Download Anti-s

FOR TUBE IAT METHOD
Anti-s
Human Polyclonal Phenotyping Reagent
METHOD SUMMARY*­
Tile
Tube
Validated Methods
No
Tube/IAT
MTP
Yes
No
No
Reagent Volume
1-2
Cell Volume
1
Cell Concentration
3-5%
Incubation Time
30 mins
Temperature
37°C
Spin (Speed/Time)
Low for 20 secs
Papain, ficin, chymotrypsin and pronase readily destroy the s antigen. However, the s antigen is resistant
to treatment by Trypsin, DTT and acid. A pH of 6.0 enhances the reactivity of some anti-s antibodies.
Gene Frequency
As the S and s antigens are allelic, all red cells from Caucasians, except the very rare Rhnull type, should
react with one or both of these reagents. However, approximately 1.5% of African-Americans (Negroes)
will be negative for both S and s antigen.
The expected results and frequencies of the phenotypes in the Australian blood donor population are
given below:
Other
*Refer to the ‘Recommended Methods’ for detailed procedures.
For in vitro
Diagnostic Use
Only
Caution:
Handle as if Capable
of Transmitting
Infection
READ LEAFLET
CAREFULLY
REAGENT DESCRIPTION
CSL Anti-s (little s) polyclonal phenotyping reagent is prepared from human serum containing the
appropriate antibodies active by the Indirect Antiglobulin Test (IAT). When used by the recommended
method this reagent will cause agglutination of red blood cells carrying the specific s antigen. CSL Anti-s
phenotyping reagent has been tested with S+s+ red cell samples to ensure adequate potency. Specificity
is assured by testing each reagent against a panel of red cells known to be negative for the appropriate
antigen. This panel includes cells representing most of the common inherited blood group antigens.
Reactivity to antigens of very low incidence is not necessarily excluded, but tests with red cells containing
such antigens are performed whenever suitable cells are available. The reagent contains Bovine Albumin,
macromolecular potentiators and Sodium Azide as a preservative. The reagent has been optimised for
use without further dilution.
STORAGE CONDITIONS
Store at 2° to 8°C (Refrigerate. Do Not Freeze).
PRINCIPLE OF THE TEST
The agglutination of red cells by a specific reagent indicates the presence of the corresponding antigen
on those cells, whilst a negative reaction signifies the absence of the corresponding antigen. Red cells
expressing the s antigen will agglutinate in the presence of the corresponding specific antibody in CSL
Anti-s.
BACKGROUND
Blood Group System
Walsh and Montgomery first reported the S antigen in 1947 and it was realised that the gene producing S
was linked to the allelic genes producing M and N antigens. In 1951 Levine et al. discovered the s antigen
and recognised it as being allelic to S. The biochemistry of the MNSs system is fully reviewed by Lisowska
and Dahr and there is a more detailed study by lssitt. Due to the population incidences, the MNSs blood
group system is the most useful of all red cell antigen systems for exclusion in parentage testing.
CSL Limited
45 Poplar Road
Parkville Victoria
3052 Australia
ABN 99 051 588 348
Phone +61 3 9389 1911
Fax +61 3 9389 1646
Antigen Characteristics
The s antigen is located on a minor sialoglycoprotein that is very similar to MN, called
Ss-sialoglycoprotein, δ-sialoglycoprotein, or glycophorin B. The Ss antigens are well developed at birth
and appear on foetal red cells at around 12 weeks gestation. Rhnull red cells have greatly reduced Ss
antigen expression.
Anti-s has been reported as causing mild transfusion reactions, as well as severe and fatal Haemolytic
Disease of the Newborn (HDN). IgG anti-s is more common than IgM. No ‘naturally occurring’ anti-s has
been reported. Anti-s is usually optimally reactive by Indirect Antiglobulin Test (IAT), at 22°C or below.
Anti-s rarely binds complement.
Reactions obtained with
Anti-S
Anti-s
0
+
Phenotype
Frequency
S-s+
0.4797
+
+
S+s+
0.4261
+
0
S+s-
0.0942
0
0
S-s-
<0.01
SPECIMEN COLLECTION AND PREPARATION
Blood samples should be withdrawn aseptically with or without the addition of anticoagulants. Tests
should be performed as soon as possible after collection of the sample. If testing the blood samples is
delayed, samples should be stored between 2° to 8°C. Samples collected into EDTA or Heparin may be
tested up to 7 days from the date of withdrawal provided storage has been at 2° to 8°C. Clotted samples
may be tested up to 14 days from the date of withdrawal provided storage has been at 2° to 8°C.
Samples collected in Citrate may be tested up to 42 days from the date of withdrawal provided storage
has been at 2° to 8°C. Cells also may be stored in CSL CelpresolTM at 2° to 8°C for up to 42 days.
RECOMMENDED METHODS
Tube Method - Indirect Antiglobulin Test (IAT)
1.Prepare a 3-5% suspension of test red cells in buffered or unbuffered isotonic saline, or in CSL
CelpresolTM.
2. Add 1 or 2 drops of CSL Anti-s phenotyping reagent to an appropriately labelled, clean glass test
tube (10x75mm or 12x75mm).
3. Add 1 drop of the suspension of test red cells.
4. Mix well and incubate at 37°C for 30 minutes.
5.Wash the cells with 4 changes of buffered or unbuffered isotonic saline, ensuring that the saline
is decanted completely after each wash and that the cells are completely resuspended between
washes.
6. To the ‘dry’ button of cells remaining after the fourth wash, add 1 or 2 drops of CSL EpicloneTM AHG
Poly reagent.
