ISLH

Laboratory Hematology 6:1–7

© 2000 Carden Jennings Publishing Co., Ltd.

Official Publication

Blood Film Preparation and Staining Procedures

BEREND HOUWEN
Department of Pathology and Human Anatomy, Loma Linda University School of Medicine, Loma Linda, California

ABSTRACT sis of blood films are often final and definitive in clinical situations,
whether for case finding, diagnosis, or monitoring of disease.
The blood film is one of the world’s most widely used labora- For easier reference use, this article uses a special format to describe
tory tests for screening, case finding, diagnosis, and monitoring of the various steps that involve blood film preparation and staining.
disease. This article provides guidelines that should enable clini-
cal laboratories to prepare and stain good-quality blood films. BLOOD FILM PREPARATION
Objective criteria have been applied as much as possible, although
there remains some “art” in how a blood film is prepared manu- Microscope Slides
ally. Troubleshooting of fixation and staining artifacts is included Slides should be made from the highest purity, corrosion-resis-
in the guidelines. Lab Hematol. 2000;6:1–7. tant glass; other material such as plastic is mostly not acceptable.
Glass slides typically measure 75 × 25 mm, are approximately 1 mm
KEY WORDS: Blood film preparation · Staining in thickness, must be flat and free from distortions and ripples, and
must be clear and colorless (“water-white”). For fluorescence
microscopy, glass that does not show autofluorescence must be used.
INTRODUCTION It is preferable to use precleaned slides, but at minimum, labora-
tories must ensure that slides are free from scratches; are clean; are
The blood film is one of the world’s most widely and fre- free of dust, lint, and fat (from fingerprints); and are dry. This may
quently used tests, and yet there appears to be no comprehensive mean that in humid environments, slides must be hydration resis-
document that lists requirements, procedures, potential prob- tant and, after their sealed container is opened, should be kept in a
lems, etc., for blood film preparation. Although it is a simple desiccator or container with water-free (methyl) alcohol or a mix-
procedure, there are many reasons that as a test it could easily fail ture of 3 parts alcohol and 1 part acetone until used.
or be less effective than it should be. This article was prepared at Slides should always be stored in sealed containers to be opened
the request of the Cytometry Panel of the International Council only immediately before use. They may be plain or have a frosted
for Standardization in Haematology (ICSH) as a guideline for or coated area for writing. Slides with rounded or beveled edges are
laboratories. It is intended to give direction and some standard- safer to use than those with squared edges and reduce the chances
ization in the preparation and staining of blood films for mor- of cut or punctured skin and gloves.
phological evaluation in the clinical laboratory. Microscopic
analysis procedures and interpretation are not within the scope Cleaning of Slides
of this article. Dirty slides need to be cleaned by soaking in a detergent at
Methods include state-of-the-art techniques as well as method- 60°C for 15 to 20 minutes, then rinsing in hot water before dry-
ologies applicable for laboratories in developing countries. When ing. New slides that need cleaning should be placed in a potas-
targets are stated, they are not at the maximum level but are targets sium dichromate cleaning solution (20 g Cr2K2O7 in 100 mL
that should be attained under all conditions lest diagnostic test water with 900 mL concentrated H2SO4) for 48 hours. This pro-
quality and therefore patient care be compromised. This is cedure is followed by a thorough rinse in running tap water. The
especially important since results obtained from microscopic analy- slides should be stored in 95% methanol and carefully wiped
clean and dried before use [1].

Correspondence and reprint requests: Berend Houwen, Department of Blood Film Preparation Methodologies
Pathology and Human Anatomy, AH309, Loma Linda University School Typical blood film preparation methodologies are: manual
of Medicine, Loma Linda, CA 92354 (e-mail: houwhem@[Link]). (wedge, coverslip), semi-automated (wedge, spinner), and auto-
(Received May 2, 2000; accepted May 5, 2000) mated (wedge).

