131

Clinica Chimica Acta, 62 (1975) 131-136 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 7140

AN IMPROVED METHOD FOR PLASMA FIBRINOGEN THROMBIN TIME MEASUREMENT

ABDUS SALEEM

and KAY FRETZ

Division of Clinical Pathology, Department of Pathology, Milton The Pennsylvania State University, Hershey, Pa. 17033 (U.S.A.)

(Received

January

BASED ON

S. Hershey

Medical

Center,

15, 1975)

Summary Methods for plasma fibrinogen based on thrombin time are technically simple, rapid, and sensitive, although “false low” results may occur due to heparin interference. The present modification incorporates Polybrene into buffer to eliminate this heparin interference. The proposed method shows excellent agreement with a reference procedure based on clottable protein, and excellent day-to-day precision (C.V. 3.5%). The present method is easily adaptable to semi-automated measurements.

Introduction An ideal plasma fibrinogen procedure should be technically simple, rapid, specific, precise, and not subject to interference from heparin, bilirubin, hemolysis, hyperlipemia, or fibrinogen degradation products. Salt fractionation methods [l-6] are technically simple and rapid, but lack specificity [ 71. Clottable protein methods [8-111 though accurate and specific, are time consuming [ 121, and hence of limited value in emergency situations. Clot density methods [12-141 are rapid, but may be insensitive in low fibrinogen range [13,14]. Thrombin time methods [15-171 are rapid, sensitive, and adaptable to semiautomation [17] ; however, the presently available procedures are affected by heparin [ 7,171 and fibrinogen degradation products [ 71. We have recently reported a clot density procedure for plasma fibrinogen [ 141, incorporating hexadimethrine bromide (Polybrene obtained from Abbott Laboratories, South Passadena, Calif., U.S.A.) to neutralize plasma heparin. The same principle may be applied to thrombin time procedures to eliminate heparin interference.

132

Materials and Methods Blood samples

Nine parts of blood are mixed with one part of sodium citrate (3.8 g~lO0 ml); plasma is removed and stored at 4” C until ready for use. Reagents

Barbital buffer 0.1 M, pH 7.2: Sodium barbital 5.71 g, sodium chloride 2.93 g, pH adjusted to 7.2 with 1N HCI, volume made up to 1000 ml. Polybrene barbital buffer: 1 mg Polybrene/lOO ml barbital buffer. Phosphate buffer, 0.067 M pH 6.4. Thrombin 1000 units/ml obtained from Parke Davis, Hunt Valley, Md., U.S.A. Heparin sodium 1000 units/ml obtained from Riker Laboratories Inc., Northridge, Calif., US.A. Fibrinogen de~adation products (FDP) were prepared according to the method of Marder [IS]. Quantitation of FDP was done by Thrombo-Wellcotest, obtained from Burroughs Wellcome Co., Research Triangle Park, N.C., U.S.A. Plasma pool

A plasma pool was prepared and standardized as follows.

from titrated

plasma of 10 normal

donors,

Standardization of plasma pool. Reference method for fibrinogen-modified from Ware et al. [ 111 and Foster et al. [ 81. 1 ml of plasma was diluted with 6 ml of physiological saline and 3 ml of phosphate buffer in a 25 ml flask and clotted with 0.2 ml of thrombin (1000 units/ml). The clot was removed on an applicator stick, washed and blotted dry with filter paper. The clot was then scraped off the stick into a mixture of 1 ml Biuret reagent and 0.9 ml of NaOH (3 g/100 ml), and dissolved in a 56°C water bath. The absorbance was read at 545 nm against a reagent blank consisting of 1 ml of Biuret reagent and 1 ml of NaOH (3 g/100 ml). Calibration curve was constructed with bovine albumin (Fraction V, obtained from Technicon Inc., Tarrytown, N.Y., U.S.A.) Procedure for standardization. Fibrinogen concentration was measured ten times on the plasma pool using the reference method. The mean fibrinogen value of these ten observations was assigned to the plasma pool. This mean value was used to prepare a calibration curve for the proposed thrombin time procedure. Thrombin

time procedure

The procedure followed: Plasma pool was diluted 1 : 5, 1 : 10, 1 : 20 and 1 : 40 with Polybrene buffer. Using the Fibrometer (BBL, Division of Bioquest, Cockeysville, Md., U.S.A.) 0.2 ml of thrombin (50 units/ml) was added to 0.2 ml of each of the above dilutions and the clotting time was obtained. A calibration curve was drawn between clotting time and fibrinogen value of each dilution. The test sample was diluted 1 : 20 with Polybrene buffer and clotting time on the test sample was obtained as described above. The result was read off the calibration curve and multiplied by the dilution factor (X 20).

