EXAMINATION OF THE PERIPHERAL BLOOD FILM

Microscopic examination of the peripheral blood is done by preparing, staining, and

examining a thin smear of blood on a slide. With the use of automatic counting devices
that determine hemoglobin, hematocrit, and red cell, white cell, and platelet counts,
together with MCV, MCH, MCHC, RDW, WBC differential, and histograms, there is a
tendency to place less emphasis on the routine examination of the peripheral blood film

Pemeriksaan darah tepi secara mikroskopis dapat dilakukan melalui

proses persiapan, pewarnaan dan pengamatan apusan tipis darah pada
slide kaca. Penggunaan alat otomatis untuk menghitung kadar
hemoglobin, hematokrit serta jumlah eritrosit, leukosit maupun trombosit
hingga dapat diketahui perhitungan MCV, MCH, MCHC, RDW, Hitung
jenis leukosit serta histogramnya, menyebabkan makin jarangnya
pemeriksaan sediaan apusan darah tepi menjadi pemeriksaan rutin
utama.

EXAMINATION OF THE PERIPHERAL BLOOD FILM

Traditionally, the microscopic examination of the peripheral blood film has been used to
study the morphology of RBCs (erythrocytes), WBCs (leukocytes), and platelets. The
percentage of each cell type present in a peripheral blood film can be determined by
direct microscopic observation, the white blood cell (leukocyte) differential. An additional
examination of the bone marrow may be necessary in certain cases, but this is not a
routine procedure. Platelets are also routinely assessed in clinical hematologic studies by
observing their number and morphology in the peripheral blood film.

Pada asalnya, pemeriksaan mikroskopis sediaan apusan darah

telah lama dimanfaatkan untuk mengetahui morfologi eritrosit,
leukosit dan trombosit. Pemeriksaan mikroskopis langsung untuk
menentukan persentase masing-masing sel yang ada pada
sediaan darah tepi dikenal dengan nama hitung jenis leukosit.
Pada beberapa kasus tertentu, diperlukan tambahan pemeriksaan
melalui sampel sumsum tulang, meskipun ini bukan prosedur yang
rutin. Pada lingkup hematologi klinis, jumlah dan morfologi
trombosit juga bisa ditentukan secara rutin melalui sediaan apusan
darah tepi

SOURCE OF BLOOD FOR THE BLOOD FILM

Fresh blood from a finger or heel puncture can be used for morphologic examination of
the white and red cells. CLSI has deleted the use of the big toe as a site for collecting
peripheral blood because of the lack of documentation supporting or discouraging
blood collection from this site. The finger must not be squeezed excessively to obtain the
drop of blood, and it must not be touched with the slide. Only the drop of blood should
touch the slide. Any oils or moisture from the finger will lead to a poorly prepared film.

Sampel darah segar yang terambil dari punksi ujung jari ataupun
tumit bisa dijadikan bahan untuk memeriksa morfologi leukosit dan
eritrosit. CLSI tidak lagi merekomendasikan sampel darah tepi
diambil dari ibu jari kaki karena sangat minimnya data-data ilmiah
yang mendukung hal ini. Tidak dianjurkan untuk memijit-mijit jari
secara berlebihan agar darah lebih mudah keluar, begitu pula tidak
diperkenankan ujung jari menempel pada slide kaca.

PREPARATION FOR THE BLOOD FILM

Blood is most often examined under the microscope by preparing a thin film or smear of
blood on a slide or a coverglass, fixing the blood film, then staining it with a polychromatic
stain (Fig. 12-14). Smears can also be prepared by centrifugation; centrifugal force is
used to spread a monolayer of blood cells over the surface of a slide as soon as possible,
certainly within 2 hours.

Pemeriksaan darah di bawah mikroskop paling sering dilakukan

melalui pembuatan apusan tipis tetesan darah pada sebuah slide
kaca atau kaca obyek, memfiksasinya kemudian mewarnainya
dengan pewarna polikromatik (bisa dilihat pada gambar 12-14).
Sediaan apusan juga bisa dibuat melalui proses sentrifugasi,
dengan menggunakan kekuatan putaran untuk menghasilkan
selapis tipis sel-sel darah di atas permukaan lempeng kaca segera
setelah sampel di dapat dalam kisaran waktu 2 jam.

PREPARATION FOR THE BLOOD FILM

COVERGLASS BLOOD FILM

When a coverglass is used to prepare a blood film, more of the prepared film can be
examined, which reduces the sampling error. With a slide, only a relatively small counting
area can be examined. In addition, the leukocytes and platelets are more evenly
distributed on a coverglass. The disadvantages of the coverglass method are that it is
more time consuming, more difficult to learn and perform correctly, and requires more
care in handling of the preparations. Automatic staining devices are available for slides,
but not for coverglasses.

