MLS 113A: HEMATOLOGY

PRELIM | LABORATORY
Lesson 3: HEMOGLOBINOMETRY AND RBS INDICES
HEMOGLOBIN (HB) Right shift : (WONT HOLD TIGHT) > CADET faces
• (Hb or Hgb) is a protein in red blood cells that RIGHT
carries oxygen throughout the body. • indicates decreased oxygen affinity of
• In severe cases of low hemoglobin count - may hemoglobin allowing more oxygen to be
indicate anemia available to the tissues.
4 TYPES OF HEMOGLOBIN o Increase CO2
1. HbA1: the most common type, accounting for o Increase acidity (decrease pH)
about 95% of the Hb in adults. o Increase 2,3 DPG
2. HbA2: the second most common type. o increase exercise
3. HbF: the third most common type. o Increase temperature
4. HbS: the least common type. TISSUES, MUSCLE, PLACENTA (more oxygen
Note: demand; decrease affinity of oxygen to tissues)
Hemoglobin A1 is the most common type of Example during exercise or renal failure
hemoglobin found in adults, accounting for about o During exercise, muscles use up oxygen to
95%-98% of hemoglobin. It contains two alpha (α) convert the chemical energy in glucose to
chains and two beta (β) protein chains. mechanical energy (O2 comes from Hb in the
FUNCTIONS blood)
• Imparts red color to the blood o CO2 and H+ removed/released from the muscle
• Vehicle for transportation of O2 and CO2 via blood
• 1 Hb = carry 1.34mL O2 o There will be an increase CO2 and increase
• 1 Hb = 3.47mg iron acidity in the blood (decrease pH)
• Adult RBC mass with 600g Hb = carries about o Hb will bind the H+ and release oxygen to
800mL O2 maintain the pH to normal
o Increase 2,3 DPG
o Increase temp
Left Shift (WONT LET GO) > LUNGS
• indicates increased oxygen affinity of
hemoglobin allowing less oxygen to be available
to the tissues.
o Decrease CO2
o Decrease acidity (increase pH)
o Decrease 2,3 DPG
OXYGEN SATURATION o Decrease temp
• In general, hemoglobin can be saturated with o Increase Hb F – helps in oxygenation of fetus
oxygen molecules (oxyhemoglobin) or Example: HYPERVENTILATION
desaturated with oxygen molecules o Too much CO2 is expelled from the lungs
(deoxyhemoglobin). o Decrease CO2 in the blood
a) Oxyhemoglobin o Decrease acidity (increase pH)
o Oxyhemoglobin is formed during o Hb will bind to oxygen and release H+ to
physiological respiration when oxygen binds maintain blood pH
to the heme component of the protein o 2,3 DPG will be released from erythrocytes
hemoglobin in re blood cells. HEMOGLOBIN DERIVATIVES
Hb + O2 = OxyHb Protein normal but iron and nature of organic group
(in arterial blood; bright red) changed
b. Deoxygenated hemoglobin • Oxyhemoglobin
o Deoxygenated hemoglobin is the form of • Methemoglobin
hemoglobin without the bound oxygen. • Sulfhemoglobin
Hb + C02 = Deoxygenated or Reduced Hb • Carboxyhemoglobin
(venous blood ; dark red)
 OXYHEMOGLOBIN During oxidation of Hb, sulfur (from some source,
• each heme group is associated with one which may vary) is incorporated into heme rings of
molecule of O2 Hb, resulting in a green hemochrome. Further
• formed during physiological respiration when oxidation usually results in the denaturation and
oxygen binds to the heme component of the precipitation of Hb as Heinz bodies ("bite cells").
protein hemoglobin in red blood cells. SHb cannot transport O2, but it can combine with
Hb + O2 = OxyHb (in arterial blood ; bright red) carbon monoxide (CO) to form
• a bright red substance formed by the carboxysulfhemoglobin. Unlike methemoglobin,
combination of hemoglobin with oxygen, SHb cannot be reduced back to Hb, and it remains
present in oxygenated blood. in the cells until they break down.
• Hemoglobin is a protein molecule that binds to CARBOXYHEMOGLOBIN (HbCO)
oxygen. Hemoglobin forms an unstable, • CO combined with hemoglobin
reversible bond with oxygen. In its oxygen- • Hb has the capacity to combine with CO with an
loaded form, it is called oxyhemoglobin and is affinity 210 times greater than O2
bright red. In the oxygen-unloaded form it is o Found in tobacco smokers, traffic officers
called deoxyhemoglobin and is purple-blue o Cherry red color of blood
Deoxygenated hemoglobin HEMOGLOBIN IS INCREASED IN
• is the form of hemoglobin without the bound • Hyperchromia
oxygen. • High altitude
Hb + C02 = Deoxygenated or Reduced Hb • Polycythemia
(venous blood ; dark red) • Dehydration associated with burns and diarrhea
METHEMOGLOBIN (Hi) • Heart diseases
• known as hemiglobin HEMOGLOBIN IS DECREASED IN:
• ferrous iron is oxidized to the ferric state, • All anemias
resulting in the inability of Hi to combine • Leukemia
reversibly with O2 • Oligochromia
• Maybe acquired or inherited METHODS IN HEMOGLOBINOMETRY
o inherited – structural abnormality in the 1. Colorimetric method
globin chains a) Direct visual
o acquired due to certain drugs and • Acid Hematin
chemicals (ex. Nitrates, nitrites, quinones, • Alkaline Hematin
b) Photoelectric Colorimetric Method
chlorates)
c) Coulter Counter Method
• Cause chocolate brown discoloration of blood ACID HEMATIN
• Methemoglobin formation is reversible and is PRINCIPLE
normally present in the blood in concentrations
• Acid hematin method (Sahli's Method) is quite
of 1 – 2 %
easy that when the blood is added to N/10
Reversible by:
Hydrochloric acid (HCl), the hemoglobin
• Methemoglobin reductase (diaphorase) – present in RBCs is converted to acid
enzymatic conversion – metHb pathway; uses hematin which is a dark brown colored
cofactor NADH (reduced) ; NADH was produced
compound.
in Embden Meyerhof pathway…
• the normality of a solution is the gram equivalent
• Other enzymes: catalase, SOD, hydrogen weight of a solute per liter of solution
peroxide
therefore n/10 is 0.1, therefore it is o.1 normal
• Non-enzymatic: ascorbic acid and glutathione
solution which means 0.1 gram per liter of
SULFHEMOGLOBIN (SHb) solution.
• sulfur is incorporated in heme rings of Hb during
oxidation
• Irreversible
• Mauve lavender color of blood
• Not normally found in the blood
• Irreversible
• Can combine with carbon monoxide to form
carboxysulfhemoglobin
 ALKALINE HEMATIN • Drabkins solution -pH 7.0 -7.4
• Alkaline Hematin-D (AHD) Method (Cyanide- o Potassium ferricyanide
Free) o Potassium cyanide
PRINCIPLE: o Potassium dihydrogen phosphate
• When a blood sample is added to o Non-ionic detergent
an alkaline solution containing a non-ionic o Distilled water
detergent, haemoglobin is converted to alkaline • Erythrocytes are lysed producing an evenly
haematin which is a stable colour compound. distributed Hb solution.
METHOD • Potassium ferricyanide converts Hb to
• In this method the Hb is converted to alkali methemoglobin.
hematin by the addition of N/10 NaOH • Methemoglobin combines with potassium
• The alkali hematin gives a brown color that can cyanide to form cyanmethemoglobin.
be read against comparator standards or in a • All Hbs present in blood are converted to this
colorimeter form.
• Apparatus • Absorbance is measured in spectrophotometer
o Photoelectric meter with green filter at 540 nm
o N/10 NaOH • To obtain amount of unknown b sample, its
o 0.05 ml pipette absorbance is compared with the standard
• Standard (Gibson’s and Harrison’s): This is a cyanmethemoglobin solution
mixture of chromium potassium sulphate, COULTER COUNTER METHOD
cobaltous sulphate, and potassium dichromate • The Coulter method of sizing and counting
in aqueous solution. The solution is equal in particles is based on measurable changes in
color to 1 in 100 dilution of blood containing electrical impedance produced by
16.0 Hb per dl nonconductive particles suspended in an
PROCEDURE electrolyte. A small opening (aperture) between
• Dispense 2.0 mL of the reagent in a clean test electrodes is the sensing zone through which
tube and add accurately 20 μL of whole blood. suspended particles pass.
Mix well and read at 578nm (540 nm – 580 nm). Normal Range:
The colour produced is very stable. Category: Men: 13.5 to 17.5 grams per deciliter
Hemoglobin Reagents Women: 12.0 to 15.5 grams per deciliter

