PRODUCT INSERT SHEET

Download the TpP Product Insert Sheet (approx 300KB)

Intended Use
Summary and Explanation
Device Description and Principle of the Method
Contents
Cautions
Components Included in the TpP Test Kit
Storage and Stability
Additional Materials and Equipment Required
Specimen Collection and Preparation
TpP Assay Procedure
Quality Control
Important Notes
Results
Specific TpP EIA Performance Characteristics
Variance of TpP EIA
References

ABS TpP™ (Thrombus Precursor Protein) Assay
Enzyme Linked Immunosorbent Assay for TpP™Polymers in Human Plasma
For In Vitro Diagnostic Use
Store between 2-8æC

Intended Use

TpP™ EIA is an enzyme-linked immunoassay for the quantitative determination of soluble fibrin polymers in human plasma as an aid in risk assessment of thrombosis and monitoring anticoagulant (heparin) therapy.

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Summary and Explanation

One of the key events in the formation of a thrombus (blood clot) is conversion of the circulating soluble plasma protein fibrinogen to the insoluble cross-linked fibrin polymer. The penultimate step in fibrin formation is the conversion of prothrombin to thrombin by the prothrombin complex. Thrombin cleaves fibrinopeptide A from the fibrinogen molecule exposing polymerization sites on the newly formed desAA fibrin monomer units. Fibrin monomers can complex with intact fibrinogen, degradation products of fibrinogen and fibrin, and polymerize with other desAAfibrin monomers. This milieu of fibrin complexes, also known as soluble fibrin, is an indicator of the level of thrombin activity in the circulation. As the polymerization of desAAfibrin proceeds, thrombin removes another set of peptides, (fibrinopeptide B), from the fibrinogen molecule resulting in the formation of a species known as desAABBfibrin (1). These soluble polymers are the immediate precursor to insoluble fibrin and are thus referred to as Thrombus Precursor Protein (TpP™). As polymerization proceeds, the soluble fibrin polymeric entities, TpP™, become incorporated into the thrombus as insoluble fibrin polymers.

Soluble fibrin polymers have been identified by electrophoretic techniques in the plasma of patients with various clinical conditions including myocardial infarction (MI) (2) and deep vein thrombosis (DVT) (3). Elevated soluble fibrin levels, as determined by ELISA, have also been reported in other clinical conditions where intravascular fibrin formation has been indicated, including disseminated intravascular coagulation (DIC) (4, 5): and patients undergoing surgical procedures who are experiencing thrombotic complications (6, 7, 8). It has also been demonstrated that TpP™ levels are significantly lower in patients who are undergoing invasive surgical procedures (e.g. PTCA) and have been adequately anticoagulated (9).

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Device Description and Principle of the Method

The TpP™ EIA is a standard sandwich type enzyme immunoassay that employs a murine monoclonal antibody (Mab), specific for soluble fibrin polymer, as a capture antibody immobilized on a microwell strip. This Mab recognizes a conformational epitope present only on TpP™ entities but which is absent from fibrinogen and degradation products of fibrin and fibrinogen (10). During the first incubation phase, TpP™ in human plasma specimens bind to the capture antibody. Afterwards the plate is washed, and a conjugate, another murine monoclonal antibody, labeled with horseradish peroxidase (HRP) is added to the well. This peroxidase conjugated Mab binds to a different site on the TpP™ molecule during the second incubation period (11). Excess enzyme conjugated Mab is washed out and a subsequent application and incubation with tetramethylbenzidine (TMB) substrate follows. The reaction after TMB incubation is terminated with dilute sulfuric acid, producing a yellow color. The level of the TpP™ present in the specimen sample is directly proportional to the resulting color intensity. Calibrator standard is provided with the kit.

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Contents

The TpP™ EIA contains sufficient reagents and calibrator to assay up to forty-one samples in duplicate.

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Cautions:

Handle with care, potentially infectious material. The calibrator in this kit is from human source material that was tested by a FDA approved method and found non-reactive for the presence of HBsAg and antibody to HIV. Because no known test method can offer complete assurance that hepatitis B virus, human immunodeficiency virus (HIV) or other infectious agents are absent, all human blood-based products should be handled in accordance with good laboratory practices using appropriate precautions. This reagent is not for internal or external use by humans or animals. All solutions supplied should be handled as though they are potentially dangerous.

Components Included in TpPTM Test Kit:

• 1 microelisa plate (MEP):
  (96 wells) pre-coated with TpP™ murine capture Mab and post coated with blocking solution (ready to use).
  
• 1 vial Conjugate Solution:
  12 ml of murine Mab conjugated to horseradish peroxidase in a protein stabilizing buffer (ready to use).
  
