BLOOD FILM STAINING EFFECTS

Transcription

1 An Educational Supplement prepared by ALQEP May 2004 Introduction The stained peripheral blood film is one of the world s most widely and frequently used tests. Since its introduction in the late nineteenth century, basic elements of the blood film preparation and analysis have changed little. Modern technology improvements and refinements have enhanced the availability of good quality commercial Romanowsky stains, automated stainers and semi automated slide makers. Blood Film Preparation A fresh, well-made, peripheral blood film is crucial for accurate cell morphology assessment. (Refer to The Peripheral Blood Film: Staining, Cell Estimation and Review, CPSA ALQEP: May 2003.) 1 Blood films that are too thin or too thick present a problem. Extremely thin films (caused by too small a drop, too slow spreading or too low a spreader angle), may result in RBCs that appear as spherocytes and increased WBCs, such as monocytes and neutrophils, in the tails. An incorrect differential will result. Always scan the film under low power to detect this aberration. In extremely thick films, the counting area is too small. At least ten low-powered fields where fifty percent of the RBCs do not overlap are required for an accurate WBC differential. Blood films with excessive tails or gritty feathered ends indicate a spreader edge that is rough or dirty, or an accumulation of leukocytes due to either slow spreading or a very high leukocyte count. Staining the Blood Film Prior to staining, cells must be fixed to the glass slide with acetone-free methanol, either alone or in solution with dye. Addition of a buffer solution to the dye changes the ph of the solution and ionizes the reactants to initiate the phdependent staining process. Acidic cellular elements such as nucleoproteins, nucleic acids and primitive cytoplasmic proteins, react with the basic dyes, methylene blue and its oxidative products. These elements are basophilic and stain variations of blue. Basic cellular elements such as hemoglobin molecules and some cytoplasmic constituents in leukocytes, have an affinity for the acidic dye, eosin. These elements are acidophilic and stain orange-red. A neutrophil has neutral staining characteristics and stains blended shades of purple or pink, representing combinations of acidic and basic molecular groups. Azure dyes stain the primary or non-specific granules in most myeloid cells red-purple, hence, the term azurophilic granules.

2 Romanowsky Stains Romanowsky stains are universally used in hematology. They are composed of methylene blue, oxidative products of methylene blue (Azure A, Azure B, Azure C and Thionin) and eosin dyes. Giemsa, a commonly used stain does not adequately stain red blood cells, platelets or white blood cell cytoplasms when used alone. A second Romanowsky stain is therefore often used in combination with Giemsa and contains azure dyes to intensify the staining of nuclear features and of azurophilic and toxic granulation. Wright, Wright-Giemsa and May-Grünwald Giemsa are commonly used combinations of Romanowsky stains. Rapid or quick stains, developed for stat situations or for small laboratories, are adequate for assessing normal cell morphology. Historically Romanowsky stains presented large variations in staining properties between stain batches and manufacturers. This variation in stain quality is a result of the continued oxidation of methylene blue. A simple combination of pure azure B and eosin Y, as used in the new polychrome stains, is preferable as oxidation is complete in these solutions producing a standardized stain. Most laboratories purchase commercial ready-to-use staining solutions for convenience and for reproducibility, however raw materials are available for homemade stains. 2 Staining Methods Thoroughly dried blood films may be stained manually or with an automated staining instrument. Manual Staining Methods 1. Dip Method (Rapid): The dip method is a quick staining method that uses a modified Wright-Giemsa stain buffered in methanol at ph 6.8. Slides are immersed in the stain in a coplin jar for a user-determined length of time. It is important that the Wright-Giemsa stain be kept tightly sealed in coplin jar when not in use and be replaced when water artifact appears in red cells (see: Causes and Corrections of Stain Deviations). Note: Staining time may be increased for greater cellular detail as required. Automated Staining Methods 1. Hema-Tek Slide Stainer - Miles Scientific (platen-type stainer): The Hema-Tek slide stainer is a self-contained bench top slide stainer that uses a Hema-Tek Stain Pak. Three sensing switches are triggered sequentially to activate three solution pumps which deliver metered volumes of stain, buffer and rinse solution from the stain pack. A Wright-Giemsa Pak is most commonly used. Slides are advanced by two parallel conveyer spirals with the stain, buffer and rinse solutions pumped up between the blood film and platen. Note: Slides can be randomly added if required The staining process takes approximately ten minutes Time phases are constant Pump volumes are user adjusted by control knobs A stain/buffer ratio of 1:2 is desirable Cleaning procedures and daily maintenance are vitally important to prevent stain deposit artefact. Page 2 of 5 Alberta Laboratory Quality Enhancement Program May 2004

