VFE

An Enzyme Immunoassay for Quantifying von Willebrand Factor in Human Plasma

For In Vitro Diagnostic Use Only

Catalog Number: VE-98

 

 INTRODUCTION

VFE is an enzyme-linked immunoassay (ELISA) for quantifying von Willebrand factor (vWF, also called factor VIII-related antigen) in human plasma. This plasma protein factor is deficient or defective in patients with von Willebrand's disease, a relatively common inherited disorder associated with an increased tendency to bleed. Laboratory studies in these patients usually demonstrate:

1) a decrease in procoagulant activity as measured by activated partial thromboplastin time;

2) a prolonged bleeding time; and

3) decreased plasma concentration of vWF.

A major diagnostic problem concerns the differentiation of patients with von Willebrand disease from those with classical Hemophilia A (factor VIII coagulant deficiency). Patients with the latter disorder have a decrease in plasma procoagulant activity (i.e. a prolonged activated partial thromboplastin time) but have normal or increased concentration of vWF in the plasma. The VFE assay affords rapid differentiation of these two disorders.

VFE is also of use in identifying carriers of Hemophilia A, a sex-linked disorder; under most circumstances only males have bleeding problems. The affected males inherit the gene for Hemophilia A from their mothers who carry the involved gene, but have no bleeding problems.

In order to determine if a woman is a carrier of Hemophilia A, it is necessary to measure factor VIII coagulant activity and vWF concentration. The ratio of factor VIII coagulant activity to vWF concentration in carrier females is approximately 0.5 while the ratio in normal females is 1.0.

 

INTENDED USE

VFE is intended for use as an aid in the diagnosis and management of patients having an increased tendency to bleed. It is useful to differentiate patients with von Willebrand disease from those with Classical Hemophilia A (Factor VIII coagulant deficiency). The later patient group has prolonged activated partial thromboplastin time (PTT) but have normal or increased concentrations of von Willebrand Factor (vWF) in their plasma. In contrast, patients with von Willebrand disease have depressed levels of vWF.

VFE should be used whenever a patient demonstrates:

1) a decrease in procoagulant activity as measured by activated PTT; and/or

2) a prolonged bleeding time.

 

TEST PRINCIPLE

Ramco's VFE enzyme immunoassay for the non-radioactive quantitative determination of human von Willebrand Factor is a 2 step assay:

Step 1: Diluted plasma specimens, Calibrators, and Sample Diluent are pipetted into individual microtiter wells precoated with rabbit anti-vWF antibody. Horseradish peroxidase (HRP) conjugated antibody (sheep) is added to the wells and the plate is incubated. During incubation, vWF in each specimen and Calibrator diffuse throughout the well and are captured on the well surface. Concurrently, the HRP-conjugated antibody reacts with other sites on vWF antigens in the specimen and Calibrators forming a sandwich immobilized on the wall of the microtiter well.

Step 2: The mirotiter wells are washed four times to remove the non_bound reactants. The remaining HRP-labelled complex is detected by the addition of TMB, a substrate specific for the HRP enzyme. After adding the TMB, a blue color develops in proportion to the amount of vWF in each well. After a short incubation, a mild acid is added to stop the substrate conversion and change the color from blue to yellow. The yellow color is measured in a microplate reader using a 450nm filter. The amount of vWF present in each specimen is then determined by interpolating from a graph of the adsorption vs concentration for the Calibrators.

 

MATERIALS PROVIDED

This kit is intended for In-Vitro Diagnostic Use Only

Human von Willebrand Factor Calibrator: 2 vials containing 0.5ml of prediluted lyophilized human plasma with a known vWF concentration, borate saline buffer, bovine serum albumin, sheep serum, EDTA and ProClin 300TM as a preservative. The concentration of vWF in the Calibrators was determined by comparison to World Health Organization International Reference material.

At least fifteen minutes prior to use, reconstitute a vial of the Calibrator by carefully adding exactly 0.50ml of deionized, distilled water to the vial, briefly vortexing to mix and solubilize the diluted plasma, and allow to stand at room temperature for 15 minutes prior to use. The reconstituted Calibrator solution is stable at 2 - 8C for a maximum of 4 weeks.

Microtiter Wells: 8 (eight) 12 x 1 microtiter well Strips (96 total wells) coated with rabbit immunoglobulin isolated from antiserum specific for human vWF and stabilized in dry form. The Wells are provided in a resealable foil pouch containing a desiccant packet.

