INTENDED USE:
Qualitative
determination of hemoglobin S (Hbs)
in blood using a phosphate solubility method.
METHOD HISTORY:
In 1910 Herrick (I)
observed the sickling of erythrocytes from patients with sicke cell anemia.
Since that time over 250 structural variants of the hemoglobin molecule have
been describe (2.3). In the United States, hemoglobin S (Hbs) is
the most common
hemoglobin variant; it is found in about 8-11% of American Negroes (4). It is essentially peculiar to Negroes, reaching
a frequency of up to 59% in various regions of Africa. It is also found in
localized
areas in countries bordering the
Mediterranean Sea, e.g. Italy, Greece, Turkey, and Arabia.
In 1953, Itano (5)
reported poor solubility of deoxyhemoglobin S in concentrate phosphate buffer.
Several modifications of
the original
procedure have been reported (6-8). The solubility test have been adapted to
automated screening.
The Biotron procedure
is a modified Nalbandian (6) procedured based upon phosphate solubility.
METHOD PRINCIPLE:
Erythrocytes are
lysed by saponin and the released hemoglobin is reduced by dithionite in a
phosphate buffer. Reduced Hbs
is characterized by
its very low solubility and by the
formation of nematic liquid crystals (tactoids) so that in the presence of Hbs
or non-S sickling hemoglobin the system becomes turbid. With the addition of
urea the liquid crystals of Hbs are dispersed and the system clears. Urea
breaks the hydrophobic bonds that are the basis of Hbs sickling. If the system
remains
turbid after the
addition of urea, a non sticking hemoglobin is indicated. In either case,
electrophoretic confirmation is required for conclusive identification.
REAGENTS PROVIDED:
1. SICKLE CELL
REAGENT:A solution containing 0.97 M potassium phosphate monobasic, 1.33 M
potassium phosphate dibasic with timesol as the preservative. Keep tightly capped and protected from
contamination. Can be used until the
expiration date
indicated on the individual bottle. Warning:
Thimesol- Toxic.
2.TEST VIALS WITH
SODIUM HYDROSULFITE: 20mg of sodium dithionite per reaction vial. Keep tightly capped and protected from
moisture prior to using. Must be free
flowing. Can be used until the expiration date indicated on the
expiration date
indicated on the individual bottle. Warning-Saponin-
Strong Hemolytic Agent.
3. SICKEL CELL UREA
REAGENT: Urea in aqueous solution. Keep tightly capped and protected
from contamination.
Can be used until
expiration date indicated on individual bottle.
4. Two dispensing
closures.
5. Interpretive stand
REAGENT STORAGE:
Store all reagents
provide in this reagent set at room temperature (below 30C)
INDICATORS OF REAGENT DETERIORATION.
1.
Physical
appearance.
a)
Appearance of turbidity in the sickle cell reagent, which will not readily
dissolve upon mixing, may indicate reagent deterioration.
b) IF SODIUM HYROSULFITE has become
damp and lumpy prior to using, it should not be used.
2) Control assays.
Failure to obtain
accurate results in the assay of control materials may indicate reagent
deterioration.
3) Biotron
Diagnostics cannot guarantee the stability of reagents, which have been.
a)
Transferred
from their original containers
b)
Improperly stored
c)
Contaminated during use.
SPECIMEN COLLECTION:
Collect whole blood in a vial containing a suitable
anticoagulant (heparin, EDTA, oxalate, ACD, CPD, CPDA-1 and CP2D solutions) and
mix thoroughly. Blood samples that have been kept for as long as 1-2 weeks at
4-5C are reportedly
Satisfactory. No
preliminary restriction of food or fluid is required.
MATERIALS PROVIDED;
Sickle Cell Reagent,
Sickle Cell Urea Reagent. 54 vials with DITHIONITE.
ADDITIONAL MATERIALS REQUIRED BUT NOT PROVIDED.
Reagent and samples
pipettes, controls (positive and Negative)
PROCEDURE:
1. Add 2.0 ml of working sickle cell reagent to
premarked tubes labeled Unknown, Positive and Negative.
Immediately,
return working sickle cell buffer to
refrigerator.
2. Add 0.02 ml of sample or controls and mix by
inversion.
3. Place in the sicke
cell rack for 5-10 minutes.
4. Read the test by
holding the tube approximately 3 cm in front of a line scale provided. Adequate
illumination is necessary
RESULTS:
POSITIVE:
If HbS or any other
sickling hemoglobin is present, the solution is turbid and the lines behind the
test vial will not be visible.
NEGATIVE:
If no sickling
hemoglobin is present, the clear solution will permit the line to be seen
through the vial.
All doubtful test,
along with all positive test, should be submitted for electrophoretic
confirmation.
If the test is
positive, the following test may be performed.
1. Add 2.0 ml of SICKLE CELL REAGENT to a test vial.
2. Ad 6 drops of SICKE CELL UREA REAGENT and
mix.
3. Add 0.02 ml (20 ul) of the positive whole
blood specimen and mix by inversion.
4. Allow to stand at room temperature for 5
minutes.
5. Read test as before.
An originally
positive HbS test will be confirmed if the solution becomes clear upon addition
of the Sicke Cell Urea Reagent. The only known variant that will also clarify is HbC (Harlem)
PROCEDURE LIMITATIONS:
Severe anemia will
cause false negatives results, therefore, if the hemoglobin concentration is 7
g/dl or less the
samples should be
re-run on 40 ul of sample. Blood from
patients with polycythemia, multiple myeloma, cryoglobulinemia
and other dysglobulinemia cause false positives,
whereas patients with over 25% HbF
present may yield false negatives (10).
In all cases where
abnormalities are indicated or suspected, electrophoretic confirmation is
recommended.
PROCEDURES NOTES:
Controls should be
run with each series of test, Negative controls may be collected from a normal,
healthy Caucasian individual. Positive controls may be purchased or obtained
from samples determined to contain HbS by
electrophoretic
methods.
BIOGRAPHY ;
1. Herrick, JP, Arch
Intern Med. 6:517 (1910)
2. Perutz, MF and
Lehmann, H. Nature 219;902 (1968)
3. Lehmann H and
Carrell, RW Brit Med Bull, 25;14 (1969)
4. Neel JV. Blood
6:389 (1951)
5. Itano, HA, Arch
Biochem Biophys, 47;148 (1953)
6. Nalbandian, RM et
al Clin Chem. 17:1028
7. Schmidt, RM and
Wilson, SM J. Am Med Assoc. 225;1225 (1973)
8. Greenberg, MS et
al N. Eng J. Med 286;1143 (1972)
9. Tietz, NW,
Fundamentals of Clinical Chemistry WB Saunders, Phila p 418 (1976)
10. Henry RJ Clinical
Chemistry, Principles & Technics, Harper & Row , NY p 1176 (1974).