• 1 vial TpP™ Calibrator (TpPC):
  Lyophilized material containing TpP™. Reconstituted TpP™ level is indicated on the Calibrator certificate of analysis.
  
• 1 bottle Dilution Buffer A (DB):
  30 ml: A 1.0% solution of bovine albumin, fraction V in tris buffer, pH 7.2 – 7.5 (ready to use).
  
• 1 bottle Stopping Reagent:
  30 ml: 0.5 M H2SO4 (ready to use).
  
• 1 bottle Developing Solution:
  30 ml: 3,3’,5,5’ - tetramethylbenzidine (TMB, ready to use).
  
• 10X Wash Buffer Concentrate:
  50 ml of phosphate buffered saline with tween 20TM and proclin as a preservative.
  

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Storage and Stability

The expiration date of each of the reagents supplied with the kit is specified on the individual container label. The entire contents of the kit should be stored at 2-8æC. Do not freeze! The reconstituted calibrator and standards will lose activity if subjected to freezing. Do not mix or interchange reagent lots with any other kit or different kit lots.

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Additional Materials and Equipment Required

• Adjustable pipettes, single and multiple
  
• Microelisa plate reader, wavelength 450 nm
  
• Timer
  
• Plastic, polypropylene tubes or vials for preparing diluted standards
  
• One ml syringe and hypodermic needle

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Specimen Collection and Preparation

Blood samples should be collected by routine venipuncture using siliconized evacuated blood tubes containing sodium citrate as the anticoagulant. The ratio of blood to anticoagulant is 9:1. In the USA, follow NCCLS standard H21 –A2, “Collection, Transport, and Processing of Blood Specimens for Coagulation Testing and Performance of Coagulation Assays.” Mix blood and anticoagulant by gentle inversion six (6) times immediately after drawing, place samples in ice (or at 4æC) and centrifuge at 1500-2000 g for 15 minutes at 4æC within one hour of draw. Plasma should be removed and analyzed immediately or frozen at -80æC until analysis. Do Not use plasma samples that have been frozen and thawed more than once. The use of EDTA and/or heparin anticoagulants is not recommended for use in this product.

10X Wash Buffer Concentrate: Directions for Preparation

1. Prior to preparation, warm 10X Wash Buffer Concentrate to ambient temperature by allowing it to stand for one hour on the benchtop, or by briefly warming it in a 37æC water bath. Gently mix before use.
   
2. Dilute the Wash Buffer Concentrate 1:10 with distilled/deionized water. To dilute the entire bottle, add the contents of the bottle to
   450 ml of distilled/deionized water. Using HCl or NaOH, adjust the pH of the diluted buffer to 7.5 (±0.1) prior to use (with constant stirring). The working dilution should be stored at 2-8æC. The expiration date is on the concentrate label. Smaller quantities may be prepared by diluting 1 volume of 10X Wash Buffer Concentrate with 9 volumes of distilled/deionized water.

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TpP™ Assay Procedure

Preparation of the MEP:

Allow the coated plate to equilibrate to room temperature (22±3†C). Wash the plate once with wash buffer. Tap plate dry on blotting paper.

Preparation of TpP™ Calibrator Standard Dilutions

Begin preparation 15 (±5) minutes before application of samples to plate. Reconstitute TpP™ Calibrator with one ml of double distilled water. Add water through the vial’s rubber septum utilizing a syringe fitted with a hypodermic needle. Remove syringe leaving needle in septum to equilibrate air pressure. Allow calibrator to incubate at room temperature (22±3æC) for 15 minutes mixing regularly. Reconstituted calibrator and standards must be stored at 2 - 8æC and will be stable for 30 days after reconstitution and dilution. Avoid prolonged time at room temperature and do not freeze. Visually inspect vial to ensure complete solubilization of calibrator. If calibrator does not reconstitute properly, contact manufacturer (ABS) before proceeding. Refer to TpP™ Calibrator certificate of analysis, provided with kit, for concentration (X) after reconstitution.
Use syringe to remove reconstituted material. Prepare a 1:13 dilution of the reconstituted calibrator with the Dilution Buffer (DB) to obtain the starting standard solution, A (X ÷ 13), the highest calibrator value on the standard curve. Then use the Dilution Buffer to prepare successive two-fold serial dilutions, mixing thoroughly between dilutions, to obtain the remaining standards according to the following scheme:

Preparation of Samples and Controls

Prepare samples and controls 15 (±5) minutes prior to application on the plate. Thaw frozen plasma samples in a 37†C water bath for 5 (±2) minutes. Any sample aliquot found to contain a visible clot upon thawing should not be tested. Use dilution buffer A (DB) as the blank absorbance sample.