7. Mix well and centrifuge at low speed (500 rcf) for 15 to 20 seconds*.
8. Gently agitate the tube to dislodge the red cells and examine for agglutination. Record results.
9. Add 1 drop of CSL AHG Control Cells 3% to all negative test tubes to validate the results.
10. Repeat steps 7 and 8.
Note: *Or centrifuge at a speed and time appropriate for the centrifuge in use.
CSL recommends the use of buffered isotonic saline solutions (buffered to pH 7.0 to 7.2).
02160000F
Gosia Kolasinski
F2
Actioned Approval 31/07/07
INTERPRETATION OF RESULTS
Agglutination of the test red cells constitutes a positive result and indicates the presence of the appropriate antigen
(subject to the cells giving a negative Direct Antiglobulin Test (DAT)). No agglutination of the test red cells indicates the
absence of the relevant antigen.
CONTROLS
The use of controls is essential in the performance of all blood grouping tests. Control samples should be tested in parallel
with the test sample.
Positive Control – red cells known to be heterozygous for the s antigen, i.e. (S+s+).
Negative Control – red cells known to lack the s antigen.
11. Green R, et al. Basic Blood Grouping Techniques and Procedures. 2nd Ed. Victorian Immunohaematology Discussion
Group 1992.
12. Scientific Subcommittee of the Australian and New Zealand Society of Blood Transfusion Inc. Guidelines for
Pretransfusion Laboratory Practice. 5th Ed. 2007.
13. Daniels G. Human Blood Groups. 2nd Ed. Blackwell Science. Carlton, Victoria 2002.
14. Race R, Sanger A. Blood groups in man. 6th Ed. Oxford: Blackwell Scientific Publications 1975.
15. Reid ME, Lomas-Francis C. The Blood Group Antigen FactsBook. 2nd Ed. Academic Press Limited. UK 2004.
16. Schenkel-Brunner H. Human Blood Groups. Chemical and Biochemical Basis of Antigen Specificity. 2nd Ed. Springer
Wien. New York 2000.
17.United Kingdom National Blood Service. Guidelines for the Blood Transfusion Services in the United Kingdom. 7th Ed.
2005.
Following the interpretation of each test, one volume of sensitised cells (e.g. CSL AHG Control Cells 3%) should be added
to all negative tests and the mixtures re-examined to ensure that agglutination occurs. This control is based on the
principle that in a truly negative test the Anti-Human Globulin reagent will have remained unconsumed after exposure to
the particular washed cell suspension and therefore agglutinate the AHG Control Cells. Its use safeguards against error
caused by imperfect technique, poor washing and inadequately reactive antiglobulin reagents.
Caution: Red cells giving a positive Direct Antiglobulin Test (DAT) cannot be typed by the Indirect Antiglobulin Test (IAT). If
cells are typed for a single antigen only, it is necessary to perform a Direct Antiglobulin Test (DAT) in parallel with the test.
A positive DAT will invalidate any positive result obtained. The direct test may be omitted if the sample of cells is being
tested simultaneously with reagents of other specificities by the IAT technique, and negative results are obtained.
LIMITATIONS OF PROCEDURE
False results may occur due to:
1. Incorrect technique.
2. Presence of gross rouleaux.
3. Use of aged blood samples, reagents or supplementary materials.
4. Contaminated blood samples, reagents or supplementary materials.
5. Red cells that have a positive Direct Antiglobulin Test (DAT).
6. Other deviation from the recommended test methods.
7. Incorrect concentrations of red cells.
8. Incorrect reading of results.
9. Enzyme techniques are not recommended as they may denature the s antigen.
PRECAUTIONS
1. For in vitro diagnostic use only.
2. The material from which this product was derived was found to be non-reactive for specified markers for HIV 1 and
2, Hepatitis B and C, HTLV and Syphilis by currently approved methods. However no known method can assure that
products derived from human blood will not transmit infectious agents.
3. Sodium Azide 0.1% w/v is added as a preservative. Users should be aware of the toxicity and cumulative explosive
nature of Sodium Azide and take appropriate precautions when handling and discarding this reagent.
4. This product should be clear; turbidity may indicate bacterial contamination. The reagent should not be used if a
precipitate or particles are present.
REFERENCES
1. Walsh RJ, Montgomery C. A new human isoagglutinin subdividing the MN blood groups. Nature, London 1947; 160: 504.
2. Sanger R, Race RR. Subdivisions of the MN blood groups in man. Nature, London 1947; 160: 505.
3. Levine P, Kuhmichel AB, Wigod M, Koch E. A New Blood Factor, s, Allelic to S. Proc Soc Exp Biol NY 1951; 78: 218-20.
4. Giblett E, Chase J, Crealock FW. Haemolytic disease of the newborn resulting from anti-s antibody. Amer J Clin Path
1958; 29: 254-6.
5. Lisowska E. Biochemistry of M and N blood group specificities. Rev franc de transf et immunohaem 1981; 24: 75-84
(in English).
6. Dahr W. Serology, genetics and chemistry of the MNSs blood group system. Rev franc de transf et immunohaem
1981; 24: 85-95 (in English).
7. Issitt PD. The MN blood group system. Cincinnati: Montgomery Scientific Publications 1981.
8. Issitt PD. Applied Blood Group Serology. 3rd Ed. Montgomery Scientific. Miami 1985.
9. Harmening DM. Modern Blood Banking and Transfusion Practices. 5th Ed. FA Davis Company. Philadelphia 2005.
10. Brecher ME. American Association of Blood Banks Technical Manual. 15th Ed. Bethesda, Maryland 2005.
02160000F
02160000F
Gosia Kolasinski
F3
Actioned Approval 31/07/07
June, 2007