Jika sediaan apusan darah dibuat menggunakan kaca penutup

obyek maka akan lebih banyak sediaan yang dapat diperiksa
sehingga mengurangi kemungkinan kesalahan sampel. Berbeda
halnya jika menggunakan slide kaca, hanya sebagian kecil area
sel yang dapat diperiksa. Kelebihan lainnya adalah distribusi
leukosit dan trombosit lebih merata di atas kaca penutup obyek.
Kekurangan metode ini adalah waktu pemrosesan yang lebih
lama, lebih sulit untuk diamati dengan tepat serta membutuhkan
proses persiapan yang lebih repot. Mesin otomatis untuk
pewarnaan apusan darah hanya tersedia untuk metode slide kaca
tetapi tidak untuk metode kaca penutup obyek.

WEDGE BLOOD FILM

Although the coverglass method is recommended
by many hematologists, the slide (push-wedge)
blood film method is much more frequently used
(Fig. 12-15 and Procedure 12-3). It is the method
used by CLSI for the reference leukocyte differential
count to evaluate any leukocyte method.6 The
directions for the examination of the blood film
can also be applied to the coverglass method. The
correct interpretation of a blood film requires:
1. Correct preparation of the blood film
2. Proper staining
3. Accurate examination

BLOOD FILM

WEDGE BLOOD FILM

The equipment used for making blood films
must be meticulously clean. Use of a spreading
device is recommended; for example, a margin-free
spreader slide with ground-glass edges may be used
to spread the film of blood on the clean, lint-free
slide. The edges of the spreader slide must be clean
and free from chips. Coverglasses held by a suitable
clip or holder can also be used as spreading devices.
The spreading device must be cleaned thoroughly
with alcohol and dried between films, and it must
be discarded when chipped or broken.

CYTOCENTRIFUGED BLOOD FILM

With the use of a cytocentrifuge, a monolayer of
cells can be prepared. These centrifuges facilitate
rapid spreading of the cells across a slide from a
central point by virtue of their high-torque, low-inertia
motors. With a cytocentrifuged preparation, cellular
destruction and artifacts present in the wedge
slide method are eliminated. Only small volumes of
a sample are used, and the cells are evenly
distributed and less distorted, producing better
conditions for critical morphologic studies. They are
especially useful for other body f luids, such as
cerebrospinal, pleural, or synovial f luid or urine.

CRITERIA GOOD BLOOD FILM

1. The body of the blood film should be smooth
and not interrupted by ridges, waves, or holes.
2. The blood smear should be thickest at the
origin and gradually thin out, rather than
having alternating thick and thin areas.
Pushing the spreader slide with an uneven
motion results in thicker and thinner areas
in the body of the film.
3. A good blood film should cover half to threefourths
the length of the slide. All of the initial drop of blood
should be incorporated into the film, not just part of
it.

CRITERIA GOOD BLOOD FILM

4.
The thin end of the smear should have a
good feather edge. The film should fade
away without a defined border on the end. In
some institutions, a fairly straight feather
edge is sought; others prefer a more
tonguelike edge.

CRITERIA GOOD BLOOD FILM

5.
A defined border at the end of a blood film indicates
that most of the WBCs have piled up at the end.
When this occurs, the heavier neutrophils accumulate
at the end to a greater extent than the other WBC
types, giving an incorrect distribution of the WBC
types in the body of the smear. Platelets also tend to
accumulate at the end of a smear, decreasing the
number in the body of the smear. This will also result
in inaccurate percentages for the cell types within the
body of the film, because the relatively stickier
neutrophils and platelets tend to concentrate in such
tails.

CRITERIA GOOD BLOOD FILM

6.
Slides should be made in one motion. The drop
of blood should be placed on the slide and the
smear made immediately; drying of the blood
drop will lead to uneven distribution of cells in
the body of the film, and the larger WBCs will
accumulate at the end. Rouleaux formation of
the RBCs and platelet clumping will also occur
if the blood is not spread immediately.

CRITERIA GOOD BLOOD FILM

7.
Pressing down on the spreader slide will also
lead to an accumulation of WBCs and platelets
at the end. This is why the spreader slide
should be evenly balanced between the finger
and thumb.

CRITERIA GOOD BLOOD FILM

8.
The degree of thickness or thinness of the blood film is
also important. When a film is too thick, the cells pile up,
making them difficult to count and obscuring their
morphology. A very thin film is satisfactory for
morphologic studies, but it may be tedious to examine.
The thickness of the film is determined by the size of the
drop of blood used, the speed of the stroke used to
move the spreader slide, and the angle at which the
spreader slide is moved. A thick film results when the
drop of blood is large, the angle is greater than 45
degrees, and the spreading motion is fast. A thin film
results when the drop of blood is small, the angle is less
than 30 degrees, and the motion is slow.

