Interpreting D-dimer results

The association between elevated D-dimer levels and thrombotic disease continues to grow. When interpreting a D-dimer result there are
some clinical aspects that should be considered. The precise level of cross-linked fibrin derivatives (D-dimer) circulating in the blood at a
given time will depend on a number of parameters:

Time elapsed since the

thrombotic event.
The initial size of the
clot.

D-dimer has a half life of approximately 6 hours in the circulation of individuals with normal renal
function. Patients with stabilised clots and not undergoing active fibrin deposition and plasmin activation,
may not give detectable D-dimer elevations.
The larger the clot size, the higher the expected level of circulating D-dimer. Obviously the converse is
also true
Blood fibrinolysis is a highly regulated process and in delicate dynamic balance. Should any of the

The rate of fibrinolysis. components be compromised (hereditary or acquired deficiency or dysfunction) then the rate of
fibrinolysis will be altered.

Fibrin may be present at alternative sites other than that suspected. For example, atherosclerotic lesions

Alternative fibrin sites. or extravascular fibrin deposits. Some tumours can be encapsulated in a fibrin sheath.
Differing antibody
specificity.

All D-dimer assays are not alike - Depending on the commercial source, different antibodies used in a
test have differing specificities for fibrinogen and fibrin and their derivatives. There are still today many
FDP assays calling themselves D-dimer specific.

What is D-dimer?
D-dimer is a protein that is released into the circulation during the process of fibrin blood clot breakdown. D-dimer represents
an area of crosslinked fibrin degradation product that originated from the breaking down of the fibrin clot network during the
bodys repair mechanisms.

D-dimer present in circulation is used as an indicator of a blood clot being formed and broken down somewhere in the body.
In 1983 scientists in association with AGEN Biomedical in Brisbane, Australia invented and patented specific methods for
detecting D-dimer. The antibody DD-3B6/22 has a very high affinity for D-dimer while not cross-reacting with similar noncrosslinked proteins derived from non-clot material. The tests using this new sensitive and specific D-dimer antibody are
marketed worldwide under the name DIMERTEST and SimpliRED.

'Good' & 'Bad' D-dimer

By understanding that D-dimer is released into the circulation following an injury as part of the wound healing process, it
becomes possible to use D-dimer as a test for many medical conditions and complications. In all people (and animals) there is
very low background levels of D-dimer, usually undetectable. Every time we graze our knee or fall over and bruise ourselves,
D-dimer is produced. However, in some diseased states this level of D-dimer in the circulation may become elevated and be
evidence of some underlying abnormality. D-dimer measurement is being used by physicians to detect blood clot breakdown
that may not be outwardly visible, as in the examples below.
A Deep Vein Thrombosis is a blood clot that occurs usually in the deep veins of the legs. This painful condition can occur as a
result of an injury to the legs (sporting injury) or even lack of movement of the limbs over time, for instance in patients
confined to bed or even plane travellers confined to a seat for long trips! This can be potentially fatal if the clot migrates to the
lungs (Pulmonary Embolism) or heart or brain. For the physician trying to determine with certainty whether there may be a
potentially life threatening blood clot in the legs, accurate D-dimer measurement proves a useful tool for this diagnosis. A
negative (normal) D-dimer result can allow a patient to be quickly excluded from invasive investigation and therapy . This can
be achieved by taking a single drop of blood and running on a rapid D-dimer test in 2 minutes (see SimpliRED D-dimer).
Thrombosis is the third greatest cause of death after heart attack and stroke and whole blood D-dimer measurement is giving
renewed hope for its early diagnosis and intervention

The clot forming process

Clot formation in the blood vessels of the body occurs in both healthy people and in diseased states. Under normal
circumstances blood clots form to prevent blood loss from damaged vessels. This process must be reversible and so allowing
the normal repair of these blood vessels by the healthy body.
VASCULAR LESION (TRAUMA or Injury)
Initial injury causes vasoconstriction (veins shrinking) around the injury site. Platelets in the blood start to adhere and
aggregate at the injury site.
CLOTTING is occurring as a result of the Coagulation Cascade. Factor proteins that are present in "healthy" patients react with
each other with the end result that a fibrin clot (or "plug") is formed at the injury site. This clot is further stabilized by
crosslinking and entrapment of platelets and blood cells. At this point an effective barrier has been formed to stop the further
loss of blood and blood pressure at the injury site.
The reverse of this process is also occuring simultaneously. Here the extensive fibrin clot network is being broken down
gradually to allow the bodys repair mechanism to work on this area. As the clot is being broken down one of the proteins that
is released into the blood stream is D-dimer.
Gradually wound healing occurs at the injury site until all the damaged vessels and tissues are repaired.