1
2 B. Houwen

Wedge Method (“Push”) Anticoagulation

This method is commonly used in manual as well as in auto- Acceptable anticoagulant agents are K2EDTA or K3EDTA,
mated and semi-automated environments. When the wedge sodium citrate, and acid citrate-dextrose (ACD). Heparin is not
method is carried out properly, a sufficiently large area is available recommended because of frequently developing platelet clumps
for microscopic examination: this area shows all cells barely touch- that interfere with the morphological interpretation of platelets and
ing or separated from each other (monolayer part). The parts of the platelet count estimates. Heparin also causes the development of a
film farthest away from the start will be too thin (with morphologi- purple/blue hue on stained films.
cal alterations as a result), whereas the part proximal to the start of
the push will be too thick for microscopy. Sample Storage Effects
Automated devices are capable of providing excellent quality Blood samples should be processed as quickly as possible after
blood films, usually with greater consistency than those obtained by collection. Significant morphological changes occur on prolonged
manual methods [2]. The main concern with wedge preparations is storage and are time and temperature dependent. Best results are
the uneven distribution of different cell types [3]. Monocytes (and obtained when films are prepared within 2 hours after collection.
other large leukocytes) in particular are pushed to the end of the Storage temperatures are as follows: short-term (<8 hours):
spread film (the “feathered” edge) and to the sides. This leads to a preferably at 4°C, but storage at room temperature is acceptable;
5% to 10% underestimation of monocyte presence compared with long-term: 4°C. Always mix blood samples after prolonged storage
monoclonal antibody–based flow cytometry differential counts by a minimum of 10 complete (180°) inversions.
(upper normal limit in proportional count is 11% in normal indi-
viduals, not 10%, as measured by microscopy) [4]. Capillary Tubes
Plastic (polystyrene or similar) tubes (unbreakable) are recom-
Coverglass Method (“Pull”) mended for placing blood drops onto the slide. Glass tubes should
The small coverglass slides cannot be labeled adequately and be avoided because of the possibility of breaking, which can cause a
should therefore be avoided. In addition, the technique itself has a biohazard. Tubes should be plain and should not contain heparin.
biohazard risk higher than that for the wedge method. The method Alternatively, a perforating device for the stoppers of blood contain-
is considered obsolete and should not be used. ers specifically designed for making blood films may be used.2

Spun Blood Film Blood Volume

This method has been described as an alternative to wedge The blood volume used should allow for a wedge blood film of
methods [5,6]. By spreading blood cells via centrifugal force over an appropriate thickness and 2.5 to 4 cm in length. If a spinner is
large areas, it offers a monolayer of blood cells for microscopic operated, a micropipette should be used to ensure consistent
examination. The morphological condition of all cell types in prop- monolayers. Typically, for spun slides, 30 µL will result in a mono-
erly prepared spun films is generally excellent, although care has to layer of sufficient size, but different volumes may be required for
be taken to avoid the formation of smudge cells [7]. Dilution of different spinners.
blood with isotonic saline has been recommended [8], but excellent
preparations are generally possible without this step. There is no Position of Blood Drop
evidence that spun blood film preparations result in uneven distri- The position of the blood drop should be approximately 1 cm
bution of cell types [9]. from the end of the slide (opposite labeling end). Alternatively, a
Spinners from the past were among the most hazardous of all distance of 1 cm from the label (or frosted part of the slide) can
laboratory instrumentation because of droplet and aerosol forma- be chosen. Use the manufacturer’s instructions when determin-
tion and contamination of the spinner’s interior by blood. Recently ing the position in the spinner.
developed spinners have overcome these problems and can be used
safely1 [10]. Spreader
It is essential to apply exactly the right amount of blood to these Ideally, the spreader should be slightly narrower than the glass
spinners to obtain consistent monolayers; therefore, the use of slide to minimize distribution effects. In practice, a second slide is
micropipettes rather than capillary tubes is recommended for dis- often used as the spreader, and this procedure is acceptable if the
pensing blood. spreader slide has rounded or beveled edges, making the spread
width of the slide narrower than the actual slide width.
Type of Blood Sample Spreaders should be discarded after use or be cleaned thoroughly
The two types of blood sample are venous (anticoagulated) and and dried before reuse. The edge of a spreader should always be
capillary, as obtained by skin puncture (without anticoagulation). smooth to ensure even thickness for the entire width of the blood film.
There are several published standard documents on devices used for The presence of blood cells from a previous specimen on the
and procedures for specimen collection, and the reader is referred spreader edge can cause significant carryover of blood cells, includ-
to those texts for details [11-14]. ing malaria parasites in red blood cells and leukemic white blood
cells, into the next blood film.

