METHOD SUMMARY SPECIMEN COLLECTION AND PREPARATION
FOR IMMUNOHAEMATOLOGY IN VITRO DIAGNOSTIC TESTING
Application Tube MTP* BioVueTM (CAT)** ID-MTSTM (CAT)**
Antibody Screen Yes Yes Yes No
Antibody Identification Yes Yes Yes No
Crossmatching Yes Yes Yes No
Rapid Antibody Medium
*MTP Methods
Due to the wide variation in microplate methods and equipment, users should validate methods in
LISS Additive
routine use.
RAM
**CAT Methods
Refer to the test method recommended by the manufacturer of Column Agglutination Technology (CAT)
Systems.
PRODUCT DESCRIPTION
CSL RAM (Rapid Antibody Medium) is a phosphate buffered low ionic glycine solution. The product is
manufactured to ensure optimum pH, osmolarity and conductivity. It is serologically tested with selected
blood group antibodies to ensure the correct specificity and sensitivity in antibody screening is achieved.
CSL RAM is designed for Tube methods, is suitable for Column Agglutination Technology (CAT) Systems
and is validated for direct addition into Ortho-Clinical Diagnostics BioVueTM (BioVueTM). As the activity
of CSL RAM depends on correct pH, conductance and molarity, care should be taken not to introduce
saline or buffer solutions into test systems. CSL RAM is provided ready to use without any further
modifications or dilutions. CSL RAM will retain optimum sensitivity until the expiry date, providing there
is no contamination with foreign solutions, fungi or bacteria.
STORAGE CONDITIONS
Store at 2° to 8°C (Refrigerate. Do Not Freeze).
PRINCIPLE OF THE PRODUCT
The low ionic environment created by CSL RAM is designed to increase the rate of antibody uptake
during test incubation, thus enhancing reactivity. This enables a reduction in the incubation time from
30 to 10 minutes and also achieves a significant increase in test sensitivity. CSL RAM is formulated to
create these conditions by the additive method. CSL RAM, when used by the recommended method
(two drops serum or plasma + one drop red cells + two drops CSL RAM), produces the optimal ionic
conditions. No washing of the screening cells or complicated cell suspension procedures are required
prior to use. Users find the LISS additive method to be rapid, sensitive and have a very low incidence of
non-specific reactions.
For in vitro
Diagnostic Use BACKGROUND
Only Low and Messeter found that when red cells were suspended in a low ionic strength solution, the
incubation time of the cells and antibody-containing serum could be significantly reduced when
READ LEAFLET performing the Indirect Antiglobulin Test (IAT), without a loss of sensitivity.
CAREFULLY
Although low ionic strength solutions have been used for many years by automated techniques, reports
of non-specific agglutination have led to a reluctance to use low ionic strength media in manual tests.
Low and Messeter showed that non-specific results were not obtained, providing the ionic strength was
not below 0.03M and the solution was buffered to pH 6.7.
Moore and Mollison confirmed these earlier findings and found that C3 and C4 reactions were also
enhanced in addition to those of IgG. They also reported that the Indirect Antiglobulin Test using a low
ionic strength solution was more sensitive in detecting low affinity antibodies. Many scientists have
added to the evidence of the advantages of using low ionic strength solution in the indirect antiglobulin
phase of compatibility testing.
Low ionic solutions may be formulated for use as a suspending medium or as an additive solution.
When used in accordance with recommended methods, both types will provide an incubation mixture
concentration of 0.06 - 0.09M. The LISS suspension method requires washing of test red cells and
resuspension to the correct cell concentration. LISS additive methods have become more commonly used
due to the lack of a cell resuspension requirement and convenience.
CSL Limited
45 Poplar Road
Parkville Victoria
3052 Australia
ABN 99 051 588 348
Phone +61 3 9389 1911
Fax +61 3 9389 1646
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 may also be stored in CSL CelpresolTM at 2° to 8°C for up to 42 days.
For compatibility testing and antibody screening, serum from freshly clotted blood should be used. The
use of stored serum or plasma from anticoagulated samples may result in failure to detect complement-
dependent antibodies.
RECOMMENDED METHODS
Tube Method – For Antibody Screen, Antibody Identification or Crossmatching
Low Ionic Strength Additive Method
A commonly used CSL RAM low ionic strength additive method is:
1. Prepare a 3-5% suspension of test red cells in buffered or unbuffered isotonic saline, or in CSL
Gosia Kolasinski Actioned Approval 22/06/07
CelpresolTM.
2. Add 2 drops of test serum to an appropriately labelled, clean glass test tube (10x75mm or
12x75mm).
3. Add 1 drop of the suspension of test red cells or Reagent Red Blood Cells. Gently, mix well.
4. Add 2 drops of CSL RAM (Rapid Antibody Medium) (see note 2 and 3). Gently, mix well.
