Instructors:

Chester Lloyd Berdan, RMT

Jan Renzo Besa, RMT

DRAWBACKS AND ERRORS

 Use of EDTA causes an in-vitro phenomenon
called platelet satellitosis
 wherein thrombocytes surround or
adhere to Neutrophils (there is
Whole blood collected in a Lavender (pink) topped antibodies produced)
tubes  Causing Pseudothrombocytopenia
 The most common and widely preferred  Can be prevented if whole blood is
specimen to be received in a Hematology anticoagulated with sodium citrate
Laboratory (whole blood in light blue topped
 Anticoagulant: disodium or tripotassium tube).
eythelendiaminetetraacetic acid (EDTA).  A 9 parts blood to 1part anticoagulant
ratio must be observed.
EDTA  This can be used for automated
 This anticoagulant chelates calcium which is analyzers.
vital for coagulation of the blood.  Clumping of platelets in a EDTA anticoagulated
 Liquid type is more preferred than its whole blood causes Pseudoleukocytosis and
powdered counterpart since it can mix with blood spurious thrombocytopenia
easily.  Coarse basophilic stippling and Dohle’s
 Good quality blood films can be obtained from a Bodies may disappear in standing in EDTA
whole blood-in EDTA if smears are prepared 2-3 anticoagulated blood
hours of drawing of specimen.  Dohle Bodies are remnants of ROUGH
 Main advantages of EDTA are that can be ENDOPLASMIC RETICULUM
made not promptly upon collection and prevents  Blood films made from EDTA tubes that
platelets from clumping, making it ideal for more remained at Room temperature for more than 5
accurate platelet estimate. hours causes echinocytic RBC,spherocytes,
 SOMETIMES USES PINK TOP BUT IT IS necrobiotic leukocytes and vacuolization of
REALLY FOR neutrophils which makes it unacceptable for
IMMUNOHEMATOLOGY(CROSS MATCHING) blood film evaluation
 Use of sodium citrate tubes may cause
NOTE: unintentional dilution of blood and must be
 Prepare EDTA after 2-3 hours of collection corrected in calculation to obtain accurate
 Preferred: 2 HOURS; The fresher earlier, the results
better  Use of Heparin produces a bluish background
 Because if mag dugay for more than 4 hours when stained with Wright’s or Leishman’s
ang blood, ga spherocytic, crenate, echinocytic stain. It causes cell clumping and distortion not
suitable for WBC and platelet count, especially
There are draw backs in using EDTA like platelet in automated cell counting
satellitosis which will be discussed later on. These can  Why heparin causes blue background?
be prevented if whole blood is anticoagulated with Heparin has carboxylic groups like
sodium citrate (whole blood in light blue topped amino acid or protein. Heparin is a
tube). A 9 parts blood to 1part anticoagulant ratio must derivative of proteins nga halin sa liver
be observed. This can be used for automated analyzers. and it is a natural anticoagulant. It is
acidic, in composition with amino acid
Skin puncture derivatives. That’s why if I stain mo ang
 Capillary puncture of finger and heel (usually for protein usually ma adapt siya blue color.
infants) This is because of the anticoagulant.
 Mixture of arterial and venous blood  Heparin is within a carboxylic functional
groups therefore acidic siya, it would
 This is made on the patient’s side right after
adapt a basic dye.(ACIDIC ATTRACTS
collection. BASIC DYE)
 But there are drawbacks in this technique such  Use of Oxalated anticoagulants and Heparin
as distorts WBCs and RBCs.
 clumping of platelets  These anticoagulants have a more
 limited availability of specimen when hypertonic concentration that’s why they
bleeding stops. would pull out water from cells and cells
 But this dilemma can be overcame with the use would tend to look like distorted cells.
of EDTA microtainer tubes.  DO NOT USE THESE
ANTICOAGULANTS
 Instructors:
Chester Lloyd Berdan, RMT
Jan Renzo Besa, RMT

