Evaluation of Three Methods for Plasma
Fibrinogen Determination
DAVID
J.
STEVENS,
B.S., MT(ASCP),
Department
AND MICHAEL
J.
SANFELIPPO,
of Laboratory Medicine, Columbia
Milwaukee, Wisconsin S3 211
M.S., M T (ASCP)
Hospital,
ABSTRACT
Stevens, David J., and Sanfelippo, Michael J.: Evaluation of three methods
for plasma fibrinogen determination. Am. J. Clin. Pathol. 60: 182-187, 1973.
The determination of plasma fibrinogen as clottable protein, as precipitable
protein, and by the reaction rate in the conversion of fibrinogen to fibrin is
evaluated in terms of the principles of analysis, normal ranges, reproducibility, reliability, and the influence of physiologic antagonists. Comparison
of the values obtained from a normal population indicated that the mean
value determined by the turbidimetric method was significantly lower than
the values obtained by either the clottable protein assay or the conversion
reaction rate. However, repeated assays performed on pooled plasma showed
comparable reproducibility of the three methods. Physiologic antagonists
such as heparin, lipemia, and antithrombin VI affected the turbidimetric
method to the greatest extent, but also significantly affected the conversion
reaction rate; the clottable protein assay appeared to be the least affected.
of plasma fibrinogen is
universally accepted as a useful biological
test for the diagnosis, treatment, and prognosis of certain hemorrhagic disorders. 1 ' 8 - 12
There is, however, little agreement on the
best method of fibrinogen measurement.
The methods most commonly used to measure fibrinogen involve direct and indirect
measurement of the clottable protein, 2 ' 4 - 5
turbidimetric methods which depend on
the precipitation of fibrinogen,6-10 the reaction rate in the conversion of fibrinogen to
fibrin,8 and immunoassay with the use of
specific antiserum.
In this study, utilizing the determination
of fibrinogen by assay of the clottable protein, 2 precipitable protein, 6 and the reaction rate in the conversion of fibrinogen to
T H E DETERMINATION
Received October 2, 1972; received revised manuscript November 6, 1972; accepted for publication
November 13, 1972.
Address reprint requests to: Mr. Michael Sanfelippo, Department of Laboratory Medicine, Columbia Hospital, 3321 North Maryland Avenue,
Milwaukee, Wisconsin 53211.
fibrin, we have investigated possible interfering substances and compared the three
procedures.
Materials and Methods
Materials
Whole blood was obtained by a clean
venipuncture and mixed nine parts to one
part of either 3.8% sodium citrate or 0.1
M sodium oxalate. The cellular fraction
was sedimented by centrifugation at 3,400
r.p.m. for 3 min.
Phosphate Buffer, (a) 40.828 Gm. of
KH 2 P0 4 ; (b) 42.594 Gm. of N a 2 H P 0 4 (anhydrous); (c) 0.5 Gm. of potassium sorbate;
(d) 500 ml. of distilled water (pH 6.5 ±
0.02).
Sodium Chloride. 0.85% in distilled
water.
Thrombin. Parke-Davis topical thrombin
was reconstituted from the lyophilized state
and made up to a concentration of 1,000
NIH units per ml. using 0.85% NaCl. It
182
August 1973
PLASMA FIBRINOGEN DETERMINATION
was then stored in lusteroid tubes at —20
C. until needed.
Biuret Reagent. Harleco biuret reagent,
Gornall-Bardwell-David, Philadelphia, Pa.
Trypsin Inhibitor.
Type I-S, Sigma
Chemical Company, St. Louis, Mo.
Beta-lipoprotein. Human Fraction III-O,
N u t r i t i o n a l B i o c h e m i c a l Corporation,
Cleveland, Ohio.
Human Protein Standard. Crystallized
Human Albumin, Dade, Miami, Florida.
Streptokinase-Streptodornase
(SK). Lederle Varidase (100,000 units streptokinase)
was reconstituted from the lyophilized state
with 0.85% NaCl to yield a concentration
of 10,000 units per ml. This solution was
then added to normal plasma for the production of fibrinogen split products.
Heparin. Sodium heparin (Lilly) was diluted with 0.85% NaCl to give a stock concentration of 400 NIH units per ml.
Late Fibrinogen Split Products (L-FSP).
