Activities 1 4 Hema1lab Manual 2022 2023

Name: ___________________________________ Date: ______________
Section: ________________ Group: _________ Score: ______________
ACTIVITY 1
BLOOD COLLECTION TECHNIQUES
Assessment Criteria for Passing
Unit Intended Learning Outcomes To achieve this unit, the learner must:
1. Disinfect properly the different areas of the a. Disinfect different areas of the laboratory
laboratory. such as the floor, sink, tables and cabinets.
2. Apply correctly the standard precautions in a. Wear complete and proper PPE (laboratory
specimen collection and handling. gown, face mask, hair net, gloves).
b. Post label the collection tubes with complete
and correct details.
3. Identify correctly the extraction sites for a. Identify the veins in the antecubital fossa as
venipuncture and capillary puncture. the venipuncture site.
b. Identify alternative sites if the veins in the
antecubital fossa are not visible.
c. Identify the collection sites for adult and
pediatric capillary puncture.
4. Demonstrate properly the collection a. Obtain blood samples correctly by
techniques of venipuncture and capillary venipuncture and capillary puncture.
puncture following the correct order of draw. b. Transfer collected venous blood in the
collection tubes following the correct order
of draw as indicated by the CLSI.
c. Transfer collected capillary blood in the
microcollection tubes following the correct
order of draw as indicated by the CLSI.
5. Determine correctly the reasons for specimen a. Enumerate at least 5 reasons for specimen
collection. rejection.
b. Explain how rejected specimens may
adversely affect the test result.
6. Perform correctly the proper waste a. Dispose sharps, infectious wastes,
segregation and disposal. biodegradable wastes, and non-
biodegradable, non-infectious waste proper
trash bins.
Clinical Hematology I – A Compilation of Laboratory Exercises | 11

Rubrics
Weight 1 – Developing 2 – Acceptable 3 - Effective 4 - Exceptional Score
Pre-Analytical (10%)
Disinfection or
Cleaning of 4 Areas in
the Laboratory All areas in the 2 areas in the laboratory 3 areas in the All areas in the
(5%) laboratory were not were disinfected before laboratory were laboratory were
disinfected before use use disinfected before use disinfected before use
(1) sink (2) table (3)
cabinet (3) floor
Wearing PPE
(5%)
1 out of 4 PPE worn 2 out of 4 PPE worn 3 out of 4 PPE worn All PPE worn
(1) gloves (2) mask (2)
hair net (4) laboratory
gown
Analytical (80%)
Venipuncture (40%)
Patient Identification
and Preparation (10%)
(1) Patient was properly

identified. (2)
Phlebotomist 1 out of 4 done 2 out of 4 done 3 out of 4 done 4 out of 4 done
introduced himself. (3)
Patient was reassured
and positioned. (4) All
equipment was
assembled.
Tourniquet Application
and Puncture Site
Selection (10%)
(1) Tourniquet was

properly tied 2 to 4
inches above the
1 out of 4 done 2 out of 4 done 3 out of 4 done 4 out of 4 done
puncture site. (2) Hands
were sanitized and
gloves were put on. (3)
Correct puncture site
selected. (4) Antiseptic
technique correctly
done.
Blood Collection (20%)
(1) Needle was inserted

bevel and angled 15 -
30° against the
puncture site. (2) Blood
was successfully
withdrawn. (3)
Tourniquet was 1 out of 4 done 2 out of 4 done 3 out of 4 done 4 out of 4 done
released, needle was
withdrawn, and
bandage was applied.
(4) Blood was
transferred to the
correct tube and
labelled with complete
and correct

information.
Capillary Puncture (40%)
Patient Identification
and Preparation (10%)
(1) Patient was properly

identified. (2)
Phlebotomist 1 out of 4 done 2 out of 4 done 3 out of 4 done 4 out of 4 done
introduced himself. (3)
Patient was reassured
and positioned. (4) All
equipment were
assembled.
Puncture Site Selection
(10%)
(1) Hands were

sanitized and gloves
were put on. (2) Correct
puncture site selected.
(3) Antiseptic technique
correctly done. (4) Area
was warmed.
Blood Collection (20%)
(1) Puncture was

perpendicular to the
grooves of the finger.
(2) First drop of blood 1 out of 4 done 2 out of 4 done 3 out of 4 done 4 out of 4 done
wiped off. (3) Blood was
transferred to capillary
tubes ¾ full. (4)
Capillary tubes were
properly sealed.
Post-Analytical (10%)
Disinfection or
the Laboratory All areas in the 2 areas in the laboratory 3 areas in the All areas in the
(5%) laboratory were not were disinfected after laboratory were laboratory were
disinfected after use use disinfected after use disinfected after use
cabinet (3) floor
Waste Disposal
(5%)
4 type of wastes were 2 types of wastes were 3 types of wastes were All wastes were
(1) sharps (2) infectious not disposed to disposed to appropriate disposed to disposed to
(3) biodegradable (4) appropriate bag bag appropriate bag appropriate bag
non-infectious, non-
biodegradable
Total Score:

