The following is a comprehensive description of the key factors affecting the accuracy of milk somatic cell analyzers, covering multiple dimensions from sample processing to instrument performance:
1、 Sample collection and pretreatment process
Representativeness of sampling
It is necessary to strictly follow the principle of stratified sampling, especially for large milk storage tanks or transport vehicles. Samples should be taken and mixed separately in the upper, middle, and lower layers to avoid uneven distribution of somatic cells caused by fat layer separation. Insufficient single sampling volume (<50mL) will amplify random errors. It is recommended to take at least 100mL of composite sample according to ISO standards.
Storage and transportation conditions
The sample needs to be refrigerated at 4 ℃ and tested within 6 hours. Overdue storage can cause autolysis and rupture of somatic cells, releasing DNA fragments that interfere with counting. During long-distance transportation, harmless preservatives (such as sodium azide) should be added and severe shaking should be avoided.
Homogenization treatment
Insufficient homogenization of milk samples can lead to the accumulation of fat globules, which can obstruct the microscopic field of view or block the flow cell. Standardized ultrasonic treatment (20kHz, 30 seconds) is required to disperse particles, while controlling the temperature below 40 ℃ to prevent protein denaturation.
2、 Core performance indicators of the instrument
Optical system accuracy
Flow cytometry relies on the intensity of laser scattered light to distinguish cell types, and the stability of laser wavelength (± 0.5nm) and the sensitivity of photomultiplier tubes directly determine the signal-to-noise ratio. Perform sheath fluid blank verification and eliminate background noise interference when starting up daily.
Reasonable threshold setting
The dual parameter gating of forward scattering (FSC) and lateral scattering (SSC) should avoid the impurity signal region. For example, limiting the active cell community within a specific scattering angle range to exclude shed epithelial cells and microbial contamination.
Fluid Dynamics Control
Excessive shear force in the flow pool can lead to cell breakage in fragile bodies, and the flow rate should be controlled at 5-10 μ L/s; When the pressure fluctuation of the sheath fluid is greater than 5%, it is necessary to check the degree of aging of the pump tube and the sealing of the gas path.
3、 Reagent and consumables management
Dyeing agent quality
Live staining agents such as Taipan Blue need to be prepared and used immediately, and stored away from light. Invalid staining solution can cause abnormal staining of dead cells, resulting in false positive counts. Each batch of newly prepared reagents requires a standard cell suspension to calibrate the staining efficiency (>95%).
Residual cleaning solution
Residual cleaning solution (such as sodium hypochlorite) on the inner wall of the injection needle can lyse the white blood cell membrane, leading to missed detection. Rinse the pipeline with deionized water three times before and after each test, and replace residual liquid with air.
Cleanliness of counting pool
The protein deposits attached to the inner wall of microcapillaries will adsorb cells, and it is necessary to soak and clean them with enzymatic solution (trypsin+EDTA) every week. Scratching is prohibited to avoid scratching and capturing cells.
4、 Environmental and human factors
Temperature and Humidity Control
A temperature deviation of 25 ℃± 2 ℃ in the laboratory can cause changes in the reaction rate of reagents, and electronic components are prone to short circuits when the humidity exceeds 70% RH. The constant temperature and humidity system needs to be equipped with an independent dehumidification module.
Operator skill differences
The uneven thickness of manually applied glass slides (ideal thickness 10-20 μ m) can lead to local accumulation effects, which can be eliminated by automated smear machines. The training should include an assessment of cell morphology recognition ability.
Verification frequency of quality control products
Daily use of standard milk powder with known concentration (containing 500000/mL of somatic cells) for recovery rate testing, with the measured value required to fall within the range of ± 10% of the labeled value. After monthly maintenance, a calibration curve needs to be re established.