Application of Standards, Controls and Samples

Pipette 25 mL in duplicate of each standard, control, and sample into the appropriate wells as indicated in the ELISA template work sheet. Pipette 100 mL of Dilution Buffer into each well with a multichannel pipetor. Cover plate and incubate for 1 hour at room temperature. To avoid “end of run effects” it is necessary to avoid any delays from the time the sample is pipetted until the dilution buffer is added. At the end of the 1 hour incubation shake out the plate and wash 3 times with the wash buffer. Tap dry on blotting paper after final wash.

Application of the Conjugate

Allow the Conjugate Solution to equilibrate to room temperature (22±3†C). Pipette 100 mL of conjugate solution into each well. Incubate for 20 minutes at room temperature. Shake out plate and wash 3 times with the wash buffer. Tap plate dry after final wash.

Preparation and Application of Developing Solution

Prior to conjugate application, allow the TMB Developing Solution and Stopping Reagent to equilibrate to room temperature (22±3†C). Apply 100 µL Developing Solution to each well. Incubate in the dark at room temperature (22±3†C) in an area free from major temperature fluctuations for 15 minutes. Stop reaction by adding 100 µL of Stopping Reagent to each well. Read absorbance at 450 nm immediately. Plates should be mixed promptly prior to reading absorbance. (Note: the Developing Solution is light sensitive. Normal appearance is clear or light yellow to light blue-green.)

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Quality Control

The use of a normal control and an abnormal (high) control is recommended for quality control of the assay. The normal control should produce a result of £ 6 µg/ml and the abnormal control should produce a result of > 6 µg/ml. It is recommended that each laboratory establish its own normal range using plasma obtained from healthy volunteers.

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Important Notes

• Blood Samples should be collected into tubes/syringes containing sodium citrate (0.129M) with a blood to anticoagulant ratio of 9:1
• Thaw frozen samples in a waterbath at 37°C
• Read absorbance at 450 nm
• Thrombolytic therapy degrades fibrin polymers and may affect the results obtained from the analysis

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Results

Development of a Standard Curve

Subtract mean of the blank absorbance values from each of the standards, controls, and samples to give corrected absorbance values. Calculate the mean corrected absorbance of the replicates of each TpP™ Calibrator standard dilution and plot versus the TpP™ associated concentration (i.e. the standard value of A, B, C, D, and E µg/ml). The points will approximate a straight line intercepting the X axis near zero absorbance units. A representative curve is shown below.

Calculation of TpP™ in Plasma Sample

Develop a linear equation for the standard curve. Insert the corrected absorbance values for each control and sample to calculate the TpP™ concentration of the samples and controls in units of µg/ml.

Validity of Assay

The assay results are considered valid if:

• The correlation coefficient for the regression analysis, r2 é 0.950
• Mean value of the blanks £ 0.100
• Optical Density (O.D.) value of the top value on the standard curve, A, is 1.2 – 2.0
• The CV of the values from replicates should be comparable to those reported in the performance characteristics of the TpP™ EIA.

Expected Results

An effective cutoff was determined by examination of TpP™ values in control populations tested at two sites (n=140). The best estimate of an effective cutoff value was determined to be 6.65 µg/ml by employing a percentile evaluation. Normal TpP™ values from healthy volunteers should produce results £ 6 µg/ml.
The values obtained from this assay are intended to be an aid to diagnosis only. Each physician must interpret the results in light of the patient’s history, physical findings and other diagnostic procedures.

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Specific TpP™ EIA Performance Characteristics

Linear Reportable Range and Minimum Detectable Level

The measuring range of TpP™ EIA is 0 - 30 µg/ml. The minimum detectable TpP™ concentration distinguishable from 0 µg/ml is 0.13 µg/ml. This concentration corresponds to the value of the mean absorbance plus two standard deviations for a 0 µg/ml calibrator.

Interferences

The TpP™ EIA has been evaluated with potential interfering substances. This interference study utilized a dose response method utilizing Hemoglobin (5, 2.5, 1, 0.5, 0.25 mg/ml), Bilirubin (0.2, 0.1, 0.05, 0.02, 0.01 mg/ml), Triglycerides (400, 250, 200, 125, 50 mg/dL), and Urokinase (100, 50 NIHU/ml). No significant interference was observed when normal plasma samples and normal plasma samples spiked with the noted level of analyte (interferent) were tested. No interference studies have been performed utilizing fibrinogen, fibrin monomers, fibrinogen degradation products, or fibrin degradation products as an interferent in patient samples.