Name: ___________________________________ Date: ______________
ACTIVITY 1
BLOOD COLLECTION TECHNIQUES
Review Questions
1. What are the collection sites for venipuncture?

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
2. Give the correct order of draw for venipuncture.

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
3. Give five (5) restriction sites for venipuncture.

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
4. What are the collection sites for capillary puncture?

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
5. Give the correct order of draw for capillary puncture.

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________

6. What is the rationale of wiping off the first drop of blood during capillary blood collection?
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
7. Give five (5) reasons for specimen rejection.

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
8. Why should we allow the alcohol to be air dried?

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
9. What is the alternative disinfectant if the test is blood alcohol level determination?
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________

For proper evaluation of blood cells, the well-made smear must be prepared.
Qualities of a well-made blood smear:
1. Has gradual and even transition from thick to thin area.
2. Must be finger-shaped with a smooth tail.
3. Must occupy two-thirds to three-fourths of the glass slide.
4. Must not touch the outer borders of the slide or run off the sides or ends of the slide.
5. Must smooth surface free from ridges, waves, gaps and holes.
6. Must have feathery edge.
Factors that may be altered to produce a well-made blood smear:

THIN SMEAR THICK SMEAR
small drop of blood Large drop of blood
Slow spread Fast spread
Low angle (<30°) High angle (>45°)
Heavy pressure Light pressure
FIGURE 3. WELL-MADE BLOOD SMEAR
SOURCE: RODAK, B., ET AL., 2012
FIGURE 4. UNACCEPTABLE BLOOD SMEARS
SOURCE: RODAK, B., ET AL., 2012
Common causes of a poor blood smear:

1. Drop of blood too large or too small.
2. Spreader slide pushed across the slide in a jerky manner.

3. Failure to keep the entire edge of the spreader slide against the slide while making the smear.
4. Failure to keep the spreader slide at a 30° angle with the slide.
5. Failure to push the spreader slide completely across the slide.
6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide
7. Holes in film: Slide contaminated with fat or grease
8. Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated
with water.
STAINING OF BLOOD SMEAR
To differentiate and identify cells, blood and other types of specimens can be stained using Romanowsky-
based stains (e.g. Giemsa stain, Wright stain, Leishman’s stain). These stains can be prepared in the
laboratory or purchased in a ready-to-use form. Either manual or automated techniques can be used. Fixing
of cells on the slide prior to staining is done using methanol.
Components of Romanowsky stain:

1. Eosin (acidic component) - stains basic structures, e.g. hemoglobin, eosin granules
2. Methylene Blue (basic component) – stains acidic structures, e.g. nucleic acids (RNA and DNA), cell
nuclei, ribosomes, cytoplasm and basophil granules.
Causes of Too Acidic Smear

1. insufficient staining time
2. prolonged buffering or washing
3. old stain
Causes of Too Basic Smear

1. thick blood smear
2. prolonged staining
3. insufficient washing
4. alkaline pH of stain components
Teaching and Learning Activities
Pre-Analytical Phase
1. Disinfect the working area using 10% sodium hypochlorite.

2. Wear complete and proper PPE.
Analytical Phase
Reagents, Supplies and Equipment

1. EDTA anticoagulated blood
2. Glass slides (frosted or plain)
3. Spreader
4. Cover slip
5. Methanol
6. Wright stain or Wright-Giemsa stain (Romanowsky stain)
7. Buffer Solution or Aged Distilled Water
8. Coplin Jars
9. Transfer device (applicator sticks, pipets, or capillary tubes)
10. Slide holder
11. Paper towel
12. Personal Protective Equipment (PPE)
Procedure
PREPARATION OF THIN BLOOD SMEARS
A. Push Wedge Preparation/ Two Slide Method

