Saline Replacement
1.0Principle
To differentiate rouleaux from true agglutination.
Rouleaux is a phenomenon that mimics agglutination. Red cells manifesting rouleaux appear microscopically like aggregates of stacked coins. It is caused by abnormalities in the patient’s plasma, not in the red cells. When the plasma is replaced with normal saline the rouleaux is dispersed but true agglutination remains.9.1
Rouleaux is seen in diseases associated with abnormal serum protein concentrations, such as multiple myeloma, macroglobulinemia, cryoglobulinemia, Boeck’s sarcoid, cirrhosis and hyperfibrinogenemia in infections.9.1
2.0Scope and Related Policies
2.1Saline replacement is usually done when all red cells tested at room temperature or 37°C show positive reactions with the patient’s plasma.
2.2Saline replacement is not done when all red cells tested by antiglobulin testing show positive reactions.
3.0Specimens
EDTA anticoagulated whole blood
4.0Materials
Equipment:Serological centrifuge
Block for test tubes
Microscope
Vacuum aspiration (optional)
Supplies:Test tubes – 10 x 75mm, 12 x 75 mm
Serological pipettes
Reagents:Normal saline
5.0Quality Control – N/A
6.0Procedure
6.1Read the tubes microscopically and check the agglutination for the characteristic “stacked coins” appearance.
6.2Centrifuge the tubes at 3400 rpm for 10 – 15 seconds.
6.3Using a pipette or vacuum aspiration equipment, remove all the supernatant leaving the cell button undisturbed.
6.4Add 2 drops of normal saline to each tube.
6.5Centrifuge the tubes at 3400 rpm for 10 – 15 seconds.
6.6Resuspend gently, read by initial reading method (i.e., macroscopically or microscopically).
6.7Grade and record results. See PA.006 – Reading and Recording Hemagglutination Reactions. Indicate that the reading is after saline replacement by writing “SR” above the column where the results are being recorded. If entering results in a computer footnote, “reading performed after saline replacement”.
6.8Interpret the results. See 7.0 – Reporting.
6.9When the procedure is complete, perform a clerical check.
6.9.1If the specimen has been labelled with a secondary label (e.g., addressograph, pre-printed, etc.) compare the information on the secondary label with the information on the specimen tube label. If the information differs, have specimen recollected.
6.9.2Ensure that the specimen label information for each specimen tested coincides with the information on the corresponding test tubes.
6.9.3Ensure that the specimen label information for each specimen tested coincides with the information on the corresponding request form.
6.10Sign or initial and record the completion time and date on the request form or complete computer record.
6.11Verification of results must be recorded.
7.0Reporting
7.1No agglutination after the saline replacement procedure usually indicates that rouleaux was present and has been dispersed by the normal saline. Report the reaction as negative.
7.2Agglutination after the saline replacement procedure usually indicates true agglutination. Interpret the reaction as positive and continue with appropriate follow up (e.g., NRT.003 – ABO Group Problem Solving or antibody investigation if indicated).
8.0Procedural Notes – N/A
9.0References
9.1Roback JD, ed. American Association of Blood Banks Technical Manual, 16th ed. Bethesda, MD: American Association of Blood Banks, 2008: 490-491, 903.
9.2Issitt PD, Anstee DJ. Applied Blood Group Serology, 4th ed. Durham, NC: Montgomery Scientific Publications, 1998: 1135.
/ Ontario Regional Blood Coordinating NetworkStandard Work Instruction Manual / NRT.002
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