City University of Hong Kong
香港城市大學
Department of Biomedical Sciences
生物醫學科學系
Bachelor of Science in Biomedical Sciences
生物醫學科學理學士
BMS2008 Hematology
BMS 2008血液學
Practical 1
實用1
Objectives:
目標：
To prepare blood films which are even, smooth and have an acceptable feathered edge.
製備均勻、光滑且具有可接受的羽狀邊緣的血膜。
To stain and determine the acceptability of the Wright’s stained and Giemsa stained blood smear by evaluating all cellular components under these two stains.
透過評估兩種染色下的所有細胞成分，對血液塗片進行染色並確定瑞特染色和吉姆薩染色的可接受性。
Content:
內容：
Making blood film (thin)
製作血膜（薄）
Romanowsky staining – Wright’s and Giemsa
羅曼諾夫斯基染色－賴特染色和吉姆薩染色
Recognition of cellular components in blood film
血液抹片中細胞成分的識別
Procedure:
程式:
Making Blood Film (thin)
製作血膜（薄）
A1. Blood Film Preparation
A 1.血膜製備
A small drop of blood is placed near the end of a clean glass slide by autopipette or wooden applicator (recommended). A second slide is used as a spreader. The blood is streaked in a thin film over the slide. The slide is allowed to air-dry before staining. EDTA anticoagulated blood is preferred. Blood film can also be made from finger prick blood applied directly onto a slide.
用自動移液器或木製塗藥器（建議）將一小滴血滴在乾淨的玻片末端附近。第二張投影片用作擴展器。血液在玻片上形成薄膜狀。染色前將玻片風乾。優選 EDTA 抗凝血血液。也可以將手指刺穿的血液直接塗在載玻片上製成血膜。
Use methanol / gauze to clean the glass slide surface, if necessary. Dry it before use.
如有必要，請使用甲醇/紗布清潔玻片表面。使用前將其乾燥。
Place the slide on a flat surface, and hold the narrow side of the nonfrosted edge between your left thumb and forefinger.
將載玻片放在平坦的表面上，並用左手拇指和食指握住非磨砂邊緣的窄邊。
With your right hand, place the smooth clean edge of a second (spreader) slide on the specimen slide, just in front of the blood drop.
用右手將第二張（展開器）載玻片光滑乾淨的邊緣放在標本載玻片上，就在血滴的前方。
Hold the spreader slide at a 30° -45° angle, and draw it back against the drop of blood.
以30° -45 °角握住擴展器滑塊，然後將其拉回以抵住血滴。
Allow the blood to spread almost to the edges of the slide.
讓血液幾乎擴散到載玻片的邊緣。
Push the spread forward with one light, smooth, and fluid motion. A thin film of blood with a feathered edge will remain on the slide.
以一種輕柔、平穩、流暢的動作將攤舖物向前推。載玻片上會殘留一層帶有羽狀邊緣的薄膜血跡。
Allow the blood film to air-dry completely before staining (Do not blow to dry)
染色前讓血膜完全風乾（不要吹乾）.
Points to Note
注意事項
Clean slide with methanol to remove dirt and grease if necessary.
如有必要，請用甲醇清潔載玻片以去除污垢和油脂。
Spreader slide should not be pushed across the slide in a jerky manner.
不應以急促的方式將吊具滑軌推過滑軌。
The smaller the angle or faster the action makes the film thinner and longer.
角度越小或動作越快，薄膜就越薄、越長。
The angle of the spreader slide should be increased when the hemoglobin level of blood sample is low.
當血液樣本的血紅素水平較低時，應增加擴展器滑塊的角度。
Romanowsky Staining - Wright’s and Giemsa stain
羅曼諾夫斯基染色 - 賴特染色和吉姆薩染色
B1. Wright’s Stain (pH 6.8)
B1。賴特染色劑(pH 6.8)
It is commonly used in staining of blood film. The Wright’s stain is a Romanowsky stain which is a polychromatic stain consisting of acidic dye (anionic) eosin and basic dye (cationic) methylene blue or any of its oxidation products. The acidic dye stains the pink coloration in hemoglobin (RBC) and eosinophil granules while the basic dye stains purple coloration of leukocyte nuclei and neutrophilic granules. Methanol is used as both a solvent and fixative in this procedure.
