31 Saline Replacement for Rouleaux

Principle

The saline replacement technique differentiates rouleaux from true agglutination. Rouleaux are aggregates of red cells that characteristically adhere to one another on their flat surface, which gives a “stack of coins” appearance when viewed microscopically. This is a “gold standard” method for differentiating true agglutination from rouleaux.

Specimen

Red cells –usually demonstrated in ABO reverse grouping.

Reagents

Isotonic saline

Procedure

1. Examine plasma/cell mixture under microscope. If “stack of coins” or tight, shiny refractile aggregates appear, perform saline replacement.
2. Re-centrifuge the cell/plasma (or serum) mixture.
3. Remove the plasma with a pipette, leaving the cell button undisturbed.
4. Replace the plasma with an equal volume of saline.
5. Suspend the cell button gently and observe for agglutination.

Interpretation

1. Rouleaux will disperse when suspended in saline, whereas true agglutination will remain. RC agglutination that remains after saline replacement is a positive test result and indicates the presence of antibody. Further investigation is needed.

Notes

1. Rouleaux does not usually occur in the antiglobulin phase as it is a phenomenon of serum and cell washing is used as part of the AHG method.
2. Rouleaux may be present for 24-36 hours after the administration of dextran, hydroxyl ethyl starch, fibrin, and polyvinyl pyrrolidine.
3. Rouleaux may be present in diseases which affect the albumin/globulin ratio, i.e. multiple myeloma, macroglobulinemia, cryoglobulinemia, cirrhosis, and hyperfibrinogenemia due to infection.