PHOTOELECTRIC COLORIMETRIC METHOD

CYANMETHEMOGLOBIN
• The method of choice for hemoglobin
determination (a type of colorimetric method).
PRINCIPLE
• when blood is mixed with a solution containing
potassium ferricyanide and potassium cyanide,
the potassium ferricyanide oxidizes iron to form
methemoglobin.
• Blood is mixed with Drabkins solution
 MLS 113A: HEMATOLOGY
MIDTERM | LABORATORY
LESSON 1: WBC AND RBC COUNT
HEMOCYTOMETER COUNTING RULE
Hemo: blood Do NOT count cells touching
Cyto: cell - Bottom line
Meter: measurement/counter - Right line
Thus, it is an instrument used to count the blood This is to avoid double counting
cells.
It includes:
• Neubauer’s slide- the name given to a thick
glass slide. In the center of the slide, there is an
H-shaped groove
• Coverslip
• RBC pipette
• WBC pipette
NEUBAUER’S CHAMBER- Neubauer’s slide with a
cover slip over it.
DIFFERENCE BETWEEN RBC AND WBC
PIPETTE
 DILUTING FLUIDS FOR RBC COUNT
a) Hayem’s solution
Mercuric chloride 0.5 gm
Sodium sulfate (anhydrous) 5.0 gm
Sodium chloride 1.0 gm
Distilled water 200 ml
b) Gower’s solution
Sodium sulfate 12.5 gm
Acetic acid (glacial) 33.3 ml
Distilled water 200 ml
c) NSS (0.85 % Sodium chloride)
d) Bethel–Hayem’s solution with Merthiolate
e) Toison – hayem’s solution with methyl violet
f) Dacie or Formol Saline
40 % solution of formalin 10 ml
3.8 % trisodium citrate 900 ml