常用於血膜染色。賴特染色劑是一種羅曼諾夫斯基染色劑，它是一種多色染色劑，由酸性染料（陰離子）曙紅和鹼性染料（陽離子）亞甲藍或其任何氧化產物組成。酸性染料將血紅素 (RBC) 和嗜酸性粒細胞顆粒染成粉紅色，而鹼性染料則將白血球核和嗜中性球顆粒染成紫色。在此過程中，甲醇既用作溶劑又用作固定劑。
Place the slide in the Coplin jar with methanol. Dip for 5 times and each 1 second or leave it in the jar for 1 minute
將玻片放入裝有甲醇的科普林罐中。浸泡 5 次，每次 1 秒或將其放在罐子中 1 分鐘.
Place the slide in the jar with Wright’s stain. Stain for 4 minutes.
將玻片放入帶有賴特染色劑的罐子中。染色 4 分鐘。
Transfer the slide in the jar with buffered Wright solution (pH6.8) for 4-6 minutes.
將玻片轉移到裝有緩衝賴特溶液(pH6.8)的罐子中4-6 分鐘。
Raise the slide out of the stain and allow the majority of the stain to run off the slide.
將載玻片從污漬中取出，讓大部分污漬從載玻片上流走。
Place the slide in the first jar containing deionized water. Allow to stand 10 seconds.
將載玻片放入第一個裝有去離子水的罐子中。靜置 10 秒鐘。
Remove the slide carefully and dip 5 times in the second jar containing deionized water (DI) to rinse off the excess stain.
小心取出載玻片，並在第二個裝有去離子水 (DI) 的罐子中浸入 5 次，以沖洗掉多餘的污漬。
NOTE: It may be necessary to change the DI water if many slides are being stained.
注意：如果許多玻片被染色，可能需要更換去離子水。
Wipe off excess fluid from the back of the slide. Place the slide upright on a slide drying rack allow to air dry.
擦掉載玻片背面多餘的液體。將載玻片直立放在載玻片乾燥架上風乾。
When completely dry, spread “immersion oil” over the slide and examine the smear under the microscope
完全乾燥後，將「浸油」塗在玻片上並在顯微鏡下檢查塗片.
B2. Giemsa Stain (pH 7.2)
B2.吉姆薩染色劑 (pH 7.2)
Giemsa stain is a differential stain and contains a mixture of azure, methylene blue, and eosin dye. It is specific for the phosphate groups of DNA and attaches itself to where there are high amounts of adenine-thymine bonding. Azure and eosin are acidic dye that variably stains the basic components of the cells like the cytoplasm, granules, etc. Methylene blue acts as the basic dye, which stains the acidic components, especially the nucleus of the cell. Methanol act as a fixative as well as a cellular stain. The fixative does not allow a further change in the cells and makes them adhere to the glass slide.
吉姆薩染色劑是一種鑑別染色劑，含有天藍色、亞甲基藍和伊紅染料的混合物。它對 DNA 的磷酸基具有特異性，並附著在存在大量腺嘌呤-胸腺嘧啶鍵合的位置。天青和伊紅是酸性染料，可對細胞的基本成分（如細胞質、顆粒等）進行不同的染色。甲醇充當固定劑和細胞染色劑。固定劑不允許細胞進一步變化，並使它們黏附在玻片上。
1. Fix the smear in absolute methanol for 5 minutes
1. 將塗片置於無水甲醇中固定 5 分鐘
2. Air dry
2. 風乾
3. Stain in jar of 10% Giemsa solution for 15 minutes
3. 在裝有 10% Giemsa 溶液的罐子中染色15分鐘
4. Remove the excess stain GENTLY by distilled water.
4. 用蒸餾水輕輕去除多餘的污漬。
5. Air dry and ready for examination under microscope when completely dry.
5. 風乾，完全乾燥後即可在顯微鏡下檢查。
6. Spread the immersion oil over the slide and examine the smear under the microscope.
6. 將浸油塗在載玻片上並在顯微鏡下檢查塗片。
Recognition of cellular components in blood film
血液抹片中細胞成分的識別
C1. Low power (10x) scan
C 1.低功率 (10x) 掃描
Determine the overall staining quality of the blood smear by evaluating cells.
