Some 37o C tests (e.g., saline 37o C, LISS 37o C, and enzyme 37o C) done in test tubes and performed using clotted specimens (serum) are read macroscopically for hemolysis prior to agitation of the cell button. Hemolysis is indicative of C9 binding following an antigen-antibody reaction and is considered to be positive.
There is no point in reading tests done on anticoagulated specimens such EDTA for hemolysis. (EDTA chelates Ca++ ions and the C1qrs complex needs free Ca++. Without free Ca++, complement binding stops at C1q.) Similarly, indirect antiglobulin tests (IATs) are not read for hemolysis, since the serum is washed away prior to adding antiglobulin serum.
Tests are also read macroscopically for agglutination once the entire cell button is off the bottom of the tube. This is accomplished by gentle rotation, tilting, and twirling of the tube. Agitation of the cell button must be done gently since antigen-antibody reactions are partly physical and can be dispersed by vigorous shaking.
Microscopic readings are not required for routine antigen typing. Many labs do not read microscopically except in special circumstances, e.g., if mixed-field agglutination is suspected. Instead, tests are read macroscopically using a magnifying mirror with a light source.
Some labs, however, always read antiglobulin tests microscopically, e.g., those done for antibody detection and identification (IAT tests using patient serum) and for DATs done on patient red cells. Negative tests are read microscopically to detect weak antibodies or mixed-field agglutination (MFA) that may not be apparent with the naked eye.
Reading is done on 4 - 6 fields using an inverted microscope. It is important to (1) make sure the cell button is totally resuspended prior to reading, otherwise agglutinates may remain in the button, with only free cells being read; (2) examine at least 4 - 6 fields, otherwise weak reactions or MFA may be missed; (3) read cell suspensions that are neither too heavy nor too light, otherwise weak reactions may go undetected.
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