Cat. No. 2245
Sterling Diagnostics,
Inc.
For In Vitro Diagnostic Use
Sterling Heights, MI 48312 U.S.A.
SICKLE CELL UNI-TEST
(Modified Nalbandian)
INTENDED USE
FOR IN VITRO DIAGNOSTIC USE in the qualitative determination of
hemoglobin (HbS) in blood.
CLINICAL SIGNIFICANCE (1-2)
Sickle cell disease is an inherited disease characterized by the
presence of an abnormal hemoglobin (HbS). In normal adults, 95% or
more of the hemoglobin is hemoglobin A (HbS). Hemoglobin S can be
inherited in the homozygous state (S/S), which results in sickle cell
anemia, or in the heterozygous state (A/S), which is usually the benign,
asymptomatic state (sickle cell trait). Hemoglobin S can also occur in the
presence of other abnormal hemoglobins, i.e. HbC (S/C), thalasemia (Sthal), or HbD (S/D). These are referred to as the sickle cell variants and
can produce symptoms of varying severity.
About 8% of American blacks have a single HbS gene and 0.2%
are homozygous for the gene. The HbS gene is essentially peculiar to
Negroes, reaching a frequency of up to 59% in various regions of Africa.
The HbS gene is also found in localized areas in countries bordering the
Mediterranean Sea, e.g. Italy, Greece, Turkey, and some of the Arabic
nations.
About 2-3% of American blacks carry the hemoglobin C gene.
The heterozygous state does not cause anemia or shortened red cell life
span. But one in 6000 American blacks is homozygous for HbC. This
causes a mild hemolytic anemia with striking morphologic abnormalities.
TECHNICAL SUMMARY
Erythrocytes are lysed by saponin and the released hemoglobin is
deoxygenated by dithionite in a concentrated phosphate buffer (3). HbS,
when deoxygenated, is insoluble in concentrated phosphate buffer and
produces a visible turbidity. Since almost all other hemoglobins (i.e. Hb
A, F, C, E, D) are soluble in the solution, blood specimens containing
HbS are quickly identified.
When a positive specimen is identified, the addition of urea to the
reaction mixture will cause the solution to become clear if HbS is present.
If the solution remains turbid after the urea addition, a non-S sickling
hemoglobin is indicated. Electrophoretic confirmation is required for
conclusive identification.
The method presented here is based upon a modified Nalbandian
(4-5) procedure.
REAGENTS
All of the following reagents can be used until the expiration date
indicated on the individual bottles. STORE REAGENTS AT ROOM
TEMPERATURE.
1. SICKLE CELL REAGENT: A solution containing phosphate buffer,
saponin and preservative. Keep tightly capped and protect from
contamination.
Precautions:
Sodium Dithionite (Hydrosulfite) is toxic! Wash hands after
handling. If ingested seek immediate attention. Do Not Pipet by Mouth.
For other reagents in this set, exercise the normal precautions
required for the handling of all laboratory reagents.
INDICATORS OF REAGENT DETERIORATION
1. Physical Appearance
a. Any cloudiness observed in the Sickle Cell Reagent which will
not readily dissolve upon mixing may indicate reagent deterioration.
b. If the powder in the Sickle Cell Dithionite Reagent has become
2. Control Assays
Failure to obtain accurate results in the assay of control materials
may indicate reagent deterioration.
Sterling Diagnostics cannot guarantee the stability of reagents,
which have been:
a. Transferred from their original containers;
b. Improperly stored;
c. Contaminated during use.
SPECIMEN (6-7)
Collect whole blood by venipuncture into vials or containers
containing appropriate amount of anticoagulant, e.g. Heparin, EDTA,
ACD CPD, CPDA-1 , Sodium or Potassium Oxalate, Sodium Citrate or
use blood segments of whole blood or packed cells which have been
mixed thoroughly.
Specimens that have been kept for 1-2 weeks at 4-5oC are
reportedly satisfactory for use in this period.
No preliminary restriction of food or fluid is required for this test.
MATERIALS PROVIDED
Sickle Cell Reagent, 54 Vials with Sodium Dithionite and Sickle
Cell Urea Reagent.
ADDITIONAL MATERIALS REQUIRED
Positive and Negative Sickle Cell Controls are available separately
as Cat. No.2244-0
Reagent and sample pipets
PROCEDURE
1.