Centrifugal Types
 are superior and have even cell distribution,
 Making of good quality blood films is a technique consistency of preparations, large examination
which needs practice and knowledge!!! area and fewer broken lymphoid cells (smudge
 The most important material to be used here cells) in Chronic lymphocytic leukemia (CLL)
aside from a properly collected specimen is a  This is for dasig ma destroy ang WBC, because
clean glass slide. in times of CLL and ALL ang imo cells dasig ma
 Glass sides can be cleaned by: lyse esp for CLL, damo smudge cells or basket
1. Immersing it in a warm sodium carbonate cells.
solution or detergent,  GENTLE FOR BLOOD SMEAR
2. Boiling or rinsing it with warm Extran  Drawbacks:
solution
 longer preparation time and cleaning
3. The common way is to rinse it with running
maintenance,
water. then distilled water and finally
immersing in a 1:1 ethyl alcohol mixture.  blood films cannot be done outside of
 Advantages of making a smear is that the laboratory
 it can be stored properly,  thinness of monolayer gives smaller
 can be labelled properly, amount of leukocytes,
 easy to handle and prepare and  no specific location of immature or
 it can be used in both manual and unusual cells
automated staining.  occasionally cell distortion is inevitable
due to the centrifugal force of the
COVERSLIP METHOD machine.
 This technique of blood film preparation has its
difficulties in performing but this is extensively MANUAL WEDGE TECHNIQUE (MOST COMMONLY
used for bone marrow preparations. (FOR USED)
BLOOD: TO PULL OUT TYPE)  Also called as wedge, spreader-slide or push
 Advantage smear preparation
 superior leukocyte distribution  Most convenient and often used method in
 Disadvantages making blood films.
 difficult to execute  This requires at least two 3 inch x 1 inch (75mm
 coverslips must be totally clean x 25mm) clean glass slides.
 difficulty in labelling,
 transporting and staining since it can be ADVANTAGES OF WEDGE TYPE PREPARATION:
easily broken  Technique is easier to master (able to
 uneven distribution of platelets in two understand its technique easily)
coverslips
 The slides are not easily broken
 lacks specific area for examination of
 Labelling, transporting, and staining are easier
unusual cells.
 A coverslip is not necessary, and smears can be
put in storage immediately.
 The combination of above assets makes this the
least expensive type of preparation
 Tendency of larger cells to settle at the slide
edges and the feathered end makes it easier to
find abnormal cells
AUTOMATED BLOOD FILMS  WHEN YOU PUSH THE BLOOD, THE
There are two techniques used for automated DENSER THE CELLS, THEY WOULD
preparation of blood smears it can be CLUMP/LOCATE THEMSELVES AT
THE MARGIN OR AT THE
a. Linear (Wedge or Push) type FEATHERED EDGE
b. Centrifugal (Spinner) type.

Linear Smears (COMMON)

 Shares advantages and disadvantages with the
manual wedge type but with added advantage of
consistency in blood films.
 Creates monolayer of cells, there is
consistency of blood smears
 Instructors:
Chester Lloyd Berdan, RMT
Jan Renzo Besa, RMT

DISADVANTAGES OF WEDGE TYPE PREPARATION:

