HEMA LAB QUIZ 1 6. All of the ff.

appearances of cells are observed

in an overstained smears except
1. Matching type
o Nuclear chromatin stained pale blue
o Neutrophilic segmenter – 50-70%
rather than vivid blue
o Lymphocyte – 18-42%
o Cytoplasm of the cells appear gray or
o Neutrophilic band – 0-5%
lavender
o Monocyte - 2-11%
o Eosinophilic granules become deep
o Eosinophil – 1-3%
gray or blue
o Basophil – 0-2%
o Erythrocytes stains blue or green
2. Matching type
7. Which of the ff. statements is true concerning
o Nucleus is broken into segments.
a normal WBC differential count in adults?
Cytoplasm contain small pinkish
o There are usually more monocytes
granules – Neutrophilic segmenters
than eosinophils
o Nucleus is usually indistinct and
o There are usually more lymphocytes
obscure by the granules. Cytoplasm
than any other cells
contains large purplish-black or dark
o There are usually more bands than
blue granules – Basophils
lymphocytes
o Younger form of neutrophil with C,S,
o There are usually more basophils than
U, or horse-shoe shaped nucleus –
eosinophils
Neutrophilic Bands
8. Which of the ff. expressed in chart form in
o Nucleus is spongy and sprawl with
which neutrophils are divided into 5 classes
brain- like convolutions. Cytoplasm is
according to number of segments (lobes) of
gray. Vacuoles are sometimes present
their nuclei? – Arneth’s Classification
– Monocyte
9. When performing a differential count, how
o Nucleus is usually bilobed. Cytoplasm
many leukocytes are counted?
contains large, coarse, reddish or
o 200
orange granules – Eosinophils
o 50
o Nucleus is compact and usually round.
o 100
Cytoplasm is light blue and scanty. –
o 300
Lymphocyte
10. Which of the ff. characteristic of a
3. The components of Wright’s stain –
degenerative shift to the left?
Methylene blue and Eosin
o Accompanied by low WBC count
4. Which of the following is the recommended
o Predominating cells are younger forms
and most reliable procedure, excellent for
o Increased in band cells but w/o
staining thin and thick blood films (inclusion
myelocytes and metamyelocytes
bodies and intracellular parasites as well as for
o All options are correct
staining WBCs)
11. A shift to the right leukocyte differential count
o Both items are correct
is characteristic of this condition?
o Giemsa Stain
o Any item is correct
o Wright's stain
o Chronic viral infection
o No option is correct
o Pyogenic infection
5. What staining method is frequently to stain
o Megaloblastic infection
and manually count reticulocytes?
12. The ff are examples of panoptic stains except
o Immunofluorescence
o Wright’s Giemsa
o Romanowsky staining
o Jenner-Giemsa
o Supravital staining
o May-Grunwald-Giemsa
o Cytochemical staining
o Jenner-Grunwald-Giemsa
13. If a dried smear cannot be stained
immediately, it should be preserved by
immersing in
o Methylene blue
o Eosin
o Ethanol
o Methanol
14. A blood smear was evaluated and showed
that all of the cells appear too blue. How
would you classify the staining technique?
o Overstained
o Understained
o Suitable stain
o Good stain
15. The haemoglobin content of the individual cell
appears to be increased in
o Hypochromasia
o Polycythemia
o Polychromasia
o Hyperchromasia
WHITE BLOOD CELL COUNT
The white blood cell count (WBC) denotes the number of white blood cells in in 1 liter (L) of whole blood. In a
normal, healthy individual, the WBC falls in the range of 4,000 to 11, 000 x 10 /L (or 4.0 to 11.0 x 10 /L). This count varies
6 9

with age. The WBC of a newborn baby is 10.0 to 30.0 x 10 /L at birth. It decreases to a range of 6.0 to 17.0 x 10 /L at one
9 9

year of age and drops to normal levels by the age of 21.

The WBC is a useful measurement to the physician. It is utilized to indicate infection and may also be employed
to follow the progress of certain diseases and therapies. The WBC may be elevated in bacterial infections, appendicitis,
leukemia, pregnancy, hemolytic disease of the newborn (HDN), uremia and ulcers. The WBC may drop below normal
values in viral diseases (such as measles), brucellosis, typhoid fever, infectious hepatitis, rheumatoid arthritis, cirrhosis of
the liver and lupus erythematosus.

Radiation and certain drug therapy tends to lower the WBC. The patients will have white cell counts performed
while receiving this treatment to ensure that the WBC does not become too low. A white cell count above 11.0 x 10 /L is 9

termed leukocytosis; a white cell count below normal is known as leukopenia. The white cell count in children usually
shows a greater variation in disease. For example, during infection, a child’s white blood cell reaches much higher
elevations than does an adult’s white cell count in response to a corresponding infection.

An individual’s normal WBC is subject to variations, being slightly higher in the afternoon than in the morning.
There is also an increase in the WBC following strenuous exercise, emotional stress and anxiety. In most laboratories an
electronic method (Automation) of counting white blood cells is used.

DISCUSSION
1. In certain conditions, such as leukemia, the WBC may be extremely high. If white cell count is above 30 x 10 /L, it is
9

advisable to employ a larger dilution of blood. A platelet Unopette may be used or the blood may be diluted 1:101 (0.02
mL whole blood + 2.0 mL diluting fluid). Alternatively, a Thoma red cell pipet may be used, the blood drawn up to the 1.0
mark and diluted to the 101 mark with the white cell count diluting fluid (1:100 dilution). If the white cell count is
markedly elevated, as in some leukemias, in which it may be as high as 100 to 300 x 10 /L, a 1:200 dilution is used. This is
9

accomplished by adding 0.02 mL of whole blood to 4.0 mL diluting fluid (1:201 dilution), or by drawing the blood up to
0.5 mark in the Thoma red cell pipette and diluting to the 101 mark with white count diluting fluid. The correction factor
for the dilution, however, changes accordingly.

2. Whenever the WBC drops below 3.0 x 10 /L, a smaller dilution of the blood should be used to achieve a more accurate
9

count. In this situation, the blood may be diluted 1:11 (0.02 mL whole blood + 0.2 mL diluting fluid) or drawn up to the
1.0 mark in a Thoma white cell pipet and diluted to the 11.0 mark with the white count diluting fluid for a dilution of
1:10. The white cell count then proceeds as previously outlined with the appropriate correction factor used for the
dilution.

3. It is important that the diluting fluid remain free from contamination. Often, small amounts of blood collect in the
diluting fluid, causing inaccuracies and difficulties in distinguishing and counting the WBCs.

4. It is imperative that the counting chamber and cover glass be free from dirt and lint. Again, contamination may cause
inaccuracies and difficulties in counting white blood cells. (The counting chamber and cover glass should be cleaned off
immediately after completion of the count).

5. Pipets must be free of dirt and dried blood. Never leave undiluted blood in a Thoma pipet. It quickly hardens and
plugs up the pipet. Draw water or diluting fluid into the pipet and place it in a container of 10% aqueous Chlorox
solution.

