Hematology 2 Lab Manual

Name: _________________________________________________ Date: __________________
Section: __________________ Group: __________________ Score: _________________
EXPERIMENT 1 DIFFERENTIAL COUNT
At the end of Hematology 413 student should be able to: 1.) Explain the principles of hemostasis coagulation and
fibrinolysis. 2.) Appreciate the importance of laboratory assays for the diagnosis of hemostatic disorders. 3.) Perform COURSE
the laboratory assays on hemostasis/coagulation with precision, accuracy and reliability. 4.) Manifest the following OUTCOMES
values, integrity, honesty, critical thinking, empathy and value for life.
To achieve this unit a learner must:

OUTCOMES
1. Be able to differentiate the different types of leukocyte in a normal

UNIT
peripheral smear.
2. Be able to perform the differential leukocyte count.
3. Be able to correlate the results to different disease or disorders
TEACHING AND LEARNING ACTIVITIES
Laboratory Experimentation
A. The critical examination of a blood smear includes of the following: quantitative and qualitative
study of platelets, differential count quantitating the three types of leukocytes (granulocytes,
lymphocytes, monocytes), and morphological characteristics of erythrocytes and leukocytes.
Staining the blood smears is a critical part of the examination. To accurately perform the
differential count it is necessary for a technician to recognize all the characteristics of a normal
blood cells. This includes normal biological variation. For instance, not every lymphocyte is
exactly the same size, nor do all lymphocytes have exactly the same number of azurophilic
granules.
B. Certain morphological and histochemical characteristics are utilized to differentiate blood cells.
A review of the significant features promotes a better understanding of blood differentials.
Cellular characteristics such as relative size, shape, cytoplasmic granulation, nuclear-cytoplasmic
ratio, nuclear configuration, chromatin or nucleoli are very important.
C. Experience is the foremost teacher in hematology. It is readily acquired in a busy hematology

section where the opportunity for differential analysis occur frequently. Experience can be
diversified and interesting if proficiency slides and material from cases of confirmed diagnoses
are maintained as study sets. This study material should be available to all technicians in the
laboratory.
1|CLINICAL HEMATOLOGY 2 LABORATORY MANUAL

1. Be able to differentiate the different types of leukocyte in a normal peripheral smear.
D. All routine blood smears should be kept until physicians have reviewed the differential reports.
A 1-wwek period is usually adequate. Occasionally, a review of a specific problem slide the
results in findings that were not originally apparent and reinforces confidence in the laboratory by the
medical staff. This practice also add s to the experience and proficiency of the technician.
Glossary of Terms
Eosinophilia: An increased number of eosinophils in the blood; associated with allergies, parasitic
infections, or hematologic disorder.
Lymphocytosis: Excess of normal lymphocytes in the blood.
Leukocytes: Any formed elements of the circulation blood system that comprise the cells if
immunity and inflammation; capable of amoeboid movement, whose chief function is to protect the
body against microorganisms causing disease and which comprise: granulocytes (basophils,
eosinophils, neutrophils), and agranulocytes (lymphocytes and monocytes)
Monocytosis: An increased number of monocytes in the peripheral blood.
Neutrophilia: An elevated number of neutrophils in the blood (absolute). Increased proportion of
neutrophils in the peripheral blood (relative).
PHASE
Upon the receiving of the blood sample collected by venipuncture the specimen should be
adequately labeled and logged properly for easy identification. Upon the physicians request of
performing differential count.
Cell counter Spreader slider

Glass slides Wright’s stain
Microscope
Cedar wood oil
C L I N I C A L H E M A T O L O G Y 2 L A B O R A T O R Y M A N U A L |2
OUTCOMES
UNIT
Procedure
Principle: The stained blood smear permits the study of the appearance and the identification of
the different kinds of leukocytes, and the appearance of erythrocytes and thrombocytes. One
hundred (100) leukocytes are counted, and the number of each type seen is recorded. The
proportion of each leukocyte type is reported as a decimal fraction.
1. Inspect the smear under low power magnification. Locate the thin end of the smear where there
is no overlapping of erythrocytes.
2. Switch to high power magnification. Check that the white cells are evenly distributed.
Figure 1.1
3. Switch to oil immersion. Identify and count 100 consecutive leukocytes and record each cell type
separately on the differential counter. Begin at the thin end of the smear and count the white
cells observed as the slide is moved in a vertical direction. When near the edges of the smear,
move the slide horizontally for a distance of about two fields, then proceed vertically back across
the smear. Continue this “snake-like” movement until 100 leukocytes have been counted and
classified. Figure 1.1
Figure 1.1

OUTCOMES
UNIT
4. If the WBC count is between 20,000 and 50,000 per cu mm of blood, count and classify 300
leukocytes. When the count is greater than 50,000 per cu mm of blood, count and classify 500
leukocytes.
5. The number of each type of leukocyte is expressed as a percent of the total number of white
cells counted. Absolute values may be calculated by multiplying the percent value by the total
leukocyte count.
A. Counting the leukocytes
6. Begin to count near the end of the smear (Fig 1.1), just where the red cells are beginning to
overlap.
7. Examine the strip of the film moving from one field to the next systematically. Record the type
of leukocyte using the cells counter seen in each field.
8. Count a total of 100 leukocytes.
B. Examination of leukocytes.
1. Note the shape of the leukocytes and its size in comparison with a red cell.
2. Note the shape of the nucleus and its size in relation to the total area of the cell:
Example: round, lobed, or indented
3. Note the appearance of the cytoplasm.
OUTCOMES
UNIT
3. Be able to to correlate the results to different disease or disorders
Critical Thinking
1. What is the significance of differential leukocyte count?
2. In what conditions will the neutrophil count decrease?
3. In what conditions will the neutrophil count increase?
4. In what conditions the eosinophil count decrease?
5. In what conditions the eosinophil count increase?
6. In what conditions the basophil count decrease?
7. In what conditions the basophil count increase?
8. In what conditions the monocyte count decrease?

OUTCOMES
UNIT
Outcome To achieve each outcome, the learner must

demonstrate the ability to:
1. Be able to differentiate the different types of a. Present different types of leukocyte that can be
leukocyte in a normal peripheral smear. seen on a normal peripheral smear.
2. Be able to perform the differential leukocyte b. Accurately count and perform differential count
count. on a strict time basis.
3. 3. Be able to correlate the results to different c. Correlate the results to different WBC disease and
disease or disorders disorders.
ASSESSMENT STRATEGIES
Focus for Assessment
ANECDOTAL NOTES
Observe students as they participate in the activity. Note the extent to which students are able to make and
talk about personal observation. Record anecdotal notes.
OBSERVATION CHECKLIST
Create an outcome-based checklist and share with students prior to beginning the activity. Use the
checklist to assess if students are able to make and talk about personal observations.
RUBRIC
Collaboratively create an outcome – based rubric with students. Use the rubric to evaluate how well the
students are able to understand cells.
RESOURCES
Rodak, Bernadette F., Hematology: Clinical Principles and Applications, 3rd Edition, Saunders Elsevier, Singapore,
2009
Steininger, Cheryl A., Clinical Hematology: Principles, Procedures, 1st Edition, Correlations, J.B. Lippincott
Company, Philadelphia, 1992
OUTCOMES
1. Be able to differentiate the different types of leukocyte in a normal peripheral
UNIT
smear.
RUBRICS
Critical Dimensions 1 2 3 4 5
REFER BEGINNER COMPETENT PROFICIENT EXEMPLARY Score
(No (Limited (Adequate (Remarkable (Professional
content) content) content) content) content)
Pre-Analytical phase:
PPE (lab gown, No PPE is Only 1-2 out Only 3 out of 5 Only 4 out of 5 Complete PPEs
mask, gloves, hair worn. of 5 PPEs are PPEs are worn. PPEs are worn. are worn.
cap &goggles) worn.
Materials No Incomplete Incomplete (3 Incomplete (2 are Complete

materials (4are lacking) are lacking) lacking) (Wright’s stain,
slides, whole
blood,
microscope,
dropper/
capillary tube)
Cleaning and Did not Cleaned but Cleaned and Cleaned or Cleaned, placed
disinfection of clean the did not disinfect the disinfected working table cover and
working area working disinfect the working area but area but placed disinfected
area before working area not placed table table cover before working area
starting or vice versa cover starting before starting
on the
working area
before starting
Phlebotomy:
Proper patient Do not 1 to 2 out of 5 3 out of 5 are 4 out of 5 are Complete
Identification (name, identify are properly properly done properly done patient
age, birthdate, patient done identification
requisition form, (name, age,
special consideration birthdate,
to the test if required) requisition form,
special
consideration to
the test if
required)
Blood extraction No blood Only 1 out of Only 2 out of 4 Only 3 out of 4 Complete blood
equipment (syringe, extraction 4 equipment equipment equipment extraction
vacuum tube, equipment equipment
tourniquet, cotton)
Proper blood Bevel Bevel Bevel orientation Bevel orientation is Bevel orientation

extraction technique orientation orientation is is correct, no correct, no is correct, no
is incorrect, correct, presence of air presence of air presence of air
presence of presence of air inside the inside the syringe, inside the
air inside inside the syringe, small insufficient amount syringe, correct
the syringe, syringe, little amount of blood of blood extracted amount of blood
no blood amount of extracted extracted
enters the blood enters
hub the hub, no
blood
extracted
Proper needle Remove Remove Remove needle Remove tourniquet, Remove

removal and needle needle without without remove the needle; tourniquet,
recapping without removing removing recapping done remove the
removing tourniquet; tourniquet; with hands needle and
tourniquet recapping recapping done recapping with
no done with with fishing fishing technique
recapping hands technique
done
Specimen labeling No label Only the name Only 2 out of 4 Only 3 out of 4 Complete
(patient name, age, was seen is seen on the label are seen on label are seen on specimen
date of collection, on the specimen label the specimen the specimen labeling
time of collection) specimen
Waste disposal No waste All All equipments All needles used All needles used
disposal equipments used were put in were put in a were put in a
observed used were put a yellow bag puncture proof puncture proof
in an container; others container; others
inappropriate are in a yellow bag are in a yellow
container bag labeled
"infectious"
Analytical phase:
Checking for Rough edge The edge of the

smearing slide smearing slide
was smooth and
labelled
Labelling of slides Not labelled 1 out of 4 is 2 out of 4 are 3 out of 4 are done Properly labelled
done properly done properly properly (name, age, date
and time of
collection)
Smear Preparation Placed 1 Placed 1 drop Placed 1 drop of Placed 1 drop of Placed 1 drop of
drop of of stained stained blood and blood and made a blood and made a
stained blood and made a good good smear perfect smear
blood and made a good smear (feathery (feathery edge, ¾ (feathery edge,
made a smear (spiky edge less than ¾ long to the slide or thumb or bullet
good smear edge, ¾ long long and not thumb/ bullet shaped smear, ¾
(spiky edge, or bullet/thumb shaped) long to the slide)
not ¾ long bullet/thumb shaped)
or shaped)
bullet/thum
b shaped)
Drying of specimen Dried by Dried by Dried by standing Dried by standing Air dry (fast), the
blowing blowing through air through air smear was
through through dry(slow), the dry(slow) the flattened
mouth mouth smear was slightly smear was
tilted or the (flattened) tilted flattened
smear was
destroyed
upon drying
Staining The stain Stained the Stained the

was washed specimen more specimen at the
out by than or less than desired time
water the desired time
Checking for good 1 out of 5 is 2 out of 5 are 3 out of 5 are 4 out of 5 are done Focus the
smear done done properly done properly properly microscope on
properly the 10X objective
(low power).
Scan the smear to
check for cell
distribution,
clumping, and
abnormal cells,
examine the
peripheral edge of
the smear
(increased
number of white
cells in this area,
the differential
count is
inaccurate)
Focusing Focused Focused using Focused using Focused using Focused using
using LPO HPO OIO, the field was OIO, the field
scanner focused on the was focused on
light part where the light part
the RBCs are where the RBCs
overlapping are not
overlapping
Identifying the cells Not done 1-2 cells out 3-4 cells out of 7 5-6 cells out of 7 Identified all the
of 7 cells are cells are identified cells are identified cells correctly
identified (neutrophil,
basophil,
eosinophil,
lymphocyte,
monocyte,
platelets and
RBC)
Direction in counting No pattern Begin the count Begin the count

has been in the thin area of in the thin area of
used the slide until the slide and
reaching the dark gradually the

side slide, down if
overlapping RBC
are started to see
Cell counting Not done +/-30 over +/-20 over the +/-10 over the +/-5 over the
the corrected corrected count corrected count corrected count
count
Recording of the test, Not done 1 out of 4 is 2 out of 4 are 3 out of 4 are Recorded the test,
Calculation, properly done properly done properly done calculation is
Interpretation of the correct
test and Clinical
,interpretation
Correlation of the
test and clinical
correlation are
done properly
Post-analytical Phase:
Disposal of waste Left the Disposed the Disposed the Disposed all the Properly cleaned
and cleaning of working waste in an wastes in an waste in proper and disinfect the
working area area dirty. inappropriate inappropriate container, did not working area.
container, did container, disinfect the
not disinfect disinfect the working area.
working area
Returning of Left Left some Left some Returned all Returned all
materials and glass materials on materials on materials on the materials, no materials, no
wares the working the working working area, no breakages, did not breakages, dried
area, with area, with breakages, did dry glass wares glass wares
breakages, breakages, did not dry glass
did not dry not dry glass wares
glass wares wares
Leukocyte type number fraction

Reference Range
Neutrophils: 0.50 - 0.70
Eosinophils: 0.00 – 0.04
Basophils: 0.00 – 0.02
Lymphocytes: 0.20 – 0.44
Monocyte: 0.02 – 0.09
Bands or Stabs: 0.02 – 0.06
Others:
Name: _________________________________________________ Date: __________________
EXPERIMENT 2 PLATELET COUNT Direct Method

OUTCOMES
1. Be able to perform the direct method of platelet counting.