133

Note. If the thrombin Polybrene buffer (1 : 40 or lower dilution (1 : 2 or 1 : should be multiplied by the

time is too short, a higher dilution of plasma in 1 : 80) may be used. If thrombin time is too long a 5) may be used. Result from the calibration curve dilution used.

Results Effect

of varying thrombin

concentration

Calibration curves were drawn for the plasma pool using the following concentrations of thrombin: 5, 10, 25, and 50 units/ml. The results are shown in Fig. 1. With 50 units of thrombin/ml, there is a linear log-log relationship between thrombin time and plasma fibrinogen levels from 8 mg to 124 mg per dl. With lesser concentrations of thrombin, the log-log relationship between thrombin and plasma fibrinogen is non-linear over this range. Effect

of polybrene

on fibrinogen

recovery

Polybrene was added to barbital buffer in concentrations of 0, 1.0 and 2.5 mg/lOO ml. Fibrinogen concentration of the plasma pool was measured using these buffers. Recovery of fibrinogen with no Polybrene and 1 mg/lOO ml of Polybrene was 100.0%. Recovery with 2.5 mg/lOO ml was 104.0%. The calibration curve obtained with the plasma pool dilutions using 1 mg/lOO ml Polybrene in barbital buffer is shown in Fig. 2 (solid line). For comparison, a calibration curve obtained with plasma pool dilutions using barbital buffer without Polybrene is also shown (dotted line). Interference

The results are summarized in Table I. 1. Heparin. Heparin was added to the plasma pool, obtaining plasma concentrations ranging from 0 to 80 units/ml. Plasma fibrinogen on these hepa-

40 . A BARBITAL BUFFER .

POLYBRENE BUFFER

; 20 8 % r ; IO . z 8

5-

: 3-

L.

FIBRINOGEN

18 CONCENTRATION

Fig. 1. Effect of thrombin time measurements.

Fig. 2. Calibration

IN MG/OL

concentration

curves on plasma using:

(units/ml)

1, barbital

on calibration

buffer;

16 32 FleRlNCSEN CONCENTRATION

curve for fibrinogen

2, Polybrene

buffer.

64

128

IN MG/DL

based on thrombin

134 TABLE

I

EFFECT

OF INTERFERING

SUBSTANCES

ON FIBRINOGEN

RECOVERY _.~

Interfering

substance

COihXtP tration of fibrinogen

Recovery of fibrinogen

5%

recovery

(mg/dl)

.-_____I___-___

(mg/dU -._________~___

Heparin (units/ml) 10 20 40 60 80

280 280 280 280 280

269 285 282 223 156

102 101 80 56

Hemoglobin 280 350

280 280

272 280

97 100

280 280

284 280

101 100

430 430 430 332 332

430 334 290 111 0

100 78 67 33 0

280 280

260 280 ___________-___

93 100

96%

(m@,/lOO ml)

Bilirubin (mg/lOO ml) 8.7 19.5 Fibrinogen 13 64 128 240 483 Lipemia Lipemic Lipemic .-._

degradation

products (pg/ml)

serum/plasma ratio = 1 serum/plasma ratio = 3 -_ .-

:1 :1

rinized samples was determined by the thrombin time method as described with 1 mg Polybrene/lOO ml. Heparin in concentration upto 40 units/ml did not significantly affect fibrinogen recovery; higher concentrations of heparin resulted in progressively lesser recovery. 2. Hemoglobin. Hemolysed samples with hemoglobin up to 350 mg/lOO ml did not significantly influence the fibrinogen recovery. 3. Bitirubin. Bilirubin levels up to 19.5 mg/lOO ml did not interfere with fibrinogen recovery. 4. Fibrinogen degradation products (FDP). Normal or slightly elevated levels of FDP did not affect the recovery of fibrinogen. However FDP levels of 64 pg/ml reduced the fibrinogen recovery to 67%. With higher FDP levels, recovery pf fibrinogen showed progressive decline and at levels of 483 pg/ml no end point was observed. 5. Lipemia. Grossly lipemic serum was mixed with normal plasma in various proportions. Recovery of fibrinogen was not significantly influenced by lipemia. Comparison with “reference” method Plasma fibrinogen levels of 29 different plasma samples (16 heparinized, 13 non-heparinized) were determined by the reference method and proposed thrombin time technique. Fig. 3 shows the relationship of the two methods plotted on log-log paper. The correlation coefficient is 0.92.