Plasma containing high concentrations of
L-FSP was produced by adding sufficient
SK to normal oxalated plasma to produce
a final concentration of 1,000 units per ml.
of plasma and incubated for 24 hr. at 37 C.
At the end of the incubation period, trypsin inhibitor, 1 mg. per ml. of plasma, was
added to arrest the fibrinogenolytic activity.
Beta-lipoprotein. Plasma containing betalipoprotein human fraction III-O was produced by adding sufficient beta-lipoprotein
to normal oxalated plasma to produce
final concentrations of 5, 7.5, 10, and 15
mg. per ml. of plasma.
Methods
Turbidimetric Method (Martinick and
Berry).6 Either oxalated or citrated plasma,
0.5 ml., was added to 6 ml. of phosphate
buffer (test), and to 6 ml. of 0.85% NaCl
(blank). These were mixed, allowed to
stand at room temperature for 5 min., and
the optical density (O.D.) was read at 450
nm. using a spectrophotometer (Bausch &
Lomb Spectronic 20).
Clottable Protein Assay (Bowie and as-
183
sociates 2 ). Either oxalated or citrated
plasma, 0.5 ml., was diluted with 4 ml. of
0.85% NaCl and clotted with 0.1 ml. of
1,000 N I H units of thrombin. The fibrin
clot was collected on a glass rod, compressed
against the side of the glass tube, and
thoroughly washed with 0.85% NaCl. The
fibrin was blotted dry and placed in biuret
reagent. The optical density (O.D.) was
read at 540 nm. on a spectrophotometer
(Bausch & Lomb Spectronic 20).
The Conversion Reaction Rate. Either
oxalated or citrated plasma, 0.1 ml., was
diluted with 0.9 ml. of Owren's buffer (Fibrinogen Determination Set, Dade). Then
0.2 ml. of this dilution was warmed to
37 C. on a fibrometer (BBL Fibrometer),
and 0.1 ml. of a standardized thrombin (Fibrinogen Determination Set, Dade) was
added and the clotting time recorded.
Comparison of the Three Methods. Plasmas from 32 normal laboratory employees
were assayed by the three methods described above. All determinations were
made in duplicate. The reproducibility of
the three methods was determined on
pooled oxalated plasma.
The Effect of Lipemia. Randomly selected lipemic plasmas were assayed by the
three methods described above. The lipoprotein fraction of the plasmas was assayed
according to the method of Noble. 0
The Effect of Heparin. Four concentrations of heparin were added to samples of
pooled oxalated plasma. These samples and
a sample without heparin were assayed for
fibrinogen concentration by the three methods described above.
The Effect of Antithrombin VI (Fibri7iogen Split Products). Normal oxalated
plasma obtained from a single normal
donor was mixed four parts to one part
autologous serum containing various concentrations of L-FSP. The fibrinogen concentration was then determined by the
three methods described above. The final
concentration of L-FSP was determined by
the method of Merrens and associates.7
184
AJ.CP.-VoL
STEVENS AND SANFELIPPO
59
Table 1. Normal Subject Population
Turbidimetric
Method
Conversion Reaction
Rate
Clottable protein
Assay
116-796
250-756
278-811
Mean (£), mg. per 100 ml.
392
485
482
Standard deviation (SD),
mg. per 100 ml.
Coefficient of variation (CV), %
144
133
127
37
27
26
Range, mg. per 100 ml.
Coefficient of correlation (r)
0.93
(vs. clottable
protein assay)
0.90
{vs. turbidimetric method)
0.92
{vs. conversion
reaction rate)
t test
3.43 (p < 0.01)
{vs. clottable
protein assay)
2.53 {p < 0.2)
{vs. turbidimetric method)
1.47 (J>>0.1)
{vs. conversion
reaction rate)
Table 2. Reproducibility
Turbidimetric
Method
Conversion Reaction
Rate
250-294
283-311
362-388
Mean (X), mg. per 100 ml.
276
303
376
Standard deviation (SD),
mg. per 100 ml.
12
9
8
Range, mg. per 100 ml.