UNIT
2. Be able to correlate the results to different diseases or disorders.
A. Platelets are active in blood coagulation. They perform the following functions: aid in
vasoconstriction and the formation of a hemostatic plug, thromboplastic activity, and clot retraction.
When platelets contact a wet table surface, at first they adhere to one another and then rupture,
releasing chemical factors. Platelets are the smallest formed elements in the blood, normally ranging
in size from 2-4 microns. They are actually fragments of cytoplasm that have broken off platelet
precursors. Platelets function in the coagulation of the blood.
B. Platelets are difficult to count due to:

1. Small size – often hard to differentiate from bacteria and debris
2. Adhesiveness – affinity for adhering to glass
3. Aggregation - tendency to clump together
Principle: Whole blood is diluted with Rees-Ecker diluting fluid which makes platelets readily visible
under the bright-field microscope. The platelets are then counted in the central large square of the
counting chamber (hemocytometer)
11 | C L I N I C A L H E M A T O L O G Y 2 L A B O R A T O R Y M A N U A L
OUTCOMES
UNIT
1. Be able to differentiate the different types of leukocyte in a normal peripheralsmear.
2.Glossary
Be ableoftoTerms
perform the differential leuckocyte count.
Coagulation: The sequential process by which multiple plasma enzymes and cofactors interact in
sequence, forming an insoluble fibrin clot.
Platelets: The smallest of the formed elements in blood; a disk shaped, non-nucleated cell formed in
the red bone marrow by fragmentation of the megakaryocytes. Platelets play an active role in blood
coagulation. Also called thrombocytes.
Vasoconstriction: A reactive decrease in the blood vessel diameter. Vasoconstriction controls blood
pressure, primary hemostasis, and the distribution of blood throughout the body.
PRE-ANALYTICAL PHASE
Collection of the blood in the desired amount in the EDTA evacuated tube by venipuncture.
ANALYTICAL PHASE
Materials
EDTA evacuated tube Hemocytometer and coverslip

Microscope with LPO and HPO RBC pipette with rubber tubing and mouthpiece
Rees & Ecker Diluting Fluid Tally Counter or Cell Counter
Procedure
A. Diluting the blood.

1. The capillary bore of the RBC pipette is rinsed with Rees-Ecker diluting fluid. All excess fluid
should be removed from the pipette before blood is drawn into the pipette. This step will ensure
platelet will not adhere on the wall of the capillary bore of the pipette.
2. Draw the blood into the pipette up to 0.5 mark. If excess blood was draw, adjust the blood level
to the exact 0.5 mark with NSS-moistened cotton. Clean the stem of the pipette first before any
adjustment will be made.
OUTCOMES
UNIT 1. Be able to perform the direct method of platelet counting.
3. The blood should be diluted immediately with the Rees-Ecker solution. Make a 1:200 dilution by
filling the pipette with the solution up to 101 mark. Two pipettes should be use simultaneously,
and each should be used to charge one side of the counting chamber.
4. After the dilution, the pipettes should be shaken immediately for at least 60 seconds. This step
will prevent platelet clumping and will ensure accurate counting.
B. Charging the counting chamber.

5. After mixing, discard the first 5 drops from each pipette. Note: Do not induce or blot the tip
of the pipette by any absorbent material to facilitate the process. The drop should drop
freely by gravity alone. Each side of the hemocytometer should be charged with a different
pipette.
6. After charging the chambers, the hemocytometer should be kept in covered container for about
10 to 15minutes with wet gauze or cotton pads beneath the hemocytometer to prevent
evaporation, this step will allow the platelets to settle aid in accurate counting later on.
C. Counting the platelets

7. Carefully placed the hemocytometer on the microscope stage so as not to disturb the platelets.
Count platelets in 5 RBC squares in the center of the counting chamber. Both sides of the
counting chamber should be mounted and the results averaged. Using the high power
magnification count all the platelets, they appear as small, roundish, unevenly shaped structures.
Calculation
As with the RBC and WBC counts , the important parameters for calculating the platelet counts
include (1) the average number of platelets per square millimeter; (2) the dilution factor (which is
0.20, just like the RBC count); (3) the volume of the diluted blood counted, which is the area times
depth (1mm² x 0.1 mm = 0.1 µl).
The following formula is usually used:
Platelet count = # of cells counted x Dilution Factor (0.20) = platelet x 109/L

Depth of Counting Chamber (0.10)
Thus if an average of 200 platelets were counted in each central square, then the platelet count should
be:
Platelet count = 200 x 0.20 = 400,000 per µl or (400 x 10⁹ /L)
0.1
OUTCOMES
Reference Range: 150,000 to 400,000/µl (or 150 to 400x 10⁹/L)
Numerical estimate of platelets from a blood smear

Since so many variables may interfere with manual platelet count. Accuracy of the result is questioned. A
blood smear is also frequently used to verify the manual platelet count. The blood smear is prepared
side by side in doing the direct method of platelet count.
For an estimate of platelet concentration from a blood smear to be valid, the platelets should not be
clumping to any degree. The platelets should be observed in the same area in which diffential WBC
counts are made. In a normal individual each oil immersion field should yield between 8-20 platelets.
An estimate can also be calculated from the formula.
Platelets/µl = Number of platelets per 100 RBCs x Hematocrit (%) x 100
OUTCOMES
POST-ANALYTICAL PHASE
Critical Thinking
1. Give 5 conditions wherein the platelet count is increased.
2. Give 5 conditions wherein the platelet count in decreased.
OUTCOMES

1. Be able to perform the direct method of platelet a. Able to count platelet using direct method
counting. accurately.
2. Be able to correlate the results to different b. Able to distinguish qualitative and quantitative
diseases or disorders platelet disease and disorders
ANECDOTAL NOTES
Observe students as they participate in the activity. Note the extent to which students are able to make and talk about
personal observation. Record anecdotal notes.
Create an outcome-based checklist and share with students prior to beginning the activity. Use the checklist to
assess if students are able to make and talk about personal observations.
RUBRIC
Collaboratively create an outcome – based rubric with students. Use the rubric to evaluate how well the students are
able to understand cells.
RESOURCES
2009
RUBRICS
Critical Dimensions 1 2 3 4 5
REFER BEGINNER COMPETENT PROFICIENT EXEMPLARY Score
cap &goggles) worn.
Materials and No 1-2 out of 6 3 out of 6 are 4-5 out of 6 are Complete
Instruments ( special materials are available available available materials and
consideration to the instruments
specimen if required)
disinfection of clean the did not disinfect the disinfected working table cover and
working area working disinfect the working area but area but placed disinfected
area before working area not placed table table cover before working area
starting or vice versa cover starting before starting
on the
working area
before starting
Phlebotomy:
Identification (name, identify are properly properly done properly done patient
age, birthdate, patient done identification
requisition form, (name, age,
special consideration birthdate,
to the test if required) requisition form,
special
consideration to
the test if
required)
equipment (syringe, extraction 4 equipment equipment equipment extraction
vacuum tube, equipment equipment
tourniquet, cotton)
Proper blood Bevel Bevel Bevel orientation Bevel orientation is Bevel orientation
extraction technique orientation orientation is is correct, no correct, no is correct, no
is incorrect, correct, presence of air presence of air presence of air
presence of presence of air inside the inside the syringe, inside the
air inside inside the syringe, small insufficient amount syringe, correct
the syringe, syringe, little amount of blood of blood extracted amount of blood
no blood amount of extracted extracted
enters the blood enters
hub the hub, no
blood
extracted
Proper needle Remove Remove Remove needle Remove tourniquet, Remove
removal and needle needle without without remove the needle; tourniquet,
recapping without removing removing recapping done remove the
removing tourniquet; tourniquet; with hands needle and
tourniquet recapping recapping done recapping with
no done with with fishing fishing technique
recapping hands technique
done
Specimen labeling No label Only the name Only 2 out of 4 Only 3 out of 4 Complete
(patient name, age, was seen on is seen on the label are seen on label are seen on specimen
date of collection, the specimen label the specimen the specimen labeling
time of collection) specimen
Waste disposal No waste All All equipment’s All needles used All needles used
disposal equipment’s used were put in were put in a were put in a
observed used were put a yellow bag puncture proof puncture proof
in an container; others container; others
inappropriate are in a yellow bag are in a yellow
container bag labeled
"infectious"
1. The capillary bore The The capillary The capillary The capillary bore The capillary
of the RBC pipette capillary bore of the bore of the RBC of the RBC pipette bore of the RBC
must be rinsed with bore of the RBC pipette is pipette is rinsed is rinsed with Rees- pipette is rinsed
Rees-Ecker Diluting RBC
rinsed with with Rees-Ecker Ecker Diluting with Rees-Ecker
Fluid. Excess fluid pipette is
should be discarded. not rinsed Rees-Ecker Diluting Fluid. Fluid. Excess fluid Diluting Fluid.
with Rees- Diluting Fluid. Excess fluid is is discarded. Excess fluid is
Ecker Excess fluid is not discarded discarded and
Diluting not discarded but wiped the wiped the
Fluid. outside part of outside part of
the pipette the pipette
2. Draw the blood Drawn the Drawn the Drawn the blood Drawn the blood Drawn the
into the pipette up to blood into blood into the into the pipette into the pipette up blood into the
0.5 mark. If excess the pipette pipette up to up to 0.5 mark. to 0.5 mark. Excess pipette up to 0.5
blood is drawn adjust
up to 0.5 0.5 mark. Excess blood blood drawn is mark. Excess
it with NSS-
moistened cotton. mark. Excess blood drawn is adjusted adjusted with NSS- blood drawn is
Clean the stem of the not adjusted. with NSS- moistened cotton. adjusted with
pipette. moistened Cleaned the stem of NSS-moistened
cotton. Stem of the pipette but with cotton. Cleaned
the pipette not bubbles the stem of the
cleaned. pipette.
3. Blood should be Blood is Blood is Blood is diluted Blood is diluted Blood is diluted
diluted immediately not diluted diluted immediately with immediately with immediately with
with Rees-Ecker immediately immediately Rees-Ecker Rees-Ecker Rees-Ecker
Solution. Make 1:200
with Rees- with Rees- Solution. Failed Solution. Made a Solution. Made a
dilution filling the
pipette up to 101 Ecker Ecker Solution to reach the 101 1:200 dituion filling 1:200 dituion
mark. Solution. but with mark. the pipette up to filling the pipette
bubbles 101 mark but with up to 101 mark.
bubbles
4. Pipettes should be Pipettes Pipettes was Pipettes was Pipettes was mixed Pipettes was
shaken immediately was mixed mixed below mixed immediately for 60 mixed
for at least 60 improperly 60 seconds. immediately seconds. immediately for
seconds. This will
prevent platelet above 60 60 seconds and
clumping. seconds. ensure no
diluted sample
will be spilled
5. Discard the first 5 Not done Discarded the Discarded the Discarded the first Discarded the
drops from pipette. or spilled all first 5 drops first 5 drops 5 drops from first 5 drops
Each side of the the diluted from pipette. from the pipette. pipette. Each side from pipette.
neubauer should be sample
Failed to charge of the neubauer is Each side of the
charged without
flooding. in the neubauer. charged with neubauer is
flooding. charged without
flooding.
6. Place the cover slip Placed the Placed the Placed the Placed the cover Placed the cover
and neubauer on the cover slip cover slip w/ cover slip w/ slip w/o excess slip w/o excess
microscope. (Insufficient excess amount excess amount amount of solution amount of
of solution
amount) of solution but but with bubbles solution and
and with
and bubbles and w/o bubbles and and placed bubbles and
neubauer placed placed neubauer neubauer on the placed neubauer
on the neubauer on on the microscope. on the
microscope the microscope. microscope.
microscope.
count
Recording of the test, Not done 1 out of 4 is 2 out of 4 are 3 out of 4 are Recorded the test,
Calculation, properly done properly done properly done calculation is
Interpretation of the correct
test and Clinical
,interpretation
Correlation of the
test and clinical
correlation are
done properly
Disposal of waste Left the Disposed the Disposed the Disposed all the Properly cleaned
and cleaning of working waste in an wastes in an waste in proper and disinfect the
working area area dirty. inappropriate inappropriate container, did not working area.
container, did container, disinfect the
(Factor: 5) not disinfect disinfect the working area.
working area
materials and glass materials on materials on materials on the materials, no materials, no
wares the working the working working area, no breakages, did not breakages, dried
(Factor: 3) area, with area, with breakages, did dry glass wares glass wares
breakages, breakages, did not dry glass
did not dry not dry glass wares
glass wares wares
OUTCOMES
UNIT To achieve this unit a learner must:

Date Performed Remarks
Trial 1 _____________ _____________
Trial 2 _____________ _____________
Interpretation of Result:
Platelet counts below normal indicate thrombocytopenia and are found associated with aplastic anemia,
pernicious anemia, acute leukemia and idiopathic thrombocytopenia purpura. Platelet count above
normal indicate thrombocytosis and are found associated with polycythemiavera, haemolytic anemia,
chronic myeloprofilerative disorders, and after splenectomy.
Sources of Error:
A. Light adjustment is critical. If the condenser is not lowered it will fade out the platelets.
B. Bacteria and debris can be misinterpreted as platelets, this type of artifact is generally much more
retractile than platelets.
C. Clumping of platelets sometime occurs, and if so the specimen must be recollected. EDTA is the
anticoagulant of choice for preventing platelet clumping.
D. General hemacytometer errors, i.e. overloading chamber, counting wrong borders, etc.
Name: _________________________________________________ Date: __________________
EXPERIMENT 3 PLATELET COUNT Modified Indirect Method
At the end of Hematology 413 student should be able to: 1.) Explain the principles of hemostasis coagulation
and fibrinolysis. 2.) Appreciate the importance of laboratory assays for the diagnosis of hemostatic disorders. COURSE
3.) Perform the laboratory assays on hemostasis/coagulation with precision, accuracy and reliability. 4.) OUTCOMES
Manifest the following values, integrity, honesty, critical thinking, empathy and value for life.