Precision

Variability was determined by testing two samples in 10 separate runs, with replicates of 3 in each run. The concentrations were calculated from a calibration curve, with sample A, mean=25.44 µg/ml, selected well above the pathological cutoff and sample B, mean=9.34 µg/ml, selected near the pathological cutoff. Assay variances, standard deviation and coefficient of variation are presented in the table below.

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Variance of TpP™ EIA

Results from Clinical Studies

In a population of patients undergoing surgical procedures to repair aortic aneurysm (n=20) TpP™ levels were tested pre-operatively, immediately following the procedure and at times 6, 12, 24, 48, and 72 hours post-surgery. It was shown that the TpP™ levels became elevated immediately following surgery indicating a hypercoaguable state. The levels of TpP™ remained elevated for 48 hours returning to baseline at 72 hours post surgery. As an indication of thrombosis TpP™ may be used to monitor coagulable states following surgical procedures.

In a second study the utility of TpP™ analysis for monitoring anticoagulant (AC) therapy following percutaneous transluminal coronary angioplasty (PTCA) was investigated. Systemic heparin was administered to patients, n=25, undergoing this procedure. Samples for TpP™ analysis were drawn prior to, and at 1 hour post heparinization. An additional sample was drawn 1 hour following the PTCA procedure. Sixteen patients had normal levels of TpP™ throughout the procedure. Three patients from this group had evidence of intra-coronary filling defects and were treated with urokinase. Thrombolytic therapy degrades fibrin polymers and may explain the normal TpP™ levels. Five patients with elevated TpP™ prior to heparinization demonstrated a return to baseline following heparin intervention. The remaining four patients had elevated levels throughout the procedure and all developed thrombotic complications. It is suggested that TpP™ may be used to monitor the efficacy of AC therapy.

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References

1. Harker LA, Mann KG. Thrombosis and Fibrinolysis. In: Fuster V, Verstraete M, eds. Thrombosis in cardiovascular disorders. WB Saunders 1992:1-16.
   
2. Francis CW, Conaghan DG, Scott WL, Marder VJ. Increased plasma concentrations of cross-linked fibrin polymers in acute myocardial infarction. Circulation. 1987;75(6):1170-1176.
   
3. Francis CW, Kornberg A. Fibrinogen- and Fibrin Degradation Products During Fibrinolytic Therapy. Annals of the New York Academy of Sciences, 1993; 667, 310- 323.
   
4. Wieding JU, Hosius C. Determination of soluble fibrin: a comparison of four different methods. Thrombosis Research 1992;65:745-756.
   
5. Okajima K, Koga S, Okabe H, Inoue M, Takatsuki K. Characterization of the fibrinolytic state by measuring stable cross-linked fibrin degradation products in disseminated intravascular coagulation associated with acute promyelocytic leukemia. Acta Haematol. 1989;81:15-18.
   
6. Vogel G, Spanuth E. Predictive value of fibrin monomers in postoperative deep vein thrombosis. Klin Wochenscher. 1990;68:1020-1026.
   
7. Niewenhuizen W. Soluble fibrin as a molecular marker for a pre-thrombotic state: a mini-review. Blood Coag. Fibrinol. 1993;4:93-96.
   
8. Rosenfeld B.A., Carville D.G.M., Dimitrijevic N., Spencer D.B. & Gargan P.E. The determination of soluble fibrin polymers in surgical patients. Thrombosis & Haemostasis. 1995; 73: 227.
   
9. Ogilby J.D., Lin B.L., Turco M.A., Reiley J., Carville D., A Method for Monitoring Formation of Arterial Thrombosis During PTCA, Identification of thrombus precursor protein (TpP) in the unstable PTCA patient, Submitted for publication.
   
10. Gargan P.E., Gaffney P.J., Pleasants J.R., Ploplis V.A., A Monoclonal Antibody which Recognizes an Epitopic Region Unique to the Intact Fibrin Polymeric Structure, Fibrinolysis, 1993; 7, 275-283.
    
11. Gargan P.E., DaMatta R.A., Silverman T., Ploplis V.A., Immunochemical evidence for intramolecular interaction of the carboxy terminal Aa-appendages of plasma fibrinogen, Blood Coagulation and Fibrinolysis. 1990; 1, 457-460.

TpP is a trademark of American Biogenetic Sciences, Inc.
Tween is a trademark of ICI Americas

Covered by United States Patent numbers 5,453,359; 5,120,834; 5,091,512; and additional United States and foreign patents pending.

Manufactured in the U.S.A.

TpP™ Product Insert

TIV~061099