Measurement of Plasma Fibrin D-Dimer Levels with the Use of
a Monoclonal Antibody Coupled to Latex Beads
CHARLES S. GREENBERG, M.D., DANA V. DEVINE, PH.D., AND KEITH M. McCRAE, M.D.
Recently, monoclonal antibody (DD-3B6) tofibrinD-dimer was
prepared and coupled to latex beads to provide a specific test
(Dimertest®) for fibrinolysis. The purpose of this study was to
evaluate the Dimertest assay as a clinical laboratory test for the
measurement of plasma fibrin D-dimer derivatives. The Dimer-
test assay specifically detected 2 ng/mL of purified fibrin D-
dimer or fibrin D-dimer/fragment E complex added to afibri-
nogenemia plasma but did not detect 500 ng/mL of either
fibrinogen fragments X, D, E, or 160 jtg/mL cross-linked fi-
brinogen. The fibrin(ogen) degradation product (FDP) assays of
American Dade or Wellcome Diagnostics detected 5.0 jig/mL
of fibrin D-dimer and from 1 to 10 /ug/mL of the other FDPs.
Twenty-eight percent of 150 random plasma samples assayed
from hospitalized patients were positive for fibrin D-dimer de-
rivatives. Plasma samples from 152 patients suspected of having
disseminated intravascular coagulation (DIC) were assayed for
serum FDP (Wellcome Diagnostics) and plasma fibrin D-dimer
derivatives. Samples from 69% of patients with serum FDP levels
less than 10 /tg/mL, and more than 90% of those with serum
FDP levels greater than 10 jtg/mL, were positive for fibrin D-
dimer derivatives. Dimertest results were not modified by hep-
arin, streptokinase, freeze-thawing, or clotting plasma. Serum
fibrinogen-related antigens were immunoadsorbed from Dimer-
test positive sera by anti-fibrinogen antibody and formalin-fixed
Cowan I strain Staphylococcus aureus. Analysis by sodium do-
decyl sulfate-polyacrylamide gel electrophoresis and protein
blotting with the use of monoclonal antibody DD-3B6 demon-
strated a protein band with similar mobility to purified D-dimer.
The measurement of plasma fibrin D-dimer derivatives by the
Dimertest assay is a rapid, sensitive, and specific laboratory test
forfibrinolysis.The Dimertest assay has proven to be a useful
addition to the clinical laboratory and should be helpful in the
diagnosis and management of patients with diseases associated
withfibrinolysis.(Key words: Fibrinogen degradation products;
Fibrin D-dimer) Am J Clin Pathol 1987; 87: 94-100
OVER THE PAST ten years, significant advances were
made in understanding the biochemical mechanisms of
fibrin formation and fibrin degradation. This information,
briefly summarized below, has important implications for
the development and interpretation of assays specific for
detecting fibrinolysis. During blood coagulation, thrombin
cleaves fibrinopeptides A and B from soluble fibrinogen,
converting it to a self-polymerizing fibrin molecule. 614
Received February 19, 1986; received revised manuscript and accepted fragments then are degraded to produce fibrin D-dimer
for publication March 20, 1986.
Supported in part by NIH grant HL-32342 awarded to Dr. Greenberg.
Address reprint requests to Dr. Greenberg: Box 3934, Duke University complex). High and low molecular weight cross-linked
Medical Center, Durham, North Carolina 27710.
94
Departments of Medicine and Pathology and the Coagulation
Laboratory of Hospital Laboratories, Duke University Medical
Center, Durham, North Carolina
Fibrin molecules polymerize via a half-staggered overlap-
ping pattern that forms a two-stranded protofibril.614
Protofibrils also assemble laterally forming fibrin fibers.