OUTCOMES

UNIT
A. Platelets are active in blood coagulation. They perform the following functions: aid in
vasoconstriction and the formation of a hemostatic plug, thromboplastic activity, and clot retraction.
When platelets contact a wet table surface, at first they adhere to one another bad then rupture,
releasing chemical factors. Platelets are the smallest formed elements in the blood, normally ranging
in size from 2-4 microns. They are actually fragments of cytoplasm that have broken off platelet
precursors. Platelets function in the coagulation of the blood.
B. Platelets are difficult to count due to:

4. Small size – often hard to differentiate from bacteria and debris
5. Adhesiveness – affinity for adhering to glass
6. Aggregation - tendency to clump together
Principle: A well-made smear is stained with Wright’s or Wright’s-Geimsa to visualize cellular elements
of blood including the platelet. Is done in the portion of the smear where the red cells start to
overlap. 10-OIO consecutive fields are examined for platelet and reported as cells per microliter of
blood.
OUTCOMES
Glossary of Terms
Coagulation: The sequential process by which multiple plasma enzymes and cofactors interact in
sequence, forming an insoluble fibrin clot.
Platelets: The smallest of the formed elements in blood; a disk shaped, non-nucleated cell formed in
coagulation. Also called thrombocytes.
Vasoconstriction: A reactive decrease in the blood vessel diameter. Vasoconstriction controls blood
pressure, primary hemostasis, and the distribution of blood throughout the body.
Collection of blood in EDTA evacuated tube by venipuncture and preparation and staining of well-
made smears.
ANALYTICAL PHASE
Materials
EDTA evacuated tube Hemocytometer and coverslip

Microscope with LPO and HPO RBC pipette with rubber tubing and mouthpiece
Rees & Ecker Diluting Fluid (Freshly Prepared) Tally Counter or Cell Counter
Procedure
1. Make a well-made blood smear and stain with Wright’s or Wright’s-Geimsa stain. See the product
insert for the specific instructions. Manufacturer has sometimes have modifications.
2. Focus the slide using low-power magnification. Examine the smear for areas that is suitable for
counting. Care must be taken to stay in an appropriate area of the slide. The RBC’s should barely be
touching one another in the area of the estimate.
3. Using the high magnification, check the area of smear for suitability for counting the platelets.
4. Shift the objective to the 1000x magnification. Examine the strip of the film moving from one field
to next systematically. See Figure 1.1
5. Count and record the platelets seen in then consecutive oil-immersion fields.
OUTCOMES
Calculation
1. A platelet count should be always be checked on the smear to make sure it looks accurate. To estimate a
platelet count from a blood smear you regard each platelet seen per oil power field as being equivalent
to 20,000 platelets/mm³. By determining the average number of platelets seen per oil field multiplying
this number by 20,000 you can estimate the platelet count. For example, if you see 10 platelets per oil
field your estimated could be: 10 x 20,000 or 200,000/mm³. Usually 7-20 platelets per OIF represent a
normal platelet count.
Platelet count= number of platelets counted in 10 oil immersion field x 20,000
10
Reference range: 150,000 to 350,000/mm³
Storage of Microscope
1. Prepare the microscope for storage and return it properly to storage cabinet.
2. Proper disposing of infectious materials (gloves, used cottons, etc)
3. Decontamination of designated working area.
4. Proper hand washing.
5. Accomplish laboratory exercise.
Critical Thinking
1. Enumerate possible sources of error in indirect method of platelet count.
2. Cite the artifacts seen during microscopic examination of the slide during indirect platelet
count.
OUTCOMES
ASSESSMENT CRITERIA FOR PASSING

1. Be able to perform the direct method of platelet a. Able to count platelet using direct method
counting. accurately.
2. Be able to correlate the results to different b. Able to distinguish qualitative and quantitative
diseases or disorders platelet disease and disorders

ANECDOTAL NOTES
RUBRIC
RESOURCES
Standardization and Harmonization of Complete Blood Count in the Philippines 1st edition, Grace A.
Jatico-Dela Calzada, MD,MM,FPSP
OUTCOMES
RUBRICS
Critical 1 2 3 4 5
Dimensions REFER BEGINNER COMPETENT PROFICIENT EXEMPLARY Score
cap &goggles) worn.
Materials No Incomplete Incomplete (3 Incomplete (2 are Complete

materials (4are lacking) are lacking) lacking) (Wright’s stain,
slides, whole
blood,
microscope,
dropper/
capillary tube)
disinfection of clean the did not disinfect the disinfected table cover and
working area working disinfect the working area working area but disinfected
area before working area but not placed placed table cover working area
starting or vice versa table cover before starting before starting
on the
working area
before starting
Phlebotomy:
Identification identify are properly properly done properly done patient
(name, age, patient done identification
birthdate, (name, age,
requisition form, birthdate,
special requisition
consideration to form, special
the test if required) consideration to
the test if
required)
equipment extraction 4 equipment equipment equipment extraction
(syringe, vacuum equipment equipment
tube, tourniquet,
cotton)
Proper blood Bevel Bevel Bevel Bevel orientation Bevel

extraction orientation orientation is orientation is is correct, no orientation is
technique is incorrect, correct, correct, no presence of air correct, no
presence of presence of presence of air inside the syringe, presence of air
air inside air inside the inside the insufficient inside the
the syringe, syringe, little syringe, small amount of blood syringe, correct
no blood amount of amount of extracted amount of
enters the blood enters blood extracted blood extracted
hub the hub, no
blood
extracted
Proper needle Remove Remove Remove needle Remove Remove

removal and needle needle without tourniquet, tourniquet,
recapping without without removing remove the remove the
removing removing tourniquet; needle; recapping needle and
tourniquet tourniquet; recapping done done with hands recapping with
no recapping with fishing fishing
recapping done with technique technique
done hands
Specimen labeling No label Only the Only 2 out of 4 Only 3 out of 4 Complete
(patient name, age, was seen name is seen label are seen label are seen on specimen
date of collection, on the on the on the the specimen labeling
time of collection) specimen specimen specimen
label
Waste disposal No waste All All equipment’s All needles used All needles used
disposal equipment’s used were put were put in a were put in a
observed used were put in a yellow bag puncture proof puncture proof
in an container; others container;
inappropriate are in a yellow bag others are in a
container yellow bag
labeled
"infectious"
Analytical phase:
1. Make a blood Not done 1 out of 4 is 2 out of 4 are 3 out of 4 are Made a blood
smear and stain or washed properly done properly done properly done smear, labelled,
with Wrights or out air dry and
Wright-Geimsa.
specimen is stained with
obtained Wrights or
Wright-Geimsa.
2. Focus the slide Cannot Focused the Focused the Focused the slide Focused the
using LPO. focused the slide using slide using using LPO. slide using
Examine the smear slide LPO. LPO. Examined the LPO. Examined
for a suitable
Examined the smear for a the smear for a
counting area.
Cells should be smear for a suitable counting suitable
evenly distributed. suitable area. Cells should counting area.
Switch to HPO counting area. be evenly Cells should be
Cells not evenly distributed. Did evenly
distributed. not switch to distributed.
HPO Switched to
HPO
3. Switch OIO. Switched to Switched to Switched to Switched to OIO. Switched to

Count and record OIO but OIO focused OIO. Counted Counted and OIO. Counted
the platelets seen in cannot the slide but and recorded recorded the and recorded
10 consecutive OIO focus the cannot count the platelets. platelets 10 the platelets
fields. slide the platelets Insufficient consecutive fields seen in 10
count not met. consecutive
OIO fields.
count
Recording of the Not done 1 out of 4 is 2 out of 4 are 3 out of 4 are Recorded the
test, Calculation, properly done properly done properly done test, calculation
Interpretation of is correct
the test and
,interpretation
Clinical
Correlation of the and clinical
test correlation are
done properly
Disposal of waste Left the Disposed the Disposed the Disposed all the Properly
and cleaning of working waste in an wastes in an waste in proper cleaned and
working area area dirty. inappropriate inappropriate container, did not disinfect the
container, did container, disinfect the working area.
working area
materials and glass materials on materials on materials on materials, no materials, no
wares the working the working the working breakages, did not breakages,
(Factor: 3) area, with area, with area, no dry glass wares dried glass
breakages, breakages, did breakages, did wares
did not dry not dry glass not dry glass
glass wares wares wares
Result/Computation: Interpretation of Result:
Platelet counts below normal indicate thrombocytopenia and are found associated with aplastic
anemia, pernicious anemia, acute leukemia and idiopathic thrombocytopenia purpura. Platelet count
above normal indicate thrombocytosis and are found associated with polycythemiavera, haemolytic
anemia, chronic myeloprofilerative disorders, and after splenectomy.
Name: _________________________________________________ Date: __________________
EXPERIMENT 4 BLEEDING TIME Duke & Ivy Method
Students shall be able to perform different Hematological procedures vital in COURSE

assessing the status of the patient. OUTCOME

OUTCOMES
1. Be able to perform bleeding time by Duke Method.

UNIT
Bleeding time is used to measure the duration of bleeding after a measured skin incision. Bleeding time
may be measured by one of the three methods: template, Ivy, or, Duke. The template method is the
most commonly used and the most accurate because the incision size is standardized. Bleeding time
depends on the elasticity of the blood vessel wall and on the number and functional capacity of platelets.
Although this test is usually performed on patients with a personal or family history of bleeding
disorders, it is also useful along with a platelet count for preoperative screening. The test is usually not
recommended for patients with a platelet count of less than 75,000/µl.
Principle of the test: The test is performed by using a sterile blood lancet to make a measured skin
puncture in the earlobe or forearm. The time is started immediately after the puncture. Touching a
piece of filter paper to the cut every 30 seconds until bleeding ceases and record the time and
reported as bleeding time
OBJECTIVES
UNIT
1. Be able to perform bleeding time by Duke Method
2. Be able to correlate the results to different diseases or disorders
Glossary of Terms
Platelets: The smallest of the formed elements in blood; a disk-shaped, non-nucleated cell formed in
coagulation; also called thrombocytes.
Preoperative Screening: Panel of test done in the patient to assess the status or condition before a
procedure has to be done.
1. Patient preparation that include:

A. Explain to the patient this test is used to measure the time required to form a clots and stop
bleeding.
B. Check the patient’s history for recent use of drugs of that prolong bleeding time, including
sulphonamides, thiazide diuretics, antineoplastic, anticoagulants, non-steroidal anti-inflammatory
drugs, aspirin and aspirin compounds, and some non-narcotic analgesics
ANALYTICAL PHASE
Materials
Duke Method:
1. Sterile Blood Lancet
2. Stopwatch
3. Sterile Filter Paper
4. Gauze pads or cotton balls
5. 70% Alcohol or Povidone Iodine Solution
Ivy Method:
1. Sterile Blood Lancet
2. Stopwatch
3. Sphygmomanometer
4. 70% Alcohol or Povidone Iodine Solution
OUTCOMES
UNIT 1. Be able to perform bleeding time by Duke Method
Procedure
A. Duke Method:
1. Obtain a piece of filter paper and stopwatch.
2. Moisten a piece of cotton with 70% alcohol or Povidone iodine and thoroughly cleanse the ball
of the patient’s middle or ring finger.
3. Allow the skin to air-dry.
4. Make a puncture wound 2-4mm deep in the earlobe or finger with a disposable blood lancet.
5. Start the stopwatch immediately.
6. Being careful not touch the puncture site. Blot the filter paper every 30 seconds, until the
bleeding stops.
7. Record the bleeding time.
Note: Should be the blood flow more than 15 minutes. Discontinue the test and report the test as
“greater than 15 minutes”
Reference Range: 1 to 3 minutes
B. Standardized Ivy Method:

1. Obtain a piece of filter paper, stopwatch, and sphygmomanometer.
2. Position the patient’s arm with the volar surface exposed.
3. Place the sphygmomanometer on the upper arm.
4. Moisten a piece of cotton with 70% alcohol or Povidone iodine solution and thoroughly cleanse
the puncture site.
5. Apply the cuff and inflate to 40mmHg, and hold at this exact pressure for the duration of the
test.
NOTE: the time between inflation and incision should be 30 to 60 seconds.
6. After applying the pressure cuff and preparing the test site. Make three (3) same punctures with
a disposable lancet (1mm wide and 3mm deep)
Caution: Avoid subcutaneous veins.
7. Start the stopwatch immediately.
8. After 30 seconds, wipe the flow of blood with the filter paper. (Bring the paper close to the
incision, but do not touch the paper directly to the incision, so as not to disturb the formation of
the platelet plug.)
9. Blot each site with filter paper every 30 seconds thereafter, until no blood stains the paper.
10. Stop the timer when only clear fluid is absorbed onto the filter paper. The bleeding time is
determined to the nearest 30 seconds.
11. Release the pressure of the sphygmomanometer.
OUTCOMES
12. Record the bleeding time for each of the puncture.