FOCUSING
• 4X to see the general formation of slide.
• 10X for WBC counting
• 40X for RBC counting

Q. What should be done if the patient is

polycythemic?
• Decrease the amount of blood sucked in the
pipet
• Solve for the dilution factor
Q: What should be done if the patient is anemic?
• Increase the amount of blood sucked in the
pipet
• Solve for the dilution factor
DILUTING FLUIDS FOR WBC COUNT
a) Glacial acetic acid
b) HCl
c) Turk’s solution
d) Unopette for (platelet and WBC)
 MLS 113A: HEMATOLOGY
MIDTERM | LABORATORY
LESSON 2: ESR DETERMINATION
ERYTHROCYTE SEDIMENTATION RATE (ESR) MODIFIED WESTERGREN METHOD
• Rate of settling of RBCs from their plasma after Reagents and Equipments:
the addition of anticoagulant • Applicator sticks
• A non-specific test used to identify health • Pipets, 2 and 0.5ml
disorders and for monitoring purposes • Test tubes
• Represents a non-specific response to tissue • Na chloride, 0.85% (w/v)
damage, inflammation, and denotes the • Westergren pipet, calibrated in mm(NCCLS)
presence of disease but not its severity. length – 300.5nm
IMPORTANCE OF ESR external dm – 5.5mm
• Good index for the presence of hidden but internal bore – 2.7mm
active diseases such as TB and carcinoma • Westergren pipet rack. All racks should contain
• It measures the suspension stability of RBCs a leveling bulb in order to ensure that the
• It measures the abnormal concentration of position of the tubes is vertical (+/-1degree).
fibrinogen and globulin • Specimen: whole blood, 3ml using EDTA as the
STAGES OF ESR anticoagulant
• Initial rouleaux formation (first 10 mins) Principle:
• Period of rapid settling (the next 40 mins when • Well-mixed, whole blood is diluted with 0.85%
settling is constant) Na chloride, placed in a Westergren pipet, and
• Period of final settling or packing (during the last allowed to stand for exactly 1 hr in a vertical
10 mins) position. The number of mm the RBCs fall
METHODS OF ESR DETERMINATION during the timed period constitutes the ESR.
WINTROBE & LANDSBERG METHOD REFERENCE RANGE:
• Anticoagulant – double oxalate or EDTA women: 0-15mm/hr
• Specimen – whole blood men: 0-10mm/hr
• Equipment children: 0-10mm/hr
o Wintrobe rack Procedure:
o disposable capillary pipet • Mix the whole blood for at least 2 mins on a
• Wintrobe tube with 2 calibrated sides: rotator (the blood should be at room temp).
o Left side-colored red; calibrated 0 on top & Check the tube for clots using 2 applicator
10 at the bottom = use for ESR sticks.
o Right side-colored white; calibrated 10 on • Place 0.5ml of 0.85%NaCl in a plane test tube.
top and 0 at the bottom = used for hct after • Add 2ml of well mixed whole blood to the test
30 mins of centri. at 3,500rpm tube.
Note: each calibration has 10mm so there is a total • Mix the tube for 2 mins.
of 100 mm in the tube. The bore is 3 mm in dm. • Make certain the Westergren ESR rack is
Principle: exactly level.
• Well-mixed, whole blood is placed in a Wintrobe • Fill the Westergren pipet to exactly the 0 mark,
tube and allowed to stand for an hour. The making certain there are no air bubbles in the
number of mm the RBCs fall during this time blood.
constitutes the ESR. • Place the pipet in the rack. Be certain the pipet
NV: women: 0-20mm/hr fits snugly and evenly into the grooves provided.
men: 0-9mm/hr • Allow the pipet to stand for exactly 60 mins.
Procedure: • At the end of 60mins record the number of mm
• Mix the whole blood for at least 2 mins on a the RBCs have fallen. This result is the ESR in
rotator or mixed by inversion several times. mm/hr.
• With a capillary pipet, fill the tube to 0 mark.
• Place the tube in an exactly vertical position in
the rack. Time for 60mins
• At the end of 60mins record the level of the
erythrocyte column. The result is in mm/hr.
DIFFERENCES BETWEEN WINTROBE METHOD resulting in an elevated ESR value. In this case,
AND WESTERGREN METHOD the tube's design and dimensions might not
conform to standard ESR testing requirements.
BLOOD MIXING
• The concentration of EDTA (ethylene diamine
tetra-acetic acid) in the tube may not be suitable
for ESR testing. EDTA is typically used as an
anticoagulant in blood collection tubes, but the
concentration should be carefully controlled for
ESR testing. An improper concentration of
EDTA might affect the blood's viscosity and
influence the ESR result.