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Once the solution contains a network of fibrin fibers, it
becomes a gel. 614 During the process of fibrin polymer-
ization, thrombin converts Factor XIII to the transglu-
taminase (Factor XIIIa), which covalently stabilizes fi-
brin. 614 A pair of 7-glutamyl-e-lysyl cross-links form in
an anti-parallel orientation 3 between glutamine (residue
397) and lysine (residue 405). 215 These cross-links form
between two 7-chains that are oriented end-to-end in the
protofibril. There are also several Factor XlH a cross-links
formed along the a-chains that produce high molecular
weight a-chain polymers. 614 In recent years, several lab-
oratories have attempted to prepare specific antibodies to
Factor XIIIa-cross-linked domains in fibrin that are re-
ferred to as fibrin D-dimer in this article.''4'7'17,18'20'22
Many of these antibodies also cross-react with fibrinogen
and plasmin-derived fibrinogen degradation products. 20
Plasmin is the protease responsible for degrading fibrin
at the site of blood clotting. Plasmin also degrades fibrin-
ogen to form fragments X, Y, D, and E.9 The plasmin-
derived degradation products from non-cross-linked fibrin
are very similar to the fibrinogen degradation products
except fibrinopeptides A and B are absent.9
Plasmin degradation of cross-linked fibrin is more
complex and involves several stages to completely solu-
bilize fibrin.8'9 In the earliest stages of fibrin degradation,
soluble fibrin degradation products are not released be-
cause the fibrin strands are held together by covalent
bonds. However, once plasmin cleaves at complementary
sites between the D and E domains in the two-stranded
fibrin structure, soluble two-stranded complexes are re-
leased.9 The four smallest complexes are DD/E, DY/YD,
YY/DXD, and YXD/DXY. 9 These cross-linked fibrin
complexed to fragment E (fibrin D-dimer/fragment E
910
fibrin degradation products have been identified in patient
Vol. 87 • No. 1 MEASUREMENT OF FIBRIN D-DIMER LEVELS
plasmas with the use of various combinations of immu-
nologic and electrophoretic methods. 5 ' 812,26
The current latex agglutination assays for detecting fi-
brinogen) degradation products do not distinguish be-
tween fibrinogenolysis and fibrinolysis because the poly-
clonal antibodies that are employed recognize both fi-
brinogen and fibrin degradation products." A specific
enzyme immunoassay for fibrinolysis has been developed
with the use of monoclonal antibodies directed against
the D-dimer domain of cross-linked fibrin, fibrin D-di-
m e r 7,23,25 These monoclonal antibodies do not react with
fibrinogen and can be used to detect cross-linked fibrin
derivatives in plasma.2s We report the results of a study
comparing the sensitivity and specificity of the Dimer-
test™ agglutination assay with the fibrin(ogen) degrada-
tion product latex agglutination assays.
Materials and Methods
Preparation of Fibrinogen and Fibrin
Degradation Products
Human fibrinogen was purchased from Kabi and was
made plasminogen- and Factor Xlll-free. 13 The fibrino-
gen migrated as a single band after separation by SDS-
polyacrylamide gel electrophoresis, and cross-linked 7-
chains were not detected when electrophoresis was carried
out under reducing conditions.
Human plasmin was obtained from Kabi and recon-
stituted as described by the manufacturer. Factor XIII
was purified from platelets and stored at - 7 0 °C, as re-
ported previously.13 Factor XIIIa was prepared by incu-
bating platelet Factor XIII (30 Mg/mL) with thrombin (10
U/mL) for 30 minutes at 37 °C. Thrombin was inhibited
by PPACK 1 mM (Calbiochem, La Jolla, CA).
Factor XIIIa-crosslinked fibrinogen (10 mg/mL) was
prepared by incubating fibrinogen with Factor XHIa (15
Mg/mL) and calcium chloride (5 mM) for 15 minutes at
22 °C. Approximately 10% of the 7-chains of fibrinogen
were cross-linked, as demonstrated by SDS-polyacryl-
amide gel electrophoresis and scanning densitometry (data
not shown).