13. Get the average of the bleeding time in minutes and seconds.
Sources of Error
1. If body hair will interfere, lightly shave the area.

2. Patient should be advised of a potential to produce a scar. This can usually be avoided by the use
of a butterfly bandage applied for 24 hours.
3. Aspirin and aspirin containing products may cause a prolonged bleeding time for up to 2 weeks.
4. A standardized cut is necessary for valid results. Too little pressure on the device and the wound
will be shallow or nonexistent. Too much pressure and the wound will be too deep. This is the
one area where standardization has not been completely controlled.
5. Low skin temperature produces constriction of the capillary vessels, resulting in decreased blood
flow.
Precaution: Be sure to maintain a cuff pressure of 40 mmHg throughout the test. If bleeding does not
diminish after 15 minutes, discontinue the test. Report the test as “greater than 15 minutes”.
Reference Range: 3 to 6 minutes Borderline: 6 to 11 minutes
OUTCOMES
Critical Thinking
1. Give conditions wherein the bleeding time would be increased.
2. Give conditions wherein the bleeding time would be decreased.
3. Give the conditions wherein the bleeding time would falsely decreased.
4. Give the conditions wherein the bleeding time would be falsely increased.
OUTCOMES
UNIT
1. Be able to perform bleeding time by Duke Method

1. Be able to perform bleeding time by Duke a. Able to perform bleeding time by Duke
Method Method and Ivy Method
2. Be able to correlate the results to different b. Able to correlate the results to different
diseases or disorders diseases or disorders,
ANECDOTAL NOTES
RUBRIC
RESOURCES
2009
OUTCOMES
RUBRICS
Critical 1 2 3 4 5
Dimensions REFER BEGINNER COMPETENT PROFICIENT EXEMPLARY Score
cap &goggles) worn.
disinfection of clean the did not disinfect the disinfected table cover and
working area working disinfect the working area working area but disinfected
area before working area but not placed placed table cover working area
starting or vice versa table cover before starting before starting
on the
working area
before starting
DUKE METHOD
Analytical phase:
1. Obtain a piece of Not done Obtained a Obtained a Obtained a clean Obtained a

filter paper and piece of filter piece of filter piece of filter clean piece of
stopwatch. paper. paper and ready paper but did not filter paper and
stopwatch. ready the ready
stopwatch. stopwatch.
2. Moisten a piece . Not done Moistened a Moistened a Moistened a piece Moistened a

of cotton with 70% piece of piece of cotton of cotton with piece of cotton
alcohol or cotton with with 70% 70% alcohol or with 70%
Povidone iodine
70% alcohol alcohol or Povidone iodine alcohol or
and cleanse the
patient’s middle or or Povidone Povidone and cleansed the Povidone
ring finger. iodine. iodine. patient’s middle iodine and
Allow the skin to Puncture site or ring finger. cleansed the
air dry. not cleansed. patient’s middle
Failure to air dry or ring finger.
skin.
Allowed the
skin to air dry.
3. Make a puncture Not done Made a Made a Made a puncture Made a

wound 2 to 4mm puncture puncture wound 2 to 4mm puncture
deep on the earlobe wound but wound 2 to deep on the wound 2 to
or finger with a not enough. 4mm deep on earlobe or finger 4mm deep on
disposable lancet. the earlobe. with a disposable the earlobe or
Start the stop watch lancet. Failed to finger with a
immediately.
start the disposable
stopwatch. lancet. Started
the stopwatch
immediately.
4. Being careful not Not done Touched the Did not touch Did not touch the Did not touch
to touch the puncture site. the puncture puncture site. the puncture
puncture site. Blot site. Blotted the Blotted the site. Blotted the
the puncture site
puncture site puncture site with puncture site
with filter paper
every 30 seconds with filter paper filter paper every with filter paper
until bleeding less than 30 30 seconds until every 30
stops. Record the seconds. bleeding stops. seconds until
bleeding time. Failure to record bleeding stops.
bleeding time. Recorded the
bleeding time.
IVY METHOD
1. Obtain a piece of Obtained 1 Obtained 2 Obtained 3 out Obtained 4 out Obtained a
filter paper, out of 5 out of 5 of 5 properly of 5 properly piece of filter
stopwatch, and properly properly paper,
sphygmomanomete stopwatch, and
r sphygmomano
Position the meter.
patients arm with Positioned the
the volar surface patients arm
exposed, place the with the volar
sphygmomanomete surface
r on the upper arm. exposed,
placed the
sphygmomano
meter on the
upper arm.
2. Moisten a piece Not done 1 out of 4 2 out of 4 were 3 out of 4 were Moistened a
of cotton with 70% were done done properly done properly piece of cotton
alcohol, cleanse the properly with 70%
puncture site. alcohol, cleanse
the puncture
site. Using
aseptic
technique with
patients care
3. Apply the cuff Not done Applied the Applied the cuff Applied the cuff Applied the
and inflate at or Applied cuff and and inflated at and inflated at cuff and
40mmHg, and hold the cuff inflated at 40mmHg, and 40mmHg, hold it inflated at
and
at this exact 40mmHg. hold it. while observing 40mmHg, hold
inflated but
pressure for the in wrong the patient. it while
duration of the test. pressure. observing and
applying
patients care
4. After applying After After applying After applying After applying the After applying
the pressure cuff applying the pressure the pressure cuff pressure cuff and the pressure
and preparing the the cuff and and preparing preparing the test cuff and
pressure
test site, make preparing the the test site, site, made three preparing the
cuff and
three small preparing test site, made made three small small punctures test site, made
punctures with the test three small punctures with with disposable three small
disposable lancet. site, made punctures disposable lancet. Started the punctures with
Start the stop watch less than with lancet. Started stop watch disposable
immediately. three small disposable the stop watch immediately. And lancet, and
punctures lancet. Failure immediately. But applied patients dispose it
with
to start watch. did not applied care properly.
disposable
lancet and patients care Started the stop
failed to watch
start watch. immediately.
And applied
patients care
5. After 30 seconds, Not done After 30 After 30 After 30 seconds, After 30
wipe the flow of seconds, seconds, wiped wiped the flow of seconds, wiped
blood with the filter wiped the the flow of blood with the the flow of
paper. Blot each flow of blood blood with the filter paper. blood with the
site with filter with the filter filter paper. Blotted each site filter paper.
paper every 30 paper. Blotted each site with filter paper Blotted each
seconds thereafter, with filter paper every 30 seconds. site with filter
until no blood less than 30 paper every 30
stains the paper. seconds. seconds until
no blood stains
the paper.
6. Stop the timer Did not Stopped the Stopped the Stopped the timer Stopped the
when only clear stop the timer when timer when only when only clear timer when
fluid is absorbed in timer when only clear clear fluid is fluid is absorbed only clear fluid
only clear
the filter paper. fluid is absorbed in the in the filter paper. is absorbed in
fluid is
Release the absorbed in absorbed in filter paper. Released gently the filter paper.
pressure of the the filter the filter Released the the pressure of Released gently
sphygmomanomete paper and paper but did pressure of the the the pressure of
r. did not not release the sphygmomanom sphygmomanome the
release the sphygmoman eter ter sphygmomano
sphygmom ometer right meter while
anometer
away. having patients
right away.
care
7. Record the Recorded Recorded the Recorded the Recorded the
bleeding time for the bleeding bleeding time bleeding time bleeding time
each of the time. for each of the for each of the for each of
puncture, get the puncture. puncture, got the puncture,
average of the
the average fo got the
bleeding time in
minutes and
the bleeding average of the
seconds. time. bleeding time
in minutes
and seconds.
test, Calculation, properly done properly done properly done test, calculation
Interpretation of is correct
the test and Clinical
,interpretation
Correlation of the
test and clinical
correlation are
done properly
Disposal of waste Left the Disposed the Disposed the Disposed all the Properly
and cleaning of working waste in an wastes in an waste in proper cleaned and
working area area dirty. inappropriate inappropriate container, did not disinfect the
container, did container, disinfect the working area.
working area
materials and glass materials on materials on materials on materials, no materials, no
wares the working the working the working breakages, did not breakages,
(Factor: 3) area, with area, with area, no dry glass wares dried glass
breakages, breakages, did breakages, did wares
did not dry not dry glass not dry glass
glass wares wares wares
Result:
Trial 1 ______________________________________________
Trial 2 ______________________________________________
Remarks:
OUTCOMES
1. The bleeding time depends primarily on extra-vascular and vascular factors and, to a lesser
degree, on the factors of coagulation. The chief factor controlling bleeding from a small cut is
the constriction of the minute vessels following injury. Accuracy in this test is enhanced by
blotting the drops of blood at shorter intervals of time as the drops of blood become
progressively smaller.
2. Thrombocytes play an important part in the formation of the hemostatic plug that seal off a
wound. In thrombocytopenic purpura there is a decrease in platelets resulting in a prolonged
bleeding time due to a defective platelet plug. An additional factor prolonging the bleeding time
in this condition is a defect in capillary contraction. In can be hereditary and acquired platelet
dysfunction.
3. In hemophilia, the bleeding time is normal. This explained by the fact that there are no vascular
or extravascular or extravascular abnormalities. However, the test should not be performed on a
known hemophiliac, for delayed oozing blood is a read hazard.
4. Elevated in, thrombocytopenia, capillary wall abnormalities, platelet abnormalities (Bernard-
Soldier disease, Glanzmann’s disease), drugs (aspirin, warfarin, anti-inflammatory medications,
streptokinase, urokinase, dextran, B-lactam antibiotics, moxolactam), disseminated intravascular
coagulation, cirrhosis, uremia, myeloproliferative disorders, von Willerbrand’s disease.
Name: _________________________________________________ Date: __________________
EXPERIMENT 5 CLOTTING TIME Slide, Wright’s, and Lee & White Method
the laboratory assays on hemostasis/coagulation with precision, accuracy and reliability. 4.) Manifiest the following OUTCOMES

OUTCOMES
1. Be able to perform different methods of blood clotting time determination.

UNIT
Clotting time is the interval between the moment when bleeding starts and the moment when the
fibrin clot thread is first seen. Bleeding time and clotting time are not the same. Bleeding time depends on
the integrity of platelets and vessel walls, whereas clotting time depends on the availability of coagulation
factors. When the blood vessel ruptures, in a few minutes blood losses its fluidity and set into a semisolid
mass called clot. This process in call blood coagulation. In vivo, blood clots outside the body on cuts and
injuries. In vivo, blood clots inside the blood vessels. In coagulation disorders like hemophilia, clotting time
is prolonged but bleeding time remains normal.
OUTCOMES
UNIT 1. Be able to perform different methods of blood clotting time determination.
Blood Clotting (Extrinsic pathway)
Glossary of Terms
Coagulation: The process of stopping the blood flow from the wound. This process involves the
harmonious relationship of the blood-clotting factors, the blood vessels, and the fibrin-forming and
fibrin-lying system.
Hemophilia: A genetic disorder where longer clotting time due to absence of some clotting factors.
In vivo: Process that occurs within the living organism.
In vitro: An artificial environment outside the living organism
OUTCOMES
ANALYTICAL PHASEANALYTICAL PHASE
MaterialsMaterials
Patient preparation, setting up of working area and extraction of blood via capillary method and syringe
method.
A. Slide Method or Drop Method

1. Blood lancet
2. Glass slide
3. Timer / Stopwatch
4. Cotton
5. 70% ethyl alcohol
B. Wright’s Method or Glass Capillary Method

1. Capillary tube, non-heparinized
2. Blood lancet
4. Cotton
5. 70% ethyl alcohol
C. Lee and White Method

1. Glass test tubes, 3 pieces (13 x 100mm)
2. Syringe, G21/G23 needle
3. Water bath 37° C
Procedure
A. Slide Method
1. Disinfect site of puncture with 70% ethyl alcohol. Air dry.