2
3
4
Coefficient of variation (CV), %
Clottable Protein Assay
t test
4.33 {p < 0.01)
{vs. clottable
protein assay)
3.37 {p < 0.01)
{vs. turbidimetric method)
3.60 {p < 0.01)
{vs. conversion
reaction rate)
F-test
1.84 {p < 0.01)
{vs. clottable
protein assay)
1.70 {p < 0.01)
{vs. turbidimetric method)
1.08 {p < 0.01)
{vs. conversion
reaction rate)
Table 3. Lipemic Plasmas
Plasma
Plasma
Plasma
Plasma
Plasma
Plasma
t test
1
2
3
4*
5
6
Turbidimetric
Method
(mg. per 100 ml.)
Conversion
Reaction Rate
(mg. per 100 ml.)
Clottable
Protein Assay
(mg. per 100 ml.)
88
144
37
86
33
150
260
225
209
286
206
500
462
370
257
306
219
581
6.72 {p < 0.01)
{vs. clottable
protein assay)
5.33 {p < 0.01)
{vs. turbidimetric method)
2.85 {p > 0.01)
{vs. conversion
reaction rate)
* Subjected to lipoprotein fractionation.*
August 1973
185
PLASMA FIBRINOGEN DETERMINATION
Table 4. Beta-lipoprotein Interference
Conversion
Reaction Rate
(mg. per 100 ml.)
Clottable
Protein Assay
(mg. per 100 ml.]
466
411
377
377
377
480
472
474
480
479
Turbidimetric
xVIethod
( m g.. per 100 ml.)
Conversion
Reaction Rate
(mg. per 100 ml.)
Clottable
Protein Assay
(mg. per 100 ml.)
130
130
120
120
120
120
106
106
106
100
100
98
311
311
288
288
288
288
272
272
275
275
261
261
336
336
338
340
336
336
336
333
333
333
328
326
10
6
1
Turbidimetric
Method
mg. per 100 ml.
Concentration of beta-lipoprotein
fraction I I I - 0
(mg. per 100 ml.)
0
5.0
7.5
10
15
206
222
209
206
201
Table 5. Effects of Heparin
Conceiitration of heparin (NIH
units per ml.)
0
0.5
1.0
1.5
2.0
5.0
Coefficient of van ation (CV),
%
Table 6. Effect of Antithrombin VI
Control sample
Sample 1
Sample 2
Sample 3
Sample 4
Coefficient of variation, %
t-test
Concentration
of L-FSP
(Mg. per ml.)
Turbidimetric
Method
(mg. per 100 ml.)
Conversion
Reaction Rate
(mg. per 100 ml.)
Clottable
Protein Assay
(mg. per 100 ml.)
4.0
4.0
22
30
180
192
187
129
120
120
206
206
178
178
152
279
263
261
261
261
25
8.2;! (p < 0.01)
(vs. clottable
protein assay)
12
4.09 (p > 0.01)
(vs. turbidimetric methods)
3
9.59 (p < 0.01)
(vs. conversion
reaction rate)
186
STEVENS AND SANFELIPPO
Results
Comparison of the Three Methods
A plasma sample was obtained from each
of 32 normal laboratory employees and the
fibrinogen concentration determined by the
three methods described above. The clottable protein assay and the conversion reaction rate method gave the highest mean
values, 482 mg. per 100 ml. and 485 mg.
per 100 ml., respectively. By contrast, the
turbidimetric method gave a mean value
of 392 mg. per 100 ml. The t test was used
to ascertain the statistical significance of
these differences in mean values (Table 1).
In comparing the reproducibility of the
three methods, the clottable protein assay
had the best coefficient of variations, 2%,
followed by the conversion reaction rate,
3%, and then the turbidimetric method,
4% (Table 2). The t test demonstrated a
significant difference in the mean values
and the F test demonstrated a significant
difference in the precision of the three
methods. 13
Effect of Lipemia
As seen in Table 3, lipemia affected the
values obtained by the methods under investigation. The greatest interference was
seen in the turbidimetric method. Lipoprotein fractionation 9 of these plasmas revealed an essentially normal pattern, with
the presence of a small amount of chylomicrons. A series of in vitro studies revealed
that the addition of the beta-lipoprotein
fraction did not significantly affect the turbidimetric method or the clottable protein
assay method; the effect on the conversion
reaction rate was minimal (Table 4).