Cross-linked fibrin was prepared by incubating human
fibrinogen (5 mg/mL) with thrombin (5 U/mL), purified
Factor XIII (15 Mg/mL), and 5 mM calcium chloride for
one hour at 37 °C. The cross-linked fibrin clot was then
incubated with plasmin (12.5 CU/mL) for 18 hours at 37
°C. Plasmin was inactivated by adding p-amidinophen-
ylmethylsulfonyl fluoride (APMSF), and the sample was
dialyzed against 0.13 M NaCl, 0.02 M TRIS, pH 7.4 (TBS),
for 16 hours at 4 °C. Samples were electrophoresed in a
native discontinuous gel to demonstrate that both fibrin
D-dimer and fragment E remained noncovalently com-
plexed.10
95
Plasmin digests of non-cross-linked fibrin were prepared
by incubating human fibrinogen (5 U/mL), 10 mM EDTA
and 5 U/mL) of thrombin for one hour at 37 °C. The
fibrin clot was then suspended in TBS containing 1 mM
EDTA and plasmin (12.5 CU/mL) for 16 hours at 37 °C.
Plasmin was inhibited with 1 mM APMSF and the sample
dialyzed against TBS and stored at - 7 0 °C.
Early and late plasmin digests of fibrinogen were pre-
pared by incubating Factor Xlll-free fibrinogen (5 mg/
mL) in TBS buffer containing EDTA (5 mM) and 12.5
CU/mL plasmin at 37 °C. Early digests were obtained by
stopping the reaction after 30 minutes with 1 mM APMSF,
whereas the late digests were prepared by incubating for
18 hours. The plasmin digests were dialyzed in TBS buffer
and stored at - 7 0 °C.
Fragment X was supplied by Dr. Salvatore V. Pizzo,
Department of Pathology, Duke University Medical Cen-
ter. Fragments D and E were purified after plasmin-cat-
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alyzed digestion of fibrinogen in 5 mM calcium chlorides,
as described.3 Fibrin D-dimer was isolated from cross-
linked fibrin.1
Sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) was performed with the use of a
3% stacking gel and 8% separating gel.13 Purified fi-
brinogen) degradation products (25 Mg) were separated
by SDS-PAGE and stained with Coomassie Blue. Stained
gels were dried between dialysis membranes and scanned
with the use of a Hoefer transmission densitometer. Pu-
rified proteins migrated with molecular weights similar to
those previously reported1 and were greater than 85% pure.
Clinical Samples
Patient samples were obtained from the Duke Univer-
sity Coagulation Laboratory after being collected by hos-
pital personnel. Samples were collected for fibrin(ogen)
degradation product assays in tubes containing soybean
trypsin inhibitor and thrombin (tube #6478, Becton-
Dickinson, Rutherford, NJ). Serum was assayed by the
fibrin(ogen) degradation product assays according to the
manufacturers' recommendations. The protamine sulfate
precipitation assay for fibrin monomer was performed, as
described.16 Thrombin clotting time was performed ac-
cording to the method of Peden and McFarland.19
Thrombin was purchased from Parke-Davis and used at
concentrations adjusted to clot normal pooled plasma in
17-20 seconds. Quantitative fibrinogen levels were per-
formed with the use of the method of Ratnoff and Men-
zies.21 Clinical diagnoses were established by either C.S.G.
or K.M.M. after the charts of the patients were reviewed.
Preparation of Platelet-Poor Plasma
Blood was collected in 3.2% citrate, one part citrate to
nine parts blood. Platelet-poor plasma was prepared by
centrifuging at 2,500 g for 20 minutes at 22 °C. Bentonite
 GREENBERG, DEVINE, AND McCRAE
96
(15 mg/mL) was incubated twice with the platelet-poor
plasma for 30 minutes at 22 °C to remove fibrinogen.
After the second incubation, the bentonite was removed
by centrifugation and the supernatant filtered through
Millex-GF® Filters (Millipore Corporation). The benton-
ite-adsorbed plasma wasfibrinogenfree when assayed un-
diluted by a radial immunodiffusion kit (Calbiochem, La
Jolla, CA) (data not shown). Bentonite-adsorbed plasma
was used for experiments testing the sensitivity and spec-
ificity of the Dimertest® and fibrin(ogen) degradation
product assays.
