SPECTRO vWF

An Enzyme Immunoassay Procedure for the Quantitative Analysis of Human von Willebrand Factor

Catalog Number: V-46

For In Vitro Diagnostic Use Only

 

INTRODUCTION

Spectro vWF is an enzyme linked immunoassay (ELISA) for the quantitation of von Willebrand factor (vWF, also called factor VIII-related antigen). This factor is deficient or defective in patients with von Willebrand's disease, a relatively common inherited disorder associated with 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 Spectro vWF assay affords rapid differentiation of these two disorders.

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

In order to determine if a woman is a carrier 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

Spectro vWF is intended to be used for identifying patients with von Willebrand's disease in whom the vWF antigen is decreased. In addition, the assay is to be used in combination with an assay for factor VIII coagulant activity to identify female carriers of Classical Hemophilia A (factor VIII coagulant activity).

 

TEST PRINCIPLE

The enzyme immunoassay for the quantitative determination of human vWF is a two stage reaction.

STAGE 1 The binding of human vWF to a solid phase antihuman vWF and the simultaneous binding of purified antihuman vWF conjugated with alkaline phosphatase to the insoluble immune complex.

STAGE 2 Reaction of the alkaline phosphatase immune complex with a substrate solution consisting of phenylphosphate disodium and 4-amino-antipyrine. Following the addition of potassium ferricyanide a red color develops, the optical density (490nm-510nm) of which is directly proportional to the concentration of vWF. The concentration is determined by comparing the optical density of the patient sample to that of a calibrator.

 

KIT CONTENTS

This Kit is Intended for In Vitro Diagnostic Use Only

Prediluted Human von Willebrand Factor Calibrator Solution:   1 vial containing 2ml of von Willebrand Factor in a concentration equivalent to 50% of the vWF concentration found in the 3rd International Standard for Factor VIII von Willebrand Factor in plasma established by the World Health Organization. The vWF is dissolved in borate buffer containing bovine serum albumin, rabbit serum, sodium chloride and inert coloring agents with sodium azide as a preservative.  Store at 2 - 8C.   Do Not Use After Expiration Date on Vial.

Solid Phase Antihuman von Willebrand Factor:    64 plastic beads coated with rabbit antihuman vWF stored in borate buffer containing bovine serum albumin, rabbit serum and sodium chloride with sodium azide as a preservative. Store at 2 - 8C.  Do Not Use After Expiration Date on Vial.

Sample Diluting Buffer:    1 vial containing 60 ml of borate buffer with bovine serum albumin, sodium chloride, ethylenediaminetetraacetate, inert coloring agents and sodium azide as a preservative.  Store at 2 - 8C.  Do Not Use After Expiration Date on Vial.

Conjugated Antihuman von Willebrand Factor:    1 vial containing 15 ml of alkaline phosphatase conjugated antihuman vWF dissolved in 0.15 M phosphate buffered saline with 1% rabbit serum and sodium azide as a preservative.  Store at 2 - 8C.  Do Not Use After Expiration Date on Vial.

Substrate:    1 vial containing 15 ml of phenylphosphate disodium, 4-amino_antipyrine in 10% diethanolamine with sodium azide as a preservative.  Store at 2 - 8C.  Do Not Use After Expiration Date on Vial.

Potassium Ferricyanide (Concentrate):   1 vial containing 20 ml of potassium ferricyanide (2.4%) in water. This Reagent MUST be diluted to a final Volume of 200 ml before use.  Store at 2 - 8C.  Do Not Use After Expiration Date on Vial.

 

PRECAUTIONS

Human Specimens:   The human vWF contained in the Calibrator solution was tested by FDA approved methods for the presence of antibodies to HIV-1 as well as for hepatitis B and found to be negative. However, since no test method can absolutely guarantee the absence of these or other infections, the Calibrator solution and human sample 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:   Reagents in this kit contain sodium azide. Contact with copper or lead drain pipes may result in the formation of explosive azide deposits. It is important during disposal to flush drains with copious amounts of water to prevent azide accumulation. Plumbing that may be contaminated with azides can be flushed with 10 percent sodium hydroxide solution.

WARNING:   A component of this kit contains potassium ferricyanide. Avoid ingestion or contact with skin.

Other Precautions:

  • Avoid splashing or generating aerosols.
  • Follow kit recommendations for incubation times and temperatures to avoid possibly erroneous results.
  • Microbial contamination of reagents may cause erroneous results.
  • Do not use reagents with those from other lots or manufacturers.
  • Do not use kit reagents after expiration date.

 

MATERIALS PROVIDED

One vial of each reagent listed under "KIT CONTENTS", 4 plastic reaction trays, 8 handles and 64 bead grippers.

 

MATERIALS REQUIRED BUT NOT PROVIDED

  • Deionized or distilled water.
  • Precision pipettes capable of delivering volumes of 20 ul, 50 ul, 200 ul and 1 ml.
  • Spectrophotometer capable of reading a red color at 500 +/- 10 nm, with a "sipper" cell or tube reading option.
  • Clinical rotator table or vibrator table.
  • 12 X 75 mm glass or plastic test tubes if tube reading option is used. 

 

REAGENT PREPARATION

Dilute the contents of the 2.4% Potassium Ferricyanide to a final volume of 200 ml in a separate container with distilled or deionized water. Label the diluted potassium ferricyanide as 0.24% potassium ferricyanide and store at 4 - 8C.

