Download Ep iclo ne A H G Po ly A H G A nti-Ig G

FOR TUBE, TUBE IAT & MICROPLATE METHODS
Anti-IgG Monospecific
Polyclonal Reagent
Polyclonal and Monoclonal Reagent
AHG Anti-IgG
Anti-IgG-C3d Polyspecific
TM
Epiclone AHG Poly
METHOD SUMMARY*
For in vitro
Diagnostic Use
Only
Caution:
Handle as if Capable
of Transmitting
Infection
READ LEAFLET
CAREFULLY
CSL Limited
45 Poplar Road
Parkville Victoria
3052 Australia
ABN 99 051 588 348
Phone +61 3 9389 1911
Fax +61 3 9389 1646
Validated Methods
Reagent Volume
Cell Volume
Cell Concentration
Incubation Time
Temperature
Spin (Speed/Time)
Tube/DAT
Yes
1 or 2
1
3-5%
Immediate Spin
Room Temp
Low for 20 secs
Validate Negatives with
CSL AHG Control Cells 3%
Tube/IAT
Yes
1 or 2
1
3-5%
10-30 mins**
37°C
Low for 20 secs
Validate Negatives with
CSL AHG Control Cells 3%
Other
* Refer to the ‘Recommended Methods’ for detailed procedures.
**Incubation time when CSL RAM added.
MTP
Yes
1
1
3-5%
Immediate Spin
Room Temp
Low for 40 secs
Validate Negatives with
CSL AHG Control Cells 3%
REAGENT DESCRIPTION
CSL EpicloneTM AHG Poly, Anti-IgG-C3d polyspecific, polyclonal and monoclonal reagent is prepared by blending the
serum of rabbits that have been immunised with purified human IgG and murine monoclonal antibodies specific
for an epitope on the human C3d molecule. If correctly stored and used according to the recommended methods,
CSL EpicIoneTM AHG Poly will detect human immunoglobulin and/or complement (C3d) on red cells. The reagent has
been shown to cause agglutination of red cells weakly coated with IgG and human complement component C3d
by serological methods recommended by the USA Office of Biologics Research and Review (OBRR). The reagent is
formulated to give clear positive reactions with red cells coated with significant amounts of either IgG or C3d, but not to
react with trace amounts of C3d that are present on stored red cells. The clone used to produce the Anti-C3d antibodies
is BRIC 8. The reagent is colour coded green.
CSL AHG Anti-IgG monospecific polyclonal reagent is prepared from the same Anti-IgG as used in the EpicIoneTM AHG
Poly reagent. If correctly stored and used according to the recommended methods, CSL AHG Anti-IgG will detect IgG
present on red cells. The reagent has been shown not to agglutinate red cells sensitised with complement components
by low-ionic methods recommended by the USA Office of Biologics Research and Review (OBRR). The reagent is colour
coded clear or green.
Both reagents contain sodium chloride, Bovine Albumin and macromolecular potentiators. Sodium Azide has been added
as a preservative. Both reagents have been optimised for use without any further dilutions or additions.
STORAGE CONDITIONS
Store at 2° to 8°C (Refrigerate. Do Not Freeze).
PRINCIPLE OF THE REAGENTS
When IgG antibodies attach to their appropriate red cell antigens, they may fail to cause agglutination of the cells, but will
remain firmly bound even when the cells are thoroughly washed in saline. This process is called red cell sensitisation. The
presence of the bound antibody may then be detected by the addition of an Anti-Human Globulin (AHG) reagent. Certain
antibody-antigen reactions may also cause binding of complement components to the red cell. A Direct Antiglobulin Test
(DAT) is used to determine in vivo coating of red cells, whilst an Indirect Antiglobulin Test (IAT), is used to detect antibodies
(present in the serum) after in vitro adsorption onto red cells. Following the detection of a positive DAT, CSL’s AHG Anti-IgG
and EpicloneTM Anti-C3d reagents are used to identify whether IgG or complement (C3d) has sensitised the cells.
CSL EpicloneTM AHG Poly is a polyspecific, polyclonal and monoclonal reagent which will react with red cells having human
immunoglobulin (IgG) or complement component C3d on their surface. CSL has chosen a polyclonal Anti-IgG component in
its EpicloneTM AHG Poly reagent to ensure that all clinically significant IgG isotypes are detected.
