Preparation of Blood Films Nada Mohamed Ahmed , MSC, MT (ASCP)i

Preparation of Blood Films Values: To study morphology of RBC. To study morphology of WBC. To study morphology of Platelets. To confirm diagnosis of blood cells disorders. To examine hemoparasites(Malaria, Trypanosoma, Babesia) Requirements: 1-Blood Sample: -EDTA ant coagulated (venous whole blood). -Finger prick blood (Capillary blood). 2- Stationary clean slide 3-Spreader slide

Procedure: Use clean standard size glass slides (3 inch x 1 inch = 7.5 cm x 2.5 cm), wiped from dust just immediately before use. Place a small drop of well mixed anticoagulated whole blood, in the center line of the slide, about 1.5 to 2 cm from one end, with the aid of a capillary tube. Immediately, without delay, with the aid of a second clean slide with uniform smooth edges (spreader slide), with a 30 –40 degrees angle, move back so blood drop will spread along the edge of the spreader slide, when this occurs, spread, or smear move forward to make ideal . Thin blood film consist of thick Head, body and tail.

Improper blood film A: Blood film with jagged (with rough, sharp points )tail made from a spreader with achipped end. B: Film which is too thick C: Film which is too long, too wide, uneven thickness and made on a greasy slide. D: A well-made blood film.

Examples of unacceptable smears

Examples of unacceptable smears

Quality Control for blood film preparation : 1-Before preparing the films, you must check that blood samples are free from clotsIf clots are present the specimen is not used. 2-Films can be labeled with patient’s name and /or Lab. No. on the thick end of the film itself, after being dried, by using a pencil. 3-Thin blood film must be done immediately as long as two to three hours after venous blood collection.  

principle of Romanosky stains Group of blood cells stains each consists of Azure B (the basic dye stains the acid portion of the cell) and Eosin Y ( the acidic dye stains the basic portion of the cell).

Romanovsky stains include: 1-Giemsa Stain 2-Wright’s Stain 3-Leishman Stain

Leishman Stain: Requirements Adequate air dried thin film Buffer (PH 8.6) instead pure distilled water. Procedure 1-Put the film on a staining trough rack. 2-Flood the slide with the stain. 3-After 2 minutes ( or more, if the stain in newly prepared), add double volume of buffer or distilled water, and mix the stain with water, until a shiny layer is seen. 4- After 8 minutes, wash with a stream of tap water. 5-Wipe the back of the slide with gauze. 6-Set the films in upright position on a dryer rack then examine macroscopically and microscopically.

Romanovsky Stain Blood Cells Characteristics Cell Structure Staining characteristics 1 Red cells Red or pinkish red 2 Nuclei of all cell types Purple/violet 3 Lymphocyte cytoplasm Blue 4 Monocyte cytoplasm Grayish blue(or glassy gray) 5 Platelets cytoplasm Light blue 6 Neutrophilic granules Violet-pink 7 Eosinophilic granules Orange-red 8 Basophilic granules Purplish black/ Deep blue 9 Platelets granules Purple

Staining characteristics of a correctly stained normal film: Nuclei Purple Cytoplasm Erythrocytes Deep pink Neutrophils Orange-pink Lymphocytes Blue; some small lymphocytes deep blue Monocytes Grey-blue Basophils Blue Granules Neutrophils Fine purple Eosinophils Red-orange Basophils Purple-black Monocytes Fine reddish (azurophil) Platelets Purple

Eosinophilic granules Basophilic granules Blue nucleus

Staining faults Too faint: Staining time too short. Excessive washing after staining. Stain deposit: Stain solution left in uncovered jar. Stain solution not filtered. Dirty slides.

Other factors which affects the staining results include : 1) Staining time, 2) pH of the staining solution, while   Sources Of Errors In Staining Stain Precipitate or deposit: May obscure cell details, and may cause confusion with inclusion bodies. Eliminate by use of the stain before use. pH of the buffer or water: Too acidic pH causes too pinkish slides. Too basic pH causes too bluish slides. Improper stain timing may result in faded staining or altered colors: Too long staining time causes too blue slides (overstaining). Too short staining time causes too red slides. Forced drying may alter color intensities and/or distort cell morphology.

TOO ACIDIC SUITABLE TOO BASIC

Non-stain related errors: 1-EDTA causes crenation of the cells after blood collection. 2-Severely anemic blood samples causes slower drying (before staining) due to excessive plasma. 3-Old blood specimens may cause disintegration in WBC’s and decrease in their numbers. 4-Collection of blood in heparin causes blue staining of RBC’s with bluish background, which makes heparin unsatisfactory for routine hematology testing, also heparin induces platelet aggregation and clumping , with subsequent erroneous platelet count with automated counters.