3 2. Hemastainer Automatic Slide Stainer - Miles Scientific (dip-type stainer): This is an automated staining instrument that stains up to fifty slides at one time. The slides are loaded into a slide basket and dipped into each of six stations in the staining process. Five individual, adjustable timers control the first five stations. The six stations consist of: methanol, stain, a second stain, buffered-water rinse, a phosphate buffer rinse and lastly, forced-air drying. The two stains commonly used in the stainer are May- Grunwald and diluted Giemsa. 3. Midas II Automatic Slide Stainer - EM Diagnostic (dip-type stainer): This instrument is a completely automatic slide stainer that stains up to twenty slides at one time. The slides are loaded into a bucket and are cycled through six stations: methanol, stain, a second stain, water rinse, phosphate buffer rinse, followed by the drying station. The first five stations are on individual, adjustable timers. The Midas II stainer is similar in operation to the Hemastainer. Note: Slides cannot be added once the staining process is started. The staining process takes approximately twenty minutes. Produces good quality, reproducible staining. Evaluating the Stain Quality Macroscopically, a properly prepared and well-stained blood film should appear pink in the thin area and have a purplish-blue tint in the thicker area. Optimal microscopic staining characteristics with a Romanowsky stain are as follows: Cell/Component; Red blood cells Nuclei of neutrophils Specific granules of neutrophils, granules of lymphocytes, granules of platelets Specific granules of basophils Specific granules of eosinophils Chromatin (including Howell-Jolly bodies) Dohle bodies Promyelocyte granules and Auer rods Cytoplasm of lymphocytes Cytoplasm of monocytes Cytoplasm of neutrophils Cytoplasm of platelets Color; Salmon pink Deep blue-purple Light purple or violet Deep purple Orange Purple Blue-grey Purplish-red Blue Blue-grey (ground glass appearance) Light pink Purple-blue to lilac Page 3 of 5 Alberta Laboratory Quality Enhancement Program May 2004

4 Causes and Corrections of Stain Deviations Stain Deviation Causes Correction 1. Stain too acidic (red) Buffer or stain too acid Correct ph, remake buffer -RBCs are bright red-orange Excess buffer for stain Shorten buffer time/amount -WBC nuclei are pale blue Insufficient staining time Prolong staining time/amount -Eosin granules are brilliant orange-red Very thin films Correct film thickness Old stain (oxidized alcohol) Check expiration date/stain 2. Stain too alkaline (blue) Buffer or stain too alkaline Correct ph, remake buffer -RBCs are blue-green Insufficient buffer for stain Increase buffer time/amount -Eosin granules are gray/blue Excessive staining time Decrease staining time/amount -WBC nuclei are blue-purple Very thick films Correct film thickness -Neutrophil granules are too dark -Lymph cytoplasm is gray 3. Stain too pale -Little contrast in WBCs Weak stain solution Change stain in station 2 and 3 (Hemastainer or Midas stainer) Lengthen staining interval (manual dip-method) 4. WBC nuclei too dark Stain too concentrated Check Giemsa dilution (dip-type stainers) Adjust metered stain volumes (platen-type stainers) Staining time incorrect Check timing on stain stations (dip- type stainers) Reduce staining interval (manual dip-method) 5. Water/drying artifact Water contamination Replace methanol and/or stain Keep dishes covered (prevent water absorption) Film drying too slowly Check humidity in air/increase drying speed, if possible Check for severe anemia 6. Precipitation on slides Unclean platen or lines Clean platen/lines, do maintenance Precipitate in stains Filter or replace stains Insufficient rinsing Check rinsing time Check rinse filter (dip-type stainers) Summary A properly made, properly stained and correctly assessed blood film is a critically important diagnostic tool. A poorly made or poorly stained blood film is of little value and may result in erroneous patient results and missed or incorrect diagnosis. Page 4 of 5 Alberta Laboratory Quality Enhancement Program May 2004

5 References 1. Clarke, Dr. Gwendolyn, The Peripheral Blood Film: Staining, Cell Estimation and Review, CPSA ALQEP: May, Dacie, Sir J.V., Lewis, S.M., Practical Haematology, 7 th edition, pages 77 to 81, Churchill Livingstone, Stiene-Martin, E.A., Lotspeich-Steininger, C.A., Koepke, J.A., Clinical Hematology - Principles, Procedures and Correlations, 2 nd edition, pages 22 to 34, Lippincott, O Connor, Barbara, H., A Color Atlas and Instruction Manual of Peripheral Cell Morphology, pages 15 to 18, Williams & Wilkins, Bain, Barbara, J., Blood Cells - A Practical Guide, 3 rd edition, pages 11 to 13, Blackwell Science, Dynacare Kasper Medical Laboratories, Staining Procedures, and Operating Instruction Manuals. Page 5 of 5 Alberta Laboratory Quality Enhancement Program May 2004