5. Incubate at 37°C for 10 minutes.
6. Centrifuge at low speed (500rcf) for 15 to 20 seconds*.
7. Gently agitate the tube to dislodge the red cells and examine macroscopically for agglutination.
Record results.
8. Wash the cells with 4 changes of isotonic saline, ensuring that the saline is decanted completely
31/05/07 2.10pm
after each wash and that the cells are completely resuspended between washes.
9. 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**.
01090000K
10. Mix well and centrifuge at low speed (500rcf) for 15 to 20 seconds*.
11. Gently agitate the tube to dislodge the red cells and examine macroscopically for agglutination.
Record results.
12. Add 1 drop of CSL AHG Control Cells 3% to all negative test tubes to validate the results.
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13. Repeat steps 10 and 11.
Notes: *Or centrifuge at a speed and time appropriate for the centrifuge in use.
**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.
1. It is preferable to use glass test tubes rather than plastic for two reasons:
(a) To ensure standard drop volumes. Moore and Mollison point out that plastic tubes attract
drops by electrostatic force and drop volume can vary by greater than ±40% of the mean
value.
(b) To ensure rapid warming of reagents to 37°C. Plastic tubes are less efficient than glass at
heat transfer. To obtain optimum results, it is essential that the incubation mixture remains
at 37°C for at least 7 minutes.
2. A four drop technique is sometimes used in an attempt to improve the sensitivity of a 30
minute normal ionic strength test. Unlike most other blood group serology tests, the addition of
extra serum to a LISS additive technique may actually decrease sensitivity by raising the ionic
strength of the incubation mixture. It is therefore most important to use the correct ratio of
serum, cells and CSL RAM.
3. The cells should not be suspended directly in CSL RAM, as this may result in non-specific
aggregation due to the very low ionic strength of the product. Therefore, the order of addition
of sample and product is important.
4. When using this method for crossmatching, the test may be read following Step 5 to detect
ABO incompatibility.
Microplate Method
Note: CSL RAM is validated for use in microplates. Due to the variation in methods and equipment, microplate users should
validate this product using their method.
BioVueTM (CAT) Method
CSL RAM is suitable and validated for the BioVueTM Column Agglutination Technology (CAT) System and may be used directly from
the vial without further modification. The manufacturer’s instructions for CAT system processing should be followed.
1. Prepare a 3-5% suspension of test red cells in buffered or unbuffered isotonic saline, or in CSL CelpresolTM.
2. Appropriately label either a BioVueTM AHG Polyspecific or BioVueTM AHG Anti-IgG cassette.
3. Add 40µL of test serum or plasma to the appropriate reaction chamber.
4. Add 10µL of the suspension of test red cells or Reagent Red Blood Cells to the appropriate reaction chamber.
5. Add 40µL of CSL RAM to the appropriate test reaction chambers.
6. Observe that the contents of the reaction chambers are combined. If necessary, tap gently. The contents must stay within the
reaction chamber and not fall through to the column to obtain valid results.
7. Incubate in the BioVueTM System Incubator at 37 +/- 1°C for 15 minutes.
8. Centrifuge the cassette in the BioVueTM System Centrifuge at the automatic preset speed setting of the centrifuge.
9. Read both front and back sides of the individual columns for agglutination and/or haemolysis under illumination. Record
results.
INTERPRETATION OF RESULTS
7. Incorrect concentrations of red cells.
8. Incorrect reading of results (ie. failure to detect haemolysis, etc).
PRECAUTIONS
1. For in vitro diagnostic use only.
2. The material from which this product was derived is from non-human sources, there is no risk of HIV or HBsAg
infection. However, good laboratory practice requires safe handling procedures are used.
3. Thiomersal 0.01% w/v has been added to retard fungal and bacterial contamination. Users should take appropriate
precautions when handling and discarding this product.
4. Bacterial or fungal contamination may occur as a result of incorrect storage of opened containers. Take care to avoid
contamination. The product should not be used if a precipitate or particles are present.
5. As the activity of CSL RAM depends on correct pH, conductance and molarity, care should be taken not to introduce
saline or buffer solutions.
REFERENCES
1. Vengelen-Tyler V, Choy C. A comparative study of antibody enhancement techniques. [Abstract] Transfusion 1986; 26:
570.
2. Low B, Messeter L. Antiglobulin Test in Low-Ionic Strength Salt Solution for Rapid Antibody Screening and Cross-
matching. Vox Sang 1974; 26: 53-61.
3. Moore HC, Mollison PL. Use of a Low-Ionic Strength Medium in Manual Tests for Antibody Detection. Transfusion