Plate Sealing Tape: 2 Sheets

Microplate Frame: 1

Specimen Diluting Buffer: 1 vial containing 45 ml of borate-saline buffer, bovine serum albumin, normal rabbit serum, normal sheep serum, ethylenediaminetetraacetate (EDTA) and ProClin 300TM as a preservative.

HRP-Conjugated Anti-vWF: 1 vial containing 22 ml of HRP-conjugate sheep antibody in a diluent of saline, normal sheep serum, a commercial stabilizing agent and ProClin 300TM as a preservative.

Substrate: 1 amber plastic bottle containing 22 ml of 3,3',5,5'-tetramethylbenzidine (TMB) one component substrate, in an acidic buffer, with 0.01% hydrogen peroxide. Because of its extreme sensitivity, avoid exposing this reagent to light, borosilicate glass and elevated temperatures. As supplied, the Substrate should be colorless, slightly amber or slightly bluish tinted.  DO NOT USE THIS SUBSTRATE IF IT IS DEEPLY BLUE IN COLOR BEFORE ADDITION TO THE MICROWELLS.

At the final stage of incubation, remove from refrigerator and measure the amount of Substrate required for the current assay, using only plastic pipet materials, recap the bottle and return to 2 - 8C storage. The material removed should be stored in the dark and allowed to slowly come to ambient temperature prior to addition to the washed microwells.

Stop Solution: 1 vial containing 12 ml of 1% hydrochloric acid.

Caution:  Caustic material. Wear eye, hand, face and clothing protection.

5X Wash Solution: 1 vial containing 50ml of concentrated buffer solution with surfactant.

Graph Paper: One sheet of 3-cycle logarithmic graph paper

 

REAGENT STORAGE

All reagents in this kit should be stored at 2 - 8C.

 

REAGENT STABILITY

Refer to the expiration date on the label of each reagent.

Upon reconstitution, the Calibrators are stable for a maximum of 4 weeks, when stored a 2 - 8C.

 

 

PRECAUTIONARY STATEMENT

Human Specimens: Calibrators provided in this kit contain plasma obtained from human donors. Each donor's serum was tested by FDA approved methods for the presence of antibodies to HIV-1 and hepatitis B surface antigen and found to be negative for both. However, since no test method can absolutely guarantee the absence of these or other infectious agents, all Calibrators and human plasma tested with this assay should be handled in accordance with NCCLS guidelines for preventing the transmission of blood-borne infections during laboratory procedures. Wear gloves and avoid contact with skin and mucous membranes

WARNING: The Stop Solution in this kit contains hydrochloric acid. Avoid ingestion or contact with skin or eyes.

 

MATERIALS REQUIRED (But Not Provided)

  • Distilled, deionized water
  • 20 ul, 50 ul, 200 ul, and 500 ul precision pipettes
  • Microplate Reader capable of reading at 450nm (Optional: a background correction filter set at 570 to 650 nm)
  • Clinical rotator or vibrator table
  • 12 x 75 mm plastic culture tubes - DO NOT USE GLASS TUBES
  • Vortex mixer
  • Wash bottle
  • Graduated cylinder capable of measuring 250 ml of liquid
  • Quality controls containing known levels of vWF in human plasma

 

 

GENERAL INFORMATION ON PROCEDURE

The intensity of the color developed during the 2nd Step is directly proportional to both time and temperature; therefore an increase in the 2nd Step incubation time will increase the intensity of the color, while a decrease in the 2nd Step incubation time will decrease the intensity of the color. The same applies for an increase or a decrease in the incubation temperature. It is recommended that time rather than temperature be used to optimize absorbance.

 

SAMPLE COLLECTION AND PREPARATION

1. Remove all reagents, except the Substrate, from cold storage and allow to warm to room temperature.

2. Use freshly collected plasma specimens anticoagulated with citrate or EDTA.

3. Centrifuge to separate the plasma from the red cells (if the assay is to be performed later, freeze the plasma and store at -20C or lower). Thaw frozen plasma samples by rapidly moving the storage vial/tube in a 37C water bath until thawed (approximately 30 - 60 seconds for a 50 ul sample) followed immediately by a brief vortex.

4. Carefully remove the aluminum seal and rubber stopper from the Calibrator vial. Be careful not to remove or lose any powder from the vial. If any material is lost prior to reconstitution, discard and use another vial of Calibrator. Add exactly 0.50 ml of distilled, deionized water to the vial. Replace the rubber stopper and mix well, including vortexing for 5 - 10 seconds. Allow the reconstituted Calibrator to stand for at least 15 minutes.