1. An example of an adequate slide spinner is the instrument developed by Statspin

Technologies, Norwood, MA. 2. eg, Diff-safe; Alpha Scientific, Southeastern, PA.
 Blood Film Preparation and Staining 3

Blood Pick-Up by Spreader Blood Film Preparation Methods for Malaria Detection
As soon as a blood drop has been placed on the slide, the It is recognized that morphological identification of malaria para-
spreader should be moved slowly backwards at an angle of approxi- sites in (thick or thin) blood films is not the most sensitive method
mately 30° to 45° toward the blood drop. The drop should spread for malaria detection. Molecular methods are much more sensitive
quickly along the spreader’s edge. Once the blood is spread along but also much more expensive. For some time to come, therefore,
the entire edge, the spreader should be moved forward immediately the morphological identification of malaria will remain in many lab-
at a steady rate, at a fairly fast speed, and at a 45° angle until all oratories a major method for detection and monitoring of malaria.
blood has been spread into a film. The spreader should be held more Both thin and thick blood film preparations should be used for
upright for samples from anemic patients, creating a thicker film. morphological detection of malaria parasites. Identification of
malaria is ±10 times more sensitive in thick preparations than in
Rate of Spreading/Spinning thin preparations, but the actual identification of the type of
The faster the blood is spread on the slide, the thicker the film malaria parasites is more difficult in thick preparations.
will be. Training and experience are required for consistent and For thick film preparation, a small drop of blood is placed on a
acceptable results. It is notoriously difficult to produce acceptable glass slide and spread to approximately 4 times its original surface.
wedge blood films from certain specimens such as those from new- After extensive drying, best done at 50°C to 60°C for 7 to 10
borns. For slide spinners, centrifugation speeds and times should be minutes, the slides can be stained. The cells will wash off the slide
available from the manufacturers. if insufficiently dried.
Two staining procedures have been described: a rapid procedure
Thickness of Blood Film taking <30 seconds for regular blood films, and a Giemsa-based
The thickness of the blood film is influenced by the size of the procedure for thick drop preparations, which takes approximately
blood drop, the patient’s hemoglobin level, the angle of the 30 minutes. As an alternative to thick drop preparations, a method
spreader (the greater the angle, the thicker and shorter the blood has been described that uses blood cell lysis by saponin followed by
film), and the speed of spreading. centrifugation to remove debris [15].

Drying of Blood Film Reticulocyte Preparation

Normally, air-drying without forced air circulation is sufficient. Anticoagulated blood must first be incubated in a supravital dye
In humid conditions, forced air-drying is recommended, with that will stain the (mainly ribosomal) RNA in reticulocytes and
proper precautions taken for aerosol formation. When forced dry- cause it to aggregate into the morphologically typical dark blue net-
ing is applied, it is recommended to do so in biohazard hoods work (reticulum) and granules.
equipped with high-efficiency particulate air (HEPA) filters. Aliquots of staining solution and blood are mixed in a test tube
and incubated for 15 minutes at an ambient temperature. After
Preparation Artifacts remixing by 10 complete inversions, at least 2 blood films are pre-
The most common artifacts are due to poor spreading tech- pared on glass slides and air-dried before microscopic analysis.
niques, slow drying in humid conditions, insufficient or late fixa-
tion, and water containing fixing solutions. Slides that are not dry Fixation/Staining
result in poorly or irregularly spread blood films, often with poor Optimal results are obtained by fixing and staining immediately
morphology. after the blood film is completely air-dried. Fixation of blood films
Certain cell types can be easily damaged by blood film prepara- before staining is recommended, although many laboratories have a
tion. Specific conditions include those with large numbers of atypi- practice of staining immediately after air-drying of blood films.
cal lymphocytes, chronic lymphocytic leukemia (CLL), and acute This procedure usually yields acceptable results. However, if slides
leukemia. Sometimes these ruptured cells (“smudge” cells or cannot be stained immediately, fixation in methanol is necessary
“Gumprechtse Schollen”) can be prevented by the addition of albu- within 4 hours, but preferably 1 hour after air-drying; otherwise,
min to the blood sample before blood film preparation. the plasma will cause gray/blue background effects. Staining and
Slow drying causes cells to contract, whereas water in excess of fixing solutions must be as free of water as possible (<3%) to pre-
3% in the methanol fixing solution can cause gross morphological vent morphological artifacts.
artifacts, such as decreased “crispness” of cellular appearance If manual staining procedures are used, it is recommended
(especially red blood cell and nuclear) and the development of that slides be immersed in reagent-filled (Coplin) jars rather than
artificial vacuoles. covering slides with staining solution because formation of pre-
Degenerative changes such as cytoplasmic vacuolization in mono- cipitate by evaporation may occur. If staining is performed under
cytes and neutrophils, nuclear lobulation or fragmentation of nucleated extremely hot conditions, care must be taken to prevent evapora-
cells, and apoptotic changes are not so much the result of preparation tion during staining, eg, by performing staining in a closed jar or
but are often caused by prolonged or inadequate sample storage. in a closed petri dish.
Automated staining devices have their own specific staining pro-
Possible Interferences cedures that are provided by the manufacturers. Users may modify
Patient conditions influencing the preparation and quality of these procedures to satisfy local requirements for cell staining.
blood films are: anemia, polycythemia, cord blood, platelet clump- Staining protocols vary between laboratories, and no generally
ing, cold agglutinins, anti–red blood cell antibodies in severe accepted routine staining method or result is available. Guidelines for
hemolytic anemia, severe rouleaux formation, and blood specimens staining and staining results can be found in hematology and labora-
from newborns. tory guidelines and textbooks. ICSH has published a “reference”
4 B. Houwen