Transfer 2.0 ml of Sickle Cell Reagent to vials containing Sodium
Dithionite, swirl to mix and label them:
UNKNOWN, (+) CONTROL, (-) CONTROL.
2. Add 20 microliters of well-mixed whole blood to its respective vial and
mix by gentle swirling.
2. 54 VIALS WITH SODIUM DITHIONITE: powdered sodium dithionite.
Keep tightly capped and protected from moisture prior to reconstitution.
3. Allow to stand at room temperature for 5 minutes.
3. SICKLE CELL UREA REAGENT: Urea in aqueous solution. Keep
tightly capped and protect from contamination.
4. Read the test by holding the test tube approximately 3-cm from the
line scale provided with the test. Adequate illumination is necessary.
8.
RESULTS
Whitehead, T.P., Quality Control in Clinical Chemistry, John Wiley
& Sons, New York (1977).
READER SCALE
POSITIVE: If HbS or any other sickling hemoglobin is present, the
solution will be sufficiently turbid to prevent reading of the scale.
NEGATIVE: If no sickling hemoglobin is present, the solution will be
clear enough to allow the scale to be seen through the test vial.
All doubtful tests, as well as all positive tests, should be confirmed by
electrophoresis.
FOR ALL POSITIVES, THE FOLLOWING STEP IS RECOMMENDED:
NOTES
1. Transfer 2.0 ml of Sickle Cell Reagent to a vial containing
Sodium Dithionite and mix by swirling.
2. Add 5 drops of Sickle Cell Urea Reagent and mix.
3. Add 20 microliters of positive whole blood and gently swirl to
mix.
4. Let stand for 5 minutes and read against scale.
The presence of HbS will be confirmed if the solution becomes
clear in the presence of Urea Reagent. The only known variant that will
also clarify is HbC (Harlem).
PROCEDURE LIMITATIONS
Severe anemia can cause false negatives; therefore, if the
patient's hemoglobin is below 7.0 Gm/dl, the test should be repeated
using 40 microliters of sample.
False negative results may occur when HbS concentration is
<15% of total hemoglobin, which may be the case when a patient is
transfused with blood from a donor with HbS trait.
The rare sickling hemoglobins C-Harlem, C-Georgetown, S-Travis,
C-Ziguinchor and Hb Barts (if present in the blood of a fetus with hydrops
fetalis due to a-thalasemia) reportedly (7) also give positive test results
with this procedure.
In patients who have had a splenectomy and have unstable
hemoglobins, the test may appear positive due to the presence of
numerous insoluble erythrocyte inclusions.
In all cases where abnormalities are suspected or indicated,
electrophoretic confirmation is recommended.
PROCEDURE NOTES
Samples that are highly lipemic or are borderline positive should
have the following procedure performed:
Centrifuge the whole blood specimen at 1500xg for 10 minutes
and carefully remove the supernatant plasma and discard. Repeat the
test using 20 microliters of the packed erythrocytes.
QUALITY CONTROL
Quality control samples should be used routinely to monitor test
performance (8). We strongly recommend the use of Positive and
Negative Sickle Cell Controls (Cat. No. 2244-0) for this purpose.
BIBLIOGRAPHY
1.
2.
3.
4.
5.
6.
7.
Widmann, F.K., Clinical Interpretation of Laboratory Tests, F.A.
Davis Co., Philadelphia, pp. 57-60 (1983).
Neel, J.V., Blood, 6:389 (1951).
Itano, H.A., Arch. Biochem. Biophys., 47:148 (1953).
Nalbandian, R.M., Nichols, B.M., Camp, F.R. et al, Clin. Chem.,
17:10281032 (1971).
Nalbandian, R.M., Nichols, B.M., Camp, F.R. et al, 17:1033 (1971).
Calam, R.R. in Selected Methods of Clinical Chemistry, Vol. 9, Ed.
by W.R. Faulkner and S. Meites, AACC Publications, Washington,
pp. 3-10
(1982).
Fairbanks, V.F. and Klee, G.G. in Textbook of Clinical Chemistry,
Ed. N.W. Tietz, W.B. Saunders Co., Philadelphia, pp. 1540-1541
(1986).
Product Availability
Sickle Cell Reagent Set, 54 Tests, Cat. No. 2245-0:
110 ml
54 vials
10 ml
Sickle Cell Reagent
Sodium Dithionite
Sickle Cell Urea Reagent
Lit. No. 2245-0 (Rev B)
Copyright 1986