6. There is an approximate 15% error for a manual WBC that falls within the normal range. It is advisable to count at
least 100 WBC on each side of the counting chamber. Generally, the more cells counted, the lower the percentage of
error.
7. The diluting fluids used for the white cell count destroy or hemolyze all non-nucleated RBCs. In certain disease states,
nucleated red blood cells (NRBCs) are present in the peripheral blood. These cells, because they contain a nucleus,
cannot be distinguished from the white blood cells. Therefore, any time there are five or more nucleated red blood cells
per 100 WBCs in a differential, the WBC count should be corrected as follows:
Corrected WBC = Uncorrected WBC x 100
100 + # of NRBC/100 WBC

The white cell count is then reported as the “Corrected” WBC.

8. Once the hemacytometer is filled, the counting of cells must proceed without delay. If too much time elapses, the
fluid in the chamber begins to evaporate, causing inaccuracies in the count.

VARIATION IN TECHNIQUE:
1. In leukopenia, draw blood to mark 1 and the diluents to 11; the ratio of dilution then is 1:0. In the formula followed
instead of using 20 as the dilution factor, use 10.

2. In leukocytosis, draw blood to mark 1 or 0.5 of the RBC pipette and the diluent to 101; the ratio of dilution is 1:200 (if
blood is up to 0.5) and 1:100 (if the blood is up to 1.0). Change the dilution factor accordingly in the formula.

3. If 8 big squares were used (4 in each ruled area), that means the cells were counted in 8 sq mm and the corresponding
area correction factor is1/8 and in the formula change the area correction factor to 8 accordingly or first get the average
of the counts made on the upper and lower chamber.

Reference Ranges for WBC Count

Normal Values Conventional Unit SI Unit
Adults 4,500 to 11,000/cu mm 4.5 – 11.0 x 10 /L
9

Infant 10,000 to 25,000/cu mm 10.0 – 25.0 x 10 /L 9

1 year 8,000 to 15,000/cu mm 8.0 – 15.0 x 10 /L

9

Correction of WBC count is done if there are 5 or more NRBC/100 red cells when examined in the blood smear.
VARIATION IN THE WBC COUNT:
1. There is an hourly rhythm of number of white blood cell. Low level is observed in the morning and goe higher in the
afternoon.

2. Food intake, moderate physical or emotional activity will cause a high increase in the number of leukocytes.

3. Highest peak is observed after meals.

SOURCES OF ERRORS IN MANUAL CELL COUNTING

1. Failure to mix blood specimens prior to sampling.
2. Failure to immediately mix blood and diluents in the Thoma pipettes.
3. Using diluents contaminated with blood.
4. Bubbles in the Thoma pipette.
5. Drawing blood too far past the appropriate dilution mark and subsequent drawing out.
EOSINOPHIL COUNT
Although the relative and absolute number of eosinophils in the blood may be determined from the
WBC and differential white cell count. It is sometimes necessary to more accurately determine the total
number of eosinophils/L of blood. The direct method for counting eosinophils is similar to the method used
for the red and white blood cell counts.

The normal range for the eosinophil count is 50 to 350 x 106/L. A low eosinophil count (eosinopenia) is
found in hyperadrenalism (Cushing’s syndrome), shock, and following the administration of
adrenocorticotropic hormone (ACTH). Increased numbers of eosinophils (eosinophilia) occur in allergic
reactions, parasitic infestations, brucellosis and certain leukemias. There may be considerable variation in the
eosinophil count over a 24-hour period, with the lowest count generally present during late morning and the
highest count present during the night (midnight and later).

There are two general methods for determining the absolute eosinophil count: indirect method (WBC
count multiplied by the percentage of eosinophils in the differential) and the direct method. The most widely
used procedure is to perform the eosinophil count
by the direct method and double-check these results using the indirect method.

Principle:
Whole blood is diluted with stain solution. The phloxine present in the diluting fluid serves to stain the
eosinophil red, the sodium carbonate and water help to lyse the white blood cells (except the eosinophils) and
the red blood cells are lysed by the prophylene glycol. Heparin, if present in the diluting fluid prevents
clumping of the white blood cells. The sodium carbonate also enhances the staining of the eosinophil granules.

How to double-check the direct eosinophil count?

a. Perform a white blood cell count on the specimen of blood. Make two blood smears and Wright
stain.
b. Perform a 200-cell differential white cell count on the blood smear.
c. Calculate the indirect eosinophil count as follows:
Eosinophil/L = Percent eosinophils in differential x WBC/L
d. The results obtained should correlate with the eosinophil count by the direct method.
If there is too large a variation, repeat the direct and indirect eosinophil counts.

Discussion.
1. A single eosinophil count may be ordered on a patient, or, the eosinophil counts may be ordered, in
conjunction with the Thorn test for adrenal cortical function This test, however, is rarely performed.

2. The indirect method for counting eosinophils is not as accurate as the direct method. Therefore, a close
correlation between the results of the two methods is not always possible.
3. Once the eosinophil count is diluted, it should be counted within 30 minutes. The eosinophils will easily
disintegrate in the diluting fluid if left diluted for too long a period of time. If the Unopette is used, the count
should be completed within 1 hour of being diluted.

4. To improve the accuracy of the direct eosinophil count at least 100 eosinophils shoud be enumerated. In
order to do this, the counting chamber may be filled and counted as many times as necessary. The C. V. of the
eosinophil count when at least 100 cells are counted is about 10%.

Diluting Fluids:
1. Phloxine – Propylene glyol, phloxine, sodium carbonate, distilled water
2. Pilot’s – same as phloxine except for the addition of heparin
3. Dunger’s – eosin, acetate, distilled water
4. Manner’s – urea, phloxine, trisodium citrate, distilled water
5. Randolph’s – phloxine, calcium chloride, propylene glycol
6. Tannen’s – neutral red, iodide solution, NaOH
7. Hinklemann’s – eosin yellow, formaldehyde, 95% phenol, distilled water

INDIRECT METHOD
Hansel stain used to stain smear for indirect eosinophil count
Stock solution 1 methylene blue in propylene glycol
Working solution 1 mix 2.5 mL of stock solution 1 with 2.0 mL of distilled water

Stock solution 2 phloxine in propylene glycol

Working solution 2 mix 2.5 mL of stock solution 1 with 2.0 mL of distilled water

Computation for eosinophils/µL:

Multiply total WBC count/µL by % eosinophil examined from the blood smear
e.g. 4% eosinophil was observed in the blood smear
15,000/µL = total WBC count
Absolute eosinophil count = % eosinophil x WBC count
100
= 4 x 15,000
100
= 600 eosinophils/µL

Thorn’s ACTH Test

This based on the fact that ACTH produces in 4 hours a decrease by 50% or more in eosinophil count of
persons with a normally functioning adrenal cortex. This is so because eosinophils migrate to the tissues.
It is used as a diagnostic test in Addison’s disease as a test for adrenal cortex reserve before surgical
procedures and as a test to distinguish functional hypopituitarism from organic disease of the adrenal cortex.

Procedure:
1. Determine eosinophil count in the fasting state (first eosinophil count).
2. The patient is injected with 25 mg ACTH intramuscularly.
3. Determine second eosinophil count four hours after injection.

Result:
NV = second eosinophil count at least 50% below the first eosinophil count
Hypoadrenalism: No change or no difference between the first and second eosinophil count.
Differential Cell Count
The manual differential white blood cell count is performed to determine the relative number of each
type of white blood cell present in the blood. At the same time, a study of red blood cell, white blood cell and
platelet morphology is performed.

An approximation of platelets is also made. The differential and smear review should be performed
after the blood counts have been completed. In this way, examination of the smear may also be used to
double check the white blood cell count.