staining method for blood films based on purified azure B and eosin Step 4: Transfer slide from jar without washing (or remove staining
Y solutions [16]. solution by holding slide vertically) into staining solution II
As a general rule for judging the quality of a stained blood film, that has been freshly diluted with 9 parts buffer for 10 to
the laboratory must ensure that all cell types in a blood film can be 15 minutes.
identified reliably by the staining procedure. This rule applies Step 5: Transfer slide to jar with buffer for 1 rinse after removing stain.
equally to manual and automated methods. Step 6: Wash slide with ample water.
Blood films are typically stained by Romanowsky dyes (consist- Step 7: Transfer slide to a jar containing water for 2 to 5 minutes.
ing of a variety of thiazines and eosins). A number of methods have Step 8: Dry the slide in a tilted position; do not blot dry.
been described and include the following: Wright, Wright-Giemsa, Step 9: Mount a coverglass if desired.
May-Grünwald-Giemsa, and Leishman. Examples of commonly
used methods for staining of air-dried blood films follow. Comment
It is essential in this method that slides are not allowed to dry or
Wright Stain evaporate between steps to prevent staining artifacts and precipitates
from forming.
Reagents
(1) Absolute methanol. (2) Staining solution (which can be pur- Malaria Stain
chased as a ready-made solution or as a powder from commercial
sources). Typically, 0.3 g of Wright stain powder is dissolved in Regular Blood Film, Rapid Method
100 mL absolute methanol and left in a closed container at room Reagents
temperature for 24 hours. It must be filtered before use. (3) Sörensen’s Stain I: 1.3 g eosin Y in 500 mL distilled water containing 12.6 g
buffer solution at pH 6.4; KH2PO4, anhydrous 6.63 g; Na2HPO4, Na2HPO4·12H2O and 6.25 g KH2PO4. Let stand at room temper-
anhydrous 2.56 g; distilled water up to 1000 mL. ature for 24 hours in a closed container. Alternatively, stain I may
be made by dissolving 0.8 g methylene blue and 0.5 g methylene
Method azure (azure I—an oxidated derivative of methylene blue) in 500
Step 1: Fix for at least 30 seconds in absolute methanol. mL distilled water containing 12.6 g Na2HPO4·12H2O and 6.25 g
Step 2: Remove methanol by tilting the slide. KH2PO4. No evaporation step is necessary. Stain II: 1.3 g methyl-
Step 3: Apply staining solution for 2 minutes on a horizontally ene blue dissolved in 500 mL distilled water containing 12.6 g
positioned slide. Na2HPO4·12H2O and boiled to dry powder in order to “poly-
Step 4: Add aliquot of the buffer solution without any of the stain chrome” the dye. The powder is resuspended in 500 mL distilled
running off the slide. Gently mix the buffer and stain with- water, and 6.25 g KH2PO4 is added. Both staining solutions must be
out touching the surface of the blood film on the slide filtered before use and kept in a closed container to prevent oxidation.
(a metallic sheen will appear on the surface of the staining Method
solution mixture). Step 1: Fix slides for at least 30 seconds in absolute methanol.
Step 5: Let stand for 3 minutes. Step 2: Place 12 drops of diluted stain I on blood film (1 part stain
Step 6: Rinse the slide with (distilled) water for 30 seconds. diluted by 4 parts distilled water).
Step 7: Dry the slide in a tilted position; do not blot-dry. Step 3: Immediately add 12 drops of undiluted stain II and mix
Step 8: Mount a coverglass if desired. with the diluted stain I already on the slide.
Step 4: Let stand for 1 minute.
May-Grünwald-Giemsa Stain Step 5: Drain slide and place in Sörensen’s phosphate buffer,
pH 6.6, for 5 seconds (for full development of staining).
Reagents Step 6: Wash with water.
(1) Absolute methanol. (2) Staining solution I: 0.3 g May- Step 7: Dry by tilting the slide; do not blot dry.
Grünwald powder in 100 mL absolute methanol; leave in closed
container at room temperature for 24 hours. It must be filtered Field’s Stain
before use. Staining solution II (Giemsa stain): 1 g Giemsa stain Dried but otherwise unfixed thin films and thick drop prepara-
powder is dissolved in 66 mL glycerol and heated to 56°C for 90 to tions can be dipped into stain I for 1 to 2 seconds followed by a rinse
120 minutes. After addition of 66 mL absolute methanol and thor- in Sörensen’s buffer, pH 6.8, until no more stain is released from the
ough mixing, the solution is left at room temperature in a closed blood film. Next the film is dipped for 1 second in stain II and rinsed
container. It must be filtered before use. (3) Buffer: Sörensen’s immediately in the same buffer. The slide is dried in vertical position
buffer solution. The pH must be at 6.8 for the May-Grünwald- after excess water has been removed by shaking, not blotting [17].
Giemsa stain instead of 6.4 as in the Wright stain.
Thick Drop Method (Giemsa)
Method Reagents
Step 1: Fix for at least 30 seconds in absolute methanol. The staining solution is Giemsa stain, which can be purchased
Step 2: Remove methanol by tilting the slide or by simply remov- directly or made up by the laboratory (see “Method” under “May-
ing from the fixing jar. Grünwald-Giemsa Stain”).
Step 3: Apply staining solution I freshly diluted with an equal part Method
of buffer for 5 minutes on a horizontally positioned slide Step 1: Immerse thoroughly dried slides in a jar or, keeping the
or in a jar. slides in a horizontal position, stain with diluted Giemsa
 Blood Film Preparation and Staining 5