CLINICAL CORRELATION OF SEDIMENTATION

RATE
INCREASE
Physiological
• Pregnancy
• Menstruation
• Cold temp
• Adults above 60 years of age
Pathological
• Inflammatory
• Degenerative
• Necrobiotic cell state
TESTS CONSIDERATIONS
TEMPERATURE
• ESR is temperature-dependent, and maintaining
the correct temperature is critical. Deviations in
temperature or timing, such as exposure to
room temperature, can lead to faster
sedimentation rates, causing an artificially
elevated ESR value.
• Test should be done at 20-25⁰ C
• Higher temperature cause false high results due
to reduction in plasma viscosity
• Always bring refrigerated blood at RT
TIME
• Test should be done within two hours of
collection
• EDTA sample should be kept at 4°C if delayed
but should be performed within 6 hours
POSITION OF TUBE
• Tube should be perfectly vertical
• ESR increase as the RBC slide down along the
lower side
• Angle of 3⁰ from vertical can increase ESR by
30℅
TUBE SIZE & DIAMETER
• The use of a disposable tube attached directly
to an EDTA tube could affect the tube's size and
diameter. A smaller or differently shaped tube
may allow red blood cells to settle more quickly,
 1st Semester Midterm
STAINING
phosphate buffer, pH 6.4.
Introduction Buffer separates the
thiazineeosinate complex into
*Purpose: to make the cells more visible methylene blue and eosin.
and allow their morphology to be • Eosin and methylene blue stains
evaluated. *When: within 4 hours of different compounds.
smear preparation *Longer than 4 hours • Alternative buffer: Aged-
will affect the morphology of the cells. distilled water (distilled water
*Staining cells is pH-dependent. stored in a glass bottle for
24 hours, pH 6.4 to 6.8)

4. Staining of Acidic and Basic Parts of the

Cells
A. Free methylene blue (basic)
*Stains acidic (basophilic) components (stains
blue). → RNA

B. Free eosin (acidic)

*Stains basic (eosinophilic) components (stains red-
orange/ salmon pink). → Hemoglobin & eosinophilic
ROMANOWSKY STAIN
granules
Wright Stain Both are
Wright-Giemsa Stain polychromatic stains Problems in Staining
(eosin and methylene
blue) *Water or drying artifact
Giemsa Stain Giemsa stains have • Appearances:
methylene blue azure o Moth-eaten, heavily demarcated central
pallor
o Refractive (shiny) blotches on the RBCs.
1. Fixing of Cells o Echinocytes (areas of the slide
*Methanol in the stain that dried most slowly)
• Fixative → fixes the cells to the slide • Water or drying artifact factors
o Humidity → destroys
2. Staining of Neutral Parts of the the morphology of the cells. o
Cells High ratio of plasma to RBCs
*Methylene Blue and Eosin (extremely anemic patients) o Water is
• The oxidized methylene blue absorbed from the air into the alcohol-based
and eosin form a thiazine- stain.
eosinate complex (stains the • Troubleshooting steps to avoid water/ drying
neutral cellular parts purple- artifacts:
lilac) o Dry the slides as quickly as possible
• E.g., Neutrophils’ cytoplasmic o Do not use your breath/ blow on the
slide.
granules
o Stopper the container of the stain
3. Application of Buffer
Materials for Staining
*Buffer
• We need to differentiate between
1. Dropper and Tweezers
acidic and basic parts of the cell.
• To differentiate these parts of the
cell, we need 0.05 M sodium
 8. Flood the slide with distilled water to remove the
excess stain and buffer.
9. Air-dry the stained smear.

Figure. Dropper and tweezers/ tongs respectively

for flooding/ horizontal method
Hematology 1 Laboratory MLS 113A 1st
Semester Midterm
2. Staining Rack and Coplin Jars