Obtaining an accurate manual white blood cell differential is somewhat difficult because of the fact
that the white blood cells are not always randomly distributed. For routine testing, the wedge smear is the
most widely used.

Smears that are poorly made and/or are thin will show an increased concentration of
polymorphonuclear white cells, monocytes, and large abnormal cells at the edges and tail of the blood film.
This will then cause a relative increased concentration of lymphocytes in the in the middle of the smear. It is
therefore of utmost importance that the blood film be well prepared.

The normal range for the white blood cell differential should be determined by each laboratory. During
infancy and childhood a mild lymphocytosis may be present. Adult normal values are reached by the age of 21.

In disease states, a particular white blood cell type ma arey show an absolute increase in number in the
blood. Common diseases showing an increased number of a specific cell type are:
1. Neutrophilia (absolute increase in the number of neutrophils)
a. Appendicitis
b. Myelogenous leukemia
c. Bacterial infections
2. Eosinophilia (absolute increase in the number of eosinophils)
a. Allergies and allergenic reactions
b. Scarlet fever
c. Parasitic infections
d. Eosinophilic leukemia
3. Lymphocytosis (absolute increase in the number of lymphocytes)
a. Viral infections
b. Whooping cough
c. Infectious mononucleosis
d. Lymphocytic leukemia
4. Monocytosis ( absolute increase in the number of monocytes)
a. Brucellosis
b. Tuberculosis
c. Monocytic leukemia
d. Subacute bacterial endocarditis
e. Typhoid
f. Rickettsial infections
g. Collagen disease
h. Hodgkin’s disease
i. Gaucher’s disease

Procedure for examination of the Stained Blood Smear

1. Check the blood smear to ensure that it is well made. The tail of the film should be smooth. Place the slide
(smear side up) on the microscope stage. (For consistency, when the wedge smear is used place the thick end
of the smear on the same side of the microscope stage each time a differential is performed).

2. Examine the blood smear using the low power objective.

a. The red and white blood cells and platelets must be correctly stained.
b. Check that there is even distribution of white blood cells on the smear.
c. Estimate the white blood cell count (by noting the number of white cells/field and the number of
white cells in relation to the red blood cell count). It should agree with the test result obtained. If it does not,
the white count should be repeated.
d. Examine the thin peripheral edge of the smear (the tail) (if the wedge smear is being used). If there
is an increased number of white cells in this area, the differential cell count may be inaccurate. If there are
clumps of platelets, the body of the smear may then show a decrease in platelets. In such situations, the blood
smear should be discard and another made.
e. When scanning the blood smear, it is important to note anything unusual or irregular, such as large,
abnormal looking cells or rouleaux formation of the red blood cells.
f. Choose the area of the blood smear where the differential counting is to begin, place a drop of oil on
the slide and carefully change to the oil immersion objective.

3. Perform the differential cell count and, at the same time, examine the morphology of the white blood cells.
There are several counting methods used, three of which are described:
a. Using the cross-sectional or crenellation technique the white cells are counted in consecutive fields
as the blood film is moved from side to side. Counting should begin in thin area of the smear where the red
blood cells are slightly overlapping and proceed into the thicker area. However, do not progress too far into
the thick area if the white cells are not sufficiently spread for easy identification.

b. In the longitudinal method the white cells are counted in consecutive fields from the tail toward the
head of the smear. This is the ideal method if the smear is thin enough so that the white cells may be
identified all the way to beginning (head) of the smear. In this case, the strip of smear examined represents
one complete section of blood. As many strips as necessary are counted until the desired numbers of white
blood cells are counted.

c. The battlement method uses a pattern of consecutive fields beginning near the tail on a horizontal
edge; count three consecutive horizontal edge fields (moving away from the tail),
count two fields toward the center of the smear, count two fields horizontally (moving away from the tail),
count two fields vertically to the edge. Continue this pattern until the desired number of cells have been
counted.

4. Identify each white blood cell seen and record on a differential cell counter until 100 white blood cells have
been counted. If any nucleated red blood cells (NRBC) are seen during the differential count, enumerate them
on a separate counter. These cells are not to be included in the 100-cell differential count, but are reported as
the number of NRBC/100 WBC. (If any megakaryocytic cells or fragments, smudge cells, or epithelial cells are
seen, these cells should also be enumerated in the same manner as the NRBC, and reported as the
number/100 WBC).

5. Examine the red blood cell morphology in a thin area of the slide where only a few of the red blood cells
slightly overlap. Note any variations from normal and classify these irregularities as slight, moderate or
marked (1+, 2+, 3+).
6. Examine the platelets on the smear for morphology and number present using the same fields on various
parts of the smear, as in step 5 above for the red blood cell morphology, determine the approximate number
of platelets per field. A normal (wedge) blood smear (normal red blood cell count and normal platelet count)
should show approximately 8 to 20 platelets per field in this area. One method for reporting platelet estimates
is to determine the average number of platelets per field (using 5 to 10 different fields) and multiply this resu
by 20,000 (for a wedge smear) to obtain an approximation of the platelet count.

A patient with a red blood cell count of 5 x 10 /L and a platelet count of 300 x 10 /L has 30 platelets for
12 9

every 500 red blood cells. This must be kept in mind when performing a platelet estimate and adjustments
made when the patient’s red cell count is greater or less than 5.0 x 10 /L.
12

For example, if a patient’s red cell count is 2.5 x 10 /L and the platelet count is 300 x 10 /L there are 30
12 9

platelets for every 250 red blood cells. It is therefore, helpful to know the patient’s red blood cell count
(hemoglobin or hematocrit) when performing a platelet estimate.

Discussion:
1. When reporting a manual differential white blood cell count the results are reported as the percentage of
each cell type present. Automated differential counters report the cells in percent and also as the actual
number of each cell type/L of blood (absolute count). The most accurate and also the most preferred method
of reporting is the absolute because the result is expressed in percent is a relative number and can be
misleading to the clinician. For example, a differential showing 15% monocytes with a 9 x 10 /L white cell
9

count would be considered abnormal. However, if the white cell count is 1.5 x 10 /L , the actual number of
9

monocytes present is within the normal range. The absolute count is calculated as follows:
Absolute number of cells/L = % of cell type in differential x WBC/L

2. When performing a diffeerential the following outline may be followed:

a. White blood cells
1) Check for even distribution and estimate the number present (also look for
any gross abnormalities present on the smear).
2. Perform the differential count.
3. Examine for morphologic abnormalities.
b. Red blood cells: Examine for
1. Size and shape
2. Relative hemoglobin content
3. Polychromatophilia
4. Inclusions
5. Rouleaux formation or agglutination
c. Platelets
1. Estimate number present
2. Examine for morphologic abnormalities

3. When studying a stained smear, do not progress too far into the thick area of the slide. The morphologic
characteristics of the cells are difficult to distinguish in this area. Conversely, do not use the very thin portion
of the smear where the red blood cells appear completely filled with hemoglobin and show no area of central
pallor. The cells in this area are generally distorted and do not show a true morphologic picture.

4. When the white blood cell count is below 1.0 x 109/L, it may be difficult to find many white blood cells on
the stained smear. In this situation, a differential may be performed by counting 50 white cells. A notation on
the report must then be made that only 50 white blood cells were counted. (Alternatively, a buffy coat smear
may be prepared.)