stain (1:10 with Sörensen’s phosphate buffer at pH 6.8) for peratures, may show nuclear changes corresponding with apo-
20 to 30 minutes. ptosis (karyorrhexis).
Step 2: Wash the slide with the same buffer (gently, because other- Precipitate may form as a result of evaporation of methanol
wise the blood film floats off the slide). from the staining solution or because of improper washing proce-
Step 3: Leave the slide in a tilted or upright position to dry; do not dures, particularly not keeping the slide absolutely flat during the
blot dry. initial phase of washing. Inadequate filtration of homemade stain-
Comment ing solutions before use, use of unclean slides, and allowing dust to
Because one of the goals of this technique is to lyse the red settle on the blood film or slide can also cause precipitation.
blood cells and make malaria parasites visible through several cell
layers, morphology of most blood cells in thick drop preparations is Coverglass
poor. The prolonged immersion in the diluted staining solution In general, the use of coverglass (coverslipping) is not necessary.
makes these preparations prone to float off the slides. However, if blood films need to be reviewed by several individuals
or need to be stored long-term, coverslipping may provide protection
Reticulocyte Stain against mechanical damage (repeated wiping) and stain deteriora-
New methylene blue (1 g) is dissolved in 100 mL diluent (cit- tion from exposure to air. A potential added advantage of covering
rate-saline: 20 mL of 30 g/L sodium citrate plus 80 mL of 9 g/L the entire area of patient blood (stained and unstained) with cover-
sodium chloride). After the dye is dissolved, the stain must be fil- glass is a reduction in biohazard from handling blood films.
tered before use. Coverglasses should be made from the highest purity colorless glass
(“water-white”) and must be hydration resistant, especially in environ-
General Comments on Staining and Staining Materials ments with high humidity. They should have uniform surface quality,
Stains and fixing materials must be kept in tightly closed con- be free from any irregularities, and be perfectly flat. Coverglasses typi-
tainers at room temperature; freezing temperatures destroy cally measure 25 × 25 mm and have a thickness ranging from 0.13 to
Romanowsky stains. Blood film staining reagents should have labels 0.17 mm (a range of other sizes is available from several distributors
that record when containers were opened; expiration dates should and manufacturers). Extra-thick coverglasses (0.17 to 0.25 mm) reduce
be on containers when applicable. the chances of breaking but place some restrictions on microscope use.
Although the raw materials are available and all stains can be The requirements for cleanliness, hydration, and storage are
made up by the laboratory, it should be noted that this is a labori- the same as for glass slides. To prevent accumulation of dust and
ous process, and in general, more consistent staining results will be hydration, coverglasses should be kept in a closed container
obtained when ready-to-use stains are applied. when not in use.
Automated coverslipping instruments are available for high-
Evaluation of Stained Blood Films volume laboratories.
Macroscopically, a properly prepared and stained blood film
should be pink in its thin part and show a purple/blue tint in the Mounting Media
thicker parts. Microscopically, the red blood cells should be pink For permanent mounting of coverglasses, synthetic resins dis-
and the nuclei of the white blood cells more purple than blue. solved in inorganic solvents (toluene and xylene) have largely
There should be no or minimal precipitation, and staining should replaced Canada balsam (Canada turpentine or balsam of fir), which
be uniform throughout the slide. The blood cells should be free is unsatisfactory because of yellowing and incomplete drying.
from vacuoles (see “Fixation/Staining”). The mounting material should have low viscosity to ensure a
thin layer of adhesive and to prevent the formation of bubbles. It
Staining Artifacts and Potential Staining Problems should not affect optical analysis by diffraction problems or shift
Excessively pink staining can be the result of using a low pH of the color of biological stains. Antioxidants in the mounting
the buffer, insufficient staining, or excessive washing/rinsing. Staining medium can prevent discoloration of stains and of the medium
solution that is more than 4 weeks old, especially when exposed to itself. The material should not lose its adhesiveness over time.
air, may also cause pink staining because of the formation of formic The use of mounting medium as a cover for blood films with-
acid in methanol. Countermeasures: check the buffer pH, use freshly out coverglass is not recommended because of the tendency of these
made staining solutions with methanol that has not been exposed to preparations to gather dust and to be not as resistant as glass to
air, and make sure staining ingredients are correct. Sometimes the scratching and fingerprints when stored over long periods and
reagent lot may need to be changed. If commercial staining solutions when viewed multiple times.
are used and buffer pH and staining times are correct, it may be nec-
essary to try another lot. In cold climates, make sure the staining Labeling
solution did not freeze during transport. Blood films should be labeled clearly and labels should be resis-
Excessively blue staining can be the result of an alkaline pH of tant to smudging and the effects of fixing, staining, and cleaning
the buffer used, prolonged staining, or insufficient washing. blood films with inorganic solvents. The label should contain at
Thick films will also cause cells to appear more blue. Countermea- minimum patient name, date and time of collection, and sample
sures: check the buffer pH, use more diluent, and/or shorten the number. It is recommended that a definitive label also contain a
staining time. patient-specific identification number. Labeling by a printed label
Inadequately stained nuclei are often the result of insufficient at the time of preparation of the blood film is recommended
staining, whereas nucleated cells in aged specimens (>8 hours (applying the label to the back of the film may avoid staining of the
after blood collection), especially when stored at ambient tem- label if a platen-type stainer is used).
6 B. Houwen