1. Place a drop of blood (about 2 to 3 mm in diameter) to the stationary slide about 0.25 inch
(1 cm) from the end or near the frosted area.
2. Position the spreader (or a second slide) before the drop of blood at angle between 30 to 45
degrees. Allow the drop of blood to spread evenly across the width of the slide.
3. Quickly and smoothly push the spreader along the length of the slide with an even and
gentle pressure.
4. Allow the smear to air-dry.
FIGURE 5. PUSH WEDGE PREPARATION/ TWO SLIDE METHOD
SOURCE: RETRIEVED FROM: HTTP://WWW.KEYWORD-SUGGESTIONS.COM/YMXVB2QGC21LYXIGCHJVY2VKDXJL/
B. Two-Cover Slip/ Erlich’s Two-Coverglass Method

1. Get two coverglasses. Hold one coverglass on its adjacent corners with the thumb and index
finger.
2. Place one drop of blood in top of the held coverglass.
3. Position the other cover glass on top of the coverglass with blood so that the two form a 16-
sided figure. As this is done, the blood begins to spread.
4. Before the blood completely spreads, separate the two coverglasses by doing a rapid, even,
horizontal and lateral pull. Avoid squeezing the coverglasses together.

FIGURE 6. TWO-COVER SLIP/ ERLICH’S TWO-COVERGLASS METHOD
SOURCE: RETRIEVED FROM: HTTPS://WWW.STUDYBLUE.COM/NOTES/NOTE/N/

LEC-3-BLOOD-SMEAR-WRIGHT-STAIN-INTRO-TO-CELLS-OF-NORMAL-BLOOD/DECK/5364623
PREPARATION OF THICK BLOOD SMEAR

1. Place a small drop of blood (approximately 2 cm in diameter) on a clean slide.
2. Spread it with an applicator stick or the corner of another slide until small prints are just visible
through the blood smear.
STAINING OF PERIPHERAL BLOOD SMEAR
1. Ensure that the blood smears are completely air dried.

2. Prepare the staining solutions by placing them in separate coplin jars. Filter the stains if
necessary to avoid artifacts.
a. Solution 1 – methanol (fixative)
b. Solution 2 – eosin (acidic dye)
c. Solution 3 – methylene blue (basic dye)
3. Dip the blood smear in each solution for 30 seconds (or as stated by the manufacturer).
4. Rinse off the excess stain by dipping the blood smear a few times in a buffer solution or aged
distilled water.
Post-Analytical Phase
1. Segregate and dispose all infectious and biohazardous waste in their proper waste bins.
3. Remove all used PPE after the activity.

Name: ___________________________________ Date: ______________
ACTIVITY 2
SMEAR PREPARATION AND STAINING

2. Wear complete and proper personal a. Wear PPE such as gloves, mask, laboratory
protective equipment. gown and hair net.
3. Execute correctly the different methods of a. Prepare blood smears using wedge technique
preparing a blood smear. and cover glass method.
4. Identify correctly the macroscopic and a. List down all the 6 characteristics of a well-
microscopic characteristics of a well-prepared done blood smear.
blood smear. b. Prepare a well-done blood smear.
5. Determine correctly the factors contributing a. List down the at least 3 factors contributing
to a too thin or a too thick blood smear. to a too thin smear and 3 factors contributing
to a too thick blood smear.
6. Stain properly the blood smear. a. Stain a blood smear adequately (not too
acidic, not too basic).
biodegradable, non-infectious wastes in
correct trash bins.
Rubrics
Weight 1 – Developing 2 - Acceptable 3 – Effective 4 - Exceptional Score
Disinfection or
the Laboratory All areas in the 2 areas in the 3 areas in the All areas in the
(5%) laboratory were not laboratory were laboratory were laboratory were
disinfected before use disinfected before use disinfected before use disinfected before use
cabinet (3) floor

Wearing PPE
(5%)
gown
Analytical (80%)
Preparation of Smear (40%)

Push-Wedge
Preparation (30%)
(1) Has gradual and

even transition from
thick to thin area. (2)
finger-shaped with a
0 - 1 out of 6 2 -3 out of 6 4 - 5 out of 6
smooth tail (3) occupy 6 out of 6
two-thirds to three-
characteristics of a characteristics of a characteristics of a
fourths of the glass characteristics of a well-
well-made smear well-made smear well-made smear
slide (4) does not touch made smear achieved
achieved achieved achieved
the outer borders of the
slide or run off the sides
or ends of the slide (5)
has smooth surface free
from ridges, waves,
gaps and holes (6) has
feathery edge.
Cover Slip Method
(10%)
1) Smear was made. (2) 1 out 4 characteristics 2 out 4 characteristics 3 out 4 characteristics
4 out 4 characteristics
Blood was evenly achieved achieved achieved
achieved
distributed to the cover
slip. (3) No holes or cuts
in the smear. (4) Cover
glass not broken.
Staining of Smear (40%)
Staining (40%)
(1) Smear was air dried.

