Evaluation of Immunoglobulins (IgG and IgM) and Complement (C3) Bounded to Erythrocyte Using the Flow Cytometric Technique on Healthy Beagles
The direct flow cytometric erythrocyte immunofluorescence assay (FC) is a sensitive method which permits multiparameter correlation. Specific characteristics of cell sub-populations may be identified and quantified through this method. The FC is used in several fields of immunology to evaluate surface antigens.
A FC was developed to detect erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in 21 healthy Beagles. The animals were obtained at the nutrition kennel of Universidade Estadual Paulista-Jaboticabal. The dogs ranged from 18-month to 4-year-old. Twelve dogs were female and nine were male. Hemograms, serial biochemical probes, agglutination and direct antiglobulin tests (DAT) were performed on such animals, where all results were considered in a normal standard basis. The FC was performed at Flow Cytometric Laboratory of Medical College, Ribeirão Preto, Universidade de São Paulo. The samples were collected on tubes in a vacuum condition. The red cells were separated, washed three times with PBS buffer and suspended up to 1.5%. Three quotes of suspended red cells were separated and mixed with specific serum for anti IgG, IgM or C3. Samples were analyzed by flow cytometer. The analyses were qualitative (determination of which erythrocyte-bound immunoglobulins) and quantitative (determination of the amount of erythrocyte-bound immunoglobulins) in healthy dogs.
The results were obtained through the percentage of anti-immunoglobulin test (anti-IgG and anti-IgM) and anti-C3 erythrocyte-bound immunoglobulins of these animals. All animals showed any amount of IgG, IgM and C3 on erythrocytes. The average and standard deviation values for IgG, IgM and C3 in the 21 healthy Beagles were 4.89, 2.99, 2.89 and 3.6, 2.26, 2.34, respectively.
The FC showed to be a more sensitive method than DAT, because DAT detects a level above of 200 to 300 molecules of immunoglobulins bounded on erythrocyte membranes, and on these dogs, all DAT tests were negatives. Another important fact is that in healthy dogs it is possible to find immunoglobulins bounded to erythrocytes (low amount), what acquires more strength that the FC result must be quantitative rather than positive or negative.