Printed labels should be checked for possible fading over time Disposal of Slides
and for sensitivity to immersion oil and solvents commonly used in Used slides to be discarded should be put in designated contain-
the laboratory. Labeling of blood films by hand can be done using ers specifically labeled and used for discarding both biohazards and
pencil, crayon (not sensitive to alcohol or other solvents), perma- sharp objects.
nent marker pen, or diamond pencil.
Microscopy and Slide Handling
Biohazard For the reasons mentioned under “Biohazard,” the use of gloves
General precautions apply to handling all patient specimens is recommended while handling blood films and performing
including blood films, whether air-dried, fixed, or stained. microscopic analysis. Slides with square edges are more prone to
cut or puncture the skin or glove material than slides with
Specimen Handling and Blood Film Preparation rounded or beveled edges. Broken slides should be handled with
The preparation of blood films need not be especially hazardous, extreme care, and unless the blood film is irreplaceable, such slides
but care must be taken on opening blood containers, especially when should be discarded.
closed by a rubber stopper and not a screw cap. The removal of a rub-
ber stopper frequently leads to droplet or microdroplet formation and Utilization of Blood Films
must take place while covering the stopper with a wipe or similar, Blood films can be used in a number of ways. Most are used for
preferably in a biohazard hood and away from the operator’s face [9]. the routine clinical quantitative and/or qualitative analysis of
When properly handled, microdroplet and aerosol formation formed blood elements. Their use for morphological screening is
does not occur in a measurable manner when tubes with screw caps probably stronger than for purely quantitative purposes now that
are used [9]. Blood can also be safely obtained from sample tubes electronic white blood cell (WBC) differential analysis has been
by using a perforating device for the stopper of a blood container much improved. Blood films are also used for quality control/qual-
specifically designed for blood film preparation. ity assurance purposes and for the evaluation of automated instru-
ment-based methods [18,19].
Stained Blood Films
Intercalating dyes such as Romanowsky dyes are carcinogenic and Role of Manual Differential Count as a Reference Method
should be handled with care, avoiding skin or mucosal contact. It is For the role of blood film–based WBC differential counts as a
often assumed that staining blood cells with Romanowsky stains deletes reference method, please refer to National Committee for Clinical
infectivity, and in general, completely stained blood films are not con- Laboratory Standards (NCCLS) document H20-A [20].
sidered a biohazard. However, methanol fixation does not provide pro-
tection against HIV or hepatitis B and is unlikely to provide protection Considerations Apply
for certain types of other infections, such as those caused by prions. One consideration is that of cell distribution on wedge blood
film preparations; large cells such as monocytes may be pushed to
Partially Stained Blood Films the periphery and the feathered edge of the blood film.
Often, only part of the blood film present on the slide is
stained, and the unstained part is therefore a direct biohazard. General Morphology Issues
Poor recognition of certain blood cell types (monocytes, nucle-
Coverglass Mounting Media ated red blood cells, band forms, etc.) can lead to underreporting
Some of the materials and solvents used in mounting cover- and/or misclassification. Cell types such as hematologic progenitor
glasses are potentially carcinogenic and should be handled accord- cells or lymphocyte subsets are difficult or impossible to classify by
ing to the manufacturer’s instructions. morphological means alone.

