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Blood Film Preparation and Staining Procedures

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Blood Film Preparation and Staining Procedures INTERPRETATION OF THE PERIPHERAL 00. ם BLOOD FILM 0272–2712/02 $15.00 BLOOD FILM PREPARATION AND STAINING PROCEDURES Berend Houwen, MD, PhD The blood film is one of the world’s most widely and frequently used tests and has undergone remarkably few changes since its introduction as a clinical diagnostic tool in the late 1800s. The origins of stained blood film microscopy are somewhat obscure, and a clear reference for the ‘‘first person or persons’’ to describe its use as a clinical laboratory procedure cannot be established with certainty. Antonie van Leeuwenhoek, in the seventeenth century, was the first to describe blood cells, using whole blood preparations and not blood films for his observations on blood corpuscles. In fact, he would not have been able to use blood film–based microscopy because it requires considerably more sophisti- cated optics than were available in his day. A second technologic innovation enabling blood film microscopy was the introduction of aniline dyes in the second half of the nineteenth century. This made it possible to study individual blood cells by light microscopy after a small amount of blood had been placed and smeared onto glass slides, dried, fixed, and then stained. Paul Ehrlich introduced eosin as the first of these dyes for staining blood films in 1856, followed by hematoxylin in 1865, and later by the metachromatic Romanowsky dyes. A few refinements have since been made, consisting mainly of improve- ments in dye quality, staining procedures, automation of slide preparation, and staining, but the basic elements of blood film preparation and analysis have not changed for over a century. This article gives direction and some standardization in the preparation of blood films used for morphologic evaluation in the clinical laboratory. Micro- scopic analysis procedures and interpretation are not within the scope of this ar- ticle. Methodologies that can be labeled as being state of the art and methods applicable for laboratories in developing countries are quoted. When targets are stated these are at minimum level, and should be attained under all conditions, From Beckman Coulter, Inc., Fullerton, California CLINICS IN LABORATORY MEDICINE VOLUME 22 • NUMBER 1 • MARCH 2002 1 2 HOUWEN lest diagnostic test quality and patient care be compromised. This is especially important because results obtained from microscopic analysis of blood films are often used as final and definitive in clinical situations, whether for screening, case finding, diagnosis, or monitoring of disease. BLOOD FILM PREPARATION Microscope Slides Slides must be made from highest purity, corrosion-resistant glass; other material, such as plastic, is mostly not acceptable. Glass slides typically measure mm; are approximately 1 mm in thickness; must be flat and free from 25ן75 distortions and ripples; and must be clear and colorless (water-white). For fluorescence microscopy applications glass must be used that does not show autofluorescence. It is preferable to use precleaned slides, but at a minimum laboratories must ensure that slides are free from scratches; are clean; free of dust, lint, and fat (from fingerprints); and dry. Slides should always be stored in sealed containers to be opened only immediately before use. In humid environments slides should be kept in a desiccator or container with 95% (methyl) alcohol or a mixture of three parts water-free alcohol and one part acetone until used after the sealed container has been opened. Slides may be plain or have a frosted or coated area for writing. Slides with rounded or beveled edges are safer to use than those with squared edges and reduce the chances of cut or punctured skin and gloves. Cleaning of Slides Dirty slides need tobe cleaned by soakingin a detergent at 60 ЊCfor15to 20 minutes, then rinsing in hot water before drying. New slides that need cleaning should be placed in a potassium dichromate cleaning solution (Cr2K2O7, 20 g in 100 mL water with 900 mL of concentrated H2S4O added) for 48 hours. This is followed by a thorough rinse in running tap water. The slides should be stored in 95% (methyl) alcohol or in a mixture of three parts water-free alcohol and one part acetone before being used. They should be wiped clean and dried carefully before use.5 Blood film preparation methodologies include (1) manual (wedge or coverslip); (2) semiautomated (wedge or spinner); and (3) automated (wedge). Typically, for wedge film preparations, a small amount of blood is placed on a glass slide, which is then spread (by a hand-held spreader slide). Moving the spreader slide forward creates a blood film of varying thickness on the initial glass slide. The other methods mentioned use different techniques to arrive at the same goal: a blood film or smear with a sufficiently large area that is adequate for morphologic analysis. Wedge Method (Push) This method is commonly used in manual and semiautomated environ- ments. When