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A Practical Guide to Haemostasis


Fibrinolysis:
Fibrinogen Degradation Products [FDPs]


Introduction

Fibrinogen consists of three pairs of polypeptide chains: two Aα, Bβ and γ.  These are linked together by 29 disulphide bonds in such a way that N-terminal regions of the 6-polypeptide chains meet to form a central E-domain. The C-terminal regions [Aα, Bβ and γ] form the D-domain and these are joined by α-helical ropes to the central E-domain to give the characteristic Fibrinogen structure.



Activation of Fibrinogen by Thrombin [IIa] cleaves the two short peptides from the N-terminal regions of the Aα and Bβ chains - these peptides are known as Fibrinopeptide A [FpA - 16 amino acids] and Fibrinopeptide B [FpB - 14 amino acids] respectively.  Removal of the N-terminal sequences from the Aα and Bβ chains chains reveals new N-terminal sequences in the Aα and Bβ chains located within the E domain, known as 'knobs.'  These knobs can interact spontaneously with the D-domains to form Fibrin polymers.  Under the influence of factor XIIIa, cross-linking of these Fibrin polymers [between Glutamine and Lysine amino acids] occurs to form cross-linked Fibrin polymers.

D-dimer


To recap the sequence is:
   Fibrinogen → Fibrin monomer → Soluble Fibrin polymer [non cross-linked] → Cross-linked Fibrin polymer

Thrombin activation of Factor XIII is accelerated by the presence of non–cross-linked Fibrin but is inhibited by fully cross-linked Fibrin. This auto-regulation serves to help limit Factor XIIIa activity to sites of Fibrin clot formation. The principal sites of cross-linkage induced by FXIIIa are between the γ-domains in the D domains of adjacent Fibrin monomers and between the carboxyl-terminal ends of the α chains of Fibrin monomers.

Fibrinogen Degradation Products [FDPs]

Fibrinogen Degradation Products [FDPs] consisting of Fragments X, D, Y and E are generated when Fibrinogen or non-cross linked Fibrin [soluble Fibrin] is broken down by Plasmin and are, therefore, fundamentally different from D-Dimers which are generated when cross-linked Fibrin [Fibrin that has been cross-linked by Factor XIIIa] is broken down.

1. Degradation of Fibrinogen and non-cross Linked Fibrin

Degradation of Fibrinogen


Fibrinogen [and soluble, non-cross linked Fibrin] is degraded by Plasmin generating a series of fragments [see above].
Partial degradation of the γ chain gives rise to Fragment X and then further digestion of this fragment gives rise to Fragment Y.  Digestion of Fragment Y gives rise to Fragment E and two D Fragments.

2. Degradation of Cross-Linked Fibrin
Digestion of cross-linked Fibrin by Plasmin gives rise to a variety of fragments including D-dimers. These define the breakdown of cross-linked Fibrin [i.e. Fibrin that has been cross-linked by FXIIIa] as the cross-linking links D-fragments. Digestion by Plasmin cleaves cross-linked Fibrin in a random order to generate a series of soluble fragments [so called 'X-oligomers'] of varying molecular weights and including D-dimers. In each of these breakdown steps, the γγ cross-links are retained and so D-dimers are present. Although the diagram below shows D-dimers in isolation, in practice they exist as a variety of D-Dimer containing oligomers e.g. EDD. The process is dynamic and more fragments are generated as the process proceeds. D-dimers create a neo-epitope and it is this that is detected by specific antibodies used in the various detection systems.

D Dimer generation by Plasmin



Principles & Method


1. Latex particle agglutination assays. In this assay Latex particles coated with anti-FDP antibodies

There are assays for FDPs in both serum and plasma. Serum FDP assays use polyclonal antibodies that cross-react with intact fibrinogen (that has not been lysed by plasmin), necessitating its removal. This is accomplished by the use of specialized collection tubes containing Botrox atrox venom (Reptilase) and inhibitors of fibrinolysis (aprotonin or soyabean trypsin inhibitor). All serum FDP assays use latex beads that are coated with antibody raised against human fibrin(ogen) degradation products, usually fragments D and E. The antibodies cross-react well enough with the fibrin(ogen) fragments of animals to make the test useful for veterinary patients. 2 mLs (as little as 0.5 mls can be used if the patient is very small) of blood from the patient is put immediately into one of the tubes provided with the test kit

There are newer latex-agglutination kits that are based on monoclonal antibodies that do not cross-react with intact fibrinogen and can thus be used on citrated plasma samples. This is advantageous compared to serum FDP assays, as a specialized collection tube for FDP assay is not required and a single citrated blood sample can be used for all coagulation tests. This is the procedure performed by the Clinical Pathology laboratory at Cornell University and it has only been validated for the dog. The plasma FDP assay is performed similarly to the serum FDP assay, except dilutions of 1:2 and 1:8 are made, with results being reported as < 5 µl/mL, 5-20 µl/mL and > 20 µl/mL. Results > 5 µl/mL are abnormal.




Test principle of the ELISA Kit for Fibrinogen Degradation Product (FDP)

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Fibrinogen Degradation Product (FDP). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Fibrinogen Degradation Product (FDP). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Fibrinogen Degradation Product (FDP), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Fibrinogen Degradation Product (FDP) in the samples is then determined by comparing the O.D. of the samples to the standard curve

Interpretation

1.

  • DIC
  • Eclampsia
  • Carcinoma
  • Post-operative
  • Cardiac or hepatic disorders
  • Fibrinolysis
  • Pulmonary embolism
  • Deep vein thrombosis

High levels of rheumatoid factor (RF) may lead to false agglutinations. 

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EnGLISH DEUTSCH
I. Intended Use
Pacific Hemostasis FDP Assay Kit, Control Set, Latex Reagent, Glycine Buffered Saline (GBS), and
Sample Collection Tubes are intended for use in the detection of Fibrinogen/Fibrin degradation
products (FDP) in serum or urine.
II. Summary and Principles
FDP are found at low levels in the sera of all healthy individuals because of normal fibrinolytic
mechanisms. The mean normal level in resting adults is 4.9 ± 2 8 μg/mL. 1 Slight elevation can occur
after stress, exercise, or anxiety.2 Higher serum FDP levels are associated with disseminated
intravascular coagulation (DIC)3, deep vein thrombosis4, some pregnancy disorders5,6, myocardial
infarction7, and pulmonary embolism.8
Urine normally contains less than 0 25 μg/mL FDP.9 Elevated levels can appear in renal disease such
as glomerulonephritis10, and in acute graft rejection.11
The Pacific Hemostasis FDP Assay Kit uses direct latex agglutination to detect elevated FDP in
serum or urine. Latex particles coated with anti-human fibrinogen are mixed with defibrinated serum
or urine and examined for macroscopic agglutina


Reference Ranges

It is