Fibrinolysis defined
D-dimer is the name given to one of a family of fibrin fragments which form and circulate in the blood stream for several days
immediately following a thrombotic event. It contains cross-linked regions introduced during clot stabilization. These fragments
are released from the clot by the action of the enzyme plasmin.

D-dimer is produced naturally as part of the wound healing process. The formation of blood clots and their subsequent lysis is
part of the normal healthy functions of the body, these events occur naturally every time a skin cut or graze is effected.
However when clots are formed at the wrong time and place as a result of some underlying disease, D-dimer becomes a
valuable marker because the presence of cross-linked species indicates the occurrence of unwanted thrombotic events.
HAEMOSTASIS is the physiological mechanism by which the body controls bleeding after injury. Hemostasis occurs in both
health and in certain disease states or vascular injury, the latter culminating in extensive fibrin clot formation forming an
insoluble barrier at the injury site. This is called Coagulation, or Fibrin formation. Tissue healing occurs with gradual clot
digestion or Fibrinolysis. Fibrinolysis completes the haemostatic process.
FIBRIN CLOT FORMATION and its subsequent dissolution by the enzyme PLASMIN yields a variety of crosslinked FIBRIN
breakdown products into the bloodstream.

The presence of these fragments, the best characterized of which is D-DIMER, indicates a thrombotic event and subsequent
lysis of that clot (secondary fibrinolysis).

Disadvantages of FDP tests

Traditional "FDP" (Fibrinogen Degradation Products) tests were used for DIC diagnosis and management.
These assays lacked specificity, utilizing polyclonal antibodies against fibrinogen and its fragments, which
necessitated the requirement for serum samples.

Deficiencies of the old FDP style tests

Specific Antibody detection?

No

Artifact Free?

No

Sample Utility?

No

Special sample collection tubes are required

Use sample directly?

No

Predilution required at the lab

Sensitivity for
Thromboembolism?

No

Too much non specific background

Diagnostic Ability in DIC?

No

It is well documented that FDP assays only detect 75-80 % of DIC patients, ie
non-diagnostic in this application

Specific for Thrombosis?

No

There is a 25% false positiverate even in normal blood donors!

Polyclonal antibodies for FDPs

Crossreactivity caused by Incomplete clotting, Heparin and dysfunctional
fibrinogen

The advantages of the specific D-dimer test over the older FDP testing is now well documented and is rapidly expanding the
growth into new Clinical Applications.

Advantages of D-dimer
D Dimer-3B6/22 is the world's first patented D-dimer specific monoclonal antibody, detecting crosslinked Fibrin
Degradation Products in plasma or whole blood samples.
DD-3B6/22 has become internationally recognized as the GOLD STANDARD monoclonal antibody for fibrin clot detection, with
in excess of 300 publications utilizing DD-3B6/22 in various assay formats. (see Clinical Paper Search later)

ADVANTAGES OF D-DIMER

Uses a specific Monoclonal antibody (3B6/22), specific only for D-dimer. No

Fibrinogen crossover.

Specific antibody detection?

Yes

Artifact free?

Yes

Sample utility?

Yes

Can use sample from any anticoagulant tube including EDTA , Heparin and
Citrate

Sensitivity for
Thromboembolism?

Yes

Now endorsed by Descriptive and Management trials support the Negative

Predictive value of D-dimer.

Diagnostic ability in DIC?

Yes

If the D-dimer is negative there can not be DIC

Specific for Thrombosis?

Yes

In normal patient samples specificity is greater than 98%

Standardisation Issues

No interference from clotting agents or Fibrinogen

D-dimer exists in plasma as a complex variety of cross-linked fibrin derivatives of molecular weight in excess of 2 x 106 daltons, and
rarely as free D-dimer. Commercially available D-dimer kits differ in reactivity toward D-dimer. Standardisation of D-dimer assays has
been under review of the Fibrinogen and DIC sub-committee of the International Society on thrombosis and Haemostasis (ISTH) for
several years, involving three international surveys (1992-1995). The reactivity of different commercial kits to various standards is widely
variable and this implies that an international D-dimer standard may not be feasible.4,40 However, this review continues, using clinical
plasma samples with high D-dimer levels which are identified as pathological by most commercial D-dimer kits.
REPORTING
* Range or specific value?
As a primary D-dimer reference standard is not available, latex results are best reported as a D-dimer range (ng/mL). Commercial ELISA
formats have D-dimer standards which can only be termed secondary standards which are prepared to each manufacturer's specifications.