Figure. Wright Staining Method

2. Giemsa Staining Method

*Process: Dipping Method
Figure. Staining rack and coplin jars respectively 1. Fix the air-dried smear in methyl alcohol
for dipping/vertical method for 2-3 mins.
2. Air-dry and immerse in dilute Giemsa stain
3. Romanowsky Stains in a Coplin jar for 15 mins to 1 hour.
*Consist of methylene blue/azure B and eosin, dissolved 3. Wash in distilled water.
in acetone-free methanol. 4. Air dry the stained smear.
*No buffer
*Types:
1. Wright stain 3. Wright-Giemsa Staining
2. Giemsa stain Method A. Flooding
3. Wright-Giemsa stain (modified) Method *Process:
4. Diff Quick 1. Fix the air-dried smear with methanol for
2 minutes
Staining Methods 2. Apply the Wright’s stain for 2 ½ minutes
3. Apply buffered Giemsa stain for 7-10
*How stains are applied onto the smear: minutes
• Dip/ Vertical Method 4. Rinse in phosphate-buffered water (pH
• Flooding/ Horizontal Method 6.8) for 2 minutes.
5. Drain and air dry
1. Wright Staining Method
*Procedure: Flooding Method B. Dipping Method
1. Put the glass slide with the smear on the staining *Process:
rack while waiting for the smear to dry. 1. Fill a Coplin jar with 50 ml
2. Pour the wright stain from the bottle through a WrightGiemsa stain
filter onto the slide. 2. Fill another jar with phosphate buffer
3. Flood the slide with the stain, completely water
covering the smear. 3. Dip the dried smear into the
4. The stain should remain on the slide at least for WrightGiemsa stain (feather edge
1-3 mins to fix the cells on the glass. down) for 30 seconds.
5. Add approximately an equal amount of buffer 4. Remove the slide from the stain and
onto the slide. dip it into phosphate buffer (feather
6. A metallic sheen (or green “scum”) should appear edge down) for 1-10 minutes (do not
on the slide if the mixing is correct. agitate the slide).
7. The mixture is allowed to remain on the slide for 5. Rinse with deionized water and air-dry.
3 mins.
4. Diff-Quick Staining Method
*Is a fast-acting variation of Romanowsky stain. 6. Rinse the slide with distilled
*15 seconds water and air-dry.

Hematology 1 Laboratory
MLS 113A 1st Semester
Midterm
4. Diff-Quick Staining Method
Continuation *Process:
1. Dip the slide into the diff-quick
fixative for 5 seconds.
2. Retrieve the slide and drain the
excess fixative onto a tissue.
3. Dip the slide into Solution I
(Xanthene dye: Eosin Y/Eosin
G + buffer) for 5 seconds.
4. Retrieved the slide and drain the
excess Solution I onto a tissue.
5. Dip the slide into Solution II Figure. (Good Stained Smear) Macroscopic: Film→ pink
(Thiazine dye: Methylene to purple
blue/azure A + buffer) for 5
seconds.

MICROSCOPIC EXAMINATIO N

RBCs orange to salmon pink

WBCs Nuclei purple to blue
Neutrophils Cytoplasm pink to tan
Granules violet/lilac
Eosinophils Refractile granules bright orange
Lymphocytes Granules Light purple/violet
Cytoplasm Blue
Basophils Granules Deep purple
Monocyte Cytoplasm Blue-grey (ground glass)
Platelets Granules Light purple/violet
Cytoplasm Purple-blue to lilac

POORLY STAINED BLOOD SMEARS

Problems Causes
RBCs appear gray. Stain or buffer too alkaline (most common)
WBCs are too dark. Inadequate rinsing
Eosinophil granules are gray, not orange Prolonged staining
RBCs are too pale or red. Heparinized blood sample
RBCs are too pale or red. Stain or buffer too acidic (most common)
WBCs are barely visible. Under buffering (too short) Over-rinsing
 MLS 113 LAB – MIDTERM – FIRST SEMESTER

 If the ESR stands for more than 60 minutes, the results will International Committee for Standardization in
be falsely elevated. If the test is timed for less than 60 Hematology.
minutes, invalidly low values are obtained.  When reading the ESR results on bloods with an extremely
 A marked increase (or decrease) in room temperature leads high WBCs or platelet count, the buffy coat should be
to increased (or decreased) ESR result. excluded from the reading.
 Tilting of the ESR tube increases the ESR.  If there is poor separation of the RBCs and plasma layers
 Bubbles in blood cause invalid results. this may be due to an increased number of reticulocytes
 Fibrin clots present in the blood invalidate the test results. and has been termed “stratified sedimentation”.
 The internal bore and the length of the graduated scale on
 The ESR should be set up within 2 hours of blood the Westergern pipette are critical measurements.
collection. If EDTA is used as the anticoagulant, the test
may be set up within 6 hours if the blood has been DISPOSABLE ESR
refrigerated.  commercial kits are now available for a disposable ESR
 The Wintrobe ESR technique is thought to be more test.
sensitive when the ESR is low, whereas the Westergren  Several kits include safety caps for the pipettes that allow
procedure more accurately reflects the patient’s disorder the blood to precisely fill to the zero mark.
when the results are high. The Westergern method  This makes the pipette a closed system and eliminates the
has been chosen as the standard method by the error involved with manually setting the blood at the zero
mark.

MAKING A BLOOD SMEAR

 If there is no frosted edge in your slide, estimate around 1
BLOOD SMEAR and ¼ inches away from the edge of the slide to put a drop
of blood.

