CHAPTER 5 Preparation of Blood Smear Blood Plasma

CHAPTER 5 Preparation of Blood Smear, Blood Plasma and Serum

Acknowledgements n n n n Addisa Ababa University Jimma University Hawassa University Haramaya University of Gondar American Society for Clinical Pathology Center for Disease Control and Prevention-Ethiopia

Objectives After completion of this chapter, the students will be able to: n State the importance of a properly made blood smear n Prepare thin blood films on slides and cover glasses n Identify the desirable qualities of a good thin blood film n Prepare thick blood films n Describe the effects of the various situation that create unacceptable blood smears n Prepare plasma and serum n Describe the difference between plasma and serum n Apply quality control methods

Outline n n Preparation of Thin film Preparation of thick blood smears Preparation of other types of smears Preparation of plasma and serum

Introduction i. peripheral blood smear n Examining peripheral blood smears is an important procedure performed in the The validity or reliability Hematology laboratory. of the information obtained from blood film n It is useful in: evaluation depends ¨ Providing diagnostic heavily on well-made information and well- stained films!! ¨ Providing additional data ¨ Guiding the selection and monitoring of therapy ¨ Indicating adverse effects of treatment

Introduction cont’d n There are two kinds of blood films ¨ Thin blood film ¨ Thick blood film

Cont’d n ii. Clinical significance of blood films: ¨ It is important in the investigation and management of: n anemia, n infections, n and other conditions which produce changes in the appearance of blood cells and differential white cell count.

5. 1 Preparation of Thin Blood Films n n A thin blood film is a drop of blood that has been systematically spread/smeared on a slide and which has more or less a monolayer of cells Blood samples for smear preparation can be obtained from: ¨ a free-flowing capillary blood or ¨ well mixed EDTA anticoagulated blood It can be prepared using: ¨ glass slides (Wedge method) or ¨ cover glasses method The most common method is the wedge or push slide smear technique

5. 1. 1. Wedge method (Two-slide method) n Compared to the cover glass method, preparation of blood films on glass slides have the following advantages ¨ Slides are not easily broken ¨ Slides are easier to label ¨ When large numbers of films are to be dealt with, slides will be much easier to handle

Materials Needed n n n Clean glass microscope slides Capillary/Well-mixed EDTA blood sample Device to make a drop of blood Marking pen or pencil Gloves Waste and sharps disposal containers

Procedure Use a glass slide that is free of dust, grease and debris Note: It is essential to ensure slides are washed free from traces of detergent and the surface of the slide is completely clean and not greasy. 1. 2. Place a small drop of well mixed EDTA blood (about 23 mm), 1. 0 cm from the end of the glass slide, using either a plain capillary tube or other type of blood dropping device ¨ when the blood is from an anemic patient larger drop of blood should be used ¨ If capillary blood to be used, follow SOP for proper collection of capillary blood

Procedure cont’d ¨ If using an anticoagulated blood sample, be sure the specimen is well-mixed ¨ The 3. drop should be placed (opposite from the end that is frosted, if that type of slide is provided) and in the middle of the slide the spreading slide is placed in front of the drop of blood at an angle of about 30 o -40 o C to the slide and then is moved back to make contact with the drop

Cont. . 4. 5. 6. 7. The drop will spread out quickly along the line of contact of the spreader with the slide ¨ As the drop of blood spreads, be careful not to let it spread to both edges of the spreader The spreader is advanced with a smooth steady motion so that a thin film of blood is spread over the slide Allow the smear to air-dry ¨ Do not blow on the smears as this can disrupt cellular morphology and cause the formation of unwanted artifacts, like target cells ¨ Also do not use heat for drying Label the name of the patient and date or reference number is written on the head of the film using a lead pencil, a diamond marker or a graphite

Technique for Making a Wedge Smear n n n Thickness and length of the film are affected by: n speed of spreading n the angle at which the spreader slide is held When the blood is from an anemic patient: n increase the angle of spreading and n spread the blood more quickly. When the blood is thick and viscous n reduce the angle of spreading and n spread the blood more slowly.

Technical tips for blood film preparation n n n Mix EDTA anticoagulated blood properly before making the smear Use capillary tubes to deliver the original drop of blood – they are easier to control The angle of push may need to be adjusted depending on the viscosity of the blood Slides must be clean, not damaged, with no chips or cracks A feather edge or bullet shaped appearance indicates a good smear The faster the film is spread the thicker and shorter it will be The bigger the angle of spreading the thicker will be the film

Technical tips cont’d Caution! During smear preparation, after applying the small drop of blood, if the drop sits for longer than 3 -5 seconds before spreading, clumping of platelets and white cells, and rouleaux formation of the RBCs occurs!