透過評估細胞來確定血液塗片的整體染色品質。
Cell Type | Appropriate Appearance on Well-Stained Slide |
RBCs | reddish pink. |
Lymphocytes | dark purple nuclei with varying shades of blue cytoplasm |
Neutrophils | dark purple nuclei with reddish, granular cytoplasm. |
Monocyte | lighter purple nucleus with a gray-blue cytoplasm. |
Eosinophils | bright red/orange granules |
Basophils | dark purple nuclei and granules. |
Stain should not be too dark or too pale.
污漬不應太深或太淺。
There should be no stain precipitate present on smear.
塗片上不應有染色沉澱。
Determine if there is proper distribution of the cells on the smear.
確定塗片上細胞的分佈是否正確。
Scan the edges and center of the slide to ensure there are no clumps of RBCs, WBCs or platelets. Note clumps of similar cell types in the feather because they may be representative of an abnormal population.
掃描玻片的邊緣和中心，確保沒有紅血球、白血球或血小板團塊。注意羽毛中相似細胞類型的團塊，因為它們可能代表異常群體。
WBCs pulled to the feather end of the blood slide should not be too much as it indicates the slide preparation is not good. Reject slides and repeat the slide preparation for an acceptable slide for review
拉到血玻片羽毛端的白血球不應該太多，因為這表示玻片準備不好。拒絕載玻片並重複載玻片準備以獲得可接受的載玻片以供審查.
C2. Examine the slide with all cell type under microscope (X100)
C2。在顯微鏡下檢查包含所有細胞類型的玻片 (X 100 )
Draw the cell type seen under microscope
畫出顯微鏡下看到的細胞類型
Describe the cell type morphology and appearance using two stains
使用兩種染色描述細胞類型形態和外觀
List out the advantage and disadvantage using Wright’s stain and Giemsa stain for blood cell morphology examination
列出使用瑞氏染色和吉姆薩染色進行血球形態檢查的優缺點
Reference list:
參考清單：
Drews, R. C. & Edelmann, C. M. Jr. (1956). The disinfectant action of concentrated acetone. Am. J. Ophthalmol., 42(5):726-730.
RC 德魯斯和小 CM 埃德爾曼 (1956)。濃丙酮的消毒作用。是。眼科雜誌，42(5)：726-730。
Lewis, S. M., Bain, B. J. & Bates, I. (2017). Dacie and Lewis Practical Haematology. 12th ed. Churchchill Livingstone.
劉易斯， S 。米。 ，貝恩， B . J. ＆貝茨， I.（20 17 ） 。達西和劉易斯實用血液學。 1第2版。丘吉爾·利文斯頓。
Video Reference:
影片參考：
How to make a Blood Film in 3 steps
如何三步驟製作血片
Wright and Giemsa Stain
賴特和吉姆薩染色
https://www.youtube.com/watch?v=9xBcm-1NMqk&pp=ygUXd3JpZ2h0IGFuZCBnaWVtc2Egc3RhaW4%3D
Basic Microscope Setup and Use
顯微鏡的基本設定和使用
https://www.youtube.com/watch?v=RJe577AQqvA&pp=ygUeQmFzaWMgTWljcm9zY29wZSBTZXR1cCBhbmQgVXNl
Contents of Blood
血液成分
https://www.youtube.com/watch?v=VSVYgivfs9c&pp=ygURQ29udGVudHMgb2YgQmxvb2Q%3D
What blood looks like down the microscope
顯微鏡下的血液是什麼樣子
--End of Practical 1--
--實踐 1 結束--