2. Puncture to a depth of 3mm using a blood lancet.
3. Start timer as soon as the first drop of blood appears.
4. Transfer the three drops of blood onto a clean glass slide. Careful not to touch the skin.
OUTCOMES
5. Pass the tip of the lancet through the first drop of blood every 30 seconds and not for the
formation of fibrin strands. Repeat with second drop and then the third drop.
6. Stop the timer immediately as soon as fibrin strands are seen clinging at the tip of the lance on
the third drop.
Reference Range: 2-4 minutes
Procedure
B. Wright’s Method or Glass Capillary Method
1.
Apply 70% alcohol to the clean finger with cotton swab. Allow it to dry naturally.
2.
Prick the finger with usual aseptic precautions. Immediately start the stopwatch.
3.
Dip one end of the capillary into blood drop gently without pressure.
4.
Allow to fill the capillary with blood by lowering the end of fitted capillary. (Do not stuck the
blood around ¾ of its length undipped.
5. After about two minutes start snapping off small lengths of the tube, at intervals of 15 seconds,
each time noting whether the fibrin thread is formed between the snapped ends.
6. Repeat breaking at regular time intervals, till fibrin thread appears at the broken end of capillary
tube. Do not pull away the catted pieces.
7. Record the time interval between pricking finger and first appearance of fibrin thread at the
broken ends of capillary tube. That is clotting time of blood.
Diagram:
OUTCOMES
C. Modified Lee and White Clotting time
Principle of the Test: The coagulation of blood is the length of time required for a measured amount
of blood to clot under certain conditions. The test is based on the fact that when venous blood is put
into a glass tube (foreign surface), it will form a solid clot. The time required for this response is a
measure of the overall intrinsic and common pathways of coagulation.
1. Label three, 13 x 10mm test tubes with Px’s name, and number them.
2. Perform aseptically a venipuncture using a 20-gauge needle syringe and withdraw 4 mL of blood.
3. After obtaining the blood, remove the needle from the syringe, and carefully place 1 mL of the
blood in test tube #3, then 1 mL in tube #2, and 1 mL to tube #1. The last 1 mL must be
discarded (follow universal safety procedures on the disposal of needles, etc.). Start the
stopwatch as soon as the blood is placed in tube #3.
4. Place the three tubes in a 37°C water bath.
5. At exactly 5 minutes, tilt test tube #1 gently to a 45-degree angle. Repeat this procedure every 30
seconds until the test tube can be completely inverted without spilling the content (that is, until
the blood is completely clotted).
6. Record the time it took the blood in test tube #1 to clot.
7. Thirty seconds after the blood in test tube #1 had clotted, proceed with tube #2, and repeat the
preceding procedure tilting the tube every 30 seconds, until it is completely clotted. Record the
result. Repeat this procedure for test tube #3.
8. Since agitation and handling, speed up coagulation, the coagulation time of test tube #3 is the
result to be reported.
OUTCOMES
Critical Thinking
1. Enumerate conditions wherein the clotting time would be decreased.
2. Enumerate condition wherein the clotting time would be increased.
3. Cite the possible causes of falsely decreased clotting time.
4. Cite the possible cause of falsely increased clotting time.
OUTCOMES

1. Be able to perform different methods of blood a. Able to perform different methods of blood
clotting time determination. clotting time determination.
2. Be able to correlate the results to different b. Able to correlate the results of different disease
diseases or disorders and disorders.
ANECDOTAL NOTES
RUBRIC
REFERENCE
2009
OUTCOMES
RUBRICS
Critical 1 2 3 4 5
Dimensions REFER BEGINNER COMPETENT PROFICIEN EXEMPLA Score
(No (Limited content) (Adequate T RY
content) content) (Remarkable (Profession
content) al content)
PPE (lab gown, No PPE Only 1-2 out of 5 Only 3 out of 5 Only 4 out of 5 Complete
mask, gloves, is worn. PPEs are worn. PPEs are worn. PPEs are worn. PPEs are
hair cap worn.
&goggles)
Cleaning and Did not Cleaned but did not Cleaned and Cleaned or Cleaned,
disinfection of clean the disinfect the disinfect the disinfected placed table
working area working working area or vice working area but not working area cover and
area versa on the placed table cover but placed disinfected
before working area before table cover working area
starting starting before starting before
starting
Analytical phase:
Slide method
1. Disinfect the Not done Disinfected the Disinfected the Disinfected the Disinfected
puncture site puncture site with puncture site with puncture site the puncture
with 70% 70% Alcohol. 70% Alcohol. Air with 70% site with
Alcohol. Air Dry. Dry. Alcohol. Air 70%
Puncture to Dry. Puncture Alcohol. Air
depth of 3mm not deep Dry.
using a blood enough. Punctured to
lancet. depth of
3mm using a
blood lancet.
2. Start the timer Failed to Failed to start the Started the timer but Started the Started the
as soon as the start the timer immediately failed to notice the timer as soon timer as soon
first drop of timer but notice the first first drop of blood. as the first as the first
blood appears. immediat drop of blood. drop of blood drop of
ely and appeared. blood
didn’t appeared
notice while having
the first patient care
drop of
blood.
3. Transfer three Wasn’t Transferred less Transferred less Transferred Transferred
drops of blood able to than three drops than three drops three drops three drops
onto a clean glass extract of blood onto a of blood onto a of blood onto of blood
slide. Do not blood. clean glass slide clean glass slide. a clean glass onto a clean
touch the skin.
and skin contact is The finger is ½ slide but skin glass slide.
made. inch away from contact is The finger
the slide made. is ½ inch
away from
the slide.
4. Pass the tip of Not done Passed the tip of Passed the tip of Passed the tip Passed the
lancet through lancet through the lancet through the of lancet tip of lancet
the first drop of first drop of blood first drop of blood through the through the
blood every 30 less than 30 every 30 seconds first drop of first drop of
seconds and note
seconds. failed to note the blood every blood every
for the formation
of fibrin strands.
formation of fibrin 30 seconds 30 seconds
Repeat with whe strands. and note for and note
2nd drop and the the formation for the
the 3rd drop. of fibrin formation
strands. of fibrin
Failed to strands.
repeat 2nd and Repeated
3rd drop. with the 2nd
drop and
the 3rd
drop.
5. Stop the timer Failed to Stopped the timer. Stopped the

immediately as stop the Failed to notice timer
soon as fibrin timer. the appearance of immediately
strands are seen fibrin strands. as soon as
clinging at the tip
fibrin
of the lancet in
strands are
the 3rd drop.
seen
clinging at
the tip of
the lancet in
the 3rd
drop.
Capillary Method
1. Apply 70% Applied Applied 70% Applied 70% Applied 70% Applied
Alcohol to the 70% Alcohol to the Alcohol to the Alcohol to 70%
finger with cotton Alcohol finger with cotton finger with cotton the finger Alcohol to
swab. Air dry. to the swab. Air dry. swab. Air dry. with cotton the finger
Prick the finger
finger Pricked the finger Pricked the finger swab. Air dry. with cotton
with precautions.
with carelessly. with precautions. Pricked the swab using
Start the watch.
cotton Failed to start the finger with aseptic
swab. precautions. technique.
Air dry. watch. Start the Air dry.
watch. Pricked the
finger with
precautions.
Start the
watch.
2. Dip one end of Dipped Dipped one end Dipped one end Dipped one Dipped one
the capillary into one end of the capillary of the capillary end of the end of the
blood drop w/o of the into blood drop into blood drop capillary into capillary
pressure. Allow capillary w/o pressure. w/o pressure. blood drop into blood
to fill the
into Blood in the Blood in the w/o pressure. drop w/o
capillary with
blood by
blood capillary tube less capillary tube is Allowed to pressure.
lowering the end drop than ½ length around ½ of its fill the Allowed to
of fitted capillary w/o undipped length undipped capillary with fill the
around ¾ of its pressure. blood by capillary
length undipped. Failed to lowering the with blood
fill the end of fitted by lowering
capillary capillary, the end of
tube. made 1 fitted
sample capillary,
around ¾ of made 2
its length samples
undipped. around ¾
of its length
undipped
3. After about two After After two minutes After two

minutes start two started snapping minutes
snapping off minutes off small lengths started
small lengths of started of the tube, at snapping
the tube, at
snappin intervals of 15 off small
intervals of 15
seconds, each
g off seconds, failed to lengths of
time noting small notice formation the tube, at
whether the fibrin lengths fibrin thread intervals of
thread is formed of the between ends. 15 seconds,
between snapped tube, each time
ends. less than noting
15 whether the
seconds fibrin
interval. thread is
formed
between
snapped
ends.
4. Repeat the Wrong Failed to break the Repeated the Repeated the Repeated
breaking at flow in capillary tube at breaking at regular breaking at the
regular time breaking regular time time intervals, regular time breaking at
intervals, till the intervals. failed to notice the intervals, till regular time
fibrin thread capillary fibrin thread. fibrin thread intervals, till
appears at the tube. appeared at fibrin
broken end of the broken thread
capillary tube. Do
end of appeared at
not pull away the
capillary tube. the broken
cut pieces.
Pulled away end of
the cut pieces. capillary
tube. Do
not pull
away the
cut pieces.
Modified White and Lee Method

1. Label three, No label Only the name is Only 2 out of 4 label Only 3 out of 4 Complete
13x 100mm test was seen seen on the are seen on the label are seen specimen
tubes with on the specimen label specimen on the labeling
specimen specimen
patient name,
age, date of
collection, time
of collection.
2. Perform Bevel Bevel orientation is Bevel orientation is Bevel Bevel

aseptically a orientatio correct, presence of correct, no presence orientation is orientation is
venipuncture n is air inside the of air inside the correct, no correct, no
incorrect, syringe, little amount syringe, small presence of air presence of
using a 20gauge
presence of blood enters the amount of blood inside the air inside the
needle syringe of air hub, no blood extracted syringe, syringe,
and withdraw inside the extracted insufficient correct
4mL of blood. syringe, amount of amount of
no blood blood extracted blood
enters the extracted
hub
3. After obtaining Not After obtaining After obtaining After After
the blood, done the blood, the blood, obtaining the obtaining
remove the removed the removed the blood, the blood,
needle from the needle from the needle from the removed the removed
syringe and
syringe. Failed to syringe and needle from the needle
carefully place
deliver accurate carefully placed the syringe from the
1mL of the blood
in test tube #3, measurement of 1mL of the blood and carefully syringe and
then 1 mL in tube blood to the test in test tube #3, placed 1mL carefully
#2, and 1mL to tubes. then 1 mL in tube of the blood placed 1mL
tube #1. The last #2, and 1mL to in test tube of the
1 mL must be tube #1. Failed to #3, then 1 blood in
discarded. Start discard that last mL in tube test tube
the watch as soon 1mL. Failed to #2, and 1mL #3, then 1
as the blood is start the watch. to tube #1. mL in tube
placed in tube
The last 1 mL #2, and
#3.
must be 1mL to
Place the three discarded. tube #1.
tubes in a 37° C Started the The last 1
watch as soon mL must be
waterbath. as the blood discarded.
is placed in Started the
tube #3. watch as
soon as the
Failed to blood is
place the test placed in
tubes in the tube #3.
waterbath.
Place the
three tubes
in a 37° C
waterbath.
4. At exactly 5
mins, tilt test
tube #1 gently to
a 45-degree
angle. Repeat
this procedure
every 30seconds
until the test tube
can be
completely
inverted w/o
spilling the
content.
Record the time

it took the blood
in test tube #1 to
clot.
5.30 seconds after

the blood in test
tube #1 had
clotted, proceed
with tube #2 and
repeat the
preceding
procedure titling
the tube every
30seconds, until
it is completely
clotted. Record
the results.
Repeat the
procedure for test
tube #3. Since
agitation and
handling, speed
up coagulation,
the coagulation
time of thest tube
3 is the result to
be reported.
Recording of the Not done 1 out of 4 is properly 2 out of 4 are 3 out of 4 are Recorded the
test, Calculation, done properly done properly done test,
Interpretation of calculation is
the test and
correct
Clinical
Correlation of ,interpretation
the test and clinical
correlation
are done
properly
Disposal of Left the Disposed the waste Disposed the wastes Disposed all Properly
waste and working in an inappropriate in an inappropriate the waste in cleaned and
cleaning of area dirty. container, did not container, disinfect proper disinfect the
working area disinfect the working area container, did working area.
not disinfect
(Factor: 5) the working
area.
Returning of Left Left some materials Left some materials Returned all Returned all
materials and materials on the working area, on the working materials, no materials, no
glass wares on the with breakages, did area, no breakages, breakages, did breakages,
(Factor: 3) working not dry glass wares did not dry glass not dry glass dried glass
area, with wares wares wares
breakages,
did not
dry glass
wares
Name: _________________________________________________ Date: __________________
EXPERIMENT 6 CAPILLIARY FRAGILITY TEST

OUTCOMES
UNIT
1. Be able to know and perform the methods of examination of blood and

correlate the results to different disease and disorders.
2. Be able to perform tourniquet test.
A tourniquet test (also known as Rumpel-Leede Capillary Fragility test) determines capillary fragility. It is a
clinical diagnostic method to determine a patient’s hemorrhagic tendency. It assesses fragility of capillary
walls and is used to identify thrombocytopenia.
Glossary of Terms
Petechiae: a small red or purple spot on the body, caused by a minor hemorrhage.
Patient preparation, setting up of working area and extraction of blood via syringe method.
OUTCOMES
1. Be able to know and perform the methods of examination of blood and correlate
UNIT the results to different disease and disorders.
ANALYTICAL PHASE
Materials
Stethoscope
Blood pressure cuff
Procedure
1. Examine the forearm, hand and fingers to make certain that no petechiae are present.
2. Apply a blood pressure cuff on the upper arm above the elbow, and take blood pressure reading.
3. Inflate the blood pressure cuff to a point midway between the systolic and diastolic blood pressures
for five minutes.
4. After deflating the cuff, wait for the skin to return to its normal color (usually about five to ten
minutes).
5. Count the number of petechiae visible in a one-inch square area on the ventral surface of the
forearm. Disregard any petechiae within 1.2 inch if the blood pressure cuff because this may be due
to pinching of the skin by the cuff.
6. The test result may be graded roughly as follows:
1+ = a few petechiae on the anterior part of the forearm