Effect of Heparin
With increasing concentrations of heparin, 0.5 to 5.0 N I H units per ml. of plasma,
there appeared to be a decrease in the detectable concentration of fibrinogen as
measured by the turbidimetric method and
the conversion reaction; the effect on the
clottable protein assay was insignificant
(Table 5).
A.J.C.P.—Vol.
Effect of Antithrombin
59
VI
As seen in Table 6, with increasing concentrations of L-FSP there was a significant
decrease in the ability to detect fibrinogen
by the turbidimetric method and the conversion reaction rate. Comparing the values of the control diluted with autologous
serum alone with that of the samples diluted with autologous serum containing
various levels of L-FSP, there was a difference in values which is not accountable to
the effect of dilution. The turbidimetric
method appears to be the most susceptible
to interference by L-FSP. This phenomenon was observed in both citrated and oxalated plasma.
Discussion
The recent interest in consumption coagulopathy and the fibrinolytic syndrome has
greatly increased the demand for an accurate, reliable determination of the plasma
fibrinogen concentration. T h e reliability of
a chemical determination is dependent
upon the utilization of reactions that are
specific for the component of interest. The
analysis of fibrinogen as a clottable protein
utilized two such reactions. First is the specific proteolysis of fibrinogen by thrombin
and second is the unique polymerization of
the resulting fibrin monomers. 11 In the turbidimetric method of analysis, this reliability is compromised by the fact that the
phosphate reagent produces a turbidity
that is nonspecific and semiquantitative. 4 ' 8
The data obtained in this study confirm
that the amount of turbidity produced by
the phosphate reagent is unreliable and in
practice yields erroneous results. This study
demonstrates the need for a fasting specimen, in that the presence of lipids in the
plasma has a direct effect upon the fibrinogen determinations (Table 3). However, the
addition of beta-lipoprotein human fraction III-O did not demonstrate a significant interference with the determinations
(Table 4). This finding, in addition to the
detection of chylomicrons in the lipemic
August 1973
PLASMA FIBRINOGEN DETERMINATION
plasma, suggests the possibility that the
interfering substance is the chylomicron
fraction. In pathologic conditions where
there are increases in fibrin or fibrinogen
split products, such as localized intravascular clotting, the need for accurate determination of fibrinogen concentration is critical. This study demonstrates (Table 6) that
the products of fibrinolysis directly affect
the values obtained by the turbidimetric
method. The presence of heparin can also
affect the values obtained by the turbidimetric method (Table 5). Thus, the reliability of the turbidimetric method is in
question in conditions where a reliable
value is essential.
The data from both the clottable protein assay of Bowie and colleagues 2 and
the conversion reaction rate show good correlation between the two methods when
used on plasma from normal subjects
(Table 1). Since both methods utilize the
conversion of fibrinogen to fibrin, both are
subject to the antagonistic effects of antithrombins such as heparin, and the effects
of fibrin or fibrinogen split products on
the polymerization of fibrin monomers.11 In
principle, these antagonistic effects should
be overcome to a large extent by the significant dilutions which both methods employ. In practice, however, it is evident
that the conversion reaction rate is more
sensitive to the effects of fibrin or fibrinogen split products (Table 6), and the effects
of heparin (Table 5), than the clottable
protein assay. There appears to be a lower
degree of correlation between the conversion reaction rate and the clottable protein assay in these studies than in the
study dealing with the 32 normal subjects
(Table 1).
Of the three fibrinogen methods studied, the clottable protein assay appears to
be the most specific and the least influenced by fibrin or fibrinogen split products, heparin or lipoproteins. Much emphasis has been placed on the need for a
rapid and precise method for determining
187
plasma fibrinogen levels. T h e question
arises whether precision should suffer for
the sake of a rapid laboratory test, or the
converse. The total time required for the
turbidimetric method was not more than
15 min. The total time required for the
conversion reaction rate was not more than
15 min., and the total time required for
the clottable protein assay was not more
than 30 min. Therefore, because none of
these assays required great amounts of
time, it seems that selection of the most
useful method should be made on the basis
of their accuracy and reliability.
Acknowledgment.
Dr. Lyle R. Heim gave valuable suggestions in the preparation of this manuscript, and Dr. Dann B. Claudon, Director, Department of Laboratory Medicine, Columbia Hospital,
encouraged us in this project.
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