carried out properly a sufficiently large area is available for microscopic examination: this area shows red blood cells barely touching or separated from each other (monolayer part). The parts of the film farthest away from the start are too thin (with morphologic alterations as a result), whereas BLOOD FILM PREPARATION AND STAINING PROCEDURES 3 the part nearer to the start of the push is too thick for microscopy. Automated devices are capable of providing excellent-quality blood films, usually with greater consistency than those obtained by manual methods.18 A serious concern with wedge preparations is the uneven distribution of different cell types.22 Monocytes (and other large leukocytes) are pushed to the end of the spreaded film (the feathered edge) and to the sides. This leads to a 5% to 10% underestimation of monocyte presence when compared with monoclonal antibody-based flow cytometry differential counts (upper normal limit in pro- portional count is 11% in normal individuals, not 10% as measured by micros- copy).13 Coverglass Method (Pull) The small coverglass slides cannot be labeled adequately and should be avoided. In addition, the technique itself has a biohazard risk that is higher than for the wedge method. The method is considered obsolete and should not be used. Spun Blood Film This method has been described as an alternative to wedge methods.1, 16 It offers a monolayer of blood cells for microscopic examination by spreading blood cells by centrifugal force over large areas. The morphologic condition of all cell types in properly prepared spun films is generally excellent, although centrifugal force must be controlled carefully to avoid the formation of morpho- logic artifacts such as smudge cells.23 Dilution of blood with isotonic saline has been recommended17 but excellent preparations are possible without this step. There is no evidence that spun blood film preparations result in uneven distribu- tion of cell types.24 Spinners from the past were among the most hazardous of all laboratory instrumentation, however, because of droplet and aerosol forma- tion and contamination of the spinner’s interior by blood. Recently developed spinners have overcome these problems and can be used safely (an example of an adequate slide spinner is the instrument developed by Statspin Technologies, Norwood, MA). For consistent results it is essential to apply exactly the right amount of blood to these spinners; the use of micropipettes for dispensing blood is recommended. Type of Blood Sample Used Venous (anticoagulated) and capillary blood, as obtained by skin puncture (without anticoagulation), are both acceptable. There are several published stan- dard documents on procedures and devices used for specimen collection, and the reader is referred tothese texts. 6, 7, 11, 19 Anticoagulation K2 orK3EDTA, sodium citrate, and acid citrate dextrose are all acceptable but heparin is not because of frequently developing platelet clumps that interfere with the morphologic interpretation of platelets and of platelet count estimates. Heparin also causes the development of a purple-blue hue on stained films. 4 HOUWEN Sample Storage Effects Blood samples should be processed as quickly as possible after collection, because significant morphologic changes occur on prolonged storage that are time and temperature dependent. Best results are obtained when films are prepared within 2 hours after collection. Recommended storage temperatures are as follows: short-term, less than 8 hours, preferably at 4ЊC but storage at room temperature is acceptable (room temperature is defined as not to exceed 22ЊC); long-term storage should be at 4ЊC. After prolonged storage blood sam- ples should always be mixed by a minimum of 10 complete (180-degree) inver- sions. Capillary Tubes for Delivery of Blood on Glass Slides Plastic (polystyrene or similar) capillary or microhematocrit tubes (unbreak- able) are recommended for placing blood drops onto the slide. Glass tubes should be avoided because of the possibility of breaking, causing a biohazard. Tubes should be plain and should not contain heparin. Alternatively, a perforat- ing device for the stoppers of blood containers, specifically designed for making blood films, may be used (e.g., Diff-safe, Alpha Scientific, Southeastern, PA). Blood Volume Used Enough blood should be used to allow for a wedge blood film of appro- priate thickness and of 2.5 to 4-cm length. If a spinner is operated, a micropipette should be used for blood delivery on the slide to ensure consistent monolayers. Typically, for spun slides, 30 ␮L (about one drop) results in a monolayer of sufficient size, but different volumes may be required for different spinners. Position of Blood Drop The blood drop should be placed approximately 1 cm from the end of the slide (opposite labeling end). Alternatively, 1 cm distance from the label (or frosted part of the slide) can be chosen (this does not apply if the slide label is placed on the back of the slide). For spinners, handle according to the manufac- turers’ instructions.
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