2+ = many petechiae on the anterior part of the forearm
3+ = multiple petechiae over the whole arm and the back of the hand
4+ = confluent petechiae on the anterior part and back of the hand
OUTCOMES
1. Be able to know and perform the methods of examination of blood and correlate the
UNIT results to different disease and disorders.
Critical Thinking
1. What stage in hemostasis is evaluated by Tourniquet test?
2. Explain briefly the role of the following factors in maintaining vascular integrity.
A. Platelets
B. Vitamin C
3. Give 2 examples for each of the following bleeding disorders associated with vascular abnormalities.
A. Connective tissue defects

I. Hereditary
II. Acquired
OUTCOMES
B. Altered vessel wall structure

I. Hereditary
II. Acquired
4. Give a brief description of the following terms
A. Purpura
B. Ecchymosis
5. Give 5 examples of hereditary disorders that will have a positive result in tourniquet test.
6. Give 5 examples of acquired disorders that will have a positive result in tourniquet test.
OUTCOMES

1. Be able to know and perform the methods of a. Able to know and perform the methods of
examination of blood and correlate the results examination of blood and correlate the results to
to different disease and disorders. different disease and disorders.
2. Be able to perform tourniquet test. b. Able to perform tourniquet test.
ANECDOTAL NOTES
RUBRIC
RESOURCES
2009
OUTCOMES
RUBRICS
Critical 1 2 3 4 5
hair cap worn.
&goggles)
starting
Analytical phase:
1. Examine the Not done Examined the Examined the Examined the Examined
forearm, hand forearm, hand and forearm, hand and forearm, hand the forearm,
and fingers to fingers. fingers to make and fingers to hand and
certain that no make certain fingers to
make certain that
petechiae are that no make certain
no petechiae are present. petechiae are that no
present. Apply a Failed to apply the present. petechiae are
blood pressure pressure cuff. Applied a present.
cuff on the upper blood pressure Applied a
are above the cuff on the blood
elbow, and take upper are pressure cuff
above the on the upper
blood pressure
elbow. are above the
reading. Failed to elbow, and
record the took blood
blood pressure pressure
reading. reading.
2. Inflate the Not done Inflated the blood Inflated the blood Inflated the Inflated the
blood pressure or pressure cuff to a pressure cuff to a blood pressure blood
cuff to a point inflation point midway point midway cuff to a point pressure cuff
is not between the systolic between the systolic midway to a point
midway between
successfu and diastolic blood and diastolic blood between the midway
the systolic and l pressures for less pressures for 5 systolic and between the
diastolic blood than 5 minutes. minutes. diastolic blood systolic and
pressures for 5 pressures for 5 diastolic
minutes and blood
minutes. observe the pressured for
patient. 5 minutes,
observed the
patient and
applied
patients care
3. After deflating Not done After deflating the After deflating the After deflating After
the cuff, wait for cuff, not waited for cuff, waited for the the cuff, waited deflating the
the skin to return the skin to return to skin to return to its for the skin to cuff, waited
its normal color normal color return to its for the skin
to its normal
normal color to return to
color. and observe its normal
the patient color,
observe the
patient and
applied
patients care
4. Count the Did not Counted the number Counted the
number of count the of petechiae visible number of
petechiae visible petechiae in a one-inch square petechiae
area on the ventral visible in a
in a one-inch
surface of the one-inch
square area on forearm did not square area
the ventral disregarded any on the
surface of the petechiae within ½ ventral
forearm. inch of blood surface of
Disregard any pressure cuff. the forearm.
petechiae within Disregarded
any petechiae
½ inch of blood
within ½
pressure cuff. inch of
blood
pressure
cuff.
the test and
correct
Clinical
correlation
are done
properly
not disinfect
area.
breakages,
did not
dry glass
wares
Name of Patient:_______________________________
Date Performed:_______________________________
Results:
CFT Trial 1 ____________________________________
CFT Trial 2 ____________________________________
Name: _________________________________________________ Date: __________________
EXPERIMENT 7 WHOLE BLOOD CLOTTING TIME

OUTCOMES
UNIT

correlate the results to different disease and disorders.
2. Be able to perform clotting time.
Clotting time is the interval between the moment when bleeding starts and the moment when the fibrin
thread is first seen.
PRE-ANALYTICAL PHASEPRE-ANALYTICAL
Glossary of Terms
Normal Value: 2-7 minutes.
Fibrin is a fibrinous protein involved in the clotting of blood, and is non-globular. It is a fibrillar protein
that is polymerized to form a “mesh: that forms a hemostatic plug or clot over a wound site.
OUTCOMES
ANALYTICAL PHASE
Materials
Water Bath 37° C

Test Tubes
Stopwatch
Plastic syringe (10 ml) and 20-gauge needle
Specimen: Fresh whole blood (4ml). A two-syringe technique is preferable, drawing 1-2 ml of blood in
the first syringe and discarding
Procedure
1. Label three test tubes with the patient’s name, and number them #1, #2, #3.
2. Perform a clean, untraumatic venipuncture using a 20-gauge needle and withdraw 4ml blood.
3. Remove the needle from the syringe, and carefully place 1ml of blood in test tube #3, 1ml in test
tube #2, 1 ml in test tube #1. That last 1 ml of blood is discarded. Start the stopwatch as the blood
is placed in tube #3.
4. Place the three tubes in a 37° C water bath.
5. At exactly 5 minutes, tilt test tube #1 gently to a 45° angle. Repeat the procedure every 30 seconds
until the test tube can be completely inverted without spilling the contents (until the blood is
completely clotted)
6. Record the time it took the blood in test tube #1 to clot.
7. Thirty seconds after the blood in test tube #1 is clotted, proceed with test tube#2, and repeat the
preceding procedure, tilting the test tube every 30 seconds, until a clot is formed. Record the results.
Repeat the procedure in test tube #3.
8. Since the agitation and handling speed up coagulation, the clotting time of test tube #3 is the
reported results.
OUTCOMES
Critical Thinking
1. Give 3 disadvantages of the Lee & White method.
2. What will be the effect of hemolyzed specimen on clotting time? Why?
3. What will be the effect of glass tubes on clotting time? Why?
4. Give the expected results of CT(Write P=Prolonged, N=Normal) if the patient has:
A. Vascular disorder
B. Fibrinogen deficiency
C. Prothrombin deficiency
D. Factor VII deficiency
E. Classic hemophilia
OUTCOMES

examination of blood and correlate the results examination of blood and correlate the results.
to different disease and disorders. b. Able to perform clotting time.
2. Be able to perform clotting time
ANECDOTAL NOTES
RUBRIC
RESOURCES
2009
OUTCOMES
RUBRICS
Critical 1 2 3 4 5
hair cap worn.
&goggles)
starting
Analytical phase:
1. Label three test 1 out of 5 2 out of 5 were done 3 out of 5 were done 4 out of 5 were Labeled
tube with the were properly properly done properly three test
patient’s name done tube with the
and number properly patient’s
them #1, #2, and name and
#3. number
Perform a clean them #1, #2,
venipuncture and #3.
using a 20 gauge Performed a
needle and clean
withdraw 4mL of venipuncture
blood. using a 20
gauge needle
and
withdraw
4mL of
blood.
2. Remove the Not done Removed the needle Removed the needle Removed the Removed the
needle from the from the syringe. from the syringe and needle from needle from
syringe and Failed to transfer the carefully placed 1 the syringe and the syringe
carefully place 1 desired amount to mL of blood in test carefully placed and carefully
mL of blood in the test tubes. tube #3, 1mL in in 1 mL of blood placed 1 mL
test tube #3, 1mL test tube #2, 1mL in in test tube #3, of blood in
in in test tube #2, test tube #1. The 1mL in in test test tube #3,
1mL in test tube last 1mL of blood is tube #2, 1mL 1mL in in
#1. The last 1mL discarded. Failed to in test tube #1. test tube #2,
of blood is start the stopwatch. The last 1mL 1mL in test
discarded. Start of blood is tube #1. The
the stopwatch as discarded. Start last 1mL of
the blood is place the stopwatch blood is
in tube #3. Place as the blood is discarded.
the three tubes in place in tube Start the
a 37° C water #3. Failed to stopwatch as
bath. transfer the the blood is
test tubes in place in tube
the water bath. #3. Place the
three tubes
in a 37° C
water bath.
3. At exactly 5 Not done At 5 minutes, tilt At 5 minutes, tilt At 5 minutes, At 5
minutes, tilt test test tube #1 gently test tube #1 gently tilt test tube #1 minutes, tilt
tube #1 gently to to 45° angle. Failed to 45° angle. gently to 45° test tube #1
45° angle. Repeat to repeat the Repeated the angle. gently to 45°
the procedure procedure every 30 procedure every 30 Repeated the angle.
every 30 seconds seconds. seconds until the procedure Repeated the
until the test tube test tube can be every 30 procedure
can be completely inverted. seconds until every 30
completely Spilled the contents. the test tube seconds until
inverted without can be the test tube
spilling the completely can be
contents. inverted completely
Record the time without spilling inverted
it took the blood the contents. without
in test tube #1 to Failed to spilling the
clot. record the time contents.
in tube #1. Recorded the
time it ook
the blood in
test tube #1
to clot.
4. Thirty seconds Not done Thirty seconds after Thirty seconds after Thirty seconds Thirty
after the blood in the blood in test the blood in test after the blood seconds after
test tube #1 is tube #1 is clotted. tube #1 is clotted, in test tube #1 the blood in
clotted, proceed
Failed to proceed to proceeded w/ test is clotted, test tube #1
w/ test tube #1
and repeat the the other step. tube #1 and repeat proceeded w/ is clotted,
preceding the preceding test tube #1 proceeded
procedure, tilting procedure. and repeat the w/ test tube
the test tube preceding #1 and
every 30 seconds, Failed to tilt the procedure, repeat the
until a clot is tube every 30 tilting the test preceding
formed. Record seconds. tube every 30 procedure,
the result, repeat
the procedure in seconds, until a tilting the
test tube #3. clot is formed. test tube
Failed to every 30
record the seconds,
result in test until a clot is
tube #3. formed.
Record the
result, repeat
the
procedure in
test tube #3.
5. The clotting The The clotting

time of test tube
#3 is the reported clotting time of test
results. time of tube #3 was
test tube the reported
#3 was results.
not the
reported
results.
not disinfect
area.
breakages,
did not
dry glass
wares
Name of Patient:__________________________
Date Performed:__________________________
Result:
Clotting time Trial 1:_______________________
Clotting time Trial 2:_______________________
Interpretation of Results:
Name: _________________________________________________ Date: __________________
EXPERIMENT 8 CLOT RETRACTION

(McFarlane Method)

OUTCOMES
UNIT

correlate the results to different diseases or disorders.
2. Be able to perform clot retraction time using McFarlane method.
This test measures the amount of time it takes for a blood clot to pull away from the walls of a test
tube. It is used to evaluate and manage blood platelet disorders, including Glanzmann’s thrombasthenia.
Glossary of Terms
Fibrin is a fibrinous protein involved in the clotting of blood, and is non-globular. It is a fibrillar protein
that is polymerized to form a “mesh” that forms a hemostatic plug or clot over a wound site.
ANALYTICAL PHASE
Materials
Test Tubes Cork

Coiled wire
OUTCOMES
Procedure
1. A test tube with a capacity of 5 ml is filled with venous blood.
2. A coiled wire is fused into the blood with the cork fitted at the neck of the tube.
3. The tube is incubated at 37° C for 1 hour.
4. The tube is examined for coagulation at 5 to 10 minutes interval. The tube is removed from the
incubator after 1 hour.
5. If retraction has taken place, the clot will be seen to have shrunken away from the wall of the tube
and
6. Is attached only to the wire.
Critical Thinking
1. Completely fill the table below:

DISEASE THROMBOCYTOPENIA, Mechanism behind
THROMBOCYTOSIS, or INCREASED or
BOTH DECREASED Platelet
count
Fanconi’s syndrome
Megaloblastic anemia
Di Guglielmo syndrome
Paroxysmal nocturnal
hemoglobinuria
May-Hegglin anomaly
Polycythemia vera
OUTCOMES
Autoimmune haemolytic anemia
Hemolytic uremic syndrome
Hodgkin’s lymphoma
Systemic lupus erthematosus

examination of blood and correlate the results to examination of blood and correlate the results.
different disease and disorders. b. Able to perform clotting time.
ANECDOTAL NOTES
RUBRIC
Collaboratively create an outcome – based rubric with students. Use the rubric to evaluate how well the
students are able to understand cells.
RESOURCES
2009
OUTCOMES
RUBRICS
Critical 1 2 3 4 5
hair cap worn.
&goggles)
starting
Analytical phase:
1. A test tube Venous A test tube with a A test tube

with a capacity of blood capacity of 5 ml is with a
5 ml is filled with transferre filled with venous capacity of 5
venous blood. A d is blood. Failed to ml is filled
coiled wire is insufficie fuse the coiled wire with venous
fused into the nt. into the blood. blood.
blood with the Coiled wire
cork fitted at the is fused into
neck of the tube. the blood
with the cork
fitted at the
neck of the
tube.
2. The tube is Tube is Tube is incubated at Tube is

incubated at 37° incubated 37° C for 1 hour. incubated at
C for 1 hour. The at 37° C The tube is then 37° C for 1
tube is then for 1 examined for hour. The
examined for hour. coagulation at 5 to tube is then
coagulation at 5 Unable 10 minutes interval. examined for
to 10 minutes to Failed to remove the coagulation
interval. The tube examine tube from the at 5 to 10
is removed from the tube incubator after an minutes
the incubator for for hour. interval. The
1 hour. coagulati tube is
on. removed
from the
incubator
after an
hour.
3. If retraction Retractio Retraction has taken Retraction

has taken place, n has place, the clot has taken
the clot will be taken should be shrunken place, the
seen to have place, away from the wall clot should
shrunken away clot is of tube. Blood not be shrunken
from the wall of attached attached to the wire. away from
the tube and is to the the wall of
attached only to wall of the tube and
the wire. the tube. is attached
only to the
wire.
the test and
correct
Clinical
correlation
are done
properly
not disinfect
area.
breakages,
did not
dry glass
wares
OUTCOMES
UNIT
the results to different disease and disorders.