PUSH-TYPE WEDGE PREPARATION

 Blood smears are needed for microscopic examination of

blood.
 They maybe prepared from venous blood or capillary blood.
 The most common blood smear is the peripheral smear
 Using the dominant hand, place the spreader before the
used for a complete blood cell count, differential.
blood and draw it back into the drop of blood. Spreader
 Blood smears are also made for tests as malarial smears
slide is held at 45-degree angle.
and special hematology procedures.
 Allow the blood to spread across the width of the slide.
 The blood is spread on a glass slide to produce a wedge or
 Quickly and smoothly PUSH the spreader slide rapidly
thin smear. The perfect slide consists of a smear that is
across the stationary slide with even stroke and pressure.
exactly one cell thick in the feathered edge (Hoeltke, 2018).
 Any pressure exerted on the spreader should be directed
across the slide in the direction that the film is made rather
SAMPLE
than down on the stationary slide.
 EDTA blood sample
 Fresh blood sample
PULL-TYPE WEDGE PREPARATION
 Heparinized blood sample is not used because heparin
gives a blue background for Wright’s stain smears.

MATERIALS FOR STAINING

WEDGE METHOD (2 GLASS SLIDE METHOD)

 The spreader slide is pushed into the blood drop and

PULLED along the length of the slide to make a film/smear.
 The faster the spreader slide is moved: longer and thinner
film
 The slower the slide is moved:
o shorter and thicker film
o Poor leukocyte distribution (bigger cells are pushed to
 Place a drop of blood 1 to 2 millimeters in diameter on one the very end of the smear).
slide.
 The drop should be in the center line approximately ¼ inch ANGLE OF THE SPREADER:
from the frosted edge.  The angle will also vary the results.
 The angle is >45 degrees  thicker smear
 The angle is <45 degrees  thinner SMEAR
FRANCES DOMINIQUE C. BENITEZ 8
 MLS 113 LAB – MIDTERM – FIRST SEMESTER

 Speed, angle, and drop size can be varied slightly to

produce a good smear. SLIDE APPEARANCES ASSOCIATED WITH THE
MOST COMMON ERRORS
HIGH OR LOW HEMATOCRIT
 High Hematocrit lower the angle of the spreader.
 low Hematocrit Raise the angle of the spreader

GLASS SLIDE-COVERSLIP METHOD (BEACOM’S)

 Place a small drop of blood near one end of the slide.
 Cover the blood with a coverslip (cover slip).
 As soon as the blood spreads, push forward the coverslip
along the surface of the slide with enough pressure to keep
the fingers form slipping the spreader.
 A. Chipped or rough edge on spreader slide.
 Air-dry the smear.
 B. Hesitation in forward motion of spreader slide.
 C. Spreader slide pushed too quickly.
TWO-COVERSLIP METHOD (EHRLICH’S)
 D. Drop of blood too small.
 E. Drop of blood not allowed to spread across the width of
the slide.
 F. Dirt or grease on the slide; may also be caused by
elevated lipids in the blood specimen.
 G. Uneven pressure on the spreader slide.
 H. Time delay; drop of blood began to dry.

 Place a small drop of blood on the center of the coverslip. Drying of Smears
 Cover the blood with another coverslip.
 As soon as the blood spreads, pull apart the two coverslips
parallel to their surfaces in a firm and steady motion.
 Air-dry the smear.

UNACCEPTABLE SMEAR

GOOD SMEAR
 The film is two thirds to three fourths the length of the slide
 The film is finger shaped.
 The lateral edges of the film are visible.  All films must be dried before staining. WHY?
 Can use a small fan to facilitate drying.
 The film is smooth without irregularities, holes, or streaks.
 Never blow on the slide
 When the slide is held up to the light, the thin portion
o moisture in breath causes RBCs to develop water
(feather edge) of the film has a “rainbow” appearance.
artifact
 The whole drop of blood is picked up and spread.
 (A) or to become echinocytic/ crenated (B).

STAINING
BUFFER
STAINING  0.05 M sodium phosphate, pH 6.4
 Aged distilled water (distilled water stored in a glass bottle
for 24 hours, pH 6.4 to 6.8)

 Purpose: to make the cells more visible and allow their

morphology to be evaluated.
 When: within 4 hours of smear preparation

WRIGHT STAIN & WRIGHT-GIEMSA STAIN

 Both are polychromatic stains (eosin and methylene blue)
 Giemsa stains have methylene blue azure

METHANOL IN THE STAIN- FIXATIVE

 (fixes the cells to the slide).
 The oxidized methylene blue and eosin form a thiazine-
eosinate complex (stains the neutral cellular parts
PURPLE-LILAC)
o Neutrophils’ cytoplasmic granules

FRANCES DOMINIQUE C. BENITEZ 9

Prepared by: Lovelyn Mae E. Cuison, RMT, MSMT, MLS(ASCPi)
 HEMOCYTOMETER
Hemo: blood
Cyto: cell
Meter: measurement/counter

• Thus, it is an instrument used to count the

blood cells.
It includes:

☑ Neubauer’s slide
☑ Cover slip
☑ RBC pipette
☑ WBC pipette
 NEUBAUER’S SLIDE
- the name given to a thick glass
slide. In the center of the slide,
there is an H- shaped groove

