Module 3 Staining Blood Smears Acknowledgments Ministry of Health Guyana Centers for Disease Control and Prevention CDC Global AIDS Program GAP Guyana Centers for Disease Control and Prevention CDC Atlanta American Society for Clinical Pathology ASCP Learning Objectives After completion of this unit of instruction the student will be able to 1 Discuss the principle and function of the Wright s stain routinely used in the Haematology laboratory 2 Using appropriate technique stain blood smears for microscopic evaluation Learning Objectives Cont d 3 Describe the effect of each of the following situations on the appearance of a stained peripheral blood smear a b c d dirty slide very thin blood film very thick blood film buffer stain or water too acidic staining reaction Objective 3 Continued buffer stain or water too alkaline f decreasing staining time g increasing staining time h excessive rinsing i insufficient rinsing j high WBC count k increased amount of plasma protein can have too much proteins which can affect how blood reacts to stain Physical feature of proteins make anything sticky Too much protein will coat slide The slide has to be fixed blood slide smear does not come in contact with WATER because water and red cells despise each other Water destroys erythrocytes e Staining Peripheral Blood Smears Once the smear is made it is stained with Wright s stain so that a differential count and morphology study can be performed Blood smears should be made within 2 hours of blood collection EDTA is the best anticoagulant because it prevents artifact formation Differential always part of CBC count each cell and ratio Principle Wright s stain composition of several dyes that are characteristic of certain characteristic features is a polychromatic stain gives multiple colors to cells and contains eosin and methylene blue dyes Methanol is used to fix cells to the glass slide Methanol is mixed in diluent Eosin dye stains basic components of cells an orange to pink color Principle Cont d Methylene blue dye stains the acid structures of cells a blue or purple color Neutral components are stained by both components of the Wright s stain Materials Needed Microscope Immersion oil gives more magnification oil lense the oil increases light refraction to magnify Wright s stain Distilled or deionized water this is okay because smear has been fixed Paper towels or blotting paper Drying rack Forceps Properly prepared blood or bone marrow smears Coplin jars or staining rack Gloves How Cells Appear When Properly Stained White cell nuclei appear purple and the cytoplasm of white cells ranges from blue gray to dusty pink Eosinophilic granules are large and orange pink Basophilic granules are large and blue black The entire smear should look pink orange Notes Proper rinsing is critical Water artifact can interfere with smear evaluation Keep stain jars tightly covered when not in use When all else fails and your stain is not working it is the size of the granules that will mark the cell It is important that participants become very familiar with the size of the granules You cannot always rely on stain to identify a cell Notes Cont d Causes of an acidic stain include insufficient staining time prolonged exposure to buffer or rinsing stain or buffer that are too acidic Causes of an alkaline stain include blood smears that are too thick prolonged staining time insufficient rinsing stain or buffer that are too alkaline Staining Problems Consider adjusting the pH of stain or buffer if Some participants may use Coplin jars as staining mechanisms If used Coplin jars must be thoroughly cleaned and not allowed to develop precipitate or debris White cells look too blue and red cells look too gray Too alkaline White cell granules are barely visible and red cells look too pale Too acidic Case Study 1 Analyzing a Smear A technician prepares to do a set of differential counts She notices that the red cells look pale and washed out and that the white cell granules are barely visible At this point identification of white cells is difficult 1 minute Question What is the proper course of action Poorly Stained Smear Notice that this stain is too pale it is difficult to tell any shading detail in the red cells Although the slide is out of focus the stain is too blue and no detail is able to be distinguished Case Study 1 Answer Analyzing a Smear Question What is the proper course of action Answer A stain that is too pale indicates stain may be too acidic due to a buffer problem Stain a second slide from the patient Increase the pH stain and then assess for color of granules and red cell color Summary Properly stained peripheral blood smears are essential for identification of cells and evaluation of cellular morphology Wright s stain is a polychromatic stain and the dyes eosin and methylene blue produce multiple colors in cells If cells do not stain as expected several adjustments can be made to enhance the staining reaction