CRITERIA GOOD BLOOD FILM

9.
Blood films with vacuoles or bubbles result
from the use of dirty slides or in some cases
from an excess of fat in the specimen (e.g.,
specimen obtained after a fatty meal).

UNACCEPTABLE SMEARS

UNACCEPTABLE SMEARS

WELL MADE SMEARS

STAINING BLOOD FILM

The stain most often used for the examination
of blood films is Wrights stain or a variation,
Wright-Giemsa stain (Procedure 12-4). Both Wrights
and Wright-Giemsa stain are adaptations of
polychrome Romanowsky stains. Such polychrome
stains produce multiple colors when applied to cells
because these stains are composed of both basic
and acidic aniline dyes. Romanowsky stains contain
methylene blue (a basic dye), eosin (an acidic dye),
and methylene azure (an oxidation product of
methylene blue also referred to as polychrome
methylene blue). Variations of the Romanowsky
stains differ in the way the methylene azure is
produced or added to the stains.

STAINING BLOOD FILM

They are called basophilic because they stain with

the basic dye. The more basic cell components,
such as hemoglobin and eosinophilic granules, are
stained orange to pink and are called acidophilic
because they stain with the acidic dye. Some
structures within cells stain with both components,
such as the neutrophilic granules, whereas the
azurophilic granules stain with methylene azure.

RAPID STAIN BLOOD FILM

Modification of Wrights stain has been incorporated
into various commercially available quickstaining
methods. Blood films are prepared in the classic
way, usually by the wedge method, and then
stained. The advantage of these products
is that they are faster to use than the traditional
Wright-staining method described earlier. Slides
are usually dipped into each of the various staining
reagents, with only a few seconds being taken
for each step. For critical morphologic studies, the
traditional Wrights stain should be used because
some of the morphologic detail is lost when certain
quick stains are used.

CHARACTERISTIC STAIN BLOOD FILM

The RBCs should appear red-orange through
the microscope. Correctly stained leukocytes
should have the following colors under the microscope.
The lymphocytes and neutrophils should have dark-purple
nuclei, and the monocyte nuclei should be a lighter purple.
The granules should be bright orange in the eosinophils
and dark blue in the basophils. The appearance of the
cytoplasm varies with the type of leukocyte. In monocytes
the cytoplasm should be blue-gray or have a faint bluish
tinge. The neutrophil cytoplasm should be light pink with
lilac granules, and the lymphocyte cytoplasm should be a
shade of blue, generally clear blue or robins-egg blue. The
platelets should stain violet to purple. If the blood film does
not meet these criteria, it should be discarded, and a new
film should be stained and examined.

PRECAUTION STAINING BLOOD FILM

1.
When the blood film is being stained, it is important that the
staining rack be level so the stain is uniform throughout the
film.
2.
It is important that the stain and buffer be made correctly.
When prepared in the laboratory, the stain should stand for
1 month before it is used. Wrights and Wright-Giemsa
stains can be purchased ready to use. These reagents
must be checked out carefully before being used for daily
staining needs.

PRECAUTION STAINING BLOOD FILM

3.
The pH of the buffer must be correct. With
every new batch of stain and buffer, the fixing
and staining times should be checked by staining
a few slides. If staining of the cells is satisfactory,
the times used for fixing and staining
should be noted and used with that batch of
reagents. If the pH is too acid or too alkaline,
the stain will give a false color and appearance
to the cells.

PRECAUTION STAINING BLOOD FILM

4.
Adequate fixing time must be allowed. A minimum
of 3 minutes is recommended for the initial
reaction of the blood film and Wright-Giemsa stain.
Inadequate fixation allows dissolution of the
nuclear chromatin, so overfixation is preferable. To
achieve the proper staining reactions in the cells,
the correct timing must be determined for each
batch of stain and buffer, and the correct staining
technique must be used.

PRECAUTION STAINING BLOOD FILM

5.
Properly applied Wright-Giemsa stain dyes both acidic
and basic components of the blood cells. The
phosphate buffer controls the pH of the staining
system. If the pH is too acid, the parts of the cell
taking up acidic dye will be overstained and will
appear too red, whereas the parts of the cells taking
up basic dye will appear pale. If the pH of the staining
system is too alkaline, the parts of the cells taking up
basic dye will be overstained, giving an overall blue
effect, with dark-blue to black nuclear chromatin
and bluish RBCs.

MICROSCOPIC EXAMINATION BLOOD FILM

Low-power examination (10 objective) includes:

1. Evaluation of the overall quality of the blood film
2. Estimate of the leukocyte count
3. Scan of the blood film for abnormal cells and
clumps of platelets

MICROSCOPIC EXAMINATION BLOOD FILM

Oil-immersion examination (100 objective)

includes:
1. Examination of the erythrocytes for alterations
and variations in morphology
2. Estimation of platelet count and evaluation
of morphologic changes
3. Differential count of the leukocytes
4. Examination of the leukocytes for morphologic
alterations