NEUBAUER’S CHAMBER
- Neubauer’s slide with a
cover slip over it.
! 14
4
Hemocytometer Chamber
 1mm

0.25mm

The four corner squares are further divided into sixteen smaller squares and
are used for WBC counting.
 Counting Rule
• Do NOT count cells
touching
NBottom line
NRight line
Ø This is to avoid
double counting.
 DIFFERENCES BETWEEN RBC
AND WBC PIPETTE
RBC pipette WBC pipette
It has a red bead It has a white bead

It has graduations upto It has graduations upto

mark 101 mark 11

Size of bulb is larger Size of bulb is smaller

Size of lumen is smaller Size of lumen is larger

RBC WBC
RBC PIPETTE

WBC PIPETTE
RBC Count
Diluting fluids for RBC count
a. Hayem’s solution
Mercuric chloride 0.5 gm
Sodium sulfate (anhydrous) 5.0 gm
Sodium chloride 1.0 gm
Distilled water 200 ml
b. Gower’s solution
Sodium sulfate 12.5 gm
Acetic acid (glacial) 33.3 ml
Distilled water 200 ml
c. NSS (0.85 % Sodium chloride)
d. Bethel – Hayem’s solution with merthiolate
e. Toison – Hayem’s solution with methyl violet
f. Dacie or Formol Saline
40 % solution of formalin 10 ml
3.8 % trisodium citrate 900 ml
 RBC/cu mm = cells counted x dilution factor
mm2 counted x 0.1 mm

JShortcut: RBC counted x 10,000 (if dilution factor is 200)

Example:
Blood was sucked up to 0.5 mark
Number of cells counted in 5 intermediate squares =550
RBC/cu mm = 550 x 200
5 (0.2x0.2) x 0.1 mm Normal range:
(M) :4.5 – 5.9 x 1012/L
= 110,000 (F) :4.0 – 5.2 x 1012/L
0.2 x 0.1

= 5,500,000 uL or 5.5x1012/L
Q: What should be done if the patient is polycythemic?
1. Decrease the amount of blood sucked in the pipet
2. Solve for the dilution factor

Q: What should be done if the patient is anemic?

1. Increase the amount of blood sucked in the pipet
2. Solve for the dilution factor
 Diluting fluids for WBC count

a.Glacial acetic acid

b.HCl
c.Turk’s solution
d.Unopette for (platelet and WBC)
 WBC/cu mm = cells counted x dilution factor
mm2 counted x 0.1 mm

Example:
Blood was sucked up to 0.5 mark
Number of cells counted 4 corner squares = 300
WBC/cu mm = 300 x 20
4 (1x1) x 0.1 mm

= 6,000
Normal range : 4 x 0.1

5,000 – 10,000 cumm (C.U) = 15,000 cumm or 15.0x109/L

5.0 – 10.0 x 109/L (S.I.)
J Shortcut : wbc count x 50
 FOCUSING
• 4X to see the general
formation of slide.
• 10X for WBC counting
• 40X for RBC counting
 4x magnification

1 mm
 10x magnification

0.2 mm
40x magnification
 ERYTHROCYTE
SEDIMENTATION RATE
(ESR)

-----------
DOOMSDAY
 ESR
• Rate of settling of RBCs
from their plasma after
the addition of
anticoagulant
• A non-specific test used
to identify health
disorders and for
monitoring purposes
• Represents a non-specific
response to tissue damage,
inflammation, and denotes the
presence of disease but not its
severity.
 Importance of ESR
• Good index for the presence
of hidden but active diseases
such as TB and carcinoma
• It measures the suspension
stability of RBCs
• It measures the abnormal
concentration of fibrinogen
and globulin
 Stages of ESR
• Initial rouleaux formation
(first 10 mins)
• Period of rapid settling (the
next 40 mins when settling
is constant)
• Period of final settling or
packing (during the last 10
mins)
Methods of ESR Determination