Assay Methods
The Fibrin(ogen) Degradation Products Detection Set
was supplied by American Dade (Currently Dade, Baxter
Travenol Diagnostics, Inc.) and the Thrombo-Wellcotest®
was purchased from Wellcome Diagnostics. The assays
were performed according to the manufacturer's recom-
mendations. The assay kits for fibrin D-dimer were sup-
plied by American Dade or were purchased from Amer-
ican Diagnostica. The Dimertest assay kit used mono-
clonal antibody DD-3B6 coupled to latex beads. The
monoclonal antibody conjugated latex beads (0.025 mL)
were incubated after mixing with platelet-poor plasma
(0.01 mL) at room temperature for 3 minutes on a rocker
platform. After 3 minutes, the slide was read against a
black background for the presence of agglutination.
Measurement of the Sensitivity and Specificity
of the Assays
Purified fibrinogen andfibrin(ogen)degradation prod-
ucts were added to bentonite-adsorbed plasma and assayed
by the three assay methods. The person recording the ag-
glutination reaction was unaware of the contents of the
sample. The lowest concentration of protein producing
agglutination was recorded. All assays were performed in
duplicate. Sensitivities were also tested by assaying the
proteins added to TBS. Platelet-poor plasma was also col-
lected in EDTA (K3), tube #6454, supplied by Becton-
Dickinson. In addition, sensitivity testing was performed
for purified fibrin D-dimer added to EDTA anti-coagu-
lated platelet-poor plasma.
Five hundred micrograms per milliliter of fragment D,
E, X, Y, or fibrinogen was incubated with 200 fig/mL
fibrin D-dimer to determine whether these proteins in-
terfered with the assay.
Identification of Plasma Fibrin D Dimer by
Immunoprecipitation and Western Blot Analysis
In order to identify the molecular species of fibrin(ogen) when 1 itg/mL of purified D-dimer was added to EDTA-
recognized by the DD-3B6 monoclonal antibody, we used anti-coagulated plasma. Two different fragment D-dimer
a combination of immunoprecipitation and Western blot
AJ.C.P. • January 1987
protein analysis. EDTA plasmas from normal donors or
patients with disseminated intravascular coagulation
(DIC) were incubated with thrombin, and clottable fi-
brinogen was removed. Twenty-five microliters of the re-
sultant sera were incubated with 10 id rabbit anti-human
fibrinogen antibody under conditions previously deter-
mined to remove allfibrin(ogen)-relatedantigens, includ-
ing fibrin D-dimer, from the sera. After incubation for
one hour at 4 °C, 200 fiL of a 10% suspension of formalin-
fixed Cowan I strain Staphylococcus aureus (Pansorbin®
cells, Calbiochem) in 0.02 M TRIS, 0.15 M NaCl, 0.5%
NP-40, pH 7.4 (TS-NP-40), was added and the incubation
continued for five hours at 4 °C. After the bacteria pellet
was washed three times in TS-NP-40, the antibody-an-
tigen complexes were dissociated by boiling the pellets in
SDS-PAGE sample buffer (12.5 mM TRIS-HC1, 20%
glycerol, 4% SDS, pH 6.8). Samples were centrifuged to
remove the bacteria, and the supernates were separated
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on 4-12% polyacrylamide gradient gels with a 3% stacking
gel under nonreducing conditions. Proteins were then
transferred to nitrocellulose with the use of the method
of Towbin and associates.24 The blots were probed first
with the DD-3B6 monoclonal, which was obtained from
the ELISA kit form of the Dimertest (American Diag-
nostica). Then, the blots were incubated with 125I-labeled
F(ab')2 sheep anti-mouse immunoglobulin (Amersham)
and autoradiographed. This method allowed the resolu-
tion of fibrinogen-related antigens and fibrin D-dimer
from other serum proteins that are often present in high
enough concentrations to confound the analysis of the
proteins of interest.