Reagent Safety Rare Event Issues

The Romanowsky stains, methanol, alcohol, acetone, xylene, Abnormal cell types are particularly prone to underreporting.
toluene, and coverslip glues mentioned in this article are highly The relatively small number of cells counted, even when a total of
flammable and should be stored in safety cabinets designated for 400 WBCs are classified, leads to significant problems in terms of
flammable materials. When handling these reagents, their volatility accuracy and reproducibility, mainly because of Poisson error. This
and potential carcinogenicity should be taken into consideration. is particularly the case for cell types that are typically present in
small numbers, such as basophils.
Long-Term Storage Because of these considerations and particularly because of cell
If blood films are stored for long periods, exposure to light classification issues, alternative methods such as monoclonal anti-
should be avoided. Although coverslipping is recommended for body–based flow cytometry have become more popular as a refer-
long-term storage, deterioration of the films is unlikely to occur if ence method, especially for rare event analysis.
stained slides without coverslips are kept closely packed in a dark,
dry environment in closed containers or cabinets. Minimum Requirements for Acceptable Blood Film and Quality
Control Procedures
Disposal of Stains and Solvents Care should be taken to properly stain and adequately label
Romanowsky dyes and volatile solvents should be disposed blood films. Areas for morphological assessment should be large
of in accordance with local waste and disposal authority rules enough that at least 100 WBCs can be identified in samples with a
and regulations. WBC count within the reference interval for a specific individual.
 Blood Film Preparation and Staining 7