5. When the differential shows an abnormal distribution of cell types (as listed below):
a. Over 10% eosinophils
b. Over 2% basophils
c. over 11% monocytes, or
d. More lymphocytes than neutrophils (except in children) a 200 cell differential may be
performed. The results are then averaged (divided by 2) and a notation made on the
report that 200white blood cells are counted.

6. Before reporting platelets as decreased, scan the slide on low power, especially the feathered edge, for
platelet clumps. Also recheck the tube of blood for a clot.

7. If the differential count shows the presence of immature granulocytic cells, this is termed a shift to the left
and may be found in such disorders as leukemias and bacterial infections. A shift to the right refers to an
increased in the number of hypersegmented neutrophils.

8. The differential is the most difficult laboratory test to learn. Learning about cells and their morphologic
features is a process that will continue for as long as you perform differentials.

9. There is relatively large range of variations in the results of the 100-cell differential count. As the number of
total cells counted increases, the amount of variability will decrease. The 95% confidence limit (±2 S.D.)
improves somewhat more remarkably when counting 200 white cells. Although the accuracy of the count
improves with a 500 or 1000 cell count, the change is not as great as seen between 100 and 200cell
differential counts.
HEMA LAB (1ST WEEK) Staining Jar/DIP method

DIFFERENTIAL WHITE BLOOD CELL COUNT 1. Dip in solution with fixative (methanol) for 30
seconds
- the linear representation of the percentage of 2. Dip in solution 2 (eosin, acidic dye) for 6
the various types of leukocytes in the seconds
peripheral or venous blood, known as the 3. Dip in solution 3 (methylene blue, basic dye) for
hemogram 4 seconds
- the determination of the percentage of each 4. Dip in buffer solution / aged distilled water for
type of WBCs in the peripheral blood 45 seconds
- consists of the enumeration of the relative 5. Air dry
proportion of the various types of WBCs as
seen as stained blood smears

Steps in Making a Differential Count

1. Making the blood smear

2. Staining the blood smear
3. Counting the cells
4. Reporting the results
Differential Counting
Fixation of Blood Smears
1. Prepare a stained blood smear
- To preserve the cell morphology, films must be 2. Place one drop of cedar oil on the feathery
fixed ASAP after they have dried edge of the stained blood smear
- It is important to prevent contact with water 3. Examine the smear using LPO of the
before fixation is complete microscope. Focus on area where the RBCs are
- Methyl alcohol (methanol) is the chose, not too overlapping or too scanty
although ethyl alcohol (absolute alcohol) can 4. Shift to OIO. Using the strip differential
be used method, count 100 WBCs while differentiating
- Methylated spirit (95% ethanol) must not be them.
used as it contains water
- To fix films, place them in a covered staining jar
in tray containing the alcohol for 2-3 minutes
- In humid climates, it might be necessary to
replace the methanol 2-3 times per day, the old
portions can be used for storing clean slides

What are Needed:

Counting the Cells
• Blood smear
• Methanol 1. Strip Differential: all the cells are counted in the
• Eosin, methylene blue longitudinal strip that is, from the head to the
• Compound light microscope tail of the smear
• Buffer solution pH 7.2/aged distilled water 2. Exaggerated battlement: the count starts at
• Differential counter one edge of the smear and counting all the
• Cedar wood oil cells, advancing inward to 1/3 of the width of
• Xylol the smear, then on the line parallel to the edge,
• Xylol-alcohol then out of the edge, then along the edge
 3. Two-field Meander method: the count is made - Younger form of
by dividing the smear into two fields and neutrophil with C, S, U, or
proceeds as exaggerated battlement method horseshoe shaped nucleus
4. Four-field Meander method: the count is made - Nucleus is continuous, no
by dividing the smear into four fields and cut or division
proceeds as exaggerated battlement method - NV of relative count: 0-5%
(CU)
Methods of Differential Counting - NV of absolute count: 0-600/cu. mm.

Lymphocyte

- Nucleus is compact or intact and

usually round
- Cytoplasm is light blue and
scanty
- NV of relative count: 18-42% (CU)
- NV of absolute count: 800-4,800/cu. mm.

Monocytes

- Nucleus is spongy and

sprawling with brain-like
convolutions
- Cytoplasm is gray. Vacuoles
are sometimes present.
- NV of relative count: 2-11%
(CU)
- NV of absolute count: 450-1,300/cu. mm.

Eosinophil
Goal: 100 WBCs
- Nucleus is usually
Neutrophilic Segmenter bilobed
- Contains large, coarse,
- Nucleus is broken into reddish, or orange
segments but still granules
connected by a fine - NV of relative count: 1-
strand 3% (CU)
- Cytoplasm contains - NV of absolute count:
small pinkish granules 0-400/cu. mm.
- NV of relative count:
50-70% (CU) Basophil
- NV of absolute count 2,300-8,100 / cu. Mm
- Nucleus is usually indistinct
and obscured by the granules
- Cytoplasm contains large
Neutrophilic Band (stab / staff) purplish-black or dark blue granules
- NV of relative count: 0-2% (CU)
- NV of absolute count: 0-100/cu. Mm
 o Most are prepared in methyl alcohol to
combine fixation and staining
o Includes Giemsa and Wright’s
✓ Giemsa stain is recommended and
most reliable procedure, excellent
for staining thin and thick blood
films (inclusion bodies and
intracellular parasites as well as for
staining WBCs)
- It is composed of eosin and
azure blue, methylene blue
Staining of Blood Smears in methanol and glycerin.
- The microscopic study of stained, peripheral The eosin component
blood smear constitutes the most important stains the parasite nucleus
part of routine haematological examination red while the methylene
- Cytochemical stains are essential for the blue component stains the
identification of hematopoietic cells cytoplasm blue.
- The most commonly used stains are ✓ Wright’s stain is a histologic stain
polychrome stains, those belonging to the that facilitates the differentiation
Romanowsky group of blood cell types.
- A polychrome stain is a stain of many colors - It is classically a mixture of
and the original polychrome stain was eosin azures and oxidized
discovered by Romanowsky methylene blue dyes.
- Polychrome methylene blue and eosin stains - It is used primarily to stain
are the outgrowth of the original time- PBS, urine samples, and
consuming Romanowsky method and are bone marrow aspirates
widely used which are examined under
- They stain differently most normally and light microscope
abnormal structures in the blood o Panoptic stains – consists of
- Intravital stain is used to stain the tissue by a Romanowsky and another dye to
dye which is introduced into a living organism improve cytoplasmic granules
and which, by virtue of selective attraction to ✓ Examples: Wright’s-Giemsa,
certain tissues, will stain these tissues. Jenner-Giemsa, May-Grunwald-
- Supravital stain is used to stain and inspect Giemsa
living cells which have been removed from the • Methylene blue
body. It enables the cells to remain alive and o Basic dye
mobile, but it does not stain the nucleus or o Has affinity for acidic component of the
cytoplasm. It does stain significant structures cell (nucleus)
in cytoplasm (ex. Reticulocyte count)
• Eosin/azure
Various Stains for Peripheral Blood Film: o Acidic dye
o Has affinity for basic component of the
• Romanowsky Stain
cell (cytoplasm)
o Employed for staining blood films
o Combinations have two essential
ingredients (i.e., methylene blue and
eosin or azure)
 Station 2 – Wright’s or Wright’s-Giemsa stain (500mL)

