Rat erythrocyte garland formation assay

B lymphocytes are involved in antibody synthesis and are effector cells of humoral immune responses. The surface of B cells carries membrane surface immunoglobulin (SmIg), Fc receptors, and complement receptors. It binds strongly to the Fc portion of the antigen-antibody complex. Chicken or sheep red blood cells are selected and sensitized into EA complexes with corresponding anti-erythrocyte antibodies. The Fc receptor of B cells can form a garland with EA, which is called EA wreath. If the EA complex is added to complement and binds to the complement receptor of B cells, an EAC rosette is formed. There are murine RBC receptors on the surface of B cells, which can bind to mouse RBCs to form mouse erythrocyte rosettes (M-RFC), which mainly bind to B cells with IgM receptors, and M-RFCs are mostly naive B cells. This test is mainly used to detect the number of B lymphocytes and to determine the level of humoral immunity. Basic Information Specialist classification: examination classification: blood examination Applicable gender: whether men and women apply fasting: fasting Analysis results: Below normal: However, the percentage of ME rosettes was reduced or absent without agammaglobulinemia and severe combined immunodeficiency. Normal value: Percentage of ME in female peripheral blood: 5%-10% Above normal: The percentage of ME wreaths in peripheral blood of patients with chronic lymphocytic leukemia is as high as 60% to 80%. negative: Positive: Tips: Please relax when checking. Normal value Results 200 lymphocytes were counted under the microscope. The lymphocytes that adsorbed 3 or more mouse red blood cells were B lymphocytes, and the percentage of mouse cell garlands (ME wreaths) was calculated. The percentage of ME rosette in normal human peripheral blood is 5% to 10%. Clinical significance The percentage of ME wreaths in peripheral blood of patients with chronic lymphocytic leukemia is as high as 60% to 80%, but there is no gamma globulinemia, severe combined immunodeficiency, etc., and the percentage of ME garlands is reduced or absent. Low results may be diseases: X-linked no-gammaglobulinemia in children, high incidence of secondary immunodeficiency in children may be disease: precautions for chronic lymphocytic leukemia (1) Incubation time: The percentage of ME rose gradually increased with the incubation time. After incubation for 1 h, the ME percentage was 7.4±3.1%, and the incubation was increased to 18.3±3.8% after 18 h. After 72 h incubation, the number of wreaths was increased. It gradually fell to 3.4 ± 7.1%. (2) Culture solution: It is most suitable to use Tc199 culture solution, and the results and repeatability are good. Washing and resuspending lymphocytes and red blood cells with Hepes-buffered RPMl1640 medium resulted in cells tending to aggregate and forming large cells, resulting in unreliable results. (3) When the optimal ratio of red blood cells to lymphocytes is about 35:1, the ratio is >60:1, or the ratio is <25:1, the number of ME wreaths can be reduced. (4) Preservation of MRBC culture solution: In Alsever solution, MRBC stored at 4 °C for 1 week still produces a stable number of garlands, while MRBC stored in PBS, saline or Tc199 culture medium, with the storage time prolonged, the number of wreaths Gradually, after 72 hours of storage, only small or garland-forming cells can be seen. Inspection process (1) Take 1 ml of the above mouse red blood cell suspension, add 3 ml of physiological saline, mix well, 1500r/min, centrifuge for 10min, wash a total of 3 times, and finally prepare 1% mouse red blood cell suspension with physiological saline (about 1.4×108 Cell/ml). (2) 1.5 ml of heparin was taken for anticoagulation, lymphocytes were separated by lymphocyte stratification, and the cell concentration was adjusted to 2 × 10 7 /ml with 20% calf serum Hank solution. (3) Take 0.1 ml of lymphocyte suspension, add an equal amount of 1% mouse red blood cell suspension, mix and place in a 37 ° C water bath for 10 min, 500 r / min, centrifuge for 10 min, put 4 ° C refrigerator for 2 ~ 3 h. (4) Pipette 0.05ml of supernatant, add 1 drop of 2g/L blue solution, gently rotate to mix, take 1 drop of the mixture on the slide, cover the coverslip, after the cells sink, Check with a high power microscope. Not suitable for the crowd There is no inappropriate crowd. Adverse reactions and risks It is a safe check and is harmless to the body.

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