* FEU
Fibrinogen equivalent units or FEU is the term used by some commercial manufacturers. It is based on the questionable assumption that
one lysed native fibrinogen molecule gives rise to two D-dimer units. It seems clear now much of the fibrinogen that clots is not
completely degraded to D-dimer. For this reason, the quantity of D-dimer reported as a value (ng/mL) remains more widely accepted.

Test Accuracy
Test Performance Characteristics - Clinical Usefulness
Considerable variation exists in reporting D-dimer results in different laboratories, surveys and in the literature. Commercially
available kits employ antibodies of different affinities and specifications to D-dimer. Hence the accuracy of these tests varies.
Fibrin specificity is of utmost importance particularly as the clinical and vascular laboratories have increasing requests for
D-dimer evaluation to exclude patients suspected of DVT or PE.
The precise clinical utility of a D-dimer test depends on two major features of the monoclonal antibody (MAb) system utilised
in the test format:

Specificity

MAb system MUST react only with cross-linked fibrin derivatives (D-dimer) structure.
MAb should NOT react with fibrinogen or fibrinogen derivatives (fragment D, X, Y).

Sensitivity

Dependent on MAb affinity for D-dimer.

Accurate results with MAb with high affinity MAb with minimal cross-reactivity with fibrinogen or its
derivatives.

Interpreting D-dimer results

The association between elevated D-dimer levels and thrombotic disease continues to grow. When interpreting a D-dimer
result there are some clinical aspects that should be considered. The precise level of cross-linked fibrin derivatives (D-dimer)
circulating in the blood at a given time will depend on a number of parameters:

Time elapsed since

the thrombotic event.

D-dimer has a half life of approximately 6 hours in the circulation of individuals with normal
renal function. Patients with stabilised clots and not undergoing active fibrin deposition and
plasmin activation, may not give detectable D-dimer elevations.

The initial size of the

clot.

The larger the clot size, the higher the expected level of circulating D-dimer. Obviously the
converse is also true

The rate of
fibrinolysis.
Alternative fibrin
sites.
Differing antibody

Blood fibrinolysis is a highly regulated process and in delicate dynamic balance. Should any of
the components be compromised (hereditary or acquired deficiency or dysfunction) then the
rate of fibrinolysis will be altered.
Fibrin may be present at alternative sites other than that suspected. For example,
atherosclerotic lesions or extravascular fibrin deposits. Some tumours can be encapsulated in a
fibrin sheath.
All D-dimer assays are not alike - Depending on the commercial source, different antibodies

specificity.

used in a test have differing specificities for fibrinogen and fibrin and their derivatives. There are
still today many FDP assays calling themselves D-dimer specific.

Products used for measuring D-dimer

Some assays that can be used for the sensitive and specific detection and monitoring of D-dimer in plasma or whole blood samples are:.
Dimertest GOLD EIA
The GOLD standard D-dimer enzyme-linked immunoassay "Dimertest Gold" is the standard by which all other tests and preparations are
judged. Dimertest Gold uses 2 Fibrin Specific monoclonal antibodies, that do not react with Fibrinogen or its breakdown products.
Dimertest Latex
The rapid latex plasma D-dimer test, which has for over 10 years being giving reliably consistant semi-quantitative D-Dimer results.
SimpliRED D-dimer
The rapid whole blood D-dimer test. Designed to be run at the Point of Care or stat lab, and can give a result on one drop of patient whole
blood in just 2 minutes.
Auto Dimertest
The 3B6/22 D-dimer monoclonal antibody has now being configured to run on various laboratory Coagulation and immunoassay systems,
delivering a truly quantitative result in minutes. This method combines the excellent low end sensitivity (65ng/ml ) of the Elisa with the
convenience and speed of latex.

Clinical Applications
D-dimer is a valuable diagnostic marker in either detecting the presence of, or monitoring the progress of a fibrin-based
thrombotic event. Such is the case in Disseminated Intravascular Coagulation (DIC), Deep Vein Thrombosis (DVT), Pulmonary
Embolism (PE), coronory heart disease and other arterial and venous thrombotic states.
Some of the main applications that D-dimer is used for currently are:.

