PHA lymphocyte transformation test
The PHA lymphocyte transformation test is a morphological and biochemical change in which T lymphocytes and mitogens are co-cultured in vitro, and some small lymphocytes are transformed into immature mother cells and subjected to mitosis. method.Basic Information
Specialist classification: Oncology examination classification: immune examination
Applicable gender: whether men and women apply fasting: not fastingTips: Pay attention to a healthy diet and maintain a good mood. Normal value
Counting lymphocytes 200, identifying lymphoblasts (more than 2 to 4 times more than normal lymphocytes, clear nuclear membrane, loose nuclear plaque, fine nucleus, obvious nucleoli, abundant cytoplasm, basophilic), transitional Lymphocytes (less change in volume, only the above-mentioned obvious nuclear changes), small lymphocytes (slightly larger than red blood cells, dense nuclear chromatin, no nucleoli), the conversion rate was counted. The normal dermatological conversion rate is greater than 58%.Clinical significance
The decrease in PHA lymphocyte transformation rate is a response to a decrease in cellular immune function. This examination, dermatology is mostly used for disease classification (such as leprosy), disease judgment (such as skin malignant tumors), drug efficacy observation, and some studies of immunodeficiency and allergic skin diseases.
Various symptoms caused by decreased immune function.
The person in need of examination is suspected of suffering from symptoms of leprosy or skin malignancy.Precautions
1. The composition of the medium has a great influence on the conversion rate, pay attention to its effective period.
2. Calf serum should be inactivated before use.
3. When culturing, ensure that there is enough gas. Generally, the total amount of liquid in the 10ml culture bottle should not exceed 2ml.
4. Excessive PHA dose is toxic to cells, too small to stimulate lymphocyte transformation. The dose of PHA conversion reaction should be determined before the test.
5. Strict aseptic operation should be carried out in the experiment to prevent pollution.
Taboo before the test: pay attention to a healthy diet and maintain a good mood.
Requirements for inspection: Actively cooperate with the doctor.Inspection process
1. Take aseptic heparin anticoagulant 0.lml, add 1.8ml cell culture medium, add 1000μg/m1 PHA0.1m1, control tube without PHA, culture the cells at 37 °C, 5% CO2 for 3d, shake 1 per day Times.
2. At the end of the culture, most of the supernatant was discarded, and 8.5 g/L NH4Cl 4 ml was added and mixed, and placed in a 37 ° C water bath for 10 min.
Centrifuge at 3.2500r/min for 10min, discard the supernatant, add 5ml of fixative to the pellet, and let it stand at room temperature for 5min.
Centrifuge at 4.2500 r/min for 10 min, discard the supernatant, leave 0.2 ml of pelleted cells for tableting, and blow dry quickly.
5. Giemsa dyeing solution is stained for 10-20 min, washed with water and dried.
6. Oil Mirror Counts the number of cells transformed in 200 lymphocytes and calculates the conversion rate.
The result is judged:
1. Morphological criteria of lymphoblasts: the size of the nucleus, the ratio of nucleus to cytoplasm, the staining of the cytoplasm and the structure of the nucleus and the presence or absence of nucleoli.
(1) Mature small lymphocytes: the size of the uncultured small lymphocytes is 6-8 μm, the nuclear staining is dense, no nucleoli, the ratio of nuclear to cytoplasm is large, and the cytoplasm is stained to mild basophilic.
(2) Transitional lymphocytes: larger than small lymphocytes, about 10 ~ 20μm, nuclear staining is dense, but nucleoli appear, which is the key point for identification of mature small lymphocytes.
(3) lymphoblasts: the cell volume increases, about 20 ~ 30μm, the shape is not neat, often small protrusion, nuclear enlargement, nuclear staining loose, there are 1 or 2 nucleoli, cytoplasm widening, often cytoplasm Empty bubble.
(4) Other cells: For example, after neutrophil culture for 72 hours, most of the decay or death is fragmentation.
2. Calculation of lymphocyte transformation rate The head, body and tail were examined in the above classification, and 200 lymphocytes were counted to calculate the percentage. Transformed lymphocytes include lymphoblasts and transitional lymphocytes, and untransformed lymphocytes refer to mature small lymphocytes. Under normal circumstances, the PHA-induced lymphocyte transformation rate is 60 to 80%, and if it is 50 to 60%, it is low, and when it is 50% or less, it is decreased.
According to the results, the correct judgment is obtained.Not suitable for the crowd
Inappropriate crowd: temporarily unknown.Adverse reactions and risks
There are no related complications and hazards.