 Inherent poor distribution of nucleated cells
 Neutrophils and Monocytes have a greater
tendency to appear on feathered end leading to
artifactually increased lymphocyte count
 Greater trauma to cells
during preparation causing
creation of numerous  Slides must be clean and grease free
“smudge” or “basket” cell  Use only a moderately sized drop of blood
(they are lymphocytes; it is  Must be made within 1-2 hours after collection (If
fragile for CLL patients) one volume only, 2 hours ang I follow. THE
 If bullet shape, UNEVEN FRESHER SPECIMEN THE BETTER)
and POOR DISTRIBUTION OF CELLS  Spreader slide should have clean and smooth
edge
PROCEDURE  Angle of the spreader slide should be decreased
1. Place a small drop of to approximately 30 to 45 degrees
blood (about 2 -3 mm in  Blood must be quickly dried
diameter or 0.05  If delayed ang drying, it would change
mL/5uL) approximately the morphology of the cells.
1 cm from the end of
the slide.
2. Place the smooth clean
edge of the spreader
slide on the specimen
slide.
3. Draw it backwards into
the drop of blood at an
angle approximately 30
to 45 degrees allowing the blood to flow evenly
across the edge of the spreader slide.  Smooth without ridges, waves or holes
(THINNER SMEAR, DECREASE ANGLE;  Thickest at the origin and gradually thin out
THICKER SMEAR, INCREASE ANGLE)  Occupies 2/3 to 3/4 the length of the slide and
4. Quickly, with a gentle pressure and an even should terminate at least 0.5 inch before the end
motion push the spreader slide over the entire of the slide
length of the specimen slide creating a wedge-  Thin end has a good feathery edge, slightly
shaped smear with thin feathery edge. rounded at the edge (finger shaped).
5. Air dry the blood film immediately.  Why bullet shaped smear is not
6. Label the film at the thick end using a lead recommended?
pencil. (If not frosted)  Uneven distribution of cells (Ang
 DO NOT USE BALLPEN iban ara sa feathered edge, ang
iba ma push sa iba na parts
sang smear)
 Makita ang abnormal cells sa
margin or lateral edges.
Regardless of method used for preparation all bone
marrow smears and blood films should be dried as  DAPAT VISIBLE ANG
quickly as possible to avoid drying artifact effect. ORIGIN/LATERAL EDGE
Blowing breath may cause echinocytic RBCs or water  Lateral edges are visible (Diri makita ang
artifact formation (drying artifact). Effective drying abnormal cells)
methods could be  Feathery edge of the film has a rainbow
 Air drying or with the aid of a small fan appearance when held up to light
 Use of low flame or oven (heat fixation)  All of the initial drop of blood are incorporated
 Immersion in Methyl alcohol or 95% Ethyl into the film
alcohol  It should contain at least 10 low power field
(correct field to view) in which 50% of the
erythrocytes do not overlap and the remainder
may overlap slightly. Single erythrocytes
have a well preserved central pallor (DIRI KA
MAISIP)
 Instructors:
Chester Lloyd Berdan, RMT
Jan Renzo Besa, RMT

Based on the picture:

a. higko ang smear, ang end san spreader may
chipped
b. ga untat-untat while spreading
 Size of drop of blood (about 2 -3 mm in c. too high ang angle
diameter) d. drop of blood is too small
 Angle between the spreader and slide (30 to 45 e. bullet shaped; uneven distribution
degree angle) f. may grease ang slides, mahigko ang slides
 Speed of the spreader (SMOOTH) g. uneven pressure
 Pressure on the spreader slide (EVEN) h. uneven pressure
 Hematocrit level

NOTE:
 2-3 mm is the approximately the correct size of
the blood Pure Wright stain or Romanowsky stain
 INCREASE ANGLE of the spreader slide –  composed of methylene blue, azures of
thicker smear methylene blue and eosin dyes
 DECREASE ANGLE of the spreader – thinner  used to stain peripheral blood film and bone
smear marrow smears.
 2/3 or ¾ of the slide must be occupied  These stains are considered a polychrome
 Ang speed mag smear is SWA-BEH – even or stains for they both contain eosin and
smooth ang motion pag smear methylene blue.
 Hematocrit level – you would adjust the angle
and volume; example if low HCT, increase the NOTE:
blood volume, increase the angle; if dehydrated,  Wright stain – mostly used stain
with polycythemia vera, you would lesser the  Staining of PBS is important in order to
amount of blood and decrease the angle visualize the blood cells.
 Polychrome stains which as a both acid and
basic stain and damo nga color ahg ma impart
Hence to the thickness and thinness of the smear is sa stain
regulated by:
 Adjusting the amount of blood used in making As the principle of electrical attraction
the drop –  Those negatively charged parts of the cell
 Altering the speed with which the drop is (anionic components) such as the
smeared (faster speed, shorter smear; slower nucleoproteins and nucleic acids of the
speed, longer smear) nucleus and other cytoplasmic proteins have
 Altering the angle at which the spreader slide is high affinity to basic dyes) such as methylene
used. (thicker: increase angle; thinner: decrease blue making them Basophilic.
angle)  Meanwhile some cytoplasmic components
(e.g. haemoglobin, mitochondria) are
positively charge making their attraction toward
acidic dyes like Eosin making them acidophilic.
 Basophilic parts appear in shades of blue
meanwhile acidophilic areas varies from
orange to red.
 Instructors:
Chester Lloyd Berdan, RMT
Jan Renzo Besa, RMT