Station 3 – stain buffer mixture, Wright’s or Wright’s-

Giemsa (80 mL), phosphate buffer (420 mL)

Station 4 – deionized water (1000 mL)

Station 5 – phosphate buffer (500 mL)

Station 6 – warm air

Staining of Two-coverglass
Other Methods of Staining

1. Staining dish method involves placing the

blood smear in a rack positioned on a dish.
2. Automated method
a. Hemastainer automatic slide stainer
- Freshly prepared staining solutions
Methods of Classification of Cells in Differential Count
are used daily or every 4-8 hours
during operations 1. Schilling hemogram
b. Hema-Tek 1000 Slide stainer - In this method, all leukocytes are
- Bottles of the Stain-Pak (stain, buffer, grouped according to maturity of cells
and rinse solutions) are opened by into
making a small hole and a cannula is • Granulocytes – neutrophils,
inserted into each solution. eosinophils, basophils
- A pump tube set is installed to • Non-granulocytes –
transport each solution lymphocyte and monocytes
- Tubing 1 = stain solution; Tubing 2 = - The PMNs are further classified
buffer solution; Tubing 3 = rinse according to myelocytes,
solution metamyelocytes, bands or stabs,
c. Hema-Tek 2000 Slide Stainer segmenters
- Stainer employs the same principle as - The Schilling hemogram may be
Hema-Tek 1000. represented as
- The innovation is the improved ✓ Basophils – 0.25-.0.5%
staining system through the use of ✓ Eosinophils – 2-4%
new pumps and volume controls. ✓ Myelocyte – 0
- The operator can electronically adjust ✓ Metamyelocyte – 0-1%
the stain, buffer, and rinse solution ✓ Stab – 2-6%
✓ Segmenters – 55-65%
Stations in Automated Method
✓ Lymphocytes – 25-35%
Station 1 – methanol (500mL) ✓ Monocytes
 ✓ 2-8%
2. Arneth’s classification
- The PMNs are classified according to
the number of lobes which their nuclei
possess. The more lobes, the older the
cells.
• Class I – with lobe or indented
nucleus (5%)
• Class II – 2 lobes (35%)
• Class III – 3 lobes (41%)
• Class IV – 4 loves (17%)
• Class V – oldest with 5 lobes
(2%)
Under the traditional unit, the results in differential
leukocyte count are reported in percentage.
Under the SI unit, the proportion of each type of cell is
reported as a decimal fraction and is called leukocyte
type number fraction.
• Regenerative shift to the left – if
predominating cells are younger forms with
the presence of myelocytes and
metamyelocytes and increase in band cells and
it is accompanied by a high leukocyte count.
• Degenerative shift to the left – if
predominating cells are younger forms, with an
increase in band cells but without myelocytes
and metamyelocytes and it is accompanied by
low WBC count.
Shifting Processes
• Shift to the left – if there is an increase in
younger forms of WBCs particularly classes I
and II; seen in pyogenic infections
• Shift to right – if there is an increase in older
forms of leukocytes particularly classes IV and
V; seen in megaloblastic anemia and in
convalescence
3. Haden’s classification
- Classifies the neutrophil according to
the presence of filaments.
- These neutrophils whose lobes are
connected by thin filaments are
classified as filamented, while those
that are not connected by filaments are
grouped under non filamented cells
• Filamented cells = 60%
• Non-filamented cells = 7%
• Eosinophils = 3%
• Basophils = 1%
• Lymphocytes = 21%
• Monocytes = 8%
Automated Differential Count
Two general principles:
1. Digital image processing – a uniformly made
and stained blood film is placed on a
microscope slide, which is driven a motor. A
computer controls the movement, scanning
the slide and stopping it when leukocytes are in
the field. The optical images are then recorded
by television camera, analyzed by computer
and converted to digital form
2. Flow through system – this system analyze the
cells suspended in a liquid. In photo-optical
system, measurement of light scattering and f
light absorption are made while the cells are
being counted
Overstained Smears
Causes:
- Too thick smears
- Insufficient washing
- Too prolonged staining time
- Excessive alkalinity of the stain, buffer or water
Appearance of Cells:
- Erythrocyte stains blue or green
- Cytoplasm of the lymphocytes become gray or
lavender
- Granules of neutrophils are intensely
overstained
- Eosinophilic granules become deep gray or
blue
Understained Smears

Causes:

- Too thin smears

- Excessive washing of the smears
- Excess acidity of the stain, buffer, or water

Appearance of Cells:

- Nuclear chromatin is stained pale blue rather

than vivid blue
- Erythrocyte stains bright red or orange rather
than pink
- Eosinophilic granules stain brilliant red

Precipitated Stain Between Cells

Causes:

- Unclean slide or coverglass

- Faulty washing because of failure to hold the
slide horizontally and to float off the scum
- Permitting dust to settle on the film

Poor Staining

- Alkaline slides and alkaline distilled water

- Acid slides and acid distilled water
- Unclean slides
- Evaporation of the stain
- Incorrect buffer pH
- Imperfect polychroming of the stain
- Incomplete reaction of the staining fluid
- Error of the operator
HEMA LAB (2nd week) • Neubauer
• Fuchs-Rosenthal
HEMACYTOMETER • Speirs-Levy
- Used to count the cellular elements of the • Buerker’s and Tuerk’s
blood, namely RBCs, WBCs, and platelets • Bass-Jones
- Consists of a counting chamber, RBC pipette, • Thoma
and WBC pipette
Improved Neubauer Counting Chamber
- Accessory devices such as suction device and
thick cover slip are also required for manual - Made of heavy colorless glass
counting of cells - Used regularly
- On the middle third of the chamber, there are
three parallel platforms extending across the
slide and separated by moats
- The central platform is subdivided by a
transverse groove into halves, each wider than
two platforms
- The central platform is exactly 0.1 mm lower
than the lateral platforms
- Each platform is exactly 0.1 mm lower than the
lateral platforms
Red tip/suction tube = RBC pipette - Each platform is 3 mm in width

White tip/suction tube = WBC pipette

Objectives:

➢ Describe the basic parts and functions of a

hemacytometer
➢ Count blood cells using hemacytometer
➢ Identify the pitfalls in filling the counting
chamber

Materials

• Microscope
• Hemacytometer
o Improved Neubauer counting chamber
o RBC pipette
o WBC pipette
• Charts of counting chambers:
o Improved Neubauer
o Neubauer
o Fuchs-Rosenthal
o Speirs-Levy

Types of Counting Chamber

- Each of the halves of the central platform has
• Improved Neubauer the improved Neubauer ruling which consists
 of a primary square measuring 3 x 3 mm (9
mm2) and subdivided into nine secondary
squares each measuring 1 x 1 mm
- The four corner secondary squares (designated
as W1, W2, W3, and W4) are used for WBC
count and are subdivided into 16 tertiary
squares
Upper left: W1
Upper right: W2
Lower right: W3
Lower left: W4
- The central secondary square is divided into 25
tertiary squares, each measuring 0.2 mm
- Furthermore, each of these tertiary squares is
subdivided into 16 smaller squares (Isang R=16
na kaagad). The total numer of smaller squares
in the central secondary square is 500.
- As a rule, RBCs are counted on five of the
tertiary squares (designated as R1, R2, R3, R4,
and R5) totalling to 80 smaller squares
Upper left: R1
Upper right: R2
Lower right: R3
Lower left: R4
Center: R5
Fuchs-Rosenthal Counting Chamber
- Considerably larger than the Neubauer
counting chamber
- Its principal use is in the performance of low
cell count, such as eosinophil count, spinal fluid
count and leukopenic blood count
- The ruled area measures 4 x 4 mm and the
depth is 0.2 mm
- The central ruling is divided into 16 smaller
squares
Speir’s Levy Counting Chamber
- Consists of four sections, two on each side of
the counting chamber
- Each ruled area consists of 10 squares each
measuring 1x1 with a toal area of 10 mm2
- The squares are arranged in two horizontal
rows of five squares, which are further
subdivided into 16 squares
- The chamber has a depth of 0.2 mm
Red Blood Pipette