(2) Fixation was done.
(3) Smear was 1 out of 4 done 2 out of 4 done 3 out 4 done 4 out of 4 done
adequately stained in
eosin. (4) Smear was
adequately stained in
methylene blue.
Disinfection or
disinfected after use disinfected after use disinfected after use disinfected after use
cabinet (4) floor
Waste Disposal
(5%)
(1) sharps (2) infectious
not disposed to disposed to disposed to disposed to appropriate
wastes (3)
appropriate bag appropriate bag appropriate bag bag
biodegradable wastes,
(4) non-biodegradable,
non-infectious wastes
Total Score:

Name: ___________________________________ Date: ______________
ACTIVITY 2
SMEAR PREPARATION AND STAINING
Review Questions
1. What are the blood smears used in the laboratory? Give their uses.
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
2. What are the qualities of a well-prepared blood smear?

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
3. List at least 3 factors that contribute to a too thin smear and at least 3 factors that contribute to a
too thick smear.
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
4. List 3 factors that contribute to a too acidic smear and 3 factors that contribute to a too basic smear.
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________

ACTIVITY 3
HEMOGLOBIN DETERMINATION
Unit Intended Learning Outcomes
At the end of the experiment, the students (should be able/are expected to):
Disinfect properly the different areas of the laboratory.

Wear complete and proper personal protective equipment.
Perform correctly hemoglobin testing different methods of determination.
Describe correctly the principle of cyanmethemoglobin method in hemoglobin determination.
Enumerate correctly the causes of increased and decreased hemoglobin levels.
Perform correctly the proper waste segregation and disposal.
Pre-laboratory Discussion
Hemoglobin is the major constituent of the red cell cytoplasm, accounting for approximately 90% of the dry
weight of the mature cell. It is comprised of heme and globin. Its primary function within the erythrocyte is
to carry oxygen to and carbon dioxide from the tissues. Hemoglobin estimation is used as a screening test
for detecting anemia.
Methods of Determination:
A. COLORIMETRIC METHODS
1. INDIRECT
a. Cyanmethemoglobin (hemiglobincyanide) method
Blood is diluted in alkaline Drabkin’s reagent containing potassium ferricyanide,
potassium cyanide, sodium bicarbonate, and a surfactant.
Principle:
potassium ferricyanide potassium cyanide
+2 +3
Hb (Fe ) → methemoglobin (Fe ) → cyanmethemoglobin
Cyanmethemoglobin is then measured spectrophotometrically at 540 nm, where the

absorbance is directly proportional to hemoglobin concentration. Cyanmethemoglobin
method is the reference method approved by Clinical and Laboratory Standards

Institute (CLSI) because all forms of hemoglobin are converted to cyanmethemoglobin,
except sulfhemoglobin.
b. Oxyhemoglobin Method
Hemoglobin is converted to oxyhemoglobin by reaction with ammonia and the color of
the solution is measured directly in spectrophotometer.
2. DIRECT/ VISUAL
a. Sahli’s or Acid Hematin Method
Blood is mixed with 0.1 N HCl resulting in the conversion of hemoglobin to acid hematin
which is yellowish brown in color. The solution is diluted until color matches with the
brown colored glass of the comparator box. Hemoglobin concentration is read directly.
b. Alkaline Hematin Method

Blood is converted to alkaline hematin by addition of alkali such as sodium hydroxide
(NaOH) and the color measured in a colorimeter at 540 nm. It gives a true estimate of
hemoglobin and is not affected by the presence of plasma proteins and lipids. However,
it is not used routinely as it is less accurate than the cyanmethemoglobin method and
some forms of hemoglobin such as HbF are resistant to alkali denaturation.
B. GASOMETRIC METHODS (Measurement of Oxygen Carrying Capacity)

1. Van Slyke Oxygen Capacity
This is a measure of the hemoglobin function. The oxygen combining capacity of blood is 1.34 ml
O2 per gram of hemoglobin. It is not used routinely as it is not practical and gives results 2%
lower than the other methods.
C. SPECIFIC GRAVITY METHODS

This is a qualitative screening test based on specific gravity. The density of the drop of blood is
directly proportional to the amount of hemoglobin it contains. A drop of blood is added to copper
sulfate solution with known specific gravity (1.052 to 1.054). If the blood drop sinks, hemoglobin
level is greater than 12.5 g/dL.
D. CHEMICAL METHODS (Iron Content of Hemoglobin)