Results
Specificity and Sensitivity of the Dimertest Assay
As can be seen in Table 1, only purified fibrin D-dimer
and fibrin D-dimer/E complex added to afibrinogenemic
plasma agglutinated the DD-3B6 coated beads. Under
these conditions, the minimum concentration of purified
fibrin D-dimer and fibrin D-dimer/fragment E complex
producing agglutination was 2 itg/mL. In contrast, purified
fragment X, D, or E or early and late plasmin digests of
fibrinogen did not cause agglutination. Similar sensitivity
results were obtained with the use of buffer instead of
bentonite-adsorbed plasma. Furthermore, single samples
from 80 healthy persons, median age 34 (18-76 range),
did not agglutinate the DD-3B6-coated beads. Although
sodium citrate was the anti-coagulant used to collect and
prepare platelet-poor plasma in most cases, EDTA plas-
mas from 10 healthy persons did not cause any aggluti-
nation. The DD-3B6 antibody-coated beads agglutinated
preparations and two different lots of DD-3B6-coated
Vol. 87 • No. 1 MEASUREMENT OF FIBRIN D-DIMER LEVELS 97
Table 1. Sensitivity and Specificity of Dimertest and FDP Assays
Protein Concentration Producing Agglutination (^g/mL)

Fibrin D-Dimer/ E Fragment Early Late Fragment Fragment

Assay D-Dimer Fragment Fibrinogen X Digest Digest D E

Dimertest® 2.0 2.0 0

FDP—American Dade 5.0 5.0 2.5 1.0 2.0 2.5 2.5 2.5
FDP—Wellcome Diagnostics 5.0 5.0 5.0 10.0 5.0 2.0 1.0 1.0

0 - no agglutination detected up to 850 mg/100 mg; — = no agglutination at concentrations The minimum concentration of protein in bentonite adsorbed plasma producing agglutination
up to 500 pg/mL. was tested as described in "Materials and Methods."

beads yielded similar sensitivity data. The sensitivity of
the Dimertest assay was stable for 12 weeks when the kits
were stored at 5 °C.
Thefibrin(ogen)degradation product assay (American
Dade) detected from 1 to 2.5 Mg/mL of either fibrinogen
or the other plasmin-derived fibrinogen degradation
products. Fibrin D-dimer (5.0 Mg/ml) was also detected
wanted to measure cross-linked fibrin derivatives (fibrin
D-dimer) in platelet-poor plasma. Fibrinogen (800 mg/
mL) added to the bentonite-adsorbed plasma did not pro-
duce agglutination in the Dimertest. The addition of pu-
rifiedfibrin(ogen)degradation products (500 Mg/mL) to
plasma containing purified fibrin D-dimer (2 Mg/mL) did
not inhibit agglutination. The concentration of purified

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by the American Dade and Wellcome Diagnostics fi- fibrin D-dimer in serum did not change after clotting in
brinogen) degradation product assays. The fibrin(ogen) the presence or absence of calcium chloride.
degradation product assay (Wellcome) detected from 1 to Plasma incubated for 24 hours at 22 °C or 37 °C did
16 Mg/mL of thefibrin(ogen)degradation fragments. The not develop a positive agglutination reaction with DD-
Wellcome fibrin(ogen) degradation product assay was 3B6-coated beads. Freeze-thawing five different samples
more sensitive to fragments D and E and less sensitive to three times did not produce fibrin D-dimer reactivity. In
fragment X and fibrinogen when compared with the addition, freeze-thawed samples that were treated with
American Dadefibrin(ogen)degradation product assay. streptokinase (250 U/mL) for 30 minutes at 37 °C did
When Factor XIIIa cross-linkedfibrinogen(160 Mg) was not develop fibrin D-dimer reactivity. The plasma and
added to afibrinogenemic plasma, agglutination did not serum levels of fibrin D-dimer were similar in 25 patients
result until after plasmin degraded the molecule to pro- studied.
duce fibrin D-dimer.
Assay of Clinical Samples
Interference with the Measurement of Plasma The two fibrin(ogen) degradation product assays were
Fibrin D-Dimer compared with the use of serum from the same patient.
The American Dade and Wellcome Diagnostics assay re-
Studies were performed with the use of afibrinogenemic sults (N = 105) were concordant in 69% of tests where
plasma prepared by bentonite adsorption because we fibrin(ogen) degradation product levels were less than 50
Mg/mL and 75% of tests where fibrin(ogen) degradation
Table 2. Relationship Between FDP and Fibrin D- product levels were greater than 40 Mg/mL. The fi-
Dimer Levels in Patients Suspected of Having the brinogen) degradation product assay from Wellcome Di-
Disseminated Intravascular Coagulation Syndrome agnostics was used for all subsequent studies.
Samples from 152 patients suspected of having DIC
Fibrin(ogen) Degradation Product
(Mg/mL)* were also tested to correlate serum fibrin(ogen) degrada-
tion product levels with fibrin D-dimer levels, fibrin(ogen)
<10 10-40 >40 levels, andfibrinmonomer levels (Table 2). The Dimertest
Assay (n = 49) (n = 56) (n = 47)
assay was positive in 92% of the cases that were positive
Fibrin D-dimer < 2 Mg/mL for fibrin monomer. Plasma fibrin D-dimer levels in-
(10%) 15(30%) 3 (5%) 5 creased as serum fibrin(ogen) degradation product levels
Fibrin D-dimer, >2, <8
/xg/mL (38%) 28 (57%) 35 (62%) 18 increased. The percentage of patients with plasma fibrin
Fibrin D-dimer > 8 Mg/mL D-dimer levels greater than 8 Mg/mL increased from 12
(51%) 6(12%) 18 (32%) 24 to 51% as the fibrin(ogen) degradation product levels
Fibrinogen < 150 mg/dL 1 3 8
Fibrin monomer positivef 3 10 27 changed from less than 10 Mg/mL to greater than 40 Mg/
mL. However, 34 out of 40 patient samples with fi-
• Fibrin(ogen) degradation products were measured with the use of thefibrin(ogen)degradation
product assay of Wellcome Diagnostics.
brinogen) degradation product levels less than 10 Mg/mL
t Fibrin monomer was assayed with the use of the protamine sulfate precipitation assay.16 had measurable fibrin D-dimer reactive material in their
 GREENBERG, DEV1NE, AND McCRAE A.J.C.P. • January 1987
98
plasma. In addition, from 5 to 10% of patient plasma Table 3. Clinical Disorders Associated with
tested had fibrin D-dimer levels less than 2 ng when fi- Measurable Fibrin D-Dimer
brinogen) degradation product levels were greater than Number
10 Mg/mL. of Patients
Fibrin D-dimer was detectable in 42 plasma samples
from 150 random plasma samples from hospitalized pa- Arterial thrombosis 31
Venous thrombosis 28
tients assayed by the Dimertest. The clinical disorders as- Postoperative state 24
sociated with a positive Dimertest assay for all patients Septicemia, abscess 20
reported in this study are listed in Table 3. We have not Carcinoma 21
Cirrhosis 14
detected any positive reactions in 80 healthy persons. Autoimmune disease 10
Plasma from 30 random hospitalized patients and 10 Pancreatitis 9
healthy persons remained negative for fibrin D-dimer de- Sickle cell crisis 6
Leukemia 2
rivatives after in vitro streptokinase treatment (250 U/mL Adult respiratory distress syndrome 2
for 30 minutes at 37 °C). In addition, the concentration Hemolytic transfusion reaction 2
of fibrin D-dimer was not modified by heparin (10.0 U/ Renal failure 2
mL) added in vitro. Total 171

Characterization of Plasma Fibrin D-dimer by

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Immunoprecipitation and Western Blot Analysis in 160 Mg/mL cross-linked fibrinogen until after it was
degraded by plasmin. Steric factors may restrict aggluti-
Whenfibrin(ogen)and D-dimer-related antigens were nation of DD-3B6 coated beads tofibrin(ogen)polymers
immunoprecipitated from serum samples from patients
with the DIC syndrome, the DD-3B6 monoclonal iden-
tified a protein that migrated in the same location as pu-
rified fragment D-dimer (Fig. 1). The monoclonal did not A B C D E FG
recognize any proteins in normal serum that were not
also present in nonimmune control lanes. Kd
DISCUSSION
With the development of monoclonal antibodies to the 200-
fragment D-dimer domain of cross-linked fibrin (fibrin
D-dimer), a sensitive and specific enzyme immunoassay IP
for fibrin degradation was created.23'25 The Dimertest ag- 92.5-
glutination assay evaluated in this study uses monoclonal
antibody DD-3B6, which is specific for fibrin D-di-
m e r 7,23,25 j n e Specificity of the assay was not affected by 69-
fibrinogen. The sensitivity of the assay to fibrin D-dimer
and fibrin D-dimer/fragment E complex was similar. In •

addition, fragment E did not modify the sensitivity of the
Dimertest assay to fibrin D-dimer. Studies by Whitaker
and associates,25 using an ELISA, have shown that purified
fibrin D-dimer has the highest affinity for the DD-3B6
antibody when compared with fibrin D-dimer/fragment
E complex or the high molecular weight products of cross-
linked fibrin. We have not found any effect of fragment
E on the measurement of fibrin D-dimer derivatives in
plasma and have not found any effect of other plasma
proteins on the sensitivity of the Dimertest assay. In ad-
dition, the antibody does not recognize fragment E in
Western blot analysis but does readily bind fragment D-
dimer. The antibody bound to latex beads may display
different sensitivities to cross-linkedfibrinderivatives and
to fragment D-dimer when compared with unbound an-
tibody. The assay did not recognize the D-dimer domain
ill
46-
30-
^yttttit
FIG. 1. Recognition offibrinogen-relatedantigens by the DD-3B6
monoclonal antibody. Immunoprecipitates prepared as described in the
"Materials and Methods" were subjected to SDS-PAGE and transferred
to nitrocellulose paper. The blot was incubated first with monoclonal
antibody DD-3B6, then with 125I-labeled F(ab')2 sheep anti-mouse im-
munoglobulin. Immunoprecipitated normal human serum (Lane A) or
DIC patient serum (Lane B) reacted with normal rabbit serum. Im-
munoprecipitated normal human serum (Lane C) or DIC patient serum
(LaneD) reacted with rabbit anti-humanfibrinogenserum. Lane E con-
tains Fragment E, Lane F contains Fragment D, and Lane G contains
fragment D-dimer. The fragment D-dimer in Lane D migrated somewhat
higher than purified fragment D-dimer because of the high concentration
of IgG migrating directly below it.
Vol. 87 • No. 1 MEASUREMENT OF FIBRIN D-DIMER LEVELS
containing D-dimer or the monoclonal antibody may
preferentially recognize an epitope on fragment D-dimer.
Further studies are needed to distinguish between these
possibilities. Although fragment D was not recognized by
the DD-3B6 monoclonal antibody in the agglutination
assay, the antibody did bind to fragments Dl, D2, and
D3 in Western blot analysis. Presumably, the epitope rec-
ognized by this antibody is not accessable to the antibody
when fragment D is in its native conformation, but it
becomes accessable after treatment with SDS or after
cross-linking two fragment D sections offibrinmolecules.
Purified fibrinogen and plasma fibrinogen concentra-
tions as high as 1,250 mg/mL did not cause agglutination
in the Dimertest assay. In addition, cross-linked fibrin
derivatives (fibrin D-dimer) did not form in plasma after
incubation for 24 hours at 37 °C or after repeated freeze-
thawing. Therefore, plasma was used to assay fibrin D-
dimer in clinical samples. The Dimertest assay remained
sensitive and specific in the presence offibrin(ogen)deg-
radation products, heparin, or streptokinase. Based on
these findings, either citrate or EDTA anti-coagulated
tubes may be used to assay fibrin D-dimer derivatives
using the Dimertest.
Fibrin D-dimer derivatives were not detected in either
citrate or EDTA anti-coagulated plasma from healthy
persons. We believe that the assay is specific for fibrino-
lysis, although it does not distinguish pathologic versus
physiologic clot lysis. The assay would not detect fibri-
nogenolysis alone, because the monoclonal antibody used
in the Dimertest assay recognizes an epitope on fibrin
fragment D-dimer. The Dimertest assay remained positive
for several days after surgery, because fibrin may be de-
graded during wound healing. Previous studies using the
fibrinogen degradation product assays and the DD-3B6
antibody in an ELISA assay also demonstrated fibrin D-
dimer reactive material postoperatively.25 A wide variety
of diseases were associated with a positive Dimertest assay
in hospitalized patients. Many of these diseases have been
reported to be associated with an increase in fibrinolytic
activity. By immunoprecipitation and Western blot anal-
ysis, monoclonal antibody DD-3B6 recognizes primarily
D-dimer in the plasma of patients with DIC.
