Special Laboratory Evaluaion For

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SPECIAL LABORATORYEVALUAION FOR

FIBRINOLYSIS

BY: DINALAGAN, DIVINAG



TESTS OF FIBRINOLYSIS

A.) Quantitative D-Dimer Immunoassay

Physiology of Fibrin Degradation Products

and D-Dimers

oDuring coagulation, fibrin polymers become cross-linked by fac

simultaneously bind plasma plasminogen and tissue plasminogen act

• Over several hours, bound TPA activates nearby plasminogen to form

bound plasmin cleaves fibrin and yields the FDPs D, E, X, andY and D



• The FDPs represent original fibrinogen domains, and D

covalently linked D domains reflecting the cross-linking effeXIIIa.

• Assays for FDPs, including D-dimer, are convenient for detfibrinolysis, which indirectly implies the occurrence of thrombo

• Normally, FDPs, including D-dimer, circulate at concentrations

ng/mL.• Fibrinolysis yields FDPs and D-dimer at concentrations grea

ng/mL.



• Increased FDP and D-dimer concentrations are characteristic ofchronic DIC, systemic fibrinolysis, deep vein thrombosis, and puembolism.

• FDPs, including D-dimer, also are detected in plasma after thro

therapy.



Principle of the Quantitative D-Dimer

Plasma D-dimer immunoassays abound, and several diagnosticoffer automated quantitative immunoassays for plasma Dgenerate results within 30 minutes. Microlatex particles in buffercoated with monoclonal anti – D-dimer antibodies. The coated agglutinated by patient plasma D-dimer; the resultant turbidity

using turbidometric or nephelometric technology.Sensitivity varies depending on the avidity of the monoclonal and the detection method; however, most methods detect conclow as 10 ng/mL.





• Because of the high sensitivity but low specificity (60% to

quantitative D-dimer assay, physicians do not use this assaydiagnose venous thromboembolic disease, but only to rule it ou

• Because any chronic or acute inflammation is accompanied bydimer concentrations, the assay cannot be used to “rule in” throdisease.

•

The upper limit of the reference interval for the quantitative Dvaries with the methodology, ranging from 250 ng/mL to 500 nD-dimer levels may reach 10,000 to 20,000 ng/mL.



B. D-Dimer Assay

• The automated quantitative D-dimer assay has largely replacedimer or FDP assays.

• The SimpliRED D-dimer assay is a manual method that usesparticles coated with monoclonal antibody.

• The SimpliRED D-dimer assay is suited to low-volume or (point-of-care) applications. The manufacturer reports the clinifor pulmonary embolus to be 94% and the specificity to be 67%



C. Fibrin Degradation Product Immun

• Although the FDP assay has largely been replaced by thequantitative D-dimer assay or the manual semiquantitative D-FDPs may be detected using a semiquantitative visible immunoassay.

• One such method is the 1972 Thrombo-Wellcotest (Remel,

Kan.).• Polystyrene latex particles in buffered saline are coated wit

antibodies specific for D and E fragments calibrated to deteconcentration of 2 mcg/mL or greater.



• The assay usually is performed on serum collected in speciapromote clotting and prevent in vitro fibrinolysis, although passays are also available.



D. Plasminogen Chromogenic Substrat

• Plasminogen, the precursor of the trypsinlike proteolytic enzymproduced in the liver and circulates as a single-chain glycoprote

• When bound to fibrin, plasminogen is converted to plasmin bynearbyTPA.

• Bound plasmin degrades fibrin, whereas free plasmin is rapidby a circulating inhibitor, α2-antiplasmin.



Congenital plasminogen deficiencies are assocthrombosis in some families. Acquired plasminogen dare seen in DIC and acute promyelocytic leukemia. Thr

therapy is ineffective when plasminogen activity

Plasminogen is readily measured in PPP using a ch

substrate assay, available from several manufacturers



Principle of the Plasminogen ChromogSubstrate Assay

• Chromogenic substrates employ synthetic polypeptides whossequences are designed to be specific for particular enzyhydrolyzes a bond in the polypeptide sequence valine-leucineLeu-Lys).