Name of Patient:_______________________
Date Performed:_______________________
Result:
Clot retaction time Trial 1:_______________
Clot retraction time Trial 2:______________
Normal Value: 44-67%

% Expressed serum = Volume of expressed serum x 100
Volume of whole blood
Name: _________________________________________________ Date: __________________
EXPERIMENT 12 CLOT RETRACTION TIME

(Hirschboek method)

OUTCOMES
UNIT

2. Be able to perform clot retraction time using Hirschboek method.
When whole blood is allowed to clot spontaneously, the initial coagulum is composed of all
elements of blood. With time, coagulum reduces in mass and the serum is expressed from the clot. This is
due to the action of the platelets on the fibrin network.
Glossary
Serum is the clear yellowish fluid obtained upon separating whole blood into its liquid and solid
components after it has been allowed to clot.
OUTCOMES
UNIT the results to different diseases or disorders.
ANALYTICAL PHASE
Materials
Test tubes
Test tube rack
Syringe
Incubator/water bath
Sahli pipette
Castor oil
Procedure
1. Place about 10ml of Castor oil in a test tube.

2. Place the blood collected into a Sahli pipette into the test tube.
3. Observe for the “dimpling” phenomenon extrusion of the blood to clot.
4. Report the result.
OUTCOMES
UNIT results to different diseases or disorders.
Critical Thinking
1. Give the expected result of clot etraction time on the following conditions. Additionally, explain
in one to two sentences the reason behind the expected result. (Write I=Increased CRT,
D=Decreased CRT, N=Normal CRT)
A. Hypofibrinogenemia
B. Low hematocrit
C. Glanzmann’s thromboasrthenia
D. ITP
E. Erthtocytosis
OUTCOMES

examination of blood and correlate the results to examination of blood and correlate the
different disease and disorders. results.
b. Able to perform clot retraction time using
2. Be able to perform clot retraction time using Hirschboek method
Hirschboek method.
ANECDOTAL NOTES
RUBRIC
RESOURCES
2009
OUTCOMES
RUBRICS
Critical 1 2 3 4 5
hair cap worn.
&goggles)
starting
Analytical phase:
1. Place about Insufficie Placed about

10mL of Castor nt 10mL of
oil in a test tube. amount Castor oil in
of castor a test tube.
oil placed
in the
test tube.
2. Place the blood Unable Placed the

collected into the to place blood
Sahli pipette into the blood collected into
the test tube. collected the Sahli
into the pipette into
Sahli the test tube.
pipette
into the
test tube.
3. Observe for the Failed to Observed for the Observed for

‘dimpling’ observe ‘dimpling’ the
phenomenon the phenomenon ‘dimpling’
extrusion of the “dimplin extrusion of the phenomenon
blood to clot. g” blood to clot. Failed extrusion of
Report the result. phenome to report the result. the blood to
non. clot. Report
the result.
the test and
correct
Clinical
correlation
are done
properly
not disinfect
area.
breakages,
did not
dry glass
wares
Name of Patient:______________________________
Date Performed:______________________________
Result:
Clot retraction time Trial 1:_____________________

Clot retraction time Trial 2:_____________________
Name: _________________________________________________ Date: __________________
EXPERIMENT 10 CLOT RETRACTION TIME

(Stefanini-Dameshek Method)

OUTCOMES
UNIT

2. Be able to perform clot retraction time using Stefanini-Dameshek method.
When whole blood is allowed to clot spontaneously, the intial coagulum is composed of all elements
of blood. With time, coagulum reduces in mass and the serum is expressed from the clot. This is due to the
action of the platelets on the fibrin network.
Glossary
Serum is the clear yellowish fluid obtained upon separating whole blood into its liquid and solid
components after it has been allowed to clot.
OUTCOMES
ANALYTICAL PHASE
Materials
Test tubes
Test tube rack
Syringe
Incubator/waterbath
Timer
Procedure
1. Three ml of blood is placed in a test tube and is incubated at 37° C.

2. Clot retraction begins in one hour and is completed within 18-24 hours.
3. Report the result.
OUTCOMES
Critical Thinking
1. Explain briefly the role platelets in clot retraction time.
2. What is thrombasthenin?
3. Aside from platelets, give 3 other factors that contribute to a normal clot retraction.
OUTCOMES

different disease and disorders. b. Able to perform clot retraction time using
Steffani-Dameshek Method.
2. Be able to perform clot retraction time using
Steffani-Dameshek Method.
ANECDOTAL NOTES
RUBRIC
RESOURCES
2009
OUTCOMES
RUBRICS
Critical 1 2 3 4 5
hair cap worn.
&goggles)
starting
Analytical phase:
1.Three ml of Three ml Three ml of

blood is placed in of blood blood is
a test tube and is is placed placed in a
incubated at 37° in a test test tube and
C. tube. is incubated
Failed to at 37° C.
incubate.
2. Clot retraction Clot Clot

begins in one retraction retraction
hour and is begins in begins in one
completed within one hour hour and is
18-24 hours. and is completed
complete within 18-24
d less hours.
than 18-
24 hours.
the test and
correct
Clinical
correlation
are done
properly
not disinfect
area.
breakages,
did not
dry glass
wares
Name of Patient:_______________________________
Date Performed:_______________________________
Result:
Clot retraction time Trial 1______________________
Clot retraction time Trial 2______________________
Reporting:
 Normal or Complete retractility
 Partial retractility
 Poor retractilty
 Very poor retractilty
Name: _________________________________________________ Date: __________________
EXPERIMENT 11 PROTHROMBIN TIME

(For In Vitro Diagnostic Use)

OUTCOMES
UNIT
results to different diseases or disorders.
2. Be able to perform prothrombin time using available High-Sensitivity Prothrombin
Time (PT-HS) Reagent.
The prothrombin time is the method of choice for monitoring oral anticoagulation therapy
and is a fundamental screening test for acquired or inherited bleeding disorders. During oral anti-coagulation
therapy, the activity of vitamin K-dependent clotting factors (II, VII, IX, X, Protein C and Protein S) is
reduced and PT time is increased. The test is used for quantitative determination of blood clotting factors in
the extrinsic (VII) and common pathways (II, V and X) of coagulation.
Principle
The capacity of blood to form a fibrin clot by way of the extrinsic hemostatic pathway
requires thromboplastin, calcium, factors I, II, V, VII and X. the reagent provides a source of tissue
thromboplastin and calcium that specifically activate factor VII in the extrinsic coagulation pathway. The
factors involved in the intrinsic coagulation pathway are bypassed. Therefore, deficiencies of intrinsic
pathway factors (VIII, IX and XII) are not detected using PT test.
Glossary
Prothrombin - a plasma protein produced in the liver in the presence of vitamin K and converted into
thrombin in the clotting of blood.
OUTCOMES
UNIT 1. Be able to know and perform the methods of examination of blood and correlate the
2. Be able to perform prothrombin time using available High-Sensitivity Prothrombin Time
(PT-HS) Reagent.
Reagent
The QuikCoag PT reagent is a lyophilized preparation of rabbit brain thromboplastin, calcium

chloride, buffer, and 0.05% sodium azide as a preservative. The International Sensitivity Index (ISI), lot
number and expiry date of the reagent are shown of the vial’s label.
PRECAUTIONS
Do not ingest. Avoid contact with skin, eye or clothing.
REAGENT PREPARATION
1. Reconstitute the contents of the vial with the specified volume of purified water.
2. Replace the stopper and thoroughly mix the vial contents. Let stand for no less than 15
minutes prior to use assure complete hydration of the contents.
STORAGE and STABILITY
The reconstituted reagent is stable for 5 days when stored in the original container at 2˚ to
8˚C.
SPECIMEN COLLECTION and PREPARATION
Test plasma should be prepared from citrated whole blood without heparin, EDTA or oxalate.
1. Blood Collection using Syringe Method:: draw venous blood into a plastic or siliconized syringe.
Immediately transfer 9.0 mL of blood into a tube containing 1.0 mL of 3.2% or 3.8% sodium citrate
solution.
2. Blood Collection using an Evacuated Blood Collection Tube: draw venous blood into a
commercial vacuum tube containing 3.2% or 3.8% sodium citrate solution. Insure that a fill draw
has been obtained since the ratio of 9 parts blood to 1 part citrate is critical. A heparinized lock or
transfer line should not be used. It is generally recommended that the second or third tube draw be
used for coagulation tests.
3. Plasma Preparation: mix well by inversion and centrifuge at 2,500 x g for 15 minutes soon after
blood collection. Unless samples are to be processed immediately, transfer the plasma into a plastic
tube. Plasma that is clearly hemolyzed or contains >10,000 platelets per cubic millimeter or red cells
is not suitable for coagulation testing.
OUTCOMES
4. Plasma Storage: plasma samples may be stored at room temperature (18 to 26˚C) for up to 2
hours; refrigerated (2 to 8˚C) for up to 4 hours; frozen at -20˚C for up to 2 months or at -70˚C for
up to 6 months. Plasma may be re-centrifuged prior to freezing to assure that all cells are removed.
Quick thaw frozen samples and test immediately. The samples must not have any contact with glass.
Do not incubate samples at 37˚C for longer than 5 minutes to avoid the loss of factors V and VII.
Loss of factor V can prolong the PT.
ANALYTICAL PHASE
Materials
Water Bath Test tube rack
Magnetic Stirring Timer
Pipette Sample Blood
Cuvette/Test tubes Reagents
Procedures
This procedure pertains to manual or semi-automated coagulation systems. Refer to your instrument
manual for more detailed instrument specific instructions.
1. Pre-incubate the QuickCoagTM Calcium Chloride (0.02M) to 37˚C for at least 10 minutes.
2. Pipette 100
QUALITY CONTROL
Reliability of test results should be monitored within each run using normal and abnormal control
plasmas such as the Control Levels 1, 2 and 3. Each laboratory should establish a control range to determine the
allowable variation in day-to-day performance of each control plasma.
OUTCOMES
UNIT correlate the results to different diseases or disorders.
2. Be able to perform prothrombin time using available High-Sensitivity
Prothrombin Time (PT-HS) Reagent.
Calculation of Results
Calculate the mean clotting time of duplicate samples and controls. Differences between duplicate
results should be less than 5%. Repeat the test if necessary.
The PT result may be reported as seconds to form a clot, ratio of patient clotting time to mean normal
clotting time, patient activity, or International Normalized Ratio (INR). The INR is recommended for use with
patients undergoing anti-coagulation therapy.
The INR is calculated using the following formula:
INR = (Patient PT/ Mean Normal PT)
ISI = Lot specific International sensitivity Index for the Reagent/Instrument system
Mean Normal PT = Lot specific mean of the normal range, as determined by each laboratory for the Reagent/
Instrument system. It is usually based upon the mean PT plus or minus 2 to 3 standard deviations using 20 or
more individuals.
Expected Values
Normal Range – 10 to 16 seconds
OUTCOMES
Critical Thinking
1. State the different factors of Intrinsic Pathway and give its common names.
2. State the different factors of Extrinsic Pathway and give its common names.
OUTCOMES
UNIT

different disease and disorders.
2. Be able to perform prothrombin time using b. Able to perform prothrombin time using
available High-Sensitivity Prothrombin Time (PT- available High-Sensitivity Prothrombin Time
(PT-HS) Reagent.
HS) Reagent.
STRATEGIES
Assessment
ANECDOTAL NOTES
RUBRIC
RESOURCES
 QuikCoag PT with Calcium BioMedica Diagnostics Inc. Product Insert
OUTCOMES
2. Be able to perform prothrombin time using available High-Sensitivity Prothrombin Time
(PT-HS) Reagent.
RUBRICS
Critical 1 2 3 4 5
hair cap worn.
&goggles)
starting
Materials No Incomplete (4are Incomplete (3 are Incomplete (2 Complete
materials lacking) lacking) are lacking) (labelled
tubes,
reagent,
water bath,
magnetic
stirring,
sample
blood,
pipette,
timer)
Analytical phase:
Pre-incubate the Incubate Incubated the Incubated the Incubated the Incubated
reconstituted d the reconstituted reconstituted reconstituted the
reagent to 37˚C reconstit reagent to less than reagent to 37˚C less reagent to 37˚C reconstituted
for at least 10
uted 37˚C for at least 10 than 10 minutes. for at least 10 reagent to
minutes.
reagent minutes. minutes. 37˚C for at
to less least 10
than minutes,
37˚C less assured the
than 10 positioning
of
minutes reconstituted
reagent was
stable
Maintain the Did not Partially maintained Maintained

suspension of the maintaine the suspension of the
reagent by d the the reagent by suspension
magnetic stirring
suspensio magnetic stirring or of the
or mixing by
inversion n of the mixing by inversion reagent by
immediately prior reagent immediately prior to magnetic
to use by use stirring or
magnetic mixing by
stirring inversion
or mixing immediately
by prior to use
inversion
immediat
ely prior
to use
Pipette100µL of 0 out of 3 1 to 2 out of 3 were 1 out of 3 were 2 out of 3 were Transferred

test or control were not done but not properly done properly done 100µL of test
plasma into a test done properly or control
cuvette
properly plasma into a
test cuvette,
ensured that
the outer
side of the
cuvette is
clean and
proper
disposal of
tips was
done
Incubate at 37˚C Not done Incubated for Incubate at 37˚C Incubate at Incubated
for 1 minute more than or less for less than 1 37˚C for more at 37˚C for 1
than 37˚C for 1 minute than1 minute minute
minute
Rapidly add Slowly Slowly/Rapidly Slowly/Rapidly Rapidly add Rapidly add