Iron found within the RBCs binds with hemoglobin. Thus, iron content can be directly related to the
hemoglobin concentration. Iron content can be measured and converted to hemoglobin by using
the formula 0.347 mg iron 100g hemoglobin.
1. Kennedy’s
2. Wong’s
E. HEMOGLOBIN ELECTROPHORESIS
Hemoglobin electrophoresis using cellulose acetate at an alkaline pH (8.2 to 8.6) is a screening test
for detecting variant or abnormal hemoglobins. Further confirmation can be achieved using citrate
agar electrophoresis.


3. Collect blood sample correctly.
Analytical Phase
1. EDTA anticoagulated blood

2. Drabkin’s reagent
3. Spectrophotometer
4. Cuvettes
5. 0.1 N HCl
6. Distilled water
7. Hemoglobinometer tube
8. Sahli’s Comparator Block
9. Copper Sulfate Solution
10. Beaker
11. Pasteur pipet or dropper
12. PPE
Procedure
A. CYANMETHEMOGLOBIN METHOD
1. Create a standard curve, using a commercially available cyanmethemoglobin standard.
a. When a standard containing 80 mg/dL of hemoglobin is used, the following dilutions
should be made.
Hemoglobin Concentration (g/dL) Blank 5 10 15 20

Cyanmethemoglobin std (mL) 0 1.5 3 4.5 6
Cyanmethemoglobin rgt (mL) 6 4.5 3 1.5 0
b. Transfer the dilutions to cuvettes. Set the wavelength on the spectrophotometer to

540 nm and use the blank to set to 100% transmittance.
c. Plot percentage transmittance on the y-axis and the hemoglobin concentration on
the x-axis. The hemoglobin concentrations of the control and patient samples can
be read from this standard curve.
Note: Standard curve should be set up with each new lot of reagents and should be checked when
alterations are made to the instrument.

2. Using the patient’s whole blood anticoagulated with EDTA or heparin or blood from a
capillary puncture, make a 1 : 251 dilution by adding 0.02 mL (20 uL) of blood to 5 mL of
cyanmethemoglobin reagent. The pipette should be rinsed thoroughly with the reagent to
ensure that no blood remains. Follow the same procedure for the control samples.
3. Cover and mix well by inversion or using a vortex mixer. Let stand for 10 minutes at room
temperature to allow full conversion of hemoglobin to cyanmethemoglobin.
4. Transfer all of the solutions to cuvettes. Set the spectrophotometer to 100% transmittance
at the wavelength of 540 nm, using cyanmethemoglobin reagent as a blank.
5. Using a matched cuvette, continue reading the patient samples and record the percentage
transmittance.
6. Determine the hemoglobin values of the control samples and the patient samples from the
standard curve.
B. ACID HEMATIN METHOD

1. Add 0.1 N HCl into the tube up to mark 2g%.
2. Mix the EDTA sample by gentle inversion and fill the pipette with 0.02 ml (20 uL) blood.
Wipe the external surface of the pipette to remove any excess blood.
3. Add the blood into the tube containing HCl. Wash out the contents of the pipette by
drawing in and blowing out the acid two to three times. Mix the blood with the acid
thoroughly.
4. Allow to stand undisturbed for 10 minutes.
5. Place the hemoglobinometer tube in the comparator and add distilled water to the solution
drop by drop stirring with the glass rod till until color matches with that of the comparator
glass. While matching the color, the glass rod must be removed from the solution and held
vertically in the tube.
6. Remove the stirrer and take the reading directly by noting the height of the diluted acid
hematin and express in g%.
C. COPPER SULFATE METHOD

1. Collect blood sample.
2. Add a drop of blood to the copper sulfate solution from a height of about 1 cm.
3. Observe the behavior of the drop until 15 seconds.
**Note: Change the copper sulfate solution after every 25 tests, daily, and if the solution
turns cloudy during testing. Keep the solution covered to prevent evaporation between tests.
FIGURE 7. HEMOGLOBIN DETERMINATION USING COPPER SULFATE METHOD
SOURCE: HTTP://WWW.EHOW.COM/HOW_4845244_DONOR-HEMOGLOBIN-USING-COPPER-SULFATE.HTML

3. Wash laboratory equipment before storage.
Name: ___________________________________ Date: ______________

ACTIVITY 3
3. Perform correctly hemoglobin testing a. Perform hemoglobin testing using
different methods of determination. cyanmethemoglobin method, acid hematin
method, and copper sulfate method.
4. Describe correctly the principle of a. Describe the effect of adding ferricyanide
cyanmethemoglobin method in hemoglobin and potassium cyanide to blood sample.
determination. b. Describe why the cyanmethemoglobin
method is the method of choice in
hemoglobinometry.
5. Enumerate correctly the causes of increased a. Identify the possible causes of an increased
and decreased hemoglobin levels. or decreased hemoglobin levels.
correct trash bins.