5. Dilute each patient sample 1:51 prior to testing. Add 1 ml of Specimen Diluting Buffer to a labeled 12 x 75 mm plastic tube for each specimen to be tested. Add 20 ul of specimen plasma sample to1 ml of Specimen Diluting Buffer in plastic test tubes. DO NOT USE GLASS TUBES

6. Label 5 separate plastic tubes as 1:2, 1:4, 1:8, 1:16 and 1:32. To each tube, add exactly 200 ul of Specimen Diluting Buffer.

7. Remove 200 ul of reconstituted pre-diluted Calibrator from the vial and carefully add to the specimen diluent in the tube labeled "1:2". Cap the tube and vortex for several seconds to mix thoroughly. Using a fresh pipet tip, remove 200 ul from this tube ("1:2") and add to the Specimen Diluting Buffer in the tube labeled "1:4". Cap the tube and vortex for several seconds to mix thoroughly. Using a fresh pipet tip, remove 200 ul from this tube ("1:4") and add to the tube labeled "1:8". Cap the tube and vortex to mix thoroughly. Repeat this process for the tube marked "1:16" and "1:32".

These five dilutions, as well as the reconstituted Calibrator, must be tested in duplicate each time the assay is performed so that an accurate calibration curve can be constructed for the determination of vWF concentrations in the patient specimens.It is recommended that each individual laboratory locate and use controls for this assay that are representative of the population upon which this test is performed. Routine use of these controls will allow each laboratory to perform ongoing quality control to identify any performance or reagent problems that may occur.

Suitable controls may be obtained as pooled plasmas from George King Bio-Medical, Inc. (Overland Park, KS 913/469 - 5464) and the College of American Pathologists (Northfield, IL 847/832- 7000).

 

ASSAY PROCEDURE

1. Determine the number of Wells/Strips that will be required for the assay run. (Note: each specimen, Calibrator dilution, and Specimen Buffershould be run in duplicate wells each time the assay is performed.)

2. Open the foil pouch and remove the number of 12-well Strips required for each assay. Place the Strips in the frame supplied, being careful to orient the strips correctly. Replace the unused Strips in the resealable foil pouch. Reseal the pouch, using the ziploc strip, and store at 2 - 8C.

3. Pipette 20 ul Specimen Diluting Buffer to each of Wells A1 and B1. With a new pipet tip, remove and add 20 ul of Calibrator from its vial to each of Wells A2 and B2. With a new pipet tip, remove and add 20 ul of Calibrator from the tube marked "1:2" and add to each of Wells A3 and B3. In the same manner, add 20 ul of Calibrators from the tubes marked 1:4, 1:8, 1:16 and 1:32.

4. Add 20 ul of the 1:51 dilutions of the patient specimens and controls, in duplicate, to the remaining Wells.

5. Pipette 200 ul of HRP-Conjugate to each well containing Calibrators, patient specimens and Specimen Diluting Buffer (0% or NSB). Cover theStrips immediately with a Plate Sealer, being careful to press the Sealer onto all Wells.

6. Place the plate on a mixing table and mix for 10 minutes at 190 - 200 rpm. Remove the plate and store undisturbed on a flat surface at room temperature (16 - 25C) for two hours.

7. Prepare a 1X Wash Solution by pouring the entire contents of the 5X Wash Solution into a clean 250 ml graduated cylinder. Bring the total volume to 250 ml with distilled water. Cover and invert to mix. Transfer to a clean wash bottle and store at room temperature.

8. After the two hour incubation, remove the Plate Sealer and dump the contents into a sink or waste receptacle. Blot the plate to remove reactants from the Well lips. Wash the Wells by filling each Well with the previously prepared 1X Wash Solution from the wash bottle. Dump the contents and repeat this wash procedure three additional times (total of four washes). After the final wash, dump the wash fluid and then vigorously tap the Strips on an absorbent surface to remove all liquids from the Wells.

Make sure there are no bubbles or liquid remaining in the Wells. If any fluid remains, continue tapping the plate on the absorbent surface until eliminated. Do not add Substrate to the Wells until the Wells have been tapped dray and are free of bubbles.

9. Using new pipet tips, add 200 ul of Substrate to each Well. Place the Wells into a dark area, such as a drawer, and incubate undisturbed for 30 minutes.

10. Carefully remove the plate from the dark and add 50 ul of the Stop Solution to each Well. Place the plate on the mixing table for 30 seconds at 190 rpm to ensure mixing. Read the absorbance of the Wells at 450nm. If possible, use a correction wavelength of 570 - 650nm.