- Has a red bead inside the mixing chamber or

a red stripe on the stem
- The stem is the portion from 0.0 to 1.0
containing one unit of volume
White Blood Cell Count
- The mixing chamber of bulb is the area from
0.1 to 101 and holds 100 units of volume - The WBC count is the number of blood cells in
- Its bore is smaller than WBC pipette 1mm3 of blood. Blood is accurately diluted with
a fluid producing complete hemolysis of RBCs
without injury to the WBCs

Objectives

➢ Demonstrate proper WBC counting techniques

using WBC pipette and counting chamber
➢ Compute the WBC count and report the result
using the conventional and SI units
➢ Identify the causes of leucocytosis (increased
WBC) and leukopenia (decreased WBC)

Materials

• Anticoagulated blood
• WBC pipette
• 1% HCl
• Improved Neubauer Counting Chamber
• Microscope
White Blood Cell Pipette
• Thick coverslip
- The stem of the WBC pipette is the portion • Aspirator
from 0.0 to 1.0 and the mixing chamber is the • Tissue Paper
portion from 1.0 to 11
Characteristics of a Good WBC Diluting Fluid
- The volume of the stem is exactly 10 times the
volume of the mixing chamber ✓ Hypotonic solution
- Thus, the bulb holds 10 units of volume and ✓ Easy to prepare
has a white bead ✓ Cheap
- Bore is bigger than RBC pipette ✓ Good preservative
 ✓ Readily available
WBC Diluting Fluid
• 1%-3% acetic acid with Gentian violet
• 1% Hydrochloric acid
• Tuerk’s (glacial acetic acid with methyl violet)
Pipette method (same with RBC count)
Procedure
A. Preparation of diluted blood
1. Aspirate blood up to 0.5 mark of the
WBC pipette
2. Wipe off the excess blood at the tip of
the pipette with a clean piece of tissue
paper, making sure the tissue paper
does not touch the opening of the
bore.
3. Place diluting fluid in clean transparent
container
4. Immerse the pipette in diluting fluid.
Use syringe aspiration to draw the
diluting fluid up to the 11 mark with
constant rotation of the pipette to
ensure proper mixing of the blood and
the diluting fluid. This makes 1:20 or
1/20 dilution
5. Gradually bring the pipette to a
horizontal position and immediately
mix the contents of the pipette. Make
sure that the pipette is held securely
using the thumb and the middle finger.
NOTE: if the diluting fluid goes beyond the 11
mark or if there are bubbles inside the bulb,
discard the mixture and start from the beginning
using a clean dry pipette
B. Filling the counting chamber (charging)
1. Place the thick coverslip on top of the
improved Neubauer counting
chamber. Both the cover slip and the
counting chamber must be free from
dirt, thumb marks, tissue strands and
the like.
2. Reshake the pipette. Discard the first 5-
6 drops of diluted blood from pipette
and carefully and exactly fill or charge
the counting chamber by capillary
action. This can be achieved by placing
the tip of the pipette on the space (V-
shaped groove) between the coverslip
and the central platform of the
counting chamber. The angle of the
pipette while charging is 30o to 35o.
NOTE: overcharging can be readily
observed by the presence of fluid on the
moats of counting chamber.
Undercharging is evidenced by failure to
cover the entire ruled area of the
counting chamber. Air bubbles in the
counting chamber indicate moisture or
dirt. If such things occur, clean the
coverslip and the counting chamber and
repeat the process.
3. Let the counting chamber stand for 5-
10 minutes for the RBCs to settle
4. Locate the ruled area for RBC counting
by focusing the microscope using the
LPO initially. Once located, shift to HPO
for actual counting.
Counting
• The four corner squares are further divided
into 16 smaller squares
• These squares are used for WBC counting
(total = 64 small squares)
• Count the WBCs on the first row of the smaller
squares from left to right, drop down to the
second row and count from right to left then to
the third row counting from left to right, and
 lastly the fourth row counting from right to
left.
NOTE: WBCs that touch any of the lines on the top
and left borders, even if they are outside of the
tertiary square, are included in the count while
those that touch any of the first lines on the right
and bottom borders are not included even if they
are inside the square.
White Blood Cells
- Blood cells that fight infection
- Elevated: leukocytosis (infections, common
cold, tuberculosis, allergy, glandular fever)
- Decreased: leukopenia (anaphylactic shock,
cirrhosis of liver, disorders of spleen,
pernicious anemia, typhoid and paratyphoid
fevers, viral infections)

Acceptable Differences Between Chambers of Precautions

Hemacytometer
❖ No overflow in the moats
➢ No more than 10 cell variation between the 4 ❖ No air bubbles and debris in the chamber area
squares for the WBC count ❖ No scratches in the ruled area of the chamber
➢ The difference between the highest and lowest
number should not be greater than 25 cell for Pipettes used must be clean and dry
the 10 tertiary squares (5 squares per side) the
RBC count

Record the count on each of the five tertiary squares

making sure that the cell difference between the
squares is 12 or less.

NOTE: All counts must be done in duplicate.

Computation

The general formula for the computation of the

number of blood cells/mm3 of blood is as follows

Normal Values: 5,000-10,000/mm3

 Normal value: 150 – 300 cells/mm3

Other Methods
Introduction
• Pilot Eosinophil Count
• Absolute eosinophil count is the number of eosinophil
• Direct Eosinophil count by Friedman
in 1mm3 of blood.
Diluting fluids
Materials
Phloxine
• Anticoagulated blood
• WBC pipette Composition
• Randolph’s diluting fluid
❖ Propylene glycol -hemolyze RBC
• Aspirator
❖ 1% phloxine - stains eosinophil only
• Tissue paper
❖ 10% Sodium carbonate - lysis of other WBC
• Microscope
(accentuator)
• Improved Neubauer counting chamber
• Thick cover slip Pilot

Randolph’s Method Composition

1. Aspirate blood up to the 1 mark of the WBC pipette. ❖ Propylene glycol

2. Draw Randolph's diluting fluid up to the 11 mark to
make a 1:10 dilution.
3. Shake Up and down (horizontal position) the pipet
well and let it stand for 10 to 15 minutes.
4. Reshake. Discard 2 to 3 drops of the diluted blood then
charge both sides of the counting chamber.
5. Count the eosinophils in all nine secondary
squares with an area of 9 square mm.
Microscopic appearance of Eosinophil (orange color)
❖ 1% water solution of phloxine
❖ 10% water solution of sodium carbonate
❖ Heparin - prevents clumping
Randolph’s
Composition (1)
❖ Acetone - fixative
❖ Distilled water- lyses RBC
❖ Eosin - stains eosinophil