200µL of the pre- add add less than add more than 200µL of the 200µL of the
incubated PT 200µL of 200µL of the pre- 200µL of the pre- pre-incubated pre-
reagent, the pre- incubated PT incubated PT PT reagent incubated
simultaneously incubate reagent, reagent, only PT reagent,
starting the timer d PT simultaneously simultaneously simultaneo
reagent, starting the timer starting the timer usly starting
simultan the timer
eously
starting
the
timer
test, Calculation, properly done properly done properly done test,
the test and
correct
Clinical
Correlation of the ,interpretatio
test n and clinical
correlation
are done
properly
Post-analytical phase:
not disinfect
area.
breakages,
did not
dry glass
wares
Name of Patient:_______________________________
Date Performed:_______________________________
Result:
Trial 1______________________
Trial 2______________________
Name: _________________________________________________ Date: __________________
EXPERIMENT 12 ACTIVATED PARTIAL THROMBOPLASTIN TIME

(For In Vitro Diagnostic Use)

OUTCOMES
UNIT
the results to different diseases or disorders.
2. Be able to perform activated partial thromboplastin time using available APTT
reagent.
The activated partial thromboplastin time (APTT) is used as a general screening test for the
detection of coagulation abnormalities in the intrinsic pathway. The APTT is sensitive to deficiencies or
abnormalities of factors VIII, IX, XI, XII, X and II, prekallikrein, high molecular weight kininogen
(HMWK) and fibrinogen. APTT is also sensitive to inhibitors of blood coagulation such as lupus inhibor
and fibrin/fibrinogen degradation products. The APTT is the most used method for monitoring
intravenous heparin anticoagulant therapy.
Principle
The capacity of blood to form a fibrin clot by way of the intrinsic hemostatic pathway requires
coagulation factors I, II, V, VIII, IX, X, XI and XII, platelet lipids and calcium. The assay is performed by
the addition of a suspension of rabbit brain cephalin with a surface activator. The APTT has proven to be a
single and highly reliable measurement of the intrinsic coagulation mechanism.
OUTCOMES
2. Be able to perform activated partial thromboplastin time using available APTT reagent.
Reagent
The QuikCoag APTT reagent is a preparation of rabbit brain cephalin and ellagic acid activator with
buffer, stabilizers and preservatives. The reagent is provided ready to use.
PRECAUTIONS
Do not ingest. Avoid contact with skin, eye or clothing.
STORAGE and STABILITY
The reconstituted reagent is stable for 5 days when stored in the original container at 2˚ to 8˚C.
SPECIMEN COLLECTION and PREPARATION
Test plasma should be prepared from citrated whole blood without heparin, EDTA or oxalate.
1. Blood Collection using Syringe Method:: draw venous blood into a plastic or siliconized syringe.
Immediately transfer 9.0 mL of blood into a tube containing 1.0 mL of 3.2% or 3.8% sodium citrate
solution.
2. Blood Collection using an Evacuated Blood Collection Tube: draw venous blood into a
commercial vacuum tube containing 3.2% or 3.8% sodium citrate solution. Insure that a fill draw
has been obtained since the ratio of 9 parts blood to 1 part citrate is critical. A heparinized lock or
transfer line should not be used. It is generally recommended that the second or third tube draw be
used for coagulation tests.
3. Plasma Preparation: mix well by inversion and centrifuge at 2,500 x g for 15 minutes soon after
blood collection. Unless samples are to be processed immediately, transfer the plasma into a plastic
tube. Plasma that is clearly hemolyzed or contains >10,000 platelets per cubic millimeter or red cells
is not suitable for coagulation testing.
4. Plasma Storage: plasma samples may be stored to a plastic tube as soon as possible and stored
refrigerated (2 to 8˚C). Plasma samples should be tested within 4 hours and should not be incubated
at 37˚ C for more than 5 minutes to avoid loss of factors V and VII.
OUTCOMES
ANALYTICAL PHASE
Materials
Water Bath
Magnetic Stirring
Pipette
Cuvette/Test tubes
Test tube rack
Timer
Sample Blood
Reagents
Procedures
This procedure pertains to manual or semi-automated coagulation systems. Refer to your instrument
manual for more detailed instrument specific instructions.
1. Pre-incubate the QuikCoagTM Calcium Chloride (0.02M) to 37˚C for at least 10 minutes.
2. Pipette 100µL of test or control plasma into a test cuvette.
3. Incubate at 37˚C for 1 to 2 minutes.
4. Add 100µL of the QuikCoagTM APTT reagent to the cuvette containing the plasma. Maintain the
suspension of the reagent by magnetic stirring or mixing by inversion immediately prior to use.
5. Incubate the mixture at 37˚C for 3 minutes.
6. Rapidly add 100µL of the pre-incubated QuikCoagTM Calcium Chloride (0.02M) and simultaneously start
the timer.
7. Record the clotting time in seconds.
QUALITY CONTROL
Reliability of test results should be monitored within each run using QuikCoagTM Coagulation Control
Plasma 1, 2 and 3. Each laboratory should establish a control range to determine the allowable variable in day to
day performance of each control plasma.
OUTCOMES
Calculation of Results
Calculate the mean clotting time of duplicate samples and controls. Differences between duplicate
results should be less than 5%. Repeat the test if necessary.
EXPECTED VALUES
Reference range: 24-39 seconds
Critical Thinking
1. If the patient has a prolonged APTT, what does it indicates?
2. What are the activators that can be used in APTT test? Give its functions.
OUTCOMES

1. Be able to know and perform the methods of a. Know and perform the methods of examination
examination of blood and correlate the results of blood and correlate the results to different
to different diseases or disorders. diseases or disorders.
2. Be able to perform activated partial b. Perform activated partial thromnoplastin time

thromboplastin time using available APTT using the available reagent.
reagent.
ASSESSMENT STRATEGIESASSESSMENT
Focus for AssessmentFocus for
ANECDOTAL NOTES
RUBRIC
RESOURCES
 QuikCoagTM APTT BioMedica Diagnostics Inc. Product Insert
OUTCOMES
RUBRICS
Critical 1 2 3 4 5
hair cap worn.
&goggles)
starting
Materials No Incomplete (4are Incomplete (3 are Incomplete (2 Complete
materials lacking) lacking) are lacking) (labelled
tubes,
reagent,
water bath,
magnetic
stirring,
sample
blood,
pipette,
timer)
Analytical phase:
Pre-incubate the Incubate Incubated the Incubated the Incubated the Incubated
QuikCoagTM d the reagent to less than reagent to 37˚C less reagent to 37˚C the reagent
Calcium Chloride reagent 37˚C for at least 10 than 10 minutes. for at least 10 to 37˚C for
to 37˚C for at
to less minutes. minutes. at least 10
least 10 minutes.
than minutes,
37˚C less assured the
than 10 positioning
minutes of was
stable
Pipette100µL of 0 out of 3 1 to 2 out of 3 were 1 out of 3 were 2 out of 3 were Transferred
test or control were not done but not properly done properly done 100µL of test
plasma into a test done properly or control
cuvette
properly plasma into a
test cuvette,
ensured that
the outer
side of the
cuvette is
clean and
proper
disposal of
tips was
done
Incubate at 37˚C Not done Incubated for Incubate at 37˚C Incubate at Incubated
for 1 to 2 minutes more than or less for less than 1 to 2 37˚C for more at 37˚C for 1
than 37˚C for 1 to 2 minutes than 1 to 2 to 2
minutes minutes minutes
Add 100 µL of the Not done Added less than Added more Added
APTT reagent to 100µL of the than 100µL of 100µL of the
the cuvette reagent to the the reagent to reagent to
containing the cuvette containing the cuvette the cuvette
plasma. the plasma. containing containing
the plasma. the plasma.
Maintain the Did not Partially maintained Maintained

suspension of the maintaine the suspension of the
reagent by d the the reagent by suspension
magnetic stirring
suspensio magnetic stirring or of the
or mixing by
inversion n of the mixing by inversion reagent by
immediately prior reagent immediately prior to magnetic
to use by use stirring or
magnetic mixing by
stirring inversion
or mixing immediately
by prior to use
inversion
immediat
ely prior
to use
Add 100 µL of the Not done Added 100 µL of Failure to add 100 Added 100 µL Added 100
pre-incubated the pre-incubated µL of the pre- of the pre- µL of the
Calcium Chloride Calcium Chloride incubated Calcium incubated pre-
and
and did not Chloride and Calcium incubated
simultaneously
start the timer. simultaneously simultaneously Chloride and Calcium
start the timer. start the timer. simultaneousl Chloride
y start the and
timer. simultaneo
usly started
the timer.
test, Calculation, properly done properly done properly done test,
the test and
correct
Clinical
Correlation of the ,interpretatio
test n and clinical
correlation
are done
properly
Post-analytical phase:
not disinfect
area.
breakages,
did not
dry glass
wares
Name of Patient:_______________________________
Date Performed:_______________________________
Result:
Trial 1______________________
Trial 2______________________

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Hematology 2 Lab Manual

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Name: _________________________________________________ Date: __________________

Section: __________________ Group: __________________ Score: _________________

EXPERIMENT 1 DIFFERENTIAL COUNT

To achieve this unit a learner must:

1. Be able to differentiate the different types of leukocyte in a normal

TEACHING AND LEARNING ACTIVITIES

C. Experience is the foremost teacher in hematology. It is readily acquired in a busy hematology

1|CLINICAL HEMATOLOGY 2 LABORATORY MANUAL

Cell counter Spreader slider

3|CLINICAL HEMATOLOGY 2 LABORATORY MANUAL

A. Counting the leukocytes

1. What is the significance of differential leukocyte count?

2. In what conditions will the neutrophil count decrease?

3. In what conditions will the neutrophil count increase?

4. In what conditions the eosinophil count decrease?

5. In what conditions the eosinophil count increase?

6. In what conditions the basophil count decrease?

7. In what conditions the basophil count increase?

8. In what conditions the monocyte count decrease?

5|CLINICAL HEMATOLOGY 2 LABORATORY MANUAL

Outcome To achieve each outcome, the learner must

Focus for Assessment

Materials No Incomplete Incomplete (3 Incomplete (2 are Complete

7|CLINICAL HEMATOLOGY 2 LABORATORY MANUAL

Proper needle Remove Remove Remove needle Remove tourniquet, Remove

Checking for Rough edge The edge of the

Staining The stain Stained the Stained the

Direction in counting No pattern Begin the count Begin the count

9|CLINICAL HEMATOLOGY 2 LABORATORY MANUAL

Leukocyte type number fraction

Eosinophils: 0.00 – 0.04

Basophils: 0.00 – 0.02

Lymphocytes: 0.20 – 0.44

Monocyte: 0.02 – 0.09

Bands or Stabs: 0.02 – 0.06

EXPERIMENT 2 PLATELET COUNT Direct Method

To achieve this unit a learner must:

1. Be able to perform the direct method of platelet counting.

2. Be able to correlate the results to different diseases or disorders.

TEACHING AND LEARNING ACTIVITIES

B. Platelets are difficult to count due to:

EDTA evacuated tube Hemocytometer and coverslip

A. Diluting the blood.

B. Charging the counting chamber.

C. Counting the platelets

The following formula is usually used:

Platelet count = # of cells counted x Dilution Factor (0.20) = platelet x 109/L

Reference Range: 150,000 to 400,000/µl (or 150 to 400x 10⁹/L)

Numerical estimate of platelets from a blood smear

1. Give 5 conditions wherein the platelet count is increased.

2. Give 5 conditions wherein the platelet count in decreased.

Outcome To achieve each outcome, the learner must

Focus for Assessment

1. Be able to perform the direct method of platelet counting.

Date Performed Remarks

Trial 1 _____________ _____________

Trial 2 _____________ _____________

EXPERIMENT 3 PLATELET COUNT Modified Indirect Method

To achieve this unit a learner must:

1. Be able to perform the direct method of platelet counting.

2. Be able to correlate the results to different diseases or disorders.

TEACHING AND LEARNING ACTIVITIES

B. Platelets are difficult to count due to:

EDTA evacuated tube Hemocytometer and coverslip

Name: _______________________________ Date:

Section: Group: Score: _

Trial 1 _ _

Trial 2 _ _