A good-quality blood film preparation should contain a sufficiently 2. Pawlick G, Relopez J. Kaiser Permanente interlaboratory abnormal cell
large area for morphological evaluation. study comparing slide quality of the Sysmex SP-100 automated slide
In a wedge blood film preparation, the best area for microscopy maker/stainer to manual technique. Sysm J Int. In press.
is where red blood cells barely touch each other. Spun films typi- 3. Stiene-Martin EA. Causes for poor leukocyte distribution in manual
cally have large monolayer areas. spreader-slide blood films. Am J Med Technol. 1980;46:624-632.
Cells should not be damaged by the preparation or staining pro- 4. Lebeck LK, Mast BJ, Houwen B. Flow cytometric white blood cell differ-
cedure or by excessive shear forces. Especially in spun films, care ential: a proposed alternate reference method. Sysm J Int. 1993;3:61-69.
must be taken to ensure that none of the cell types in the mono- 5. Bacus JW. Erythrocyte morphology and “spinner” blood film preparations.
layer are distorted by the preparative procedure. However, in cer- J Histochem Cytochem. 1974;22:506-516.
tain disease conditions such as CLL, it is difficult to prevent cellular 6. Megla GK. Automatic blood film preparation by rheologically controlled
damage to the affected cells, the lymphocytes. spinning. Am J Med Technol. 1976;42:336-342.
For repeated microscopic analysis, a coverslip should be used so 7. Wenk RE. Comparison of five methods for preparing blood smears. Am J
that removal of immersion oil does not damage the blood films for Med Technol. 1976;42:71-78.
future analysis. 8. Nourbaksh M, Atwood JG, Raccio J, Seligson D. An evaluation of blood
smears made by a new method using a spinner and diluted blood. Am J
Microscopy Clin Pathol. 1978;70:885-892.
Methods for proper microscopic analysis of blood films have 9. Wolley RC, Dembitzer HM, Herz F, Schreiber K, Koss LG. The use of a slide
been described elsewhere and are outside the scope of this article. spinner in the analysis of cell dispersion. J Histochem Cytochem. 1976;24:11-15.
10. Hull BP, Bakhiet N, Ryu J, Houwen B. Biohazard evaluation of laboratory
Quality Control hematology tests. Presented at the 7th International Symposium on Tech-
Blood films are a product of the laboratory and should therefore nological Innovations in Laboratory Hematology: Lake Tahoe, NV; 1994.
be subject to checks on their consistency and quality in terms of 11. NCCLS. Evacuated Tubes and Additives for Blood Specimen Collection. Vil-
minimum requirements for acceptability. It is the responsibility of lanova, PA: NCCLS approved standard document H1-A4; 1996.
the laboratory management to put such checking procedures in 12. NCCLS. Devices for Collection of Skin Puncture Blood Specimens. Villanova,
place. In an entirely manual environment, sufficient training and— PA: NCCLS approved guideline document H14-A2; 1990.
when necessary—remedial training should be offered to the labora- 13. NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Venipunc-
tory staff to ensure that blood films of sufficient and consistent ture. Villanova, PA: NCCLS approved standard document H3-A4; 1998.
quality are being prepared. In automated and semi-automated envi- 14. NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Skin
ronments, staff should be properly trained in instrument use, and Puncture. Villanova, PA: NCCLS approved standard document H4-A3;
records of instrument maintenance should be kept. 1991.
15. Gleeson RM. An improved method for thick film preparation using
Proficiency Testing saponin as a lysing agent. Clin Lab Haematol. 1997;19:249-251.
The laboratory can (and is recommended to) participate in 16. International Council for Standardization in Haematology (ICSH): ICSH
(external) proficiency testing programs, although such programs reference method for staining of blood and bone marrow by azure B and
mostly test the laboratory’s ability to properly stain a blood film eosin Y (Romanowsky stain). Br J Haematol. 1984;57:707-710.
and assess the morphological skills of the staff. In external profi- 17. Field JW. The morphology of malarial parasites in thick blood film prepa-
ciency testing programs, logistics and the aging of blood specimens rations. Part IV. The identification of species and phase. Trans R Soc Trop
usually prohibit the blood film preparation itself, especially when Med Hyg. 1940;34:405.
blood specimens are mailed out to the individual institutions. 18. Lewis SM. Blood film evaluations as a quality control activity. Clin Lab
Haematol. 1990;12(suppl 1):119-127.
19. Bentley SA. Quality control and the differential leukocyte count. Clin Lab
ACKNOWLEDGMENTS
Haematol. 1990;12(suppl 1):101-109.
The author is indebted for many helpful suggestions and com- 20. NCCLS. Reference leukocyte differential count (proportional) and evalua-
ments, especially from Dr. Mitchell Lewis at the Royal Postgraduate tion of instrumental methods. Villanova, PA: NCCLS approved standard
Medical School in London, England; Dr. Robert Raynor and his document H20-A; 1992.
colleagues at Beckman-Coulter Corp. in Miami, FL; and Mr. Terry 21. Henry JB, ed. Clinical Diagnosis and Management by Laboratory Methods.
Fawcett at Auscorp Marketing Consultants, Melbourne, Australia. 18th ed. Philadelphia, PA: WB Saunders; 1991.
A number of textbooks were used as general references [21-24]. 22. Chanarin I. Laboratory Haematology. Edinburgh, Scotland: Churchill Liv-
ingston; 1989.
23. Lee RG, Bithell TC, Foerster J, Athens JW, Lukens JN, eds. Wintrobe’s
REFERENCES
Clinical Hematology. 9th ed. Philadelphia, PA: Lea and Febiger; 1993.
1. Dacie JV, Lewis SM. Practical Haematology. 5th ed. Edinburgh, Scotland: 24. Beutler E, Lichtman MA, Coller BS, Kipps TJ, eds. Williams Hematology.
Churchill Livingston; 1975:604. 5th ed. New York, NY: McGraw Hill; 1995.