MCA

FIB

6.47 6.29 9.04 2.83 6.1575%

MCA

FIB

90

‘ii 3

60-

01

0

,

,

,

,

,

,

0.05 Hsparin (IU/ml)

,

,

,

60

,

pt.1

0.10

FIG. 12 The effect-of heparin on APTT (o,o), PT (0 ,I) and TT (A,A) using the MCA and Fibrometer, respectively.

PI. 2

Pt. 3

PI. 4

FIG. 13 The precision of the MCA APTT (e) versus the Fibrometer APTT (0) on 3 abnormal and 1 normal plasma samples. Note the greater scatter with the Fibrometer.

DISCUSSION The clot based methods of coagulation testing are high volume laboratory procedures. For screening and for anticoagulant monitoring purposes, the clot based tests have held their ground in the face of the newer, readily automatable coagulation tests using synthetic substrates. In fact, the increasing demand for these time honored tests inspired technology to make available automated instrumentation for clot based coagulation testing (5). The Electra 700 and Coag-A-Mate are fully automated photoelectric instruments, while the Fibrometer is a semi-automated electromechanic device dedicated to clot based

METHOD FOR CLOT BASED COAGULATION

498

coagulation determine

testing. Both Electra PTs and APTTs simultaneously

700 and (5).

the

Coag-A-Mate

Vo1.39, No.4

X2 can

be

used

to

In this work we used a general purpose, fully automated laboratory analyzer for clotting studies. The methods developed permit the simultaneous evaluation of TTs , PTs and APTTs in one run. The flexibility of this approach is such that it is equally useful in high volume, batch type, as in individual panel type operations.

“end tion gous

The idea for determining the reaction time was not based on the notion of point” or total clot formation. Rather, it was predicated on the assumpthat an initial increase in turbidity, as seen in Figure 1, would be analoto the onset of turbidity seen in manual clotting methods.

The greatest usefulness of these methods and in the follow-up of several frequently obtained during the screening.

is in coagulation screening encountered pathological

studies patterns

The addition of the TT to the coagulation screening panel was useful in revealing the adequacy of fibrinogen fibrin conversion along with the total clottable fibrinogen concentration (6). This proportionality between the maximal turbidity increase and the total clottable fibrinogen concentration, during thrombin induced clotting, was first described by Clauss (7) and confirmed by other investigators (2,8,9). Consequently, the three tests performed actually provide four important laboratory results (PT, APTT, TT and FBG cone.). The various patterns obtained as a result of the simultaneous performance of these tests can be readily followed up in a systematic logical manner (6). The follow-up of the initial screening often involves a search for inhibitors (anticoagulants) or factor deficiencies. the methods employed In general, in this work are suitable to resolve the most commonly encountered coagulation The search for inhibitors can be easily accomplished using mixtures problems . of patient plasma with normal plasma. The ratio of patient plasma to normal plasma may be varied as needed as long as the total volume of these plasma mixtures in the assays remains the same as specified in the method section (6). The search for factor deficiencies may be accomplished expeditiously with a panel of tests consisting of mixtures of the patient plasma with known factor deficient plasmas (6). The Fibrometer has been used as the reference instrument throughout these Every effort was made to assess the effect of reagent volume (Fig. 2), studies. Ca2+ concentration (Fig. 3), ionic strength (Fig. 4), pH (Fig. 5) and temperature (Fig. 6) on the results of PT, APTT and TT.

the

Excellent Fibrometer

agreement was found and the MCA methods.

in

the

PT (Fig.

7)

and TT (Fig.

8)

between

The APTT results were often longer in the MCA than in the Fibrometer (Fig. This discrepancy appears to be related to the presence of low 9). concentrations of inhibitors and/or mild factor deficiencies (Fig. 10-12, Table than hinder the 3-5) which delay the onset of the turbidity increase more

Vo1.39, No.4

METHOD FOR CLOT BASED COAGULATION

499

It was documented electromechanical effect of the polymerized fibrin strand. that both the sensitivity and the precision of the photometric MCA APTT procedure is excellent in the presence of small concentrations of heparin (Fig. 12,13). The methods developed during this work require smaller volumes of plasma and reagents than used routinely. This is advantageous from the point of view of economy and also extends the usefulness of these methods in pediatric settings. The disposable rotors may be partially filled with reagents and kept in the refrigerator until used. This may be convenient to perform panels of tests on a "stat" basis. For these reasons it is apparent that the methods constitute an important step in the automation of coagulation methods.

We wish to thank Drs. W. DiBattista, J. Sturgeon, K. Nyikes and D. Kovesdi for their technical assistance and D. James and K. Chesney for their clerical contribution. REFERENCES 1.

BOSTICK, W.D., BAUER, J., MORTON, J.M., BURTIS, C.A. Coagulation-time determination with automatic multivariable analysis, by use of a miniature centrifugal fast analyzer. In: Methods for the Centrifugal Analyses, J. Savoy, R.E. Cross (Eds) Amer. Assoc. Clin. Chem., Washington, 1978, p. 219-227.

2.

DENEGRI, E., PRENCIPE, L. Kinetic determination of fibrinogen with centrifugal analyzer. -Clin. Chem. 28, 1502-1505, 1982.

3.

HILLS, L.P., LORENZI-ANDERSON, M., HUEY, E.E., TIFFANY, T.O. Use of a centrifugal analyzer in coagulation testing. --~ Semin. Thromb. Hemost. 2, 217-229, 1983.

4.

WALENGA, J., FAREED, J., BERMES, E.W. Automated instrumentation and the laboratory diagnosis of bleeding and thrombotic disorders. -Semin. Thromb. Hemost. 9_, 172-193, 1983.

5.

WALENGA, J., HOPPENSTEADT, D., FAREED, J., SIBERMAN, S., SHEVLIN, P. Automated clot-based methods in coagulation testing: Current and future considerations. --Semin. Thromb. Hemost. 9_, 239-243, 1983.

6.

LUCAS, F. Lab tests and clinical clues equal diagnosis of bleeding disorders. Diagnostic Med. 6_, 65-77, 1983.

7.

CLAUSS, A. Gerinnungsphysiologische Schnellmethode Fibrinogens. Acta Haematol. 17, 237-246, 1957.

8.

BECKER, U., BARTL, K., WAHLEFELD, A.W. A functional photometric assay for plasma fibrinogen. -Thromb. Res. 35, 475-484, 1984.

9.

INADA, Y., OKAMOTO, H., KANAI, S., TAMAURA, Y. Faster determination of clottable fibrinogen in human plasma: An improved method and kinetic study. -Clin. Chem. 24, 351-353, 1978.

zur

Bestimmung

a

des