• A fluorophore or a chromophore such as para-nitroanilicovalently bound to the carboxyl end of the polypeptide released on digestion. S-2251, composed of H-D-Val-Leu-Lchromogenic substrate for plasmin. On plasmin digestion,released and turns yellow.



Streptokinase is an exogenous plasminoge

derived from cultures of β-hemolytic streptococci. Strepadded to patient PPP, where it binds and activates pla

The resulting streptokinase-plasmin complex reac

chromogenic substrate such as S-2251 to release a cintensity is proportional to the plasminogen concentrati

Several analogous chromogenic and substrates are suitable for plasminogen measurement

plasma is tested with the patient plasma, and the recorded.



Assay of plasma plasminogen using the chromogenmethod. Reagent streptokinase activates plasminogen to formpNA designates a chromogenic substrate, where R indicates seve

peptide sequence and pNA (para-nitroaniline) is the chromophore

In the case of plasminogen, the R represents the peptivalineleucine-lysine-pNA (Val-Leu-Lys-pNA). The Val-Leu-Lys amis recognized and hydrolyzed by plasmin.



Results and Clinical Utility of the PlasminChromogenic Substrate Assay

The plasminogen reference interval is 5 to 13.5 mg/dL. levels are decreased in thrombolytic therapy, DIC, hepatitis,Hereditary deficiencies also have been recorded. Decreased plaassociated with thrombosis. Plasminogen rises in inflammatiopregnancy, and high levels may be associated with hemorrhage.

Plasminogen levels are elevated in systemic fibrinolysis. assays are seldom offered in acute care facilities but are readilyspecialty reference laboratories



E. Tissue Plasminogen Activator AsPhysiology of Tissue Plasminogen Activat

The two physiologic human plasminogen activatoand urokinase

TPA is synthesized in vascular endothelial cells and rethe circulation, where its half-life is approximately 3 m

its plasma concentration averages 5 ng/mL .



Urokinase is produced in the kidney and vascular endothelial ce

half-life of approximately 7 minutes and a concentration of 2 to 4

Both activators are serine proteases that form ternarwith bound plasminogen at the surface of fibrin, activating the and initiating thrombus degradation. Both are covalently inact

endothelial secretion plasminogen activator inhibitorb 1 (PAI-1).



Clinical Significance of Tissue Plasmin

Activator

The reference interval upper limit for TPA activitymL, and the upper limit for TPA antigen is 14 ng/mL.

primary mediator of fibrinolysis and is the model for syn

Decreased TPA levels may indicate increase

myocardial infarction, stroke, or deep vein thrombosismore data are needed to verify a relationship. Impaired

in the form of TPA deficiency or PAI-1 excess also is asso

deep vein thrombosis and myocardial infarction.



Specimen Collection for the Tissue PlasmActivator Assay

TPA activity exhibits diurnal variation andexercise. Further, TPA is unstable in vitro because it raPAI-1 after collection. For specimen collection, patient

at rest, tourniquet application should be minimal, and

acidification of the specimen in acetate buffer is Acidification may be accomplished using the Stabilyt

citrate tube. Supernatant PPP may be frozen at −70° assay is performed.



Principle of the Tissue Plasminogen AcAssay

The level of TPA antigen may be estimated immunoassay. To measure TPA activity, a specified con

of reagent plasminogen is added to the patient plasmTPA activates the plasminogen, and the resultant plasmis measured using a chromogenic substrate. The resu

intensity is proportional to TPA activity. The sy

incorporate soluble fibrin to increase TPA activity.



To assay tissue plasminogen activator (TP

containing TPA is added to plasminogen to producPlasmin activity is measured using the same ch

substrate system as in the plasminogen assay. The icolor is proportional to TPA activity. pNA, Paranitrovariable peptide sequence.



F. Plasminogen Activator Inhibitor 1

PAI-1 is produced by vascular endothelial c

hepatocytes and circulates in plasma bound to vitronecaverage concentration of 10 mcg/L with diurnal variations

An inactive form of PAI-1 circulates in high concentplatelets. PAI-1 inactivates free TPA by covalent binding.

PAI-1 is associated with venous thrombosis and mcardiovascular risk factor.