Composition (2)

❖ Phloxine
❖ Propylene glycol
❖ Sodium carbonate
❖ Heparin or sodium citrate

Use inverted L cells shape in counting

Computation

𝑡𝑜𝑡𝑎𝑙 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

𝑎𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑒𝑜𝑠𝑖𝑛𝑜𝑝ℎ𝑖𝑙 𝑐𝑜𝑢𝑛𝑡 (/𝑚𝑚3 ) =
𝑎𝑟𝑒𝑎 𝑥 𝑑𝑒𝑝𝑡ℎ 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
𝑡𝑜𝑡𝑎𝑙 𝑛𝑜.𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡
=
9 𝑥1/10𝑥 1/10

100
= 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥
9

= 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 11.11

Bleeding Time and Clotting Time
Coagulation Screening Procedures
 The detection and diagnosis of a hemostatic disorder
should begin with a
- Physical examination
- Clinical history
- Drug or medication history – very important to
determine why bleeding doesn’t stop
 Presence of abnormality yields a clue as to the
type of disorder
- Liver disease
- Renal failure
- Cancer
- Vitamin K deficiency
- Alcoholism
- Certain disorders – secondarily to the primary
disease
 Screening procedures
- Vasculature
- Platelets
- Coagulation mechanism
- Fibrinolysis
 Comprehensive and careful clinical history – most
valuable screening test
 Abnormality In primary hemostasis may present with
easy bruisabiliy and/or bleeding of the mucous
membranes
 Tests:
- Bleeding time
- Platelet count
- Review of a peripheral blood smear – for the
presence of platelet, if the platelets are clumping
- Estimation of numbers
 The results of the bleeding time should be correlated
with the platelet count. When these results do not agree
(e.g., prolonged BT, normal platelet count). There
might be Von Willebrand’s disease or other functional
platelet disorders may be suspected and further
appropriate testing (such as platelet aggregation
studies) may be performed
- When testing bleeding time, platelet count is also
measured
 Patients with factor deficiencies will usually exhibit
bleeding from larger blood vessels (coagulation factors)
 Prothrombin time (PT) – is used to evaluate the
extrinsic coagulation system and when abnormal,
indicates a defect in the pathway
- Three pathways – extrinsic, intrinsic, and common
pathway
 Activated partial thromboplastin time (APTT) – is
sensitive to factor deficiencies in the intrinsic
coagulation pathway
 Mild deficiencies may have a normal screening test
 If there is a positive history, a normal PT or APTT does
not rule out a deficiency
 Also, a normal BT may be found in a mild case of Von
Willebrand’s disease
 If a factor deficiency is suspected, it is important to
perform a test for circulating anticoagulants in order to
rule out the presence of an inhibitor
 If this test is negative for an inhibitor, the APTT (or PT)
substitution may then be performed to identify the exact
factor deficiency
 A factor assay should be performed to determine the
activity of the deficient factor
 In the presence of a circulating anticoagulant (inhibitor)
the prothrombin and/or the APTT will be prolonged,
depending on the specific factor inhibitor present
 If there is a lupus inhibitor the APTT is more often
abnormal. The TT (thrombin time) or quantitative
fibrinogen (functional) tests for functional fibrinogen
 The presence of fibrinogen degradation products may be
detected by the use of FDP Fibrinogen Degradation
Product) test
 The D-dimer procedure is performed for the detection of
the cross-linked fibrin degradation product (D- dimer)
 Circulating fibrin-monomer-fibrinogen complexes may
be detected using the ethanol gelation test
 The euglobulin clot lysis procedure will test for
fibrinolysis
 In summary, the routine screening procedures used to
detect a coagulopathy are the
- Platelet count
- Bleeding time
- Prothrombin time
- APTT
- Thrombin time (or quantitative fibrinogen) test
 Tourniquet test may be used to test for vascular
(capillary) integrity
 Further studies are performed if any of these test results
are abnormal
 The exact tests to be performed will depend of which
test result/s (which portion of the hemostatic
mechanism) was abnormal
 Prothrombin time is used to monitor oral
anticoagulant therapy in patients with deep vein
thrombosis or with a high risk of developing thrombosis
 Heparin is frequently administered to hospitalized
patients with thrombosis and the dosage monitored by
use of the APTT
 Coagulation specimens require special attention
 Poor techniques used in collection, processing, or
storage will cause misleading and inaccurate results
 A clean venipuncture is absolutely necessary. Any
contamination of the blood with tissue fluid leads to
incorrect results
 Capillary specimens are generally not used for
coagulation testing of the difficulty in obtaining a blood
specimen without trauma and free of tissue juice
 The anticoagulant to blood ratio is important in
coagulation studies. Greater or lesser amount of blood
will yield incorrect coagulation test results
 All test tubes used for coagulation studies should have
a “noncontact surface.” That is the inside of the tubes
should not be of a material (such as glass or soda lime)
that will react or activate the coagulation factors. Most
vacutainer tubes for collecting coagulation studies have
a siliconized surface
 Blood specimens with a high hematocrit will contain
 less plasma in relation to the total volume of blood in
the tube. As a result, when the blood has been
centrifuged, the plasma fraction will contain an
increased concentration of anticoagulant (sodium
citrate)
 Each blood specimen should be checked for clots prior
to testing. Because the tubes must be centrifuged with
their tops on the red cell layer may be checked for clots
after the plasma has been removed. The presence of a
clot irrespective of how small it renders the specimen
unacceptable for coagulation testing
 Unless otherwise noted, specimen should be
centrifuged within 1 hour of obtaining the sample. It is
desirable to complete testing within 2 to 4 hours
depending on the specific procedure
 The plasma specimen should be in a stoppered
container at all times, except when testing. This
prevents loss of CO2 and a resultant pH change of the
plasma
 If the specimen will be tested within 2 hours of
collection, it may be kept at room temperature
 If there is to be a delay beyond 2 hours, it is advisable
to keep the specimen on ice. Exceptions to this rule
are specimens for prothrombin time, factor VII assay,
and platelet function studies. These specimens should
be maintained at room temperature
 Factor VII may become activated by the cold. Also,
plasma for the euglobulin clot lysis procedure must be
tested or frozen within 30 minutes of collection
 With the exception of platelet function studies, plasma
for coagulation testing should be platelet poor (plasma
platelet count less than 15,000/μL)
 An alternative to crushed ice for keeping the specimens
cold is the commercially available Kryorack
 Hemolyzed plasma is indicative of a traumatic
venipuncture and should not be used for coagulation
studies. Also, automated instruments may not be able
to detect the end point on some lipemic or icteric
plasma samples
 Most coagulation studies are carried out at 37°C. It
should be noted that specimens incubated in dry heat
take slightly longer to reach 37°C than those incubated
in a water bath
 Overheating or prolonged heating at 37°C, however,
may lead to destruction of some of the coagulation
factors and therefore, a prolonged clotting time
 Accurate timing of clotting time is extremely important.
Many of the procedures are timed to within one tenth of
a second so that the initial starting and stopping of the
stopwatch must be done precisely
 There are four general techniques for reading the
endpoint of clotting procedures
- Tilt tube method – requires gentle tilting of the
tube back and forth at the rate of about once per
second until a fibrin web is formed
- Nichrome wire loop technique – employs the use
of a wire loop that is passed through the mixture at
the rate of two sweeps per second until a formed
clot adheres to the loop
- The third and fourth procedures employ the use of
automation: a clot is detected either by use of a
moving probe immersed in the mixture that is
triggered by clot formation or by the change in
optical density of the mixture when a clot forms.
 The appearance of the clot formed frequently depends
on its rate of formation
 Normal, or only slightly prolonged clotting times show a
pronounced clouding of the mixture that is easy to see
with the human eye
 Prolonged clotting times, however, form more slowly
and may first appear as a very fine fibrin threads
Introduction to Bleeding Time
 Bleeding time measures the ability of small blood vessels
to control free flow of blood after injury
 The duration of bleeding is a measure of the function of
platelets as well as the integrity of the blood vessel wall
 It is a screening test for detecting disorders of platelet
function and von Willebrand’s disease, and is directly
affected by the platelet count and the ability of platelets
to form a plug
- Bleeding time goes hand in hand with your
platelet count
 The thickness and vascularity of the skin and the ability of
the blood vessels to constrict and retract may also affect
test results
 Coagulation mechanism, however, does not influence
the bleeding time unless there is a severe deficiency
present
 Prolonged bleeding time will result when the platelet
count is lower than 30,000 to 50,000/μL, when the
platelets are dysfunctional, in Von Willebrand’s disease,
and following ingestion of aspirin, aspirin- containing
compounds, anti-inflammatory drugs, anticoagulants,
some antibiotics and certain other drugs
 These medications should not be taken for a minimum
of 1 week prior to performance of this test. Drug
therapy is the most common cause of prolonged BT
 Historically, bleeding times were performed using the
Duke method. In this procedure, the earlobe or
fingertip was punctured using a sterile blood lancet
 The wound was blotted every 30 seconds until bleeding
ceased. This method was not precise or very accurate
 The ivy method introduced some standardization into
the procedure. Blood pressure cuff (inflated to
40mmHg) was used to exert a uniform pressure on the
blood vessels
 Two standardized punctures of the forearm were made
using disposable blood lancets. The wound was blotted
every 30 seconds until bleeding ceased
 This procedure was modified by Mielke Jr, who
introduced the, which utilized a template containing as
standardized slit in place of the disposable lancets
 A Bard-parker or similar disposable blade was placed in
a special handle such that when the template was
placed on the forearm, the blade would make an
incision having a standardized depth and length
 Simplate contains a spring-loaded blade w.in a plastic
case (Simplastin II holds a double blade. When activated
the blade springs forward from the housing and makes a
guillotine like cut in the skin
 Surgicutt utilized a slicing using a surgical blade. It is
 spring-loaded and when activated the blade moves
forward to the outside of the unit and sweeps across
the opening of the bottom of the housing. The blade
automatically retracts into the housing after it has
made a standardized cut
 The normal range for the BT will depend on the device
and method used and the angle of the incision
(perpendicular or horizontal to the bend of the elbow)
and must be determined by each laboratory
 Objectives
- Perform bleeding time determination accurately
- Describe the clinical significance of prolonged
bleeding time
 Materials
- 70% isopropyl alcohol
- Sphygmomanometer cuff
- Blood lancet
- Timer
- Cotton
- Filter Paper (in case you want to perform the tube
method)