Wintrobe & Landsberg

Method
• Anticoagulant – double oxalate
or EDTA
• Specimen – whole blood
• Equipments
-Wintrobe rack
-disposable capillary pipet
-Wintrobe tube with 2 calibrated
sides:
Left side-colored red; calibrated
0 on top & 10 at the bottom = use
for ESR
Right side-colored white;
calibrated 10 on top and 0 at the
bottom = used for hct after 30
mins of centri. at 3,500rpm
Note: each calibration has 10mm so
there is a total of 100 mm in the
tube. The bore is 3 mm in dm.
Principle:
Well-mixed, whole blood is
placed in a Wintrobe
tube and allowed to
stand for an hour. The
number of mm the RBCs
fall during this time
constitutes the ESR.
NV: women: 0-20mm/hr
men: 0-9mm/hr
Procedure:
• Mix the whole blood for at least 2
mins on a rotator or mixed by
inversion several times.
• With a capillary pipet, fill the tube
to 0 mark.
• Place the tube in an exactly
vertical position in the rack. Time
for 60mins
• At the end of 60mins record the
level of the erythrocyte column.
The result is in mm/hr.
 Modified Westergren Method
Reagents and Equipments:
• Applicator sticks
• Pipets, 2 and 0.5ml
• Test tubes
• Na chloride, 0.85% (w/v)
• Westergren pipet, calibrated in
mm(NCCLS)
length – 300.5nm
external dm – 5.5mm
internal bore – 2.7mm
• Westergren pipet rack. All racks
should contain a leveling bulb in
order to ensure that the position
of the tubes is vertical (+/-
1degree).
Specimen: whole blood, 3ml using
EDTA as the anticoagulant
Principle:
Well-mixed, whole blood is diluted
with 0.85% Na chloride, placed in
a Westergren pipet, and allowed
to stand for exactly 1 hr in a
vertical position. The number of
mm the RBCs fall during the timed
period constitutes the ESR.
REFERENCE RANGE:
women: 0-15mm/hr
men: 0-10mm/hr
children: 0-10mm/hr
Procedure:
• Mix the whole blood for at least 2 mins on a
rotator (the blood should be at room temp).
Check the tube for clots using 2 applicator
sticks.
• Place 0.5ml of 0.85%NaCl in a plane test tube.
• Add 2ml of well mixed whole blood to the test
tube.
• Mix the tube for 2 mins.
• Make certain the Westergren ESR
rack is exactly level.
• Fill the Westergren pipet to exactly
the 0 mark, making certain there
are no air bubbles in the blood.
• Place the pipet in the rack. Be
certain the pipet fits snugly and
evenly into the grooves provided.
• Allow the pipet to stand for exactly
60 mins.
• At the end of 60mins record the
number of mm the RBCs have
fallen. This result is the ESR in
mm/hr.
 Differences Between Wintrobe Method and
Westergren Method
POINT OF DIFFERENCE WINTROBE WESTERGREN
Tube Wintrobe tube Westergren tube
Bore 3mm in dm 2.5mm in dm
Graduation Up to 100mm Up to 200mm
Anticoagulant Double oxalate 3.8% Na Citrate
Dilution No dilution since anticoagulant is in dry 1 part citrate to 4 parts blood
form

Amount of blood used 1 ml 2.4ml

Reading Once after 1 hour Twice, after 1 hr & after 2 hours

Sensitiveness Less sensitive More sensitive

Correction for anemia When ESR is high & hct is low No correction for anemia
Disadvantages a. Not so sensitive because of short a. Sensitive but cumbersome
column b. Requires large amount of blood
b. Oxalates may be converted to temp c. Dilution may cause error
above 80C d. Gives no additional information
Clinical Correlation of Sedimentation Rate
INCREASE
Physiological
a. Pregnancy
b. Menstruation
c. Cold temp
d. Adults above 60 years of age
Pathological
a. Inflammatory
b. Degenerative
c. Necrobiotic cell state
 TESTS CONSIDERATIONS
1. Temperature
• ESR is temperature-dependent, and maintaining
the correct temperature is critical. Deviations in
temperature or timing, such as exposure to room
temperature, can lead to faster sedimentation
rates, causing an artificially elevated ESR value.
• Test should be done at 20-25⁰ C
• Higher temperature cause false high results due
to reduction in plasma viscosity
• Always bring refrigerated blood at RT
2. Time
• Test should be done within two hours of
collection
• EDTA sample should be kept at 4°C if delayed
but should be performed within 6 hours.
3. Position of tube
• Tube should be perfectly vertical
• ESR increase as the RBC slide down along the
lower side
• Angle of 3⁰ from vertical can increase ESR by
30℅
4. Tube size & Diameter
• The use of a disposable tube attached directly
to an EDTA tube could affect the tube's size
and diameter. A smaller or differently shaped
tube may allow red blood cells to settle more
quickly, resulting in an elevated ESR value. In
this case, the tube's design and dimensions
might not conform to standard ESR testing
requirements.
• The concentration of EDTA (ethylene diamine
tetra-acetic acid) in the tube may not be
suitable for ESR testing. EDTA is typically used
as an anticoagulant in blood collection tubes,
but the concentration should be carefully
controlled for ESR testing. An improper
concentration of EDTA might affect the blood's
viscosity and influence the ESR result.
5. Blood Mixing
• The use of a disposable tube attached directly to an EDTA
tube could affect the tube's size and diameter. A smaller or
differently shaped tube may allow red blood cells to settle
more quickly, resulting in an elevated ESR value. In this
case, the tube's design and dimensions might not conform
to standard ESR testing requirements.
• The concentration of EDTA (ethylene diamine tetra-acetic
acid) in the tube may not be suitable for ESR testing. EDTA
is typically used as an anticoagulant in blood collection
tubes, but the concentration should be carefully controlled
for ESR testing. An improper concentration of EDTA might
affect the blood's viscosity and influence the ESR result.
-end-