 

SAMPLE COLLECTION AND PREPARATION

1. Collect 1 - 5 ml of EDTA anticoagulated venous blood. (Sodium citrate treated blood may be used as well.)

2. Centrifuge to separate the plasma (if assay is to be performed later, freeze the plasma and store at -10C.)

3. Dilute patient samples 1:50 by adding 20l of sample to 1 ml of the Sample Diluting Buffer.

4. The Calibrator is supplied ready to use. DO NOT DILUTE.

 

GENERAL INFORMATION ON PROCEDURE

The intensity of the color developed during the 2nd stage incubation is directly proportional to both time and temperature; therefore an increase in the 2nd stage incubation time will increase the intensity of the color, while a decrease in the 2nd incubation time will decrease the intensity of the color. The same applies for an increase or decrease in the incubation temperature. We suggest that time, not temperature, be used to optimize the absorbance.

 

ASSAY PROCEDURE

Label and arrange the reaction trays according to the test schedule. Reagents and patient samples should be at room temperature before use.   NOTE:   Two sets of reaction trays will be required to perform the assay. One set for the STAGE 1 incubation and one set for the STAGE 2 reaction.

1. Allow all reagents to come to room temperature.

2. Set up the reaction trays, holders and grippers needed for the test.

3. Pipette 50 ul of Sample Diluting Buffer into well A1 and A2 of the first reaction tray.

4. Pipette 50 ul of Calibrator into wells A3 and A4 of the first reaction tray.

5. Pipette 50 ul of each diluted sample into the bottom of appropriate wells of the first reaction tray.

Modification to Extend the Range of the Assay

This assay has been optimized to provide maximum precision for samples with von Willebrand Factor concentration between 0 and 100%. If a greater range is desired, Step 5 should be modified as follows: Pipette 10 ul of each diluted sample into the bottom of appropriate wells.

6. Add 200 ul of the Conjugated Antihuman vWF to all wells containing either Calibrator or sample and shake for 5 minutes on a vibrator table or horizontal rotator.

7. Add an Antihuman vWF Bead to all wells.

8. Place holders so that a bead gripper is in each well and push down to attach the beads. Place on a rotating table (set speed to 200 rpm) or a vibrator table and start the incubation period.

9. Incubate at room temperature for 2 hours.

10. Pipette 200 ul of Substrate into a second set of reaction wells (or test tubes if the color is to be read in a spectrophotometer with a tube reading option).

11. Remove the beads and grippers from the Conjugate/sample mixture and wash under running deionized water. Shake to remove excess water.

12. Place the washed beads into the Substrate and start 30 minute incubation period at room temperature.

13. Develop the color by adding 1.0 ml of the diluted (0.24%) Potassium Ferricyanide to each well (or test tube). MIX WELL by raising and lowering each bead holder several times (or by vortexing if using test tubes). The beads may be discarded after this step.

14. Zero the instrument with a blank prepared with 200l u of the Substrate solution and 1 ml of the diluted (0.24%)Potassium Ferricyanide.

15. Read the absorbance at 500nm +/- 10nm and record the values.

16. Calculate the results. (See the Calculation Section).

   

CALCULATIONS

Divide the optical density of the sample by the optical density of the Calibrator. Multiply the result by 50%.

If the assay range has been extended by using a 10 ul sample size, multiply the ratio of optical densities by 250%.

 

EXAMPLE DATA

(For Demonstration Purposes Only)   

   

 

 

 

 

**The first two wells measure non-specific binding (NSB).

CALIBRATOR/SAMPLE  O.D. MEAN - Net O.D.vWF % Activity
0% or NSB**

.003  /.002

.0025
50% Calibrator.295 / .291.293.2905
Sample 1.733 / .687.710.7075121.8%
Sample 2.285 / .320.3025.30051.6%
Sample 3.131 / .130.1305.128

22.0%

    

  PERFORMANCE CHARACTERISTICS

Intra-Assay variation and Inter-Assay variation were calculated using high, borderline and low levels of vWF.

 

INTRA-ASSAY VARIATION

RUNNO. of SAMPLESMEAN S.D.  C.V.
I8125.5%9.76.2%
II850.5%3.28.5%
III822.0%1.98.0%

 

INTER-ASSAY VARIATION

MEAN S.D. S.D.
High120.6%7.66.3%
Medium50.2%5.811.5%
Low18.7%2.412.8%

 

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 .23%.

 

ACCURACY

The accuracy of the current assay is determined by comparing quantitation of von Willebrand Factor by this test procedure with quantitation by the Laurell rocket technique.

Twenty-our plasma samples were obtained from a group of patients being tested for possible coagulation defects and normal volunteers. These samples were assayed for the quantitation of vWF by the Laurell rocket technique and by this procedure. Excellent correlation was found between the procedures. The correlation coefficient of the Laurel rocket technique and this test procedure was .96 by linear regression analysis.

 

INTERPRETATION OF DATA

Plasma samples were collected from twenty normal hospital employees. These specimens were tested for the concentration of vWF by the current assay to determine the normal range. Normal vWF concentration ranged from 78% - 153%.

These values are representative only. Each laboratory should establish its own normal range based on its own patient population and considerations.

Patients with relatively common Classical (Type I) form of von Willebrand's disease have plasma vWF levels that are usually in the 5 - 40% 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 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.

 

 - NOTICE -

The 50% Prediluted Human vWF Calibrator solution contained in this kit was calibrated against the 3rd International Standard for Factor VIII and von Willebrand Factor in plasma established by the Expert Committee on Biological Standardization of the World Health Organization in 1992 (coded 91/666). The enclosed Calibrator may give higher results than the Calibrator solution contained in previous kits (kit lot numbers < V-024).

 

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, TEXAS 77477

Issue Date: February 12, 1987

Revised: May 11, 1998:11/1/00

PKGINSRT\VWF-V461.P