A few cases of PAI-1 deficiency have been reported;

hemorrhage apparently occurs only in the complete abPAI-1.



Blood is collected from patients at rest into an acidified c(Stabilyte; Tcoag US) and centrifuged immediately to make PPP; thvitro release of platelet PAI-1. Several immunometric and chsubstrate methods are available for estimation of PAI-1 antigenactivity. One enzyme immunoassay for functional PAI-1 uses urokinPAI-1. The urokinase–PAI-1 complex is immobilized with smonoclonal anti–PAI-1 and is measured using monoclonal antiurokidetecting antibody.

Most chromogenic substrate kits for PAI-1 use a

measurement approach (Spectrolyse PAI-1; Tcoag US). Patient PPwith a measured amount of reagent TPA. Residual TPA is assaplasminogen system as shown in Figure 45-15. The resulting color inversely proportional to plasma PAI-1 activity.



Confirmation of complete PAI-1 deficiency may be accomplisheserum PAI-1 assay. In serum, platelet PAI-1 is expressed in excess. absence, no PAI-1 is detectable in serum.

To assay plasminogen activator inhibitor 1 (PAI-1) activity, plasmPAI-1 is added to reagent tissue plasminogen activator (TPAconcentration. The residual TPA is assayed as shown in Figureintensity of color is inversely proportional to PAI-1 level. nitroaniline; R, variable peptide sequence.



G. Euglobulin Clot Lysis Time Tes

Excessive fibrinolytic activity occurs in a variety oInflammation and trauma may be reflected in a radical increase plasmin that has the potential to cause hemorrhage. Bone traumand surgical dissection of bone, as in cardiac surgery, may raisactivity. Fibrinolysis deficiencies occur when TPA or plasmi

become depleted, or when excess secretion of PAI-1 depresses Ttime-honored approach to measurement of fibrinolyticacteuglobulin clot lysis time (ELT) test.



Principle and Procedure of the EuglobuLysis Time Test

The ELT is a global screen of the fibrinolytic

the assay, plasma inhibitors of fibrinolysis are chemicalland the reactions of fibrinogen, plasminogen, and pl

activators are allowed to proceed uncontrolled. Their reported as a qualitative assessment using

the ELT.



Patient plasma is diluted and acidified wi

acetic acid until the solution reaches a pH of 5.35 to

refrigeration for approximately 30 minutes, a precipithat contains fibrinogen, plasminogen, plasmin, andprecipitate is termed the euglobulin fraction. Exclude

precipitate are α2-antiplasmin and PAI-1; in thefibrinolysis may proceed unchecked. The tubes are c

and the supernatant is decanted completely. The preredissolved in borate buffer, and reagent thrombin chloride is added. A clot should form within a few minute



A timer is started at the time of thrombin addition, anobserved periodically for dissolution (disappearance) over 2 to 1time required for the intrinsic plasmin to lyse the fibrin clot in hou

Results of Euglobulin Clot Lysis Time Te

If the euglobulin clot dissolves in less than 2 hours, excess

present. If the clot remains for longer than 10 hours, the fibrinolytdeficient.



Limitations of Euglobulin Clot Lysis Tim

Positive and negative plasma control specimens are inclused as the negative control, whereas PNP plus streptokinase (a

activator) is used as the positive control. The rate at which the cloin the patient fraction is compared with the rate of clot disappepositive and negative control specimens.

The scientist prepares another control, the “patientactivaThis is an aliquot of the patient’s euglobulin fraction with streptokThis control ensures against invalid results caused by depletio

plasminogen. When patient plasminogen levels are diminishelong-term DIC, clot dissolution extends beyond 10 hours. activated control indicates when this condition exists, because stactivated patient plasmin causes rapid clot dissolution. When pldepleted, the streptokinase-activated control produces no dissoluELT is prolonged.



Hypofibrinogenemia and factor XIII deficiency

ELT. In hypofibrinogenemia, there is less fibrin to be lyshort lysis time may be seen without a genuine ifibrinolytic activity. In factor XIII deficiency, the original is poor, and digestion appears normal or speedy eve

plasmin activity. The ELT also may be falsely prolonged

inflammation if the fibrinogen level exceeds 600 mg/dL.

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