11. If automatic background subtraction is not available, it is suggested that the plate be read a second time at 570 - 650nm and these readings be manually subtracted from the initial 450nm readings. Failure to compensate for the background absorbance may increase the variability of the assay and result in potentially erroneous values.

 

CALCULATION OF RESULTS

Average the duplicate readings for each specimen, Calibrator, and Specimen Diluting Buffer. Subtract the average absorbance of the Specimen Diluting Buffer from all other averages to obtain the corrected mean absorbances.

Manual Data Analysis: On the graph paper provided, plot the corrected net absorbance values for the prediluted Calibrator and its two-fold dilutions on the Y axis and plot the von Willebrand Factor concentration supplied in the kit for the prediluted Calibrator on the X axis. The vWF concentrations of the two-fold dilutions will be 1/2, 1/4, 1/8 etc. of the value reported on the label.

Draw the straightest line possible that intersects the points plotted on the graph paper. This is the assay calibration line. To determine the vWF concentration of each patient specimen, locate the mean corrected net absorbance of the specimen on the Y axis and then locate the corresponding point on the calibration line determined previously. Slide a ruler down paralleling the Y axis and determine the corresponding value of the intersect point on the X axis. This value on the X axis is the concentration of the vWF present in the undiluted patient specimen, expressed as a percentage of normal concentration (%N).

Automated Data Analysis: Best fit for a calibrator line plot can be most often described by a third order polynomial plot of the calibrator (and dilutions) absorbances versus the vWF concentrations. Using the polynomial equation for the line which results, determine the vWF concentrations in the unknown specimens by interpolation of their corrected mean absorbances. The results will be expressed as a percentage of normal values (%N).

Do not extrapolate values that are outside the highest values determined for the calibration line. If values are obtained that are greater than the highest vWF value plotted, they can either be reported as values greater than the value of the highest Calibrator, or diluted further and retested.

The 1:51 dilution may be diluted 1:2 in Specimen Diluting Buffer and retested. The 1:51 dilution may be further diluted and tested immediately or stoppered and frozen at -20C (or less) for no more than 30 days after the original dilution was assayed. If the specimens are diluted and retested, the values reported must be corrected after assay by a factor of 2 to correct for the dilution.

 

EXAMPLE DATA (for Demonstration Purposes Only)

 

STANDARD

O.D.

MEAN

Net O.D.*

VWF CONC. % of NORMAL

0% or NSB

0.032 / 0.036

0.034

0

0%

Calibrator

1.477 / 1.472

1.475

1.411

200%

1:2 Calibrator

1.038 / 1.043

1.041

1.007

100%

1:4 Calibrator

0.624 / 0.653

0.639

0.605

50%

1:8 Calibrator

0.341 / 0.368

0.355

0.321

25%

1:16 Calibrator

0.192 / 0.192

0.192

0.158

12.5%

1:32 Calibrator

0.107 / 0.112

0.110

0.076

6.25%

 

 

* Net O.D. = Mean O.D. minus Mean O.D. of 0% (NSB)

 

PERFORMANCE CHARACTERISTICS

REPRODUCIBILITY:

Within and between assay variations were calculated using repeated analysis of a panel of reference plasma samples. Within assay variance was determined by assaying and individually comparing 10 repetitions of 8 samples. Between assay variance was determined by individual comparison of 35 samples each run on 9 separate occasions.

 

WITHIN-ASSAY VARIATION (n = 10)

 

 

REFERENCE PLASMAS

VWF Ag MEAN

S.D.

C.V.

 

1

165

8.2

5.0%

 

2

145

6.2

4.3%

 

3

63

3.7

5.8%

GRAND MEAN

4

62

3.0

4.8%

C.V. = 5.2%

5

59

2.6

4.4%

 

6

37

1.8

4.8%

 

7

37

1.9

5.3%

 

8

19

1.4

7.2%

 

 

 

 

BETWEEN-ASSAY VARIATION: (n = 9)

 

 

TEST GROUPS

CONC. VWF

No. of Samples

vWF Ag Mean

S.D.

C.V.

 

1

0 - 10%

1

8

0.6

7.5%

GRAND MEAN

2

11 - 49%

11

35

2.8

7.8%

C.V. = 8.2%

3

50 - 160%

16

84

5.9

7.0%

 

4

161 - 200+%

7

211

24.5

11.5%

 

 

 

 

SENSITIVITY:

Sensitivity is defined as the smallest amount of vWF which can be distinguished from zero with a 95% confidence limit (+ or - two standard deviations). The smallest concentration of vWF that can be distinguished from zero is 0.78% of Normal.