CSL AHG Anti-IgG is a monospecific, polyclonal reagent that will react only with red cells having IgG on their surface and
not with cells sensitised with complement components. CSL EpicloneTM Anti-C3d may also be used in conjunction with this
reagent to determine if cells have complement component C3d attached to their surface.
The chart below summarises the intended use of the range of Anti-Human Globulin reagents issued by CSL.
­
Reagent
For Use In
EpicloneTM AHG Poly AHG Anti-IgG EpicloneTM Anti-C3d
Direct Antiglobulin Test
Investigation of Haemolytic Disease of the Newborn
Yes
Yes
Yes
Investigation of Haemolytic Transfusion Reactions
Yes
Yes
Yes
Detection of Drug-Induced Red Cell Sensitisation
Yes
Yes
Yes
Detection of Auto-Immune Haemolytic Anaemia
Yes
Yes
Yes
Identification of Cell Surface Coat No Yes
Yes
(Complement vs Immunoglobulin)
For Use In
Indirect Antiglobulin Test
Compatibility Testing
Donor Screening for Unexpected Antibodies
Patient Screening for Unexpected Antibodies
Detection of Antigens (Phenotyping)
Antibody Identification (Serum)
Antibody Identification (Eluates)
Note: Reagent
EpicloneTM AHG Poly AHG Anti-IgG EpicloneTM Anti-C3d
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Monospecific reagents are used most often to confirm if a positive Direct Antiglobulin Test
(DAT) is due to sensitisation by IgG, C3d or both.
BACKGROUND
The Anti-Human Globulin (Coombs) Test was first described by Coombs, Mourant and Race in 1945, although the principle
of antiglobulin reactions was reported by Moreschi in 1908. Many immune antibodies may attach to their respective
red cell surface antigens without causing direct agglutination of the red cells, but these antibodies can be detected by
antiglobulin tests. The polyspecific Anti-Human Globulin reagent contains antibodies to both immunoglobulins (mainly
IgG) and complement components (C3d) and is designed as the primary reagent for antibody detection, compatibility
tests and investigation of auto-immune haemolytic anaemias.
The first crossmatching tests were developed in 1907 to detect ABO incompatibility. These tests were performed on slides
using cells suspended in saline (saline test) and were able to detect what are now known as IgM antibodies. It was soon
realised that unexplained transfusion reactions still occurred, despite apparent ABO compatibility between the donor and
recipient. These reactions were thought to be caused by "immune" antibodies produced by an immune response after
exposure of the recipient to foreign red cells. These antibodies are usually IgG, are generally not detectable by the saline
test and are capable of causing severe transfusion reactions and Haemolytic Disease of the Newborn (HDN).
Haemolytic transfusion reactions can be either immediate or delayed in nature. In Immediate Haemolytic Transfusion
Reactions (IHTR), the red blood cells are destroyed by one of two mechanisms, either intravascular haemolysis or
extravascular haemolysis. In both mechanisms, patient’s antibody binds to incompatible transfused red blood cells forming
an antigen-antibody complex. Intravascular haemolysis occurs when complement is activated; resulting in haemoglobin,
red blood cell stroma and intracellular enzymes being released from the lysed red blood cells. Extravascular haemolysis
occurs when there is antigen-antibody formation on the red blood cells (sensitisation), with incomplete activation of
complement. These sensitised red blood cells are then removed by the reticuloendothelial system. Delayed Haemolytic
Transfusion Reactions (DHTR), can be caused by either secondary response to transfused red blood cells or primary
alloimmunisation. Extravascular haemolysis is the mechanism of red blood cell destruction in both types of DHTR.
Haemolytic Disease of the Newborn (HDN), occurs when maternal antibodies (IgG in class), directed towards foreign
antigens on foetal red blood cells, cross the placenta and attach to the foetal red blood cells, causing their destruction. This
leads to foetal anaemia of varying severity. In severe cases, an exchange transfusion may be required for foetal survival.
