网织红细胞是晚幼红细胞到完全成熟的红细胞之间的过度型细胞,由于其细胞浆中尚存在嗜碱性的RNA物质,用煌焦油蓝染色液行活体染色后,胞浆中镜检可见有浅蓝或深蓝色的网状结构。新亚甲蓝染色液由新亚甲蓝、磷酸盐、去离子水等配制而成,用于网织红细胞活体染色和计数,利用本染色液所染出的结果背景颜色明亮清晰,而且不受长时间过度染色的影响。染色后网织红细胞呈浅蓝色或深蓝色网状结构。该试剂仅用于科研领域,不适用于临床诊断或其他用途。
自备材料:
1、新鲜全血或EDTA抗凝全血
2、小试管或离心管、载玻片、显微镜 (油镜)
操作步骤 (仅供参考):
1、取小试管或离心管,加入新亚甲蓝染色液2~3滴。
2、再向其中加入新鲜全血或EDTA抗凝全血2~3滴,混匀。
3、室温下放置15~20分钟或更久。
4、取一滴混合液制成血涂片。
5、油镜下至少计数1000个红细胞中网织红细胞数。
染色结果:
染色后网织红细胞胞浆中含有浅蓝或深蓝色的网状结构。
计算:
网织红细胞百分数=计数1000个红细胞中的网织红细胞数/1000
网织红细胞绝对数(个/L)=网织红细胞百分数×红细胞数/L
注意事项:
1、该染色法仅限试管法操作。
2、染色时间要充足,混合后不易立即涂片,当室温较低时染色时间应相应延长或置于37℃恒温箱。
3、血液涂片应厚薄均匀,不使红细胞重叠,以免影响染色效果。
4、血细胞涂片染色要求新鲜全血或EDTA抗凝血。
5、试剂开封后请尽快使用,以防影响后续实验效果。
6、为了您的安全和健康,请穿实验服并戴一次性手套操作。
Reticulocytes are transitional cells between late normoblasts and fully mature erythrocytes. Since basophilic RNA substances still exist in their cytoplasm, vital staining with brilliant cresyl blue staining solution will reveal a light blue or dark blue reticular structure in the cytoplasm under microscopic examination.
New Methylene Blue Staining Solution is prepared from New Methylene Blue, phosphate, deionized water and other components, and is used for the vital staining and counting of reticulocytes. The results stained with this solution feature a bright and clear background, and are not affected by prolonged over-staining. After staining, reticulocytes exhibit a light blue or dark blue reticular structure. This reagent is for research use only and not intended for clinical diagnosis or any other applications.
Materials to Be Prepared by the User:
1. Fresh whole blood or EDTA-anticoagulated whole blood
2. Small test tubes or centrifuge tubes, glass slides, microscope (oil immersion lens)
Operating Procedures (For Reference Only):
1. Take a small test tube or centrifuge tube and add 2–3 drops of New Methylene Blue Staining Solution.
2. Add another 2–3 drops of fresh whole blood or EDTA-anticoagulated whole blood to it and mix well.
3. Incubate at room temperature for 15–20 minutes or longer.
4. Take one drop of the mixture to prepare a blood smear.
5. Count the number of reticulocytes among at least 1000 erythrocytes under an oil immersion lens.
Staining Results:
After staining, the cytoplasm of reticulocytes contains a light blue or dark blue reticular structure.
Calculation:
Reticulocyte percentage = Number of reticulocytes counted among 1000 erythrocytes / 1000
Absolute reticulocyte count (cells/L) = Reticulocyte percentage × Erythrocyte count (cells/L)
Precautions:
1. This staining method is only applicable for the test tube procedure.
2. Ensure sufficient staining time; do not prepare blood smears immediately after mixing. If the room temperature is low, appropriately extend the staining time or incubate the mixture in a 37℃ constant temperature incubator.
3. Prepare blood smears with a uniform thickness and avoid erythrocyte overlapping to prevent interference with staining effects.
4. Fresh whole blood or EDTA-anticoagulated blood is required for blood cell smear staining.
5. Use the reagent as soon as possible after opening to avoid compromising subsequent experimental outcomes.
6. For your safety and health, wear a lab coat and disposable gloves during operation.