Acceptable Smears Are: n Smooth, homogeneous and have no lines, vacuoles or holes, jerks, streaks, or ridges ¨ Note: ‘Holes’ in a blood film are usually caused by using a slide, which is not clean (greasy) ¨ Lines across a film are usually due to spreading a film jerkily n Availability of sufficient examination i. e. , the length of the film should be 1/2 to 3/4 of the length of the slide

Acceptable Smears Are: n n Straight at the feathered edge ¨ A jagged ‘tail’ to a blood film is caused by using a spreader with a chipped end or end that is not clean. Free of visible clotting Not too thick and not too thin Gradual transition to thickness from the thick to thin areas terminating in smooth ‘tail’

Date – 12/09/09 Card no- 2453/09 Pt name- Almaz Characteristics of an Acceptable Smear Wright’s stained blood smear

Sources of error In Making a Smear n n Size of blood drop ¨ Too much blood makes Caution! the blood film thicker Wrong angle ¨ Too low and the smear will be too long ¨ Too high and the smear will be too short Speed Too much pressure on the push slide

Sources of error cont’d n n n Dirty slides Delay in spreading blood drop Failure to completely spread blood drop Stopping abruptly before completely spreading the blood drop Pushing the spreader slide too quickly or too slowly

Examples of Peripheral Blood Smears

5. 1. 2 The Cover Glass Method Material: 22 mm cover glasses are required Procedure: 1. Touch a clean cover glass to the top of a small drop of blood without touching the skin (when using capillary blood) and place it down 2. Cross- wise on another cover glass so that the corners will appear as an eight-pointed star.

Cont. . - 3. 4. 5. If the drop is not too large and if the cover glasses are perfectly clean, the blood will spread out evenly and quickly in a thin layer between the two surfaces. Separate the two cover slips by pulling them in opposite direction Cover glasses should be placed film side up on a clean paper and allowed to dry in the air After they are stained they are mounted with DPX mountant film side down on glass slides

Advantage of cover slip method q q q WBC and Platelets are more evenly distributed More of the prepared film can be examined q decrease (low) sampling error Used for Bone Marrow aspiration smear

Advantage of Slide method n n n Slides are not easily broken Slides are easier to label and stain When large numbers of films are to be dealt with, slides will be found much easier to handle. Easier to learn performing the technique Unlike the cover slip method, RBCs are well distributed

5. 2. Preparation of thick blood film n n n Thick blood smears are widely used in the diagnosis of blood parasites particularly malaria. It gives a higher percentage of positive diagnosis in much less time since it has ten times the thickness of normal smears. Five minutes spent in examining a thick blood film is equivalent to one hour spent in traversing the whole length of a thin blood film.

Cont’d n n n Place a small drop of blood on a clean slide spread it with an applicator stick or the corner of another slide until small prints are just visible through the blood smear The prepared smear corresponds to a circle of approximately 2 cm diameter.

5. 3. Other types of smears Other types of smear preparation include: ¨ Automated spun smear ¨ Buffy coat smear n Buffy – coat smear ¨ Is a smear prepared from buffy coat layer (the layer that contains WBC and Platelets) ¨ Has a great value especially in leucopenia ¨ Also important in the diagnosis of malaria, amastigotes of visceral leishmaniasis

5. 4. Preparation of Plasma and Serum n n n Plasma is the fluid part of anticoagulated blood Serum is the fluid part of clotted blood ¨ Serum does not contain fibrinogen Plasma preparation: ¨ Collect the needed volume of blood (5 ml) using a vacutainer tube containing an appropriate anticoagulant n If syringe method is used for blood sample collection, dispense the collected blood carefully in a test tube, which contain the required anticoagulant

Procedure Cont. . n n Mix the blood by inverting the test tube 8 -10 times ¨ Do not shake Centrifuge the blood at 300 g for 10 minutes. Separate the upper clear fluid (plasma) using long neck pasture pipette and dispense it in a clean test tube Avoid mixing of RBCs with the fluid part

Serum preparation n Collect about 5 ml of blood using plane vacutainer tube (a tube with no anticoagulant) If syringe method is employed for sample collection, collect 5 ml blood and dispense in dry leak proof glass tube ¨ avoid plastic tubes because blood does not clot well in plastic container Allow the blood to completely clot and retract at room temperature ¨ This might take more than 30 minutes

Cont. . n n After complete clotting and retraction centrifuge the clotted blood at 300 g for 10 minutes Collect the upper clear fluid (serum) using long neck pasture pipette and dispense it in clean test tube ¨ Serum can be stored for several days if kept refrigerated ¨ For long term storage, store in deep freezers ¨ Note: Fridge temperatures should be monitored and documented

Types of Specimens n Plasma ¨ Plasma contains fibrinogen ¨ Centrifuge whole blood, separate plasma from cells

Types of Specimens n Serum ¨ Allow blood to clot for 20 - 30 minutes n Glass tube n Plastic 15 - 20 minutes ¨ Centrifuge 10 – 15 minutes, separate cells from serum ¨ Serum does not contain fibrinogen ¨ Chemistry Testing

Sources of error in plasma and serum preparation n n n Centrifugation time and speed Not waiting until blood clots in serum preparation Use of wet or unclean test tubes (contaminated with detergent) for centrifugation Improper separation of cells and plasma/serum Wringing of the clot with applicator stick to enhance clotting Any factors that cause hemolysis during specimen collection affect the quality of plasma/serum

Review Questions/Summary 1. 2. 3. 4. 5. 6. 7. 8. 9. What is a thin blood film? List types of methods for making a thin film Which technique of blood film preparation is commonly employed and how is the method of preparation? What are the desirable qualities of a thin blood film? What are the possible effects of using a blood sample that has been standing at room temperature for some time on blood cell morphology? What is the effect of delaying in spreading after applying the small drop of blood? What is the difference between serum and plasma? What is a buffy coat smear and its application? List at least five common sources of error in blood smear preparation