DISSEMINATED INTRAVASCULAR COAGULATION (DIC)

DIC is considered a systemic thrombo-hemorrhagic disorder. Although the hemorrhagic aspect of this disorder is not an
insurmountable clinical consideration, it is the small and large vessel thromboses as a consequence of excess thrombin
activation and fibrin deposition leading to reduced vascular flow, ischemia, and associated end-organ damage, that usually
lead to irreversible morbidity and mortality.
Although DIC has been described as low grade compensated or fulminant, commonly when a patient presents with suspected
DIC such a clear differentiation of the disease process is unclear. The patient may be at any stage between these extremes
with progressing severity. Early diagnosis and appropriate treatment are important in DIC prognosis. D-dimer detection has
been reported useful for early diagnosis and ongoing monitoring of DIC.
Some clinical conditions associated with DIC are:.
Pre-eclampsia is a common cause for thrombotic complications during pregnancy. HELLP (hemolysis, elevated liver enzyme
and low platelet) syndrome is a severe form of pre-eclampsia having an incidence of up to 12% in this patient group. D-dimer
is used as a pre-screen and followup test to identify those women at high risk of severe disease, warranting more intense
maternal-fetal monitoring and management.
Septicemia is frequently associated with DIC, particularly burns, trauma and crush injuries
Disseminated tumours also have a systemic effect on hemostasis. Elevated concentrations of D-dimer have been reported in
ovarian, lung, prostatic, breast and colorectal cancers .
Prosthetic devices, such as shunts and aortic balloons, may trigger DIC due to exposure to foreign surfaces activating the
hemostatic mechanism.
In addition to the hereditary defects which lead to thrombotic disease, thrombosis is frequently associated with several
medical illnesses, surgical procedures and traumatic events. It is these thrombotic complications that contribute significantly to
the morbidity and mortality of many diseases. D-dimer is finding increased value in many of the following clinical states .

FURTHER CLINICAL APPLICATIONS

Disseminated Intravascular Coagulation (DIC)
Deep Vein Thrombosis (DVT)
Pulmonary Embolism (PE)
Identify complications (high risk) to:.

Pregnancy (Pre-eclampsia, HELLP)

AMI
Post angioplasty (PTA, Balloon dilatation)
Post surgery or trauma
Metastic malignancy
Diabetes
Sickle Cell Disease crisis
Liver transplantation
Monitoring Anticoagulant and Thrombolytic Therapy
Sepsis - Gram negative
Cancer (lung, ovarian, prostatic)
Leukemia (AML: M3, M4)
Renal Disease
Liver Disease
Autoimmune Disease
Differentiation of Subarachnoid Haemorrhage from traumatic lumbar puncture

Diagnosis of Venous Thromboembolism

Venous Thromboembolism (VTE), is a common disorder particularly of the western world, and is the third most common
cardiovascular disease after ischemic syndromes and stroke. Specifically, DVT and PE are common causes of morbidity and
mortality as a result of various clinical states, having an annual incidence of 1 in 1000.
Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE)
The diagnosis of DVT and PE by clinical symptoms alone is inaccurate and insensitive with 50% of suspected cases missed.
Prevention of VTE during surgical procedures and as a consequence of medical illness is an important part of patients' health
care strategy during hospitalisation.
Existing diagnostic methods are either invasive, not widely available out of hours or are associated with their own risks. Noninvasive methods are relatively non-specific and may be inconclusive.
By comparison D-dimer is now being used more as a negative predictor to rule out DVT. While a positive D-dimer is not
diagnotic of DVT or PE, the power of the D-dimer result lies in its negative predictive value, the ability to exclude DVT/PE in
suspected cases.
The use of a rapid, simple whole blood tests such as SimpliRED D-dimer, to quickly eliminate at presentation those suspect
patients who could be excluded from unnecessary venograms or lung scans, provides a efficient convenient cost effective
strategy to VTE patient screening and management.

D-dimer as a risk marker in Myocardial Infarction

Coronory artery thromboembolism arises from interaction between platelets and vessel wall in response to vessel wall injury
commonly caused by such diseases as atherosclerosis. Platelets adhere and aggregate at injuried sites, with coagulation
activation to form a consolidated platelet-rich embolus which may ultimately occlude the vascular lumen, thus causing an
infarct.
Myocardial infarcts, thrombotic strokes, transient ischemic attacks and peripherial arterial occlusions are associated with
atherosclerosis. Prosthetic valves, particularly of nonbiological origin, are an important source of intracardiac thrombosis and
systemic embolisation as well as cardiac catheterization and bypass surgery.