Rubrics
Weight 1 - Developing 2 - Acceptable 3 – Effective 4 – Exceptional Score
Disinfection or
Cleaning of 4 Areas
in the Laboratory All areas in the 2 areas in the 3 areas in the All areas in the
disinfected before use disinfected before use disinfected before use disinfected before use
cabinet (4) floor
Wearing PPE
(5%)
(1) gloves (2) mask
(3) hair net (4)
laboratory gown
Analytical (80%)
Determination of
hemoglobin
concentration
(80%)
(1) Created a
cyanmetHgb curve.
(2) Measured Hgb 1 out of 4 done 2 out of 4 done 3 out of 4 done 4 out 4 done
concentration using
cyanmetHgb
method (3) using
acid hematin
method, and (4)
using copper
sulfate method.
Disinfection or
Cleaning of 4 Areas
in the Laboratory All areas in the 2 areas in the 3 areas in the All areas in the
cabinet (4) floor
Waste Disposal
(5%)
(1) sharps (2)

infectious wastes
not disposed to disposed to appropriate disposed to appropriate disposed to appropriate
(3) biodegradable
appropriate bag bag bag bag
wastes (4) non-
biodegradable,
non-infectious
wastes
Total Score:

Name: ___________________________________ Date: ______________
ACTIVITY 3
Laboratory Report
Cyanmethemoglobin Standard Curve

% Transmittance
Hemoglobin concentration (g/dL)

Cyanmethemoglobin Acid Hematin Copper Sulfate
Method of Determination
Method Method Method
Hemoglobin Concentration

Interpretation:
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
Review Questions
1. Describe the principle of cyanmethemoglobin in hemoglobin determination.

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
2. What is the method of choice in hemoglobin determination? Discuss briefly.

_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
3. Describe the principle of copper sulfate method in hemoglobin determination.

_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
4. Why copper sulfate is considered an obsolete method of hemoglobin determination?

_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
5. Give the clinical importance of hemoglobin determination.

_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________

ACTIVITY 4
HEMATOCRIT DETERMINATION
Unit Intended Learning Outcomes
At the end of the experiment, the students (should be able/are expected to):
Disinfect properly the different areas of the laboratory.

Wear complete and proper personal protective equipment.
List correctly the three layers of centrifuged blood.
Measure hematocrit accurately using microhematocrit method.
Determine correctly the causes of increased and decreased hematocrit levels.
Discuss thoroughly trapped plasma.
Perform correctly the proper waste segregation and disposal.
Pre-laboratory Discussion
The hematocrit (Hct) or the packed cell volume (PCV) or erythrocyte volume fraction (EVF) measures the
concentration of packed RBCs in a given volume of whole blood. It is determined after centrifugation and is
reported either as a percentage (e.g., 36%) or in liters per liter (0.36 L/L).
Hematocrit can be used in calculating RBC indices which is needed in assessing the morphologic
classification of anemia. Furthermore, it is helpful in evaluating fluid status, in determining the degree of
anemia, and monitoring other diseases such as acute hemorrhagic conditions.
METHODS OF DETERMINATION
1. Microhematocrit method
This method requires small amount of blood, 2 to 3 drops only. The blood can be obtained by finger
puncture.
2. Macrohematocrit method
A large volume of blood is required in this method. Approximately 2 to 4 ml is required.
a. Wintrobe method
This method utilizes a Wintrobe tube which is 110 mm in length and has two (2) calibrations:
0 to 10 for ESR (left side) and 10 to 0 (right side) for hematocrit determination.

FIGURE 8. WINTROBE METHOD
SOURCE: RETRIEVED FROM: HTTP://WWW.MEDICINE.MCGILL.CA/PHYSIO/VLAB/BLOODLAB/ESR.HTM
b. Haden’s modification
c. Van Allen method
d. Sanford-Magath method
e. Bray’s method
3. Automatic analyzers
They provide an indirect measurement of hematocrit.
a. Coulter counter
b. Autoanalyzer
4. Rule of Three
The “Rule of Three” is used to estimate RBC count and hemoglobin levels on the basis of the
hematocrit level. However, this rule applies is only applicable to specimens that have normocytic
normochromic erythrocytes.
Trapped Plasma
Trapped plasma is that amount of intracellular plasma remaining in the packed cell column of the
microhematocrit after centrifugation. It artificially increases the packed cell column constituting a positive
bias to the hematocrit.