Duke’s Method
 Cleanse the earlobe or the 3rd or 4th finger with 70%
isopropyl alcohol and allow to dry
 Make a relatively deep puncture with a sterile blood
lancet and start timing
 Blot the blood using filter paper every 30secs
- Note – do not allow the filter paper to touch the
wound as this will hasten the bleeding time.
 Stop timing as bleeding ceases and record the bleeding
time
- Normal value – 2 to 4 minutes
Ivy Method
 Apply a sphygmomanometer cuff on the patient’s upper
arm. Inflate it at 40mmHg. Maintain the pressure during
the entire procedure
 Cleanse an area on the polar surface of the forearm with
70% alcohol and allow it to dry. The selected area should
be free from visible veins
 Make three successive punctures in the form of a
triangle to a depth of 2-3mm using a disposable lancet.
Start timing
 Blot the blood with filter paper at 30-second interval
until the bleeding stops
 Record the time when the bleeding stops
 Normal value – 1 to 7minutes
- Sphygmomanometer then inflate until 40 mmHg then
the vein will be visible, then it can be punctured
- Two incisions are made and the time for clotting to
occur is recorded
Copley-Lalitch Method
 Cleanse the fingertip with alcohol and allow it to dry
 Make a puncture to a depth of 6mm. Start timing
 Immerse the punctured finger in sterile physiologic
saline solution warmed at 37℃ until the bleeding stops
 Record the bleeding time
 Normal value – < 3 mins
Discussion
 There is a minimal to no scarring at the incision site
 The aspirin tolerance test may be useful in helping to
distinguish functionally abnormal platelets from normal
platelets. Bleeding times are performed before and 2
hours following ingestion of 650 mg aspirin. The BT after
aspirin ingestion is usually slightly longer
 In von Willebrand’s disease, however, the BT after
aspirin ingestion will be more markedly prolonged
 When performing a bleeding time on infants using
Surgicutt newborn, the blood pressure cuff should be
maintained at 20mmHg for children weighing less than
2 pounds, at 25mmHg for body weights bet 2 and 4
pounds and at 30mmHg when the infant weighs over 4
pounds

Introduction to Clotting Time

 Clotting time measures the period for blood to clot or
solidify after it has been extracted from the body
- Coagulation or clotting time
 Objectives
- Perform different methods of coagulation time
determination
 - Discuss the advantages and disadvantages of the ABNORMAL:
different techniques Clot degrades rapidly
- Explain the significance of prolonged coagulation (Grade 1, friable clot)
time or fails to
 Materials coagulate(Grade 2)
- 70% isopropyl alcohol
- Blood lancet
- Timer
- Cotton
- Capillary tube (blue)
- Wasserman tube (3)
- Water bath 37°C Other Methods of Coagulation Time
- Syringe with needle  Drop or slide method
- Gum label - Capillary blood methods
- Clean glass slide  Perform a skin puncture. Discard the drop of
blood
Whole Blood / Lee & White Method  Place a drop of blood on a clean glass slide.
 Label three tubes (1,2,3). Place the clean, dry tubes in Start timing
a 37°C water bath  Draw the tip of the lancet across the drop of
 Obtain 3mL venous blood in a plastic syringe, blood at 30 seconds interval until fibrin threads
preferably using the two-syringe method. cling to the tip
 Place a 1mL of blood into each of the tubes starting  Stop timing and record the clotting time
with tube 1  Normal value – 2 to 4 minutes
 Start timing as blood is delivered into tubes. Put the  Capillary tube or Dale & Laidlaw's method
tubes back into the water bath - Make a skin puncture. Wipe off the first drop of
 Gently tilt tube 3 every 30 seconds until blood blood
solidifies. Handle tube 2 in the same way. Finally, tilt - Fill a non-heparinized capillary tube with ¼ blood
tube 2 until blood forms a solid clot - Start timing as soon as blood enters the tube
- Set the capillary tube aside in a horizontal position
for two minutes
 Stop timing and record the coagulation time
- Break off about 1cm of the capillary tube at 30
 Normal value – 7 to 15minutes
seconds interval until fibrin thread bridges the
broken ends of the capillary tube
- Record the coagulation time
- Normal value – 2 to 4 minutes
Collection: a blood