SILVER - NITROPRUSSIDE TEST PROCEDURE

Intended As A Screening Method for Cystinuria and Homocystinuria

SUMMARY AND EXPLANATION

Both cystinuria and homocystinuria are readily detectable by the nitroprusside test (1). The two disorders have different etiologies and clinical manifestations. In order to initiate the proper course of therapy, it becomes important then to distinguish between the two conditions after a positive result in the nitroprusside test.

Spaeth and Barber (6) have described a procedure to differentiate homocystine and cystine in urine. The method depends on the rapid reduction of the homocystine disulfide linkage by silver ions. The resulting free sulfhydryl groups can be detected then with the nitroprusside test. Cystine is not reduced under the conditons of the reaction. The method appears quite sensitive, detecting as little as 2 mg/dl homocystine. Since other disulfides or sulfhydryl groups may react, the procedure is recommended for screening purposes only. Any positve result would suggest further confirmatory testing for diagnosis.

The EAGLE SILVER-NITROPRUSSIDE TEST PROCEDURE employs a modification of the Spaeth method. Saturation of the urine specimen with solid sodium chloride is unnecessary. The SILVER REAGENT is supplied in a stable liquid form. Analysis timeis one minute. Eagle recommends the SILVER-NITROPRUSSIDE TEST PROCEDURE be employed to differentiate homocystine from cystine after a positve result has been obtained with the NITROPRUSSIDE TEST PROCEDURE.

PRINCIPLE

Silver ions reduce the disulfide linkage in homocystine to the free sulfhydryl groups. Cyanide displaces the silver ions and nitroprusside immediately forms a colored complex with the free sulfhydryl groups which appears pink to purple. The color intensity depends on the urinary concentration of homocystine.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reactive Set Cat. No. N-110 includes:

SILVER REAGENT - (Cat. No. N-21)

REACTIVE INGREDIENTS:

12 mM silver nitrate. Ammonium salts added.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing.

STORAGE AND STABILITY:

Store at 2 - 8º C. Stable until expiration date if sealed tightly. PROTECT FROM LIGHT.

DETERIORATION:

The reagent should be a clear, colorless solution. Turbidity or a gray-black coloration would indicate deterioration, and the solution should not be used.

HOMOCYSTINE SOLUTION - (Cat. No. N-22)

REACTIVE INGREDIENTS:

50 mg/dl homocystine. Stabilizer added.

PRECAUTIONS:

For in-vitro diagnostic use.

STORAGE AND STABILITY:

Store at 2 - 8º C. Stable until expiration date if sealed tightly.

DETERIORATION:

The solution should be clear and colorless. Turbidity would indicate deterioration, and the solution should not be used.

CYSTINE SOLUTION - (Cat. No. N-13)

REACTIVE INGREDIENTS:

50 mg/dL cystine. Stabilizer added.

PRECAUTIONS:

Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 2 - 8º C. Stable until expiration date if sealed tightly.

DETERIORATION:

The solution should be a clear, colorless solution. Turbidity would indicate deterioration, and the solution should not be used.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Freshly collected urine is recommended.

2. The specimen should not be contaminated with feces.

3. For screening newborns, specimens should be collected at 10 - 14 days after birth, and at 3 - 4 weeks of age.

SAMPLE STORAGE:

Refrigerate the specimen as soon as possible after collection. If testing is delayed longer than 24 hours after collection, freeze the sample.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Free sulfhydryl groups or any disulfide linkage capable of being reduced by silver may react. Creatinine, acetone and acetacetate do not interfere. Acetaminophen, salicylates, phenobarbital, ampicillin and tetracycline are unreactive. Uric acid levels above 2 g/l and urines from patients receiving abscorbic acid may interfere with the test sensitivity.

PROCEDURE

MATERIALS PROVIDED:

SILVER REAGENT (Cat. No. N-21), and HOMOCYSTINE SOLUTION (Cat. No. N-22).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. Spot plate

2. Dropper pipets

3. Applicator sticks

4. NITROPRUSSIDE TEST KIT (Cat. No. N-10)

PREPARATION OF HOMOCYSTINE REFERENCE SAMPLE:

A Homocystine Reference Sample should be prepared by adding 1 drop of the HOMOCYSTINE SOLUTION to 10 drops of a normal urine and mixing well. The homocystine concentration is approximately 5 mg/dL in the Regerence Sample. It is recommended that a normal urine and the Reference Sample be included in each test series.

MANUAL PROCEDURE:

1. Add 2 drops of urine specimen tot he spot plate cavity.

2. Add 1 drop of SILVER REAGENT . Use an applicator stick to mix well.

3. Wait 1 minute. Add 1 drop of NITROPRUSSIDE REAGENT.

4. Observe for color change.

INTERPRETATION OF RESULTS

An immediate pink to purple color upon the addition of NITROPRUSSIDE REAGENT would indicate the presence of homocystine. Color intensity depends on urinary concentration. Normal urine has a slight pink color due to the NITROPRUSSIDE REAGENT. The test should be considered positive if the color intensity equals or exceeds that of the 5 mg/dL Homocystine Reference Sample.

LIMITATIONS

The method is not completely specific for homocystine since other disulfides and free sulfhydryl groups may react. The SILVER-NITROPRUSSIDE TEST PROCEDURE is intended as a screening method only. A positive result would indicate further amino acid testing for diagnosis.

REFERENCES

1. Thomas, G.H., and Howell, R.R., Selected Screening Tests for Genetic Metabolic Disorders, Year Book Medical Publishers, Chicago, 1973 p. 30.

2. Levy. H.L., Madigan, P.M., and Shih, V.E., Pediatrics 49, 825 (1972).

3. Wong, L.T.K., Hardwick, D.F., Applegarth, D.A., and Davidson, A.G.F., Clin. Biochem. 12, 167 (1979).

4. Knox, W.E., The Metabolic Basis of Inherited Disease, 2nd ed., Edited by Stanbury, J.., Wyngaarden, J.B., and Frederickson, D.S., McGraw-Hill Book Co., New York, 1966, p. 1262.

5. Covey, R.C., Lab World 32, 23 (1981).

6. Spaeth, G.L., and Barber, G.W., Pediatrics 40, 586 (1967).