Analytical Phase
1. Capillary tubes
a. Heparinized, for capillary blood sample
b. Plain (non-heparinized), for EDTA anticoagulated blood sample
2. Microhematocrit centrifuge
3. Microhematocrit reader
4. Sealing clay
5. Soft wax
6. Gauze
7. PPE
Procedure
MICROHEMATOCRIT METHOD
1. Obtain blood sample.
a. Fill two (2) heparinized capillary tubes approximately ¾ full with blood collected through
skin puncture. Gently tilt the tubes back and forth to mix blood and the heparin coating
the sides of the tube.
b. Fill two (2) plain capillary tubes approximately ¾ full with EDTA anticoagulated blood
collected through venipuncture.
2. Wipe off excess blood outside the tube and seal the end with the colored ring using
nonabsorbent clay. The plug should be at least 4 mm long.
3. Balance the tubes in the centrifuge. Sealed ends should be positioned away from the center,
touching the rubber gasket.
4. Centrifuge the tubes at 10,000 rpm for 5 minutes.
5. Determine the hematocrit using a microhematocrit reader. The values of the duplicate
hematocrit should agree within 1% (0.01 L/L).
a. Hold the tube against the scale so that the bottom of the column of erythrocytes (not
the bottom of the tube) is aligned with the horizontal zero line.
b. Move the tube across the scale until the top of the plasma intersects the top line of the
scale.
c. The value of the hematocrit is read on the line where the RBC’s meet the plasma.

Name: ___________________________________ Date: ______________
ACTIVITY 4
3. List correctly the three layers of centrifuged a. List in correct order the three (3) layers of
blood. centrifuged blood.
4. Measure hematocrit accurately using a. Measure hematocrit accurately using
microhematocrit method. microhematocrit method.
b. Measure hematocrit in duplicate with values
that agree within 1% (0.01 L/L).
5. Determine correctly the causes of increased a. List down the physiologic and pathologic
and decreased hematocrit levels. causes of increased and decreased
hematocrit levels.
6. Discuss thoroughly trapped plasma. a. Define trapped plasma and discuss factors
contributing herewith.
7. Perform correctly the proper waste b. Dispose sharps, infectious wastes,
correct trash bins.
Rubrics
Weight 1 - Developing 2 - Acceptable 3 - Effective 4 – Exceptional Score
Disinfection or All areas in the 2 areas in the 3 areas in the All areas in the
Cleaning of 4 Areas in laboratory were not laboratory were laboratory were laboratory were
the Laboratory disinfected before use disinfected before disinfected before disinfected before
(5%) use use use

cabinet (4) floor
Wearing PPE 1 out of 4 PPE worn 2 out of 4 PPE worn 3 out of 4 PPE worn All PPE worn
(5%)

gown
Analytical (80%)
Sample collection
(20%)
(1) First drop of blood

wiped off. (2) Capillary
tube filled to ¾. (3)
Tubes gently tilted to
mix blood and
anticoagulant. (4)
Excess blood wiped off.
Sample processing
(30%)
(1) Tubes sealed

properly. (2) Tubes
were balanced in the
centrifuge. (3) Sealed
ends placed facing the
rubber gasket. (4)
Samples were
centrifuged for 5
minutes.
Microhematocrit
reading (30%)
(1) Capillary tube

positioned correctly in
the reader. (2) 1 out of 4 done 2 out of 4 done 3 out of 4 done 4 out of 4 done
Hematocrit was read
correctly. (3) Done in
duplicate. (4) Duplicate
agree within 1% of the
value.
Disinfection or
cabinet (4) floor
Waste Disposal
(5%)
4 type of wastes were 2 types of wastes 3 types of wastes All wastes were
(1) sharps (2) infectious not disposed to were disposed to were disposed to disposed to
(3) biodegradable (4) appropriate bag appropriate bag appropriate bag appropriate bag
non-infectious non-
biodegradable
Total Score:

Name: ___________________________________ Date: ______________
ACTIVITY 4
Laboratory Report
Using the “Rule of Three,” estimate the red blood cell (RBC) count and the hemoglobin level based on the
hematocrit. Show your computation in the space provided.
Sample Hematocrit Level (%) RBC count Hemoglobin (g/dL)

1
2
Computation:
Interpretation:
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________

Review Questions
1. What are the layers of centrifuged blood? Draw and label.
2. What is trapped plasma? Discuss.