 

ACCURACY:

 

The accuracy of the current assay was determined by comparing the quantity of von Willebrand Factor determined by this test procedure with the quantity of von Willebrand factor determined by an approved bead ELISA test.

One hundred and eighteen (118) plasma samples were obtained from four groups of patients and normal volunteers. These samples were assayed for the quantity of vWF by the bead ELISA and by this procedure. The correlation coefficient of the bead ELISA and this test procedure was r = 0.849 by linear regression analysis.

 

INTERPRETATION OF DATA

In the proposed National Committee for Clinical Laboratory Standards guidelines (document H39-P) for the determination of von Willebrand Factor Antigen, the expected normal reference interval was determined to be 50 -150% of normal. These values, however, are representative only. Each laboratory should establish its own normal reference range based on its own patient population and other considerations. Plasma levels of vWF antigen may increase above the normal range during pregnancy, use of birth control pills, during and following physical exercise, and during stressful situations.

Patients with the relatively common classical (Type I) form of von Willebrand's disease have plasma vWF levels that are usually in the 5 - 40% of normal range. Patients with relatively rare severe forms of von Willebrand's disease have plasma levels that are often less than 5% of normal. Some patients with von Willebrand disease have faulty molecules which do not polymerize properly (Type II). These patients may have normal von Willebrand antigen concentrations.

Individuals with classical Hemophilia A (deficiency of factor VIII coagulant activity) generally have vWF antigen levels within or above the normal range. Female carriers of Hemophilia A usually have a ratio of plasma factor VIII coagulant activity/vWF antigen that is approximately 1:2.

 

BIBLIOGRAPHY

1. Bennett, B., and Ratnoff, O.D.: Antihemophilic factor (AHF, factor VIII) procoagulant activity and AHF-like antigen in normal pregnancy and following exercise and pneumoencephalography. J. Lab. Clin. Med. 80:251, 1972.

2. Bennett, B., Oxnard, S.C., Douglas, A.S., and Ratnoff, O.D.: Studies on antihemophilic factor (AHF, factor VIII) during labor in normal women, in patients with premature separation of placenta and in a patient with von Willebrand's disease. J. Lab. Clin. Med. 84:851, 1974.

3. Parquet-Gernex, A., Maxurier, U., and Goudemand, D.: Detection of the carrier state for classic hemophilia by comparative assay of factor VIII activity and factor VIII antigen. Pathol. Biol. 22:37, 1974.

4. Ratnoff, O.D., and Steinberg, A.G.: Detection of the carrier state of classic hemophilia. Ann. N.Y. Acad. Sci. 20:95. 1975.

5. Meyer, D., Jenkins, C.S.P., Dreyfus, D., Fressinaud, E., and Larrier, M.J.: Willebrand factor and ristocetin-II. Relationship between Willebrand factor, Willebrand antigen and factor VIII activity. Br. J. Haematool. 28:579, 1974.

6. Weiss, H.J., Hoyer, L. W., Rickles, F.R., Varma, A., and Rogers, J.: Quantitative assay of a plasma factor, deficient in von Willebrand's disease, that is necessary for platelet aggregation: Relationship to decreased factor VIII procoagulant activity and antigen content. J. Clin. Invest. 52:2708, 1973.

7. Zimmerman, T.S., Ratnoff, O.D., and Powell, A.E.:Immunologic differentiation of classic hemophilia (factor VIII deficiency) and von Willebrand's disease. J. Clin. Invest. 50:244, 1971.

8. Sites, D.P., Hershgold, E.J., Perlman, J.D., and Fudenberg, H. H.: Factor VIII detection by hemagglutination inhibition: Hemophilia A and von Willebrand's disease. Science 171:196,1971.

9. Hoyer, L.W.: Immunologic studies of antihemophilic factor (AHF, factor VIII). IV. Radioimmunoassay of AHF antigen. J. Lab. Clin. Med. 80:822, 1972.

10. Jaffe E.A.: Endothelial cells and the biology of factor VIII. N. Engl. J. Med. 1977: Feb. 17;296:377-383.

 

Ramco Laboratories, Inc.

4100 Greenbriar Drive Suite 200

Stafford, TX 77477

ISSUE DATE: February 12, 1987

REVISED: November 16, 2005; April 13, 2011; January 31, 2012; September 14, 2016