 Some areas of the cell are neutral between the 5. Add twice as much buffered distilled water to the
two dyes making them display colors such as slide. (Phosphate buffer solution can also be used:
purple. for Blood and Bone marrow- pH 6.4 to 6.8, for
Blood parasites pH 7.2
6. Leave to stain for at least 3 minutes.
NOTES: 7. Wash off with tap water
 Color blue (negatively charged)– nucleus, ER, 8. Air dry in a vertical position.
ribosomes
 Color pink/red (positively charged) – cytoplasm, NOTES:
mitochondria  Stains should be filtered. Why? Kung iprolong nga
ma expose sa air, it would oxidized – makita na mag
internship ga bilog bilog sa ibabaw or may ga crystal-
crystal. So if magdip ka sang smear, there is a
possibility of obscuring of cells.
 Always filter stain to avoid stain precipitates.
1. Coating procedure (Flooding technique or rack  Fix the smear with methanol before staining para ma
method) adhere nag gin smear sa slide, kay if hindi ma fix sa
2. Immersion procedure Incubation or Dip methanol, madula sa slide ang gin smear.
technique)  Ethanol cause lysis of RBCs so methanol is used.
 Tap water ang ginagamit magremove sang stain.
 Air dry in vertical position.
NOTES:
 You would know ang ginprepare mo nga smear
 Hospitals usually use immersion technique or manami if sa green metallic sheen ang slide
dip technique
 Gram stain is a flooding technique

1. Appears more pink when examined macroscopically

1. Platen type (spiral conveyor)- uses 2. Microscopically:
polychrome stain, buffer and rinse solution a. RBCs appear red orange or salmon pink
2. Carousel type – sprays a measured amount of b. WBC
stain on smear and transported in rotating  Nuclei:
carousel  Lymphocytes and neutrophils –
3. Dip type instrument- use dip or immersion dark purple
technique similar to those of traditional histology  Monocytes - lighter purple
 Granules:
 Eosinophils - bright orange
 Basophils - dark blue
 Cytoplasm:
1. Hemacolor – rapid staining using an immersion  Monocytes – blue gray or faint
method (a fixative and two stains) bluish
 Neutrophil - light pink with lilac
2. Sangodiff staining foils – highly transparent
granules
foils, specially coated with mixture of stain  Lymphocytes- clear blue or
(Romanowsky stain) robin’s egg blue
c. Platelets: violet to purple

NOTES:
 Neutral - light pink or lilac
granules
Procedure:  Too alkaline smear
Note: Staining requirement is o RBCs – violet
subject to the availability and o WBCs – dark violet
applicability of staining materials  If there is platelet satellitosis
provided by the Medical Technology o REPEAT collecyion
Laboratory. o Use sodium citrate
1. Make a thin film and air dry
rapidly.
2. Filter the stain prior to use.
3. Place the film on a staining rack with the right side
facing upward.
4. Flood with the stain and leave for 1 - 3 minutes to fix
the cells.
 Instructors:
Chester Lloyd Berdan, RMT
Jan Renzo Besa, RMT

