SEROLIGICAL TESTING METHODOLOGIES: TUBE, GEL & SOLID PHASE

SEROLOGICAL TESTING METHODOLOGIES: TUBE, GEL & SOLID PHASE

Shan Yuan, MD (Last Updated 3/8/2011) Tube Testing The old fashioned way! Traditional format, still widely used Mix the RBC and the plasma + enhancement solution (+AHG), incubate to allow antibody-RBC binding, shake, spin and read. (See diagram below) Positive: Agglutination, or hemolysis (uncommon) Negative: No agglutinatiion or hemolysis

If hemolysis seen, this is typically seen with anti-Vel, anti-Tja, anti-Lewis antibodies. Also anti-ABO antibodies cause in vitro hemolysis? but reagent cells used in antibody identification and screening panels are all type O RBCs. Hemolysis can occasionally be seen with anti-I, IH, -Kidd antibodies.

Gel Microcolumn Testing (Gel) Utilizes a gel (e.g. dextra acrylamide )column with pores that will allow passge of free RBCs but not small enough to trap agglutinated RBCs. Column is impregnanted with antiglobulin reagent to trap antibody bound RBCs Mix patient serum/plasma sample with reagent RBCs+LISS, place at the top of the well of the gel column, where they are allowed to incubate. Then centrifuge: o Negative: free RBC pass through and go the bottom producing a "botton at the bottom" o Positive agglutinated cells are trapped in the gel, larger agglutinates (strong reaction) will stay at the top of gel as a line near where the sample was originally deposited. o May detect hemolysis by observing fluid in the well o Mixed field appearance: two layers of rBCS, the slower moving agglutinated cells and other unagglutinated cells. Suggestive of IgM antibodies. o Grading of test result: o

From Left to Right: 4+, 3+,2+,1+, 0 and 0/microscopically + reactions. Advantages of the gel format:

o Only small sample/reagent volumes needed (microliters) o Less labor intensive, can be semiautomated and performed in batches. o Less subjective reading o Reproducible, stable read-out, facilitates QC or review by another

technologist

Solid-Phase Red Cell Adherence Assay (SPRCA, or "solid phase") Solid phase testing system uses microtiter plates, with wells coated with stroma from reagent RBCs of known phenotype Wells are usually coated by manufacturer to make standard reagent RBC panels for testing, but also possible to perform the coating in-house Patient plasma and LISS added to the RBC well. The incubate,

 If antibody present, will bind to the target antigen present on the RBCs coating the well

Plate washed to remove unbound antibodies, and indicator RBCs coated with antiIgG are added,

Spin and read: o Positive: Uniform layer of indicator RBCs at the bottom of the well o Negative: Indicator RBCs from atight bottom at center. o Similar Methodology used for detecting antibody directed against platelet antigens or HLA antigens, and for platelet crossmatching.

Advantages of solid phase testing o Sensitive o Compatible with automation o Less subjective result interpretation, o Stable results, allows review by 2nd person at a later time. Results also easily captures by images taken by the instrument o Small sample and reagent volumes required.

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