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
3. When is trapped plasma increased?

__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
4. Why is it important to read the microhematocrit soon after the centrifuge stops?
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
5. List at least 5 pathologic conditions that can cause increased hematocrit levels.
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
6. List at least 5 pathologic conditions that can cause decreased hematocrit levels.
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
7. List at least 2 factors that can cause falsely increase and at least 2 factors that can falsely decrease
hematocrit levels.
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________

Activities 1 4 Hema1lab Manual 2022 2023

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Activities 1 4 Hema1lab Manual 2022 2023

Uploaded by

Copyright:

Available Formats

Name: ___________________________________ Date: ______________

Section: ________________ Group: _________ Score: ______________

BLOOD COLLECTION TECHNIQUES

Assessment Criteria for Passing

Clinical Hematology I – A Compilation of Laboratory Exercises | 11

Weight 1 – Developing 2 – Acceptable 3 - Effective 4 - Exceptional Score

(1) Patient was properly

(1) Tourniquet was

(1) Needle was inserted

Clinical Hematology I – A Compilation of Laboratory Exercises | 12

Capillary Puncture (40%)

(1) Patient was properly

(1) Hands were

(1) Puncture was

Clinical Hematology I – A Compilation of Laboratory Exercises | 13

BLOOD COLLECTION TECHNIQUES

1. What are the collection sites for venipuncture?

2. Give the correct order of draw for venipuncture.

3. Give five (5) restriction sites for venipuncture.

4. What are the collection sites for capillary puncture?

5. Give the correct order of draw for capillary puncture.

Clinical Hematology I – A Compilation of Laboratory Exercises | 14

7. Give five (5) reasons for specimen rejection.

8. Why should we allow the alcohol to be air dried?

Clinical Hematology I – A Compilation of Laboratory Exercises | 15

Factors that may be altered to produce a well-made blood smear:

FIGURE 3. WELL-MADE BLOOD SMEAR

SOURCE: RODAK, B., ET AL., 2012

FIGURE 4. UNACCEPTABLE BLOOD SMEARS

SOURCE: RODAK, B., ET AL., 2012

Common causes of a poor blood smear:

Clinical Hematology I – A Compilation of Laboratory Exercises | 17

STAINING OF BLOOD SMEAR

Components of Romanowsky stain:

Causes of Too Acidic Smear

Causes of Too Basic Smear

Teaching and Learning Activities

1. Disinfect the working area using 10% sodium hypochlorite.

Reagents, Supplies and Equipment

PREPARATION OF THIN BLOOD SMEARS

A. Push Wedge Preparation/ Two Slide Method

FIGURE 5. PUSH WEDGE PREPARATION/ TWO SLIDE METHOD

SOURCE: RETRIEVED FROM: HTTP://WWW.KEYWORD-SUGGESTIONS.COM/YMXVB2QGC21LYXIGCHJVY2VKDXJL/

B. Two-Cover Slip/ Erlich’s Two-Coverglass Method

Clinical Hematology I – A Compilation of Laboratory Exercises | 19

SOURCE: RETRIEVED FROM: HTTPS://WWW.STUDYBLUE.COM/NOTES/NOTE/N/

PREPARATION OF THICK BLOOD SMEAR

STAINING OF PERIPHERAL BLOOD SMEAR

1. Ensure that the blood smears are completely air dried.

Clinical Hematology I – A Compilation of Laboratory Exercises | 20

SMEAR PREPARATION AND STAINING

Weight 1 – Developing 2 - Acceptable 3 – Effective 4 - Exceptional Score

Clinical Hematology I – A Compilation of Laboratory Exercises | 21

Preparation of Smear (40%)

(1) Has gradual and

(1) Smear was air dried.

Clinical Hematology I – A Compilation of Laboratory Exercises | 22

SMEAR PREPARATION AND STAINING

2. What are the qualities of a well-prepared blood smear?

Clinical Hematology I – A Compilation of Laboratory Exercises | 23

Unit Intended Learning Outcomes

Disinfect properly the different areas of the laboratory.

Cyanmethemoglobin is then measured spectrophotometrically at 540 nm, where the

Name: _____________________ Date:

Section: ____________ Group: _ Score: __________

Name: _____________________ Date: