Experiment No.

1
Capillary Puncture Method of Blood Collection

Capillary Puncture

A capillary is a tiny blood vessel connecting the small arteries (arterioles) to the
small veins (venules). Capillary puncture is a safe and efficient means of collecting a
blood specimen when only a small amount of blood is required.
Capillary blood is a good specimen because the cell distribution resembles that
normally found in the circulating blood. Blood cell counts, the microhematocrit, and the
blood smear are some of the hematologic procedures than be performed using capillary
blood.

Objectives

 To be able to perform capillary puncture

 Identify the sites for capillary puncture
 To collect blood specimen from a capillary puncture

Materials

 Blood lancet
 70% alcohol
 Cotton balls

Procedure

1. Preparing the puncture sites.

2. Performing the puncture.
3. Collecting the blood sample.

Illustrate the capillary blood collection sites

Ring/great finger

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Ear lobe

Infant’s heel/great toe

The Capillary Puncture (Illustrate the procedure)

1. Preparing the puncture sites

2. Performing the puncture

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3. Collecting the blood sample

Questions
1. What is a capillary vessel?

2. Why would a capillary puncture be performed?

3. What are the usual puncture sites for adults; for infants?

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4. How is a capillary puncture site prepared?

5. What is the procedure if the patient has cold hands?

6. Why is the 1st drop of blood wiped away?

7. List the precautions that must be observed when performing a capillary puncture.

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8. Define capillary, capillary action, capillary tube, heparin, lancet, and lateral.

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 Experiment No. 2
Venipuncture Method of Blood Collection

The most common method of obtaining blood for laboratory examinations is by

venipuncture. The venipuncture is a quick way to obtain a large sample of blood on
which many different analyses can be performed. In a venipuncture, also called
phlebotomy, the blood is taken directly from a superficial vein. The vein is punctured
with hypodermic needle and blood is collected in a syringe or tube.
The venipuncture is a safe procedure when performed correctly by a skilled worker.
The procedure must be performed with care. Every effort should be made to preserve the
condition of the vein. Much observation and practice is required to become skilled and
self-confident in the art of venipuncture.

Objectives

 To be able to perform venipuncture

 Identify the sites for venipuncture
 To collect blood specimen from a venipuncture

Materials

A. Syringe method
 Needle
 Syringe
 Needle 20 gauge
 70% alcohol
 Cotton balls
 Tourniquet

B. Evacuated tube system

 Evacuated tube
 Adaptor
 Vacutainer needle
 Tourniquet
 70% alcohol

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Procedure
Illustrate the procedure (for both syringe and vacutainer method)

1. Selecting the proper equipment

Syringe method Evacuated tube method

2. Preparing the patient for venipuncture

3. Apply the torniquet

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4. Selecting the venipuncture site

5. Preparing the venipuncture site

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6. Performing the venipuncture
a) Insertion of needle
b) Extraction of blood
c) Releasing of tourniquet
d) Releasing of needle
e) Application of pressure

Syringe method

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Evacuated Tube Method

Guide for selection of vacuum tubes

Color cold Anticoagulant Examples of use
Red None Chemistry, serologic tests
Red/Gray None Serum separator tube; used for
tests that require serum
Lavender EDTA Most hematologic tests; bold
typing
Green Heparin Open heart surgery; arterial
blood gas
Light blue Sodium citrate Most coagulation studies
Gray Sodium fluoride Certain glucose methods

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Complications in venipuncture (fill in the blank)
1. Local immediate complications

2. Local delayed complication

3. General delayed complications

Questions
1. Name precautions that must be observed in performing venipuncture.

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2. What are the different sites of venipuncture for
a) Newborn infants
b) Children
c) Adults

3. Give the advantages and the disadvantages of using vacutainer method.

4. Give the advantages and the disadvantages of using syringe method.

5. What are the different methods of making non visible veins prominent?

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 Experiment No. 3
Hemocytometry

Hemocytometry or the process of enumerating blood cells like wbc, rbc, and platelets.
Other bodies that may be counted are spermatozoa, punctuate basophilic red cells,
eosinophiles and cells in the CSF.

Objectives

 Identify the parts of a hemocytometer

 Use the microscope to identify the hemacytometer areas where red cells and white
cells are counted
 Fill the hemocytometer using a blood diluting pipet
 Clean and dry the hemocytometer and coverglass
 Lists the precautions observe when using the hemacytometer

Principle

1. Selection of diluting fluid that will not only dilute the cells to manageable levels, but
will either identify them in some fashion or destroy some contaminated cellular
elements.
2. The use of a hemocytometer or an electronic counter that will present the cells in such
a way to the observer or to an electronic device that the number of cells per unit
volume of fluid can be counted. Electronic counter avoids human error and because
of larger number of cells is counted, it is statistically more accurate.

The Counting Chamber

This is made up of thick rectangular slides. In the center of the upper surface there
are ruled areas separated by moats from the rest of the slides, and two raised transversed
bars, one of which is present on each side of the ruled area. The ruled portion maybe:
1. Single Chamber – ruled portion in the central area
2. Double Chamber – ruled portion maybe in upper and lower portion

There are two types of counting chamber, the Improved Neubauer and Fuchs
Rosenthal.

IMPROVED NEUBAUER: the depth between the lower surface of the coverglass, which
is lying on the raised bars, and the ruled area is 0.1 mm. Each ruled are is a square of 3
mm divided into 9 large squares, each of 1 mm side. The central square of these 9 is

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divided by engraved lines into 400 tiny squares arranged in 25 group of 16 by triple
boundary lines. Each large square is 1 sq.mm, each of the 25 medium squares is of 0.2
mm side (0.04 sq.mm area), each of the 400 tiny squares is of 0.05mm side (0.0025
sq.mm area).

FUCHS ROSENTHAL: the depth of the chamber is 0.2 mm. Each ruled area is a square
of 4 mm divided into 16 squares of 1 sq.mm each. These squares are divided by triple
lines. Each large square is further subdivided into 16 smaller squares. The total volume
over the ruled area is 16 × 0.2 cu.mm =3.2 ul.

Illustrate the following:

1. Draw the counting chamber top view with cover glass in place.

2. Draw the side view of the counting chamber with cover glass in pla/

3. Draw the counting chamber. Label the dimensions and areas used in the erythrocytes
and leukocyte counting.

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Questions
1. List the parts of the hemacytometer.

2. Which squares are counted for WBC count ------for an RBC count?

3. Explain how to fill the hemocytometer using the pipet.

4. What is the proper procedure for cleaning the hemacytometer and cover glass?

5. List the precautions observed when using the hemacytometer.

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 Experiment No.4
Thoma Diluting Pipet

Basically, there are two types of diluting pipettes. These are the RBC and WBC
pipettes. The pipette has a bulb about 1/3 of the total length from one end of the capillary
tube. The shaft of the long end is divided into 10 equal parts. Just beyond the bulb on the
short limb of the pipette is a mark 101 for the RBC pipette and mark 11 for the WBC
pipette. When filled to this mark, the bulb holds 100 times the volume contained in the 10
divisions of the long limb. Blood from skin puncture or from blood with anticoagulants
drawn up to 0.5 mark, with care to ensure that the column of blood is uninterrupted by
bubbles. With experience, one can draw the blood just level with 0.5 mark but if too
much is drawn, the excess may be withdrawn by holding the pipette horizontally and
touching the tip with absorbent paper tissue. The tip and outside of the pipette are then
wiped clean and without delay, diluting fluid is drawn up to 101 mark for RBC and 11
mark for WBC. After mixing, the blood in the bulb will thus be diluted 1 in 200 for RBC
and 1 in 20 for WBC.

Objectives
 To be able to familiarize and know the difference between the RBC and WBC
diluting pipettes
Materials
 RBC and WBC pipettes

Illustrations
Draw the RBC and WBC diluting pipettes. Label all parts. Tabulate at least 5 differences
between two pipettes.

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Point of Difference RBC Pipette WBC Pipette
1. Color of the bead
2. Mark of the short limb
3. Dilution Factor
4. Tip
5. Diluting Fluids
6. Others

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 Experiment No. 5
RBC Count

This is the determination of the number of red blood cells present in 1 ul of blood.
Accurate hemoglobin and hematocrit determinations are more useful in determining
anemia.
The red cell count consists of dilution of blood (1:200) because of the large number
of cells present with a fluid that is isotonic and that prevents clotting, clumping and
rouleaux formation and then counting the cells in a counting chamber. The diluting fluid
does not destroy the leukocytes and therefore they are included in the count, but their
number is so small that their inadvertent inclusion in the count is negligible.

Objectives
 To perform RBC count with utmost accuracy and precision
 To correlate RBC count with clinical findings

Materials and Reagents

 Pipet shaker
 RBC pipets
 Hemocytometer
 Microscope
 Tally counter
 NSS diluting fluid
 Coverslip
 Blood collection kit

Procedure

The cover glass is placed on the chamber. If it is accurately placed and if the glass of
the chamber is transparent, then slight pressure to the ends of the cover glass will reveal
“rainbow” Newton Diffraction rings. The chamber is now ready for charging.

1. Discard a few drop of the diluted blood.

2. With the index finger forming a controlled seal over the end of the pipette, touch the
tip to the edge of the cover glass.
3. Gently release the index finger pressure and the diluted blood will flow into the
chamber by capillary action.
4. Tighten finger seal when the chamber securely on the microscope stage and allow to
stand for 2 to 3 minutes.

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5. Using a medium power (×10) objective, survey the ruled area to check cell
distribution.
6. The high power (×40) objective is then swung into position and with illumination
suitably reduced, the cells in 80 tiny squares are counted, i.e., the central and four
corners medium squares of the central large square.

Instruction:
1. Make the dilution, charge the counting chamber, count the cells.
2. Draw the area of the chamber used with the cells.
3. Record your count and computation.
4. Write the formula used for the computation.

Results

SIDE 1 SIDE 2
SQUARE CELLS COUNTED SQUARE CELL COUNTED
Ⅰ Ⅰ
Ⅱ Ⅱ
Ⅲ Ⅲ
Ⅳ Ⅳ
Ⅴ Ⅴ
TOTAL TOTAL

Computation

Write the general formula and the computation of the results in the box provided.
Interpret the results whether anemia, polycythemia or normal.

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Illustrate the area of the chamber used with the cells.

Questions
1. Lists the normal red cell count for male and female.

2. Name the compositions of Dacie’s fluid, Hayem’s diluting fluid, and Gower’s
solutions.

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3. What are the principles of other red cell counting techniques such as the Unoppete
Microcollection System and the Automated Red Cell Count.

4. Name at least three conditions associated with a decrease red cell counts and an
increase red cell counts.

5. Define erythrocytosis, hemolysis and isotonic solutions.

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 Experiment No. 6
Hemoglobinometry

This is red iron-bearing protein contained within the erythrocytes in normal blood.
Function: carrier of oxygen from molecules conjugated with one globin molecule. Heme
represents the colored component; globin is a complex protein with a molecular weight
between 63,000 and 67,000

Objectives:
 To be able to perform hemoglobinometry
 To be able to correlate clinical results.

Important Facts
1. Hemoglobin oxygen capacity: 1.34ml per gram(Hufner’s factor)
2. Hemoglobin Fe content: 0.347 per 100 g hb

A. Visual colorimetry
Sahli’s Method: based on conversion of hemoglobin to acid hematin.

Procedure:
1. place 20 ul (0.02ml) of blood into sahli’s tube containing N/10HCL up to 20
mark.
2. Mix and allow to stand for 10 minutes at room temp.
3. Add distilled water drop by drop until the color of the unknown matches that of
the standard.
4. Take reading in % and convert to grams using 14.8 grams equal 100% (accepted
standard in north America).

Results:

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B. Cyanmethehomoglobin
1. Add the reagents in the ff tubes labeled as:
Sample Tube Hb Standard Tube Blank Tube
Patients’ Blood 20 uL none none
Drabkin’s 5 ml 5ml 5 ml
Distilled Water none none 20 uL
Hb Standard none 20 uL none

2. Mix sample and stand for 10 minutes.

3. Measure absorbance at 540nm.
4. ABS Unknown X conc of STD
ABS of standard

Results
Patient’s Name:
Age:
Calculations:

Interpretation:

Questions
1. Define cyanmethemoglobin, drabkin’s reagent, globin, HiCN, and sythesis of Hb.

2. What are the possible sources of error in this activity?

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3. What are the two main components of hb?

4. What is the significance of the development of HiCN method for Hb?

5. Give the reference of Hb for adult male, female and children.

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 Experiment No 7
Hematocrit Determination

The blood hematocrit is a commonly preformed test. It may be performed

separately or as a part of a complete blood count (CBC). There are two types of
hematocrit determination.: macrohematocrit and microhematocrit. The microhematocrit is
a variation of hematocrit. It is a simple procedure in which whole blood is centrifuged in
a narrow tube. The hematocrit value reflects the red cell volume relative to a given blood
volume. It is an ideal test to follow the progress of anemic or bleeding patients.

Objectives

 To be able to perform correctly the macrohematocrit and microhematocrit

determination.
 To be able to correlate the results of hematocrit with the clinical condition of the
patient.

Reference value in hematocrit

SI Conventional Unit
Male 0.47 +/- 0.05 L/L 47 +/- 5%
Female 0.42 +/- 0.05 L/L 42 +/- 5%

A. Macrohematocrit by Wintrobe

Wintrobe tube is a thick flat bottom tube 11.5 cm long, 3mm internal bore, graduated
0-100mm with the cm marked by numbers, both ascending and descending. The left hand
side graduation descending from 0 down to 100 mm is designed for ESR determination
while the right hand side graduation ascending from 0 up to 100 mm is designed for
hematocrit determination.

Materials

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 - Wintrobe tube
- Black tap vacutainer
- Long stem Pasteur pipette
Procedure
1. Fill up the Wintrobe tube to the mark 10 cm with oxalate blood avoiding bubble
formation.
2. Centrifuge at 2500rpm for 30 minutes.
3. Observer the following:
a. fatty layer(not always discernible)
b. plasma layer
c. buffy coat layer
d. PCV layer
4. Take the hematocrit reading at the level between the lower layer of the buffy coat
and upper layer of the packed red blood cells.
5. Report in terms of volume per cm.

The buffy coat serves to give a rough idea of the white blood cell count. This is from
0.5 to 1mm thick made up leukocytes and platelets. Buffy coat less than 0.5 mm thick
means a high reduction in the total number of WBC: if greater than 1 mm thick it
indicates a higher level of WBC. If the buffy coat is subdivided into two indistinct layers,
the uppermost is rich in platelets’ The buffy coat may be aspirated and spread in case of
marked leukopenia in orders to obtain a greater concentration white cells for differential
count although this is not recommended for routine procedure. In search for LE cells, the
precipitation may be allowed to stand for 2 hours at room temperature prior to
centrifugation and then make smear out of the buffy coat. This will help in search for
magaloblasts and malignant cells in case of leucopenia.

The plasma layer may be investigated for hemolysis.

Calculation:
PCV vol.%=Net reading mm X 100
Vol. blood used (mm)

Results: Patients Name_____________________________ Reading:________________

Illustration:

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 B. Microhematocrit determination

The microhematocrit is performed using a few microliters of blood in a slender

capillary tube, approximately 7cm long and 1mm in diameter. The specimen may be
oxalated in which a plain capillary pipettes, color coded blue, may be used or capillary
blood directly from finger puncture, in which case heparinized capillary pipette, color
coded red, will be used.

Materials
- capillary tube(heparinized)
- lancet
- paraffin
- clay seal
- microhematocrit reader
- microhematocrit centrifuge

Procedure
1. Partially fill a capillary tube with blood leaving about 10 mm unfilled.
2. Seal the empty end by a small flame or by a plastic sealing compound.
3. Centrifuge for 4 minutes at a high speed in a microhematocrit centrifuge.
4. Read the cell column using a reading device.

Results: Patient’s Name_______________________Age____Sex____

Reading_________
Please attach and label the capillette used in the exercise

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Illustrate the procedure
1. Filling a capillary tube from a capillary puncture

2. Filling a capillary tube from a tube of blood

3. Sealing the capillary tube with sealing clay

4. The microhematocrit reader.

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Questions
1. What does the microhematocrit measure?

2. Name a condition that causes a decreased hematocrit level.

3. What precaution should be observed when performing a microhematocrit?

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 Experiment No. 8
Erythrocyte Sedimentation Rate (ESR)

The ESR is a simple, frequently performed hematology test, often referred to as the
“sed rate”. The ESR test is not specific for a particular disease but is used as an indicator
of inflammation, or tissue injury. The sedimentation rate results may also be used to
evaluate treatment and follow the course of certain inflammatory diseases.

Objectives
1. To be able to determine and perform the ESR accurately.
2. To be able to correlate the results of ESR with clinical condition of patients.

Principle:

The test depends on the fact that the blood to which anticoagulant has been added,
the RBC sediments until they form a packed column in the lower part of the tube or
container. The rate of this process depends on the number of factors like rouleaux
formation, concentration of alpha and beta globulin in the plasma. The test or the rate of
fall of the red blood cell is reported in milliliter at the end of one hour.

Stages of ESR:
1. Initial period of few minutes during which rouleaux formation takes place.
2. A period of approximately 1 ½ to 2 hours depending on the length of the tube
during which settling or sedimentation occurs at a more or less constant rate.
3. A slower rate of fall during which packing of the sedimentation cell column
occurs.

Materials
- Oxalated blood
- Wintrobe tube
- Pasteur pipette
- Stop watch

Procedure

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 1. Fill up the tube to the 0 mark with oxalated blood using Pasteur pipette with long
stem, avoiding bubble formation.
2. Allow to stand i9n a vertical position for one hour.
3. Take and record reading in mm.\

Results

Name of Patient______________________Age_______________Gender_______
Results for Wintrobe:

Results for Westergren:

Normal Range
Wintrobe (mm/hr) Westergren (mm/hr)
Male 0-9
<50 0-15
>50 0-20
Female 0-20
<50 0-20
>50 0-30
Children 0-13

Illustration:

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Wintrobe Tube Westergren Tube
Technical errors in ESR
False increased rate False decrease rate
Tube tilted Low temperature of blood
Vibration of tube Air bubbles in tube
Test>one hour Test<one hour
Improper bllod dilution Improper blood dilution
Improper mixing of blood Improper mixing of blood
Room temperature>25 C Room temperature< 20 C

Questions
1. What are the different physiologic and pathologic factors affecting ESR?

2. What is the importance of ZSR

3. Differentiate Westergren method from Wintrobe method.

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 Experiment No. 9
Reticulocyte Count

Reticulocytes are non nucleated immature red blood cells present in the peripheral
blood. They retained the remains of nuclear materials called basophilic spongioplasm or
reticulum, which stains a bright blue when mixed with the stain while the cells are still
alive (supravital stain). They contain aggregation of ribonucleic acid (RNA) within the
disintegrating ribosomes. The reticulocyte in 2 to 5 days, loses this reticulum along with
the RNA and becomes a mature RBC.
Reticulocyte count is the best index of erythroblastic activity of the bone marrow,
erythropoieses of blood regeneration.

Objective
- to be able to perform accurately reticulocyte count and correlate with the
clinical condition of the patient.

Materials/Reagents
- WBC diluting pipette
- Glass slide
- BCB or NMB
- NSS
- Wright’s stain

Procedure
1. Fill WBC pipette with saline brilliant cresyl blue to mark 1.
2. Puncture sterilized finger, wiped off the 1 st drop, draw blood into the pipette with stain
up to mark 1.
3. Slowly draw both dye and blood into the pipette bulb, mix and allow to stand for 10
minutes.
4. Mix again and make thin blood smear.

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5. Air dry and counter stain with wrights stain.
6. Count under OIO.
7. The reticulocyte appears as cells with a network of blue material. Care must be taken
not to confuse precipitated dye with reticulum.

Counting
1. A piece of paper the size of the eyepiece with an opening of 5 mm sq. cut in the
center is placed in the eyepiece to reduce the size of microscopic field.
2. Count 1,000 RBC nothing the number of cells showing reticulum, which appears
as blue dots filament.

Formula
% of reticulocytes = number of reticulocytes counted
10
Reticulocyte/cumm= RBC/cumm X reticulocytes counted
1,000

Normal values

Reticulocytes %=0.5 to 1.5% (infants 2-5%)

Reticulocytes/ cumm= 25,000-75,000/cumm
Reticulocyte index= 0.5-2 %

Computations:

Results:___________________________________
Name________________Age____ Sex________

Illustration:

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Questions
1. Why is reticulocyte count considered as the best index of erythropoiesis?

2. Give the clinical significance of:

a. increased count
b. decreased count

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 Experiment No. 10
Examination of Peripheral Blood Smear for RBC Anomalies

The peripheral blood smear particularly the feathery edge can be utilized to examine
the RBC anomalies. RBC anomalies can be classified according to variations in shape,
sizes and arrangement.

Objectives
 To identify different RBC anomalies
 To correlate the said anomalies with different clinical conditions of the patients

Materials
 Wright-stained blood smear
 Microscopes
 Cedarwood oil

Procedure
1. Scan the peripheral blood smear of the patient under the OIO.
2. Take note of the different RBC anomalies and tabulate as follows:

Anomalies Descriptions Illustrations Clinical Findings

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 Experiment No. 11
WBC Count

This is the determination of the number of white cells per ul of blood. There are two
possible methods for the WBC count and these are the following:

Objectives
 To perform WBC count with utmost accuracy and precision
 To correlate results with the clinical findings of the patient
Materials
 WBC pipets, WBC Diluting fluid, Hemocytomer, Microscope, Tally counter

1. Thoma Pipette method:

This pipette is similar to the red cell pipette except that the bulb is smaller, the bore is
larger and the mark at the short limb is 11 instead of 101.

Procedure

 Suck blood to mark 0.5 and diluting fluid to mark 11.

 This will give a dilution of 1:20.

2. Tube dilution:

Procedure

 Pipette 0.02ml of blood and mix with 0.38 ml of diluting fluid in a small tube or snap

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 cap plastic vial.
 Mix and charge the counting chamber.

Diluting Fluids

2-3% Acetic Acid

Glacial acetic acid.......................................2 ml
Distilled water…………………………...98 ml

Acetic acid is hypotonic, so it destroys rbc. Gentian violet permits easy identification
of the diluting fluid and renders the wbc visible by staining lightly.

Method of Charging the Counting Chamber

Same as for RBC, after standing for 1-3 minutes, the counting chamber is surveyed
with a low power objective (×10) to make sure cells are evenly distributed. Then the
number of cells in four large squares are counted.

Write the Formula for Counting of the Leukocytes:

Leukocyte Count in Blood with Large Number of Nucleated RBC:

Nucleated RBC (NRBC) are not dissolved by the wbc diluting fluid such that making
WBC count falsely elevated since NRBC are mistaken for as WBCs resulting in
pseudoleukocytosis.

This leukocyte count may be corrected in the following manner:

1. Determine the number of NRBC per 10 WBCs in the blood smear.
2. Do a WBC count – this is the uncorrected WBC count.
3. Determine the number of NRBC per ul – this is the absolute number of nucleated
RBCs.

Formula:

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 Let X be the absolute count of NRBC / ul.

X= no. of NRBC / 100 WBC × uncorrected WBC

100 + no. NRBC / 100 WBC

If the UWBC =19,500 u/l

NRBC = 25/100 WBC

X= 25 × 19,500
100 + 25
= 3,900 u/l of blood

Instructions:
1. Make the dilution, charge the counting chamber, count the cells.
2. Draw the area of the chamber used with cells on.
3. Record your count and computation. Interpret the result.
4. Write the formula used for your computation.

Results

SIDE 1 SIDE 2
QUADRANTS CELLS COUNTED QUADRANTS CELLS COUNTED
Ⅰ Ⅰ
Ⅱ Ⅱ
Ⅲ Ⅲ
Ⅳ Ⅳ
TOTAL TOTAL
Average Count:___________________

Computation

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Illustration

Questions
1. Aside from glacial acetic acid, what are the other diluting fluids can you use for
WBC count? Indicate their composition.

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2. What is leukocytosis? Give the clinical significance of such condition.

3. What is leucopenia? Give the clinical significance of such condition.

4. Give the different normal value of WBC count for adults, children and infants. Why
is it that infants have higher WBC count than that of the adults?

5. Why WBC count is higher in the afternoon?

6. Give the principle of automated method of WBC count.

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 Experiment no. 12
Preparation of Blood Smear

The examination of stained blood smear is a routine part of CBC. A blood smear
is prepared by spreading blood on a microscope slide. The smear is dried and stained. The
blood components may then be viewed microscopically identified and evaluated, as in the
differential leukocyte count. \A blood smear enables the technologist to view the cellular
components of blood in as natural as possible. The morphology, or structure of cellular
components can then be studied. The best specimen for a blood smear is a capillary
blood which has had no anticoagulant added.

Objectives:
 Discuss the purpose and importance of blood smear.
 List the components that may normally observed in a blood smear
 Be able to prepare a good blood smear.

Materials
 glass slide
 cover slip
 blood specimen
 EDTA tube
 stain

Procedure:
(Illustrate the procedure)
1. Cleaning of slide

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 2. Collecting the blood specimen

3. Making the smear

a. Two slides method

b. Cover glass method

4. Preserving and staining the smear

Illustrate the features of a good blood smear

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Illustrate the features of improperly prepared blood smear

Common problems encountered in preparing blood smear and possible causes

Problem Possible causes

Smear too thin or too long Drop of blood to small
Spreader slide at too low angle
Improper speed in making blood smear
Smear too thick or too short Drop of blood to large
Spreader slide at too high angle
Improper speed in making blood smear
Ridges or waves in smear Uneven pressure on spreader slide
Hesitation in pushing spreader slide
Holes in smear Slides not clean
Uneven or dirty edge of spreader slide
Uneven cell distribution Uneven pressure during spread of blood
Delay in spreading blood
Uneven or dirty edge of spreader slide

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Artifacts or unusual cell Smear dried too slowly
Morphology Smear not fixed within one hour after
preparation
High humidity

Staining of blood smear

Principle: Acidic dye unites with the basic components of the cell ( cytoplasm).
Conversely, basic stains are attracted and combine with the acidic part of the cell. Eosin
is an acidic dye; hence “eosinophilic” are used interchangeably in describing staining
qualities of cell components.

Wright stain
Preparation- the methylene blue is polychrome by heating with sodium bicarbonate. The
powder may be purchased and the solution is prepared by carefully dissolving 1 gram of
the powder with 600 ml of methyl alcohol.

Procedure:
1. Place the air-dried smear, film side up on the staining bridge of staining rack.
2. Avoiding slides to touch one another as it will cause drainage of the stain into the
staining rack.
3. Flood the smear with enough Wright stain solution and allow to stand for 4
minutes.
4. Wash with buffer and wipe off excess stain at the back of the slide.
5. Dry in a cleaning position.

Other Staining Methods

1. Leishman stains
2. Giemsa stain
3. Jenner-giemsa
4. May-Grunwald stain

Buffer solution, ph 6.8

Potassium phosphate, monobasic------------------6.63 g
Sodium phosphate, dibasic-------------------------2.56 g
Distilled water------------------------------------up to 1 liter

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Evaluating stain Quality
Slides that are too pink may be due to;
-Stain time too short
-Wash time too long
-pH of stain or buffer too acidic

Slides that are too blue may be due to;

-overstaining
-wash time or buffering time too short
-pH of stain or buffer too alkaline

Questions
1. What are the factors affecting the quality of a blood smear?

2. What is the purpose of blood smear?

3. What components in the blood can be viewed on a smear?

4. Discuss the different components of the Wrights Stain?

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 5. What are some errors to avoid whan making a blood smear?

Experiment No.13
Differential Leukocyte Count

The examination of WBC and its classification into the different types is the
differential count. The procedure involves counting 100-200 WBC on a stained blood
smear and recording how many each of the 5 types of WBC are seen. Information is also
obtained concerning the RBC and platelets. The RBCs are evaluated for morphology and
hemoglobin content. The platelets are evaluated for morphology and an estimation of
platelet numbers. The differential leukocyte count can be used to diagnose and monitor
the treatment of leukemias, anemias, and other diseases. For example, the viral infection
causing IM produces a characteristics differential count. Iron deficiency anemia produces
a characteristic RBC morphology with small red cell having a reduced amount of
hemoglobin.

Objectives

 To be able to differentiate leukocytes by using the peripheral blood smear

 To be able to correlate the differential count with the clinical condition of the patients

Features Evaluated in Cell Identification

1. Cell size
2. Nuclear characteristics
3. Cytoplasmic characteristics

Leukocyte Identification Guide

Segmenter Band/Stab Eo. Baso lympho mono

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Cell Size 10-15 um 10-15 um 10-15 um 10-15 um 8-15 um 12-20 um
Nucleus 2-5 lobes Sausage Bilobed Segmented Round Horseshoe
Shape
Structure Coarse Coarse Coarse Difficult to Smudge Folded
see
Cytoplasm Abundant Abundant Abundant Abundant Scanty Abundant
Amount
Color Pale-pink-tan Pale-pink- Pale-pink-tan Pale-pink-tan Clear blue Opaque
tan blue gray
Inclusions Small, lilac Small, lilac Coarse, Coarse, blue Occasional Ground
granules granules orange-red black red purple glass
granules granules granules appearance

Procedure

1. Before starting a differential count, have the following table ready for tallying the
count.
2. Focus of the smear under high power and study the cell upon pressure in the 1st
microscopic field.
3. Jot down the frequency or numbers of each cell type on the tally sheet.
4. Proceed to the next microscopic field and repeat no.3.
5. Continue with the differential count until a total of 200 cells are differentiated.
6. Sum up the individual cell type on the tally sheet and the total of each type by 2.
7. The result will be a corresponding percentage on each cell differentiation.
8. The result will be a corresponding percentage of each cell differentiated.

Results

PATIENT’S NAME:_____________________________
AGE:__________________SEX:___________________

SIUNIT
STAB…………………………………………………………..
SEGMENTERS………………………………………………..
EOSINOPHIL………………………………………………….
BASOPHIL…………………………………………………….
LYMPHOCYTE………………………………………………..
MONOCYTE…………………………………………………...

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GRAND TOTAL
Illustrate the types of WBC

Questions
1. What are the different ways of scanning blood smear. Illustrate it.

2. Give the clinical significance of an increase of each type of WBC.

3. Aside from WBC, what other cells of the blood may be observed in the smear? Give
its clinical significance.

4. Illustrate at least 10 examples of poikilocytosis.

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5. List the references ranges for WBC in the differential count.

Experiment No. 14
Osmotic Fragility Testing (OFT)

Osmotic fragility is a test to detect whether red blood cells are more likely to break
down. This test is performed to detect hereditary spherocytosis and thalassemia.
Hereditary spherocytosis makes red blood cells more fragile than normal. When exposed
to a hypotonic concentration of sodium in a solution, red cells take in more water, swell
until the capacity of the cell membrane is exceeded, and burst. RED BLOOD CELL
sensitivity to change in OSMOTIC PRESSURE.

Objectives:
 To be able to perform osmotic fragility with utmost accuracy and precision
 To correlate the results with the clinical conditions of the patients

Materials
 12 pcs test tubes
 Test tube rack
 0.5% salt solution
 Distilled water
 Heparinized blood
 Blood collecting kit

Procedure:
1. Dry some chemically pure NaCl in an oven and make an accurate 0.5% solution.
2. Place Kahn tubes in a rack.

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 3. Label the tubes in rack from 25 to 14.
4. Add to tube 0.5% salt solution and distilled water as follows:

Tube Number 25 24 23 22 21 20 19 18 17 16 15 14
Drops of 0.5% 25 24 23 22 21 20 19 18 17 16 15 14
NaCl
Drops of 0 1 2 3 4 5 6 7 8 9 10 11
distilled water
Drops of blood 1 1 1 1 1 1 1 1 1 1 1 1
% of NaCl .50 .48 .46 .44 .42 .40 .38 .36 .34 .32 .30 .28

To solve the concentration or the percentage of salt solution dilution in each tube,
multiply the number of drops of 0.5% salt solution by 0.02. Example Tube 5 x 0.02 =
0.50%

5. Obtain a heparinized blood from the vein.

6. Shake the tubes and let stand at room temperature for 2 hours.
7. Examine the tube for initial hemolysis and complete hemolysis.
8. Interpretation

Initial Hemolysis Complete Hemolysis

Normal Tube 22 Tube 17
Hereditary Spherocytosis Tube 24 Tube 20
Sickle Cell Anemia Tube 19 Tube 15

Results:
Name: Age: Gender:

Illustrate the tube set up and the results of the patient

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 Experiment No. 15
Examination of Bone Marrow Smear

The examination of bone marrow is usually done by a hematologist to assess the

blood cells populations directly from a hematopoietic tissue. Such procedure may be
vital in confirming certain blood dyscrasia.

Objectives
 To examine bone marrow smear under the supervision of a teacher
 To identify the blood cells that can be seen in a bone marrow smear

Materials
 Bone marrow smear
 Microscopes
 Cedarwood oil
 Hematology Atlas

Procedure
1. Scan a bone marrow smear under OIO.
2. Identify blood cells

Illustrate the bone marrow blood cells that you were able to identify

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 Experiment No. 16
Examination of Peripheral Blood Smear for WBC Anomalies

The peripheral blood smear particularly the feathery edge can be utilized to examine
the WBC anomalies. RBC anomalies can be classified according to variations in shape of
the nucleus, granulations, lobulations etc.

Objectives
 To identify different WBC anomalies
 To correlate the said anomalies with different clinical conditions of the patients

Materials
 Wright-stained blood smear
 Microscopes
 Cedarwood oil

Procedure
3. Scan the peripheral blood smear of the patient under the OIO.
4. Take note of the different WBC anomalies and tabulate as follows:

Anomalies Descriptions Illustrations Clinical Findings

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