Three applications that use the strength of D-dimer in this area

are:
Risk Assessment
Prognostic Marker

Epidemiological studies confirm D-dimer as a strong independant marker for ischemic heart
disease. D-dimer should be included in biochemical workup for health assessment.
D-dimer is a prognostic marker in atrial fibrillation.

Monitor Anticoagulant Theraputic anticoagulant levels will reduce the likelihood of new clots forming or the extension
of fresh clots. Monitoring D-dimer levels will confirm the effectiveness of anticoagulation.
Therapy
Monitor Thrombolytic

D-dimer is a sensitive marker of systemic fibrinolytic activity.

Therapy

Clot Imaging Agent - ThromboView

What is ThromboView?
ThromboView is an AGEN project to develop a clot imaging agent based on the proprietary D-dimer monoclonal antibody,
DD3B6/22, for diagnostic imaging of Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE).

What is DVT?
In DVT, a blood clot develops in the deep veins of the lower limbs. The clot blocks the supply of blood carrying oxygen and
nutrients to an organ or extremity and can lead to a wide variety of thrombotic complications including pulmonary embolism,
cerebral thrombosis (stroke) or myocardial infarction (heart attack). The major constituents of the blood clots are red cells,
white cells and platelets, bound together in a fibrin mesh. The DD3B6/22 antibody binds to sites that are only present on the
fibrin mesh, the D-dimer site.

How does ThromboView work?

Purified DD3B6/22 antibody is prepared and radiolabeled with a suitable radioisotope for imaging. Following injection of the
product into patients suspected of suffering from DVT or PE, the radiolabeled antibody localises to D-dimer sites present on the
clot. Subsequent imaging of the patient with a gamma camera allows for confirmation of diagnosis in suspected DVT/PE
patients. Confirmation of the imaging potential of this antibody has already been demonstrated by the nuclear imaging of
(DVT) clots in over 20 patients; the results were published in The Journal of Nuclear Medicine *. The results from this study
have been internationally recognised as the best in the field.
Monoclonal antibodies have been available for many years, but they are only now beginning to be introduced into the
pharmaceutical market. This emergence has been linked to recent technological improvements in the biotechnology industry,
which allow for "humanisation" of antibodies to reduce their immunogenicity - one of the major limitations of their use in
human medicine. Most antibodies for medical use are derived from mice and are therefore recognized as foreign molecules
when injected into humans. The humanisation process removes the mouse like sections of the antibody that are not involved
in the antigen binding, and replaces them with a human equivalent. In addition, any mouse like components that are left, are
engineered to appear more human like, reducing immunogenicity of the antibodies to humans.

Clinical Trials
Pre-clinical trials are required to characterize the effectiveness of the humanised DD3B6/22 antibody. Trials in clinical sites will
follow to image clots in patients suspected of DVT. The humanised DD3B6/22 antibody will be radiolabeled with Technetium99m (a commonly used radioisotope in nuclear medicine) and injected into patients enrolled in the study. Radioisotopic images
of the patients taken with a medical gamma camera can then be examined for vascular irregularities indicative of the presence
of blood clots. The successful outcome of this proposed human trial, will support the application of ThromboView in the
diagnosis of DVT and subsequent therapeutic management.

Clinical Benefits
The incidence of venous thromboembolism in Europe, North America and Australia is greater than four million per year. The
number of diagnostic investigations per year in the US alone is estimated to be in excess of one million per year.
Accurate diagnosis is crucial for successful clinical management of DVT and PE.
ThromboView offers improved diagnosis over current scanning procedures through the specificity of the DD3B6/22 antibody for
detection of the fibrin blood clots.
An independent commercial assessment indicates potential worldwide sales of such an imaging agent could exceed $100
million annually.

*Bautovich, G., et al 1994. Detection of deep vein thrombosis and pulmonary embolus with Technetium-99m-DD3B6/22 anti fibrin monoclonal antibody Fab' fragment. J.Nucl.Med. 35 195-202.

Products currently manufactured by AGEN are used for blood testing in medical conditions arising from cardiovascular
disorders and infectious disease in humans and companion animals.
AGEN Services include contract manufacturing and GMP Distribution of Clinical Trial Materials. See Contacts for more
information.
AGEN's R&D focus in medical and veterinary diagnostics is on developing simple and rapid blood testing products.
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All investor enquiries should be directed to:
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