Sickle cell patients with vasocclusive crisis had fibrin
D-dimer in their plasma, whereas those who were not in
crisis and did not have any other complication were un-
reactive with the Dimertest. Studies are in progress to
define the utility of the Dimertest assay in diagnosing and
managing sickle cell crisis.
We have found measurable fibrin D-dimer levels in
69% of patients suspected of having the DIC syndrome
withfibrin(ogen)degradation product levels less than 10
Mg/mL. Oncefibrin(ogen)degradation product levels were
greater than 10 Mg/mL, more than 92% of the samples
were positive for fibrin D-dimer derivatives. Although
there was a trend for patients to have higher D-dimer
99
levels as theirfibrin(ogen)degradation product levels in-
creased, there were 5-10% of patients with fibrin(ogen)
degradation product levels greater than 10 Mg/mL who
were D-dimer negative. These patients may predomi-
nantly have either fibrin(ogen) degradation or high mo-
lecular weightfibrinD-dimer derivatives that did not react
with DD-3B6-coated beads. In addition, ~30% of patients
withfibrin(ogen)degradation product levels less than 10
Mg/mL with the use of the Wellcome Diagnostics fi-
brinogen) degradation product assay had positive Di-
mertest results. These patients may be selectively degrad-
ing fibrin, and because the Wellcome fibrin(ogen) deg-
radation product assay does not detect levels of D-dimer
less than 5 Mg/mL, only the Dimertest would be reactive.
In this study, the concentration of fibrin D-dimer de-
rivatives in plasma was lower than the serum fibrin(ogen)
degradation product levels. This has also been noted by
other investigators,18,23,25 Recently, Gaffney and Perry
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demonstrated that some of the fibrin(ogen) degradation
product measured by the standard agglutination assays
may be derived from nonclottable fibrinogen." In a pre-
liminary study, Connaghan and associates demonstrated
that heparin commonly caused false positive tests for
serumfibrin(ogen)degradation product levels.5 We have
not made any attempt in our study to neutralize heparin
in the patient plasma, and this may have contributed in
part to the higher serumfibrin(ogen)degradation product
levels. Further studies are needed to define the molecular
weight of thefibrin(ogen)degradation product in patients
who have levels of serumfibrin(ogen)degradation product
greater than 10 Mg/mL.
Future studies are needed to define the stability of the
monoclonal antibody bound to the latex beads. Compar-
ative studies between the ELISA and the latex aggluti-
nation assays for fibrin D-dimer and higher molecular
weight to fibrin D-dimer derivatives are also needed to
compare the sensitivity and specificity of the two assays.
In conclusion , the Dimertest agglutination assay for
fibrin D-dimer derivatives is a sensitive and specific assay
for the detection of fibrin D-dimer and high molecular
weight fibrin D-dimer derivatives in plasma or serum. It
is a useful addition to the clinical laboratory and may be
used to monitor patients receivingfibrinolytictherapy or
to identify patients with active fibrinolysis. The fi-
brinogen) degradation product assays from American
Dade and Wellcome Diagnostics were comparable for de-
tecting fibrinogen and fibrin degradation products in
serum. However, thefibrin(ogen)degradation product as-
says do not define whetherfibrinolysisor fibrinogenolysis
is occurring, because a portion of the measured fi-
brinogen) degradation products may be nonclottable fi-
brinogen." The Dimertest assay is more rapid, sensitive,
and specific than the protamine sulfate test for fibrin
monomer and may replace this test in the clinical labo-
ratory.
100 GREENBERG, DEVINE, AND McCRAE
Acknowledgments. The authors acknowledge the technical assistance
of Charles C. Miraglia and J. R. Gilbertson.
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