The complement system, denoted by the letter C, has been found to consist of a complex group of soluble serum proteins
made up of at least nine components labelled sequentially C1 to C9. Activation of complement causes these proteins to
follow a cascading pathway that may result in a range of effects. From a serological point of view, the end result is an
active product capable of causing destruction of the red cells (called lysis). Reaction of the antigen and antibody leads to
a change in the shape of the antibody, with exposure of a site on the Fc portion of the molecule capable of binding with
the first component of complement C1. C1 has 3 parts C1q, C1r and C1s which act on C4 and in turn C2 which forms a
complex, known as C3 convertase, that is capable of reacting with C3. C3 is then split into C3a and C3b with C3b fixing
to the red cell surface. C3b reacts with C5 and subsequently C6, C7, C8 and C9 leading to cell membrane damage and
lysis. Complement fixation does not always proceed through all of the steps to lysis and often stops after a few steps
and leaves various complement components bound to the cell membrane, most often C3.
From a clinical point of view, the complement fraction of serological interest is C3b; a complex of C3d, C3g and C3c.
When the immune reaction has occurred in vivo (in the patient), C3d is usually the component that remains attached to
the cell. Therefore, AHG Polyspecific reagents need to contain Anti-C3d, which will detect only the clinically relevant C3d
complement component on red cells following both in vivo and in vitro immune red cell reactions, and not C4 and other
non-clinically relevant components or trace amounts of C3 that may normally be found on stored red cells.
Users of AHG may choose a polyspecific reagent to allow detection of IgG and complement sensitised cells, and usually
will have available Anti-IgG and Anti-C3d monospecific reagents to assist in investigating samples found positive with
the polyspecific reagent. An exception to this general rule, are users of EDTA plasma, who may choose monospecific AntiIgG as their primary reagent. This is because EDTA, a commonly used anticoagulant, prevents complement activation.
However, Polyspecific AHG is also suitable for use with EDTA plasma samples.
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 into Citrate may be tested up to 35 days from the date of withdrawal provided storage has been at
2° to 8°C. Cells also may be stored in CSL CelpresolTM for up to 42 days.
For Direct Antiglobulin Tests (DAT), blood collected into an anticoagulant (EDTA) should be used and tests should
preferably be performed within 24 hours of collection. EDTA is the recommended anticoagulant, as this will prevent
complement sensitisation in vitro.
03070000H
Gosia Kolasinski
H1
Actioned Approval 31/07/07
RECOMMENDED METHODS
CSL EpicloneTM AHG Poly and CSL AHG Anti-IgG reagents are recommended for use by the tube method and are equally applicable in both
the Direct and Indirect Antiglobulin Tests. CSL EpicloneTM AHG Poly and CSL AHG Anti-IgG may also be used with the microplate method.
Tube Methods
Direct Antiglobulin Test
The Direct Antiglobulin Test (DAT), is a one--stage procedure for the detection of in vivo red cell sensitisation by antibodies and/or complement
as found in Haemolytic Disease of the Newborn (HDN) and certain auto-immune conditions.
1. Appropriately label 2 separate, clean glass test tubes (10x75mm or 12x75mm).
2. Prepare a 3-5% suspension of test red cells in buffered or unbuffered isotonic saline, or in CSL CelpresolTM.
3. Place 1 drop of the suspension of test red cells into each tube.
4. Wash the cells in both tubes with 4 changes of isotonic saline, ensuring that the saline is decanted completely after each wash and
that the cells are completely resuspended between washes.
5. T o the ‘dry’ button of cells remaining after the fourth wash, in the first tube, add 1 or 2 drops of CSL EpicloneTM AHG Poly or CSL AHG Anti-IgG*.
6. To the ‘dry’ button of cells remaining after the fourth wash, in the second tube, add 2 drops of isotonic saline, (the purpose of this
second tube is to check that a positive result in the first is genuinely due to a reaction between the reagent and the globulin coating
the cells, as distinct from saline agglutination which has not been dispersed by the washing or aggregation due to Wharton's jelly).
7. Mix well and centrifuge at low speed (500rcf) 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 (see Control section of this leaflet).
10. Repeat steps 7 and 8.
Note:
* CSL EpicloneTM AHG Poly and AHG Anti-IgG reagents are validated for either 1 or 2 drop methods. Users may find that the 2
drop method provides a higher final liquid volume and easier reaction reading with the "tip and roll" technique.
** Or centrifuge at a speed and time appropriate for the centrifuge in use.
T here is strong evidence that red cells weakly sensitised with C3d in vivo give greatly enhanced reactions if the washed cells are
left in contact with the CSL EpicloneTM AHG Poly reagent for 5 minutes at room temperature following Step 6. It is recommended
that duplicate tests be performed so that one set may be centrifuged immediately (for detection of IgG sensitisation) and the
other set left at room temperature for 5 minutes prior to centrifugation (for detection of C3d sensitisation). The immediate
centrifugation phase should not be omitted, as some IgG/anti-IgG reactions may be weakened by incubation.
Indirect Antiglobulin Test
The Indirect Antiglobulin Test (IAT), is a two-stage procedure which can be applied to compatibility testing, the detection, titration and
identification of antibodies and to phenotyping with IgG reagents of known specificity.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Prepare a 3-5% suspension of test red cells in buffered or unbuffered isotonic saline, or in CSL CelpresolTM.
Add 2 drops of test serum to an appropriately labelled, clean glass test tube (10x75mm or 12x75mm).
Add 1 drop of the suspension of test red cells.
If desired*, add 2 drops of CSL RAM (Rapid Antibody Medium).
Mix well and incubate at 37°C for 10* to 30 minutes.
Wash the cells with 4 changes of isotonic saline, ensuring that the saline is decanted completely after each wash and that the cells
are completely resuspended between washes.
To the ‘dry’ button of cells remaining after the fourth wash, add 1 or 2 drops of CSL EpicloneTM AHG Poly or CSL AHG Anti-IgG**.
Mix well and centrifuge at low speed (500rcf) for 15 to 20 seconds***.
Gently agitate the tube to dislodge the red cells and examine for agglutination. Record results.
Add 1 drop of CSL AHG Control Cells 3% to all negative test tubes to validate the results (see the Controls section of this leaflet).
Repeat steps 8 and 9.
Note: *The addition of CSL’s RAM (Rapid Antibody Medium) results in enhancement of antigen-antibody reactions in many cases, and
allows the incubation time to be reduced from 30 to 10 minutes without loss of sensitivity.
**CSL EpicloneTM AHG Poly and CSL AHG Anti-IgG reagents are validated for either 1 or 2 drop methods. Users may find that the 2 drop method provides a higher final liquid volume and easier reaction reading with the "tip and roll" technique.
***Or centrifuge at a speed and time appropriate for the centrifuge in use.
Microplate Method
Note: CSL EpicloneTM AHG Poly and CSL AHG Anti-IgG are validated for use in microplates. Due to the variation in methods and equipment,
microplate users should validate these reagents using their methods.
A commonly used microplate method is outlined below:
1. Prepare a 3-5% suspension of test red cells in buffered or unbuffered isotonic saline containing 1% BSA.
2. Add 1 volume of test serum to the appropriate test well.
3. Add an equal volume of the suspension of test red cells to the appropriate test well.
4. Mix the contents of each well using manual means or a microplate shaker. The time required to achieve this will depend on the speed
and orbit of the shaker.
5. Incubate the microplate at a temperature and time appropriate to the test serum.
6. Centrifuge at high speed (200rcf) for 40 seconds*.
7. Discard the serum or phenotyping reagent and resuspend the cell button.
8. Add 150µL of buffered isotonic saline to each well and centrifuge as for step 6.
9. Repeat steps 6 to 8 four times.
10. To the ‘dry’ button of red cells remaining after the fourth wash, add 1 volume of CSL EpicloneTM AHG Poly or CSL AHG Anti-IgG.
11. Mix and centrifuge at low speed (100rcf) for 40 seconds*.
12. Re-suspend the red cells using a microplate shaker for an optimal time and agitation speed.
13. Read the tests macroscopically or with an automated reader.
14. Add 1 volume of CSL AHG Control Cells 3% to all negative test wells to validate the results (see the Control section of this leaflet).
15. Repeat steps 11 to 13.
Notes: *Or centrifuge at a speed and time appropriately validated for the centrifuge in use.
INTERPRETATION OF RESULTS
A positive reaction is indicated by agglutination of the test cells in the presence of CSL EpicloneTM AHG Poly or CSL AHG Anti-IgG.
A positive reaction in the Direct Antiglobulin Test (DAT), accompanied by a negative saline blank, indicates that either human
immunoglobulin (IgG) or human complement (C3d) has been adsorbed to the cells from the patient's own serum, with the proviso that cold
auto-antibodies may bind complement as a result of cooling after the blood sample has been withdrawn from the patient. Precautions are
necessary when dealing with patients in whom detectable cold auto-antibody is present. A positive DAT reaction indicates sensitisation of
the patient’s cells in vivo.
A positive reaction in the Indirect Antiglobulin Test (IAT) indicates the presence of either human immunoglobulin (IgG) or human complement
(C3d) on the red cells following incubation with serum. In the case of testing unknown serum, this means that an antibody directed at an
antigen on the test cells is present in the serum. When testing unknown cells against a phenotyping reagent that requires the use of an
IAT technique, the presence of the appropriate antigen on those cells is indicated, provided a DAT on the same cells or an auto control test
performed in parallel with the test, gives a negative reaction.
Some laboratory scientists prefer to interpret antiglobulin reactions microscopically. Whilst this practice may lend additional sensitivity to
the test, it can also be a potential source of misleading positive reactions, the predominant cause of which is the propensity of red cells to
adsorb complement during storage at refrigerator temperatures. Accordingly, it is recommended that antiglobulin tests be read with the
use of a hand lens or a concave mirror and a suitable source of illumination. Stronger magnification, such as microscopes, may be used for
investigative tests, but are not recommended for routine tests.
CONTROLS
The antiglobulin test is an exceedingly delicate procedure and the presence of even minute amounts of free human protein can result
in neutralisation of the reagent. The washing of cells should be carried out with great care and the quality and cleanliness of saline and
glassware should be maintained. The inclusion of both positive and negative controls is essential with every batch of tests, and as a final
check on the adequacy of washing and on the potency of the Anti-Human Globulin (AHG) reagent.
Following the interpretation of each test, one drop of sensitised cells (eg. 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.
Note: To control and validate the Anti-C3d component of CSL’s EpicloneTM AHG Poly reagent, real clinical examples of C3d sensitised cells
should be used. While the cold sucrose or "fruitstone" method may be used to artificially complement coat cells, it is not always effective
and may be unreliable with monoclonal Anti-C3d reagents.
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. Other deviation from the recommended test methods.
6. Incorrect concentrations of red cells or expired reagents.
7. Incorrect reading of results.
8. Red cells with a positive DAT being used in IAT.
9. Red cells being incompletely washed or glassware being contaminated with human globulins or complement.
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. Coombs RR, Mourant AE, Race RR. A new test for the detection of weak and "incomplete" Rh agglutinins. Br J Exp Pathol 1945; 26: 255-66. 2. Coombs RR, Mourant AE, Race RR. In vivo isosensitisation of red cells in babies with haemolytic disease. Lancet 1946; 1: 264-6.
3. Eyster ME, Jenkins DE Jr. Erythrocyte coating substances in patients with positive direct antiglobulin reactions. Am J Med 1969; 46: 360-71.
4. Lown JAG, Barr AL, Davis RE. The use of low ionic strength saline for cross matching and antibody screening. J Clin Pathol 1979; 32: 1019-24.
5. Clayton EM, Brown BB, Bove JR. The antiglobulin reaction on albumin enriched cell suspensions. Transfusion 1965; 5: 344-9.
6. Ahn JH, Rosenfield RE, Kuchwa S. Low ionic antiglobulin tests. Transfusion 1987; 27: 125-33.
7. Garratty G, Petz LD. The significance of red cell bound complement components in development of standards and quality assurance
for the anti-complement components of antiglobulin sera. Transfusion 1976; 16: 297-306.
8. Harmening DM. Modern Blood Banking and Transfusion Practices. 5th Ed. FA Davis Company. Philadelphia 2005.
9. Brecher ME. American Association of Blood Banks Technical Manual. 15th Ed. Bethesda, Maryland 2005.
10. Green R, et al. Basic Blood Grouping Techniques and Procedures. 2nd Ed. Victorian Immunohaematology Discussion Group 1992.
11. Scientific Subcommittee of the Australian and New Zealand Society of Blood Transfusion Inc. Guidelines for Pretransfusion Laboratory
Practice. 5th Ed. 2007.
12. Hoppe PA. The role of the Bureau of Biologics in ensuring reagent reliability. In: Considerations in the selection of reagents. Washington:
American Association of Blood Banks 1979; 29-33.
13. United Kingdom National Blood Service. Guidelines for the Blood Transfusion Services in the United Kingdom. 7th Ed. 2005.
03070000H
03070000H
Gosia Kolasinski
H3
Actioned Approval 31/07/07
June, 2007