Gosia Kolasinski Actioned Approval 22/06/07

A positive reaction is indicated by agglutination of the test cells in the presence of CSL EpicloneTM AHG Poly or CSL AHG 1976; 16: 291-6.
Anti-IgG. A positive reaction in the Indirect Antiglobulin Test (IAT) indicates the presence of either human immunoglobulin 4. Austin R. An Evaluation of a Rapid Coombs Technique. NZ J Med Lab Tech 1976; 30: 51-4.
(IgG) or human complement (C3d) on the red cells following incubation with serum. In the case of testing unknown serum, 5. Wicker B, Wallas CH. A Comparison of a Low Ionic Strength Saline Medium with Routine Methods for Antibody
this means that an antibody directed at an antigen on the test cells is present in the serum. When testing unknown cells Detection. Transfusion 1976; 16: 469-72.
against a phenotyping reagent that requires the use of an IAT technique, the presence of the appropriate antigen on those 6. Elliot M, Bossom W, Dupuy ME, Masouredis SP. Effect of Ionic Strength on the Serologic Behaviour of Red Cell
cells is indicated, provided a Direct Antiglobulin Test (DAT), on the same cells or an auto control test performed in parallel Isoantibodies. Vox Sang 1964; 9: 396-414.
with the test, gives a negative reaction. 7. Rosenfield RE, Shaikh SH, Innella F, Kaczera Z, Kochwa S. Augmentation of Haemagglutination by Low Ionic
Conditions. Transfusion 1979; 19: 499-510.
Some laboratory scientists prefer to interpret antiglobulin reactions microscopically. Whilst this practice may lend 8. Rock G, Baxter A, Charron N, Jhaveri J. LISS - an Effective Way to Increase Blood Utilization. Transfusion 1978; 18:
additional sensitivity to the test, it can also be a potential source of misleading positive reactions, the predominant cause 228-32.

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.
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9. Harmening DM. Modern Blood Banking and Transfusion Practices. 5th Ed. FA Davis Company. Philadelphia 2005.
10. Scientific Subcommittee of the Australian and New Zealand Society of Blood Transfusion Inc. Guidelines for
Pretransfusion Laboratory Practice. 5th Ed. 2007.
11. Daniels G. Human Blood Groups. 2nd Ed. Blackwell Science. Carlton, Victoria 2002.

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12. Green R, et al. Basic Blood Grouping Techniques and Procedures. 2nd Ed. Victorian Immunohaematology Discussion
CONTROLS Group 1992.
The antiglobulin test is an exceedingly delicate procedure and the presence of even minute amounts of free human protein 13. Brecher ME. American Association of Blood Banks Technical Manual. 15th Ed. Bethesda, Maryland 2005.
can result in neutralisation of the Anti-Human Globulin (AHG) reagent. The washing of cells should be carried out with 14. United Kingdom National Blood Service. Guidelines for the Blood Transfusion Services in the United Kingdom, 7th Ed.

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great care and the quality and cleanliness of saline and glassware should be maintained. The inclusion of both positive 2005.
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 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.

LIMITATIONS OF PROCEDURE
1. It is imperative that the correct ratio of serum, cells and CSL RAM is used in the test and that reagents are added to
the tube in the specified order. Direct contact of red cells and CSL RAM may cause non-specific aggregation.
2. Red cells may be damaged by prolonged exposure to a low ionic environment.
3. Variations in time and temperature of incubation, time and speed of centrifugation and reaction reading technique
may cause imprecision in results.
4. Some samples of cold reacting antibodies may not be optimally active by the recommended test method.
5. The cell button obtained following centrifugation in Step 6 may vary in appearance to that usually seen in Saline or
Albumin tests. Interpretation should be made only after gentle, but complete, resuspension of the cell button.
6. As the procedure depends on the ionicity of the incubation mixture, false results may occur when testing eluates.
7. High concentrations of protein in patient samples may cause autoagglutination or rouleaux.
8. IgM antibodies may not be detected by the CSL RAM method.
9. CSL RAM is not a resuspension medium unlike LISS. It should be used only as an additive following the methods
detailed above.

Discrepant 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. 01090000K May, 2007