FACTORS AFFECTING THE SMEAR

PROBLEM/ERROR CAUSE EFFECT TROUBLESHOOTING

Too Thick smears a. Too large drop of  Excess plasma a. Adjust amount of
blood causes nucleated blood
b. Too fast spreader cells to shrink and b. Maintain smooth
speed satin intensely, motion and even
making pressure
c. Too high angle of
identification c. Decrease angle
the spreader
difficult. RBCs form
d. Polycythemia vera more rouleaux. d. Decrease spreader
(a,b,c) slide angle and
decrease push
speed

Too thin smears a. Too small drop of  Smudge cells are a. Adjust amount of
blood increased and blood
b. Too slow spreader erythrocytes b. Maintain smooth
speed become artificially motion and even
c. Too low angle of spheroid with a pressure
distorted shape.
the spreader c. Increase angle
Nucleated cells to
d. Anemia be carried out to d. Increase spreader
the edges of the slide angle and
smear affecting decrease push
accuracy of speed
differential counts.

Gritty appearance of a. Large number of  Uneven distribution b. adjust spreading speed

feathered or tail areas leukocytes of cells (b,c) or c. pick up whole drop of
b. Slow or delay in causing holes or blood before pushing
Spreading streaks in the blood d. clean spreader slide
film or may carry before and after each
c. Spreading partially
over previous patient or better change
the drop of blood
specimen to slide spreader slide
d. Rough edge or dirty (d)
spreader

Presence of crenated a. Blowing breath on  Erythrocytes Dry smear quickly and

RBCS slide appear echinocytic thoroughly
b. Delay in drying of or develop a drying
smear artifact

Agglutinated RBCs a. Presence of IgM Blood films appears sandy Warm blood at 37 degrees
associated with cold autoantibodies with multiple agglutinated Celsius for 15 minutes
agglutinin disease RBCs before smearing

Increase viscosity a. Multiple myeloma RBCs form more rouleaux. Decrease spreader slide
angle and push speed
 Instructors:
Chester Lloyd Berdan, RMT
Jan Renzo Besa, RMT

POTENTIAL ERRORS IN STAINING AND THEIR REMEDIES

PROBLEM POTENTIAL CAUSES REMEDIES
Excessively blue or dark (too  prolonged staining  Check pH of stain, buffer and
Alkaline smear)  inadequate washing water
 RBCs appear gray  too alkaline stain and/or  Shorten staining time
 WBCs are too dark buffer (>pH 6.8)  Prolonged buffering time
 Eosinophil granules are  Thick blood smear  Check the incubation time of
gray not orange  too little buffer for stain the stain
solution
 old smear
 Protein abnormality (multiple
myeloma)
 Heparin blood sample
 Leukocytosis with many
blasts
 Low hematocrit

Excessively pink or light  Insufficient staining  Prolong staining time

 RBCs are too pale or are  Too acidic stain and/or buffer  Check the pH of the stain,
red (< pH 6.4) buffer and correct with alkali if
 WBCs are barely visible  Excess buffer for stain necessary
solution  Shorten the buffering time
 Very thin smear
 Contaminants (e.g. chlorine)
in wash water
 Exposure of buffer or stain
solution to acid fumes
 Old stain in which methanol
has oxidized to fumic acid

Presence of precipitates  unclean slides  Use clean and grease free

 drying during staining process slides
 inadequate filtration of the  Re-dissolve precipitate with
stain additional stain by covering
smear with Wright’s stain for
5 to 10 seconds and flushing
with distilled water or
deionized water
 Dip the slides 3 to 4 minutes
in a solution of 30% ethanol
in Distilled water, air dry &
examine microscopically

Blood smear being washed off the  Inadequate fixation  At least 1 minute of fixation
slide  Too thick blood smear

Drying artifact such as:  severe anemia causing  Check buffer and fixative
 moth eaten RBCs excessive plasma  Dry smears quickly
 heavily demarcated central  humid environment  Used 10% volume to volume
pallor  inadequate fixation period methanol
 refractive blotches on the  water contamination  Keep a stopper tightly on the
RBCs  excessive buffering stain bottle
 crenated RBCs