135

0 NON HEPARINIZEO

PLASMA

A HEPARINIZED PLASMA . 0

A

.

0 .A

& 0

0

\ .

I 100

200

FIBRINOGEN

300

GO0

400

CONCENTRATION

BOO

IN MG/DL

(REFERENCE METHOD)

Fig. 3. Fibrinogen

by reference

method

versus thrombin

time by the proposed

method.

Reproducibility

The fibrinogen of the plasma pool, by the proposed thrombin time method, on 50 different days showed a mean of 320 mg/lOO ml with a range of 298-340 mg/lOO ml. The SD. was 11.2 mg/lOO ml (C.V. 3.5%).

Discussion If plasma is diluted sufficiently, fibrinogen concentration becomes the limiting factor for thrombin time [16]. Thrombin time methods are rapid, simple and sensitive; however different workers have used different concentrations of thrombin [7,16,17]. In the proposed method, we recommend 50 units/ml of thrombin, which gives a linear log-log relationship between thrombin time and fibrinogen concentration from 8-124 mg/lOO ml. With lower concentration of thrombin, this linear relationship does not hold (Fig. 1). With higher concentration of thrombin, thrombin times tend to be too short in normal subjects, which increased “within run” as well as “day-to-day” variability. Thrombin time procedures for fibrinogen are subject to heparin interference [7,17]. However, in the present procedure, heparin is neutralized by Polybrene in the buffer, and plasma heparin levels upto 40 units per ml do not cause significant interference. In the concentration used (1 mg/lOO ml) Polybrene does not have any anticoagulant effect as the calibration curves obtained on plasma with or without Polybrene are almost identical (Fig. 2). Fibrinogen degradation products have been reported to interfere with thrombin time and clottable protein procedures [ 71. This is also true of the present procedure (Table I). The present method shows good agreement with a “reference” method based on clottable protein procedure (Fig. 3). The day-to-day precision in our setting was considered satisfactory, with a coefficient of variation of 3.5%.

136

References 1

W.R.

2

P.E.

Howe,

Campbell

J. Biol.

and

J. Lab.

M.I.

Hanna,

Chem..

3

R.F.

Jacox,

4

R.G.

Martinek

Clin.

5

I.A.

Parfentjev,

M.L.

Johnson

6

D.A.

Podmore.

Clin.

Chim.

and

D.J.

Stevens

and

M.J.

L.B.

Foster,

C.S.

Frings

9

R.J.

Henry,

D.C.

Cannon

Nelson,

New

10

O.D.

Ratnoff

11

A.G.

Ware,

12

B.C.

Ellis

and M.M.

and

13

H.B.C.

14

A.

15

S. Borgstrom.

16

A.

17

T. Okuno

18

V.J.

Claws.

and

K. Krieg

and

V.

J.W.

Chir.

Clin.

Selenko, Shulman

Stand., 17

G.I. 90

(1957)

Am. and

Pathol.. Clin.

60

(1973)

Chem.,

(eds).

17

46

(1953)

470

182

(1971)

Clinical

Med.,

37

Arch.

Biochem.

Med.,

J. Clin.

(1945)

(1951)

58 (1961)

Horsfield,

Am.

1216

Chemistry:

Principals

J. Clin.

316 Biophys.,

13 (1947)

477 Pathol.,

Pathol.,

63

23

(1975)

(1970)

43

426

419

237

J. Med. W.R.

Biophys.,

242

Clin.

J. Lab. Fretz.

Biochem.

p. 458

Seegers,

and

15

10

Arch.

Winkleman

1974,

K.

(1937)

(1965)

J. Clin.

W.H.

Aulton

11

Cliffton,

Hochholzer,

J. Lab.

and

Hematol.,

N.R.

E.E. Am.

York,

C. Menzie,

Acta

Acta

Marder.

and

119

885

Chem.,

4 (3959)

and J.M.

A. Stransky,

A.F.

(1954)

and Acta,

Chem.,

235

Clin.

Sanfelippo.

Guest

Burrnester,

Saleem,

44

Berry.

7

and

(1923)

Med.,

R.E.

8

Harper

.J. Biol.

57

Technol., CarroII,

38

J. Biol.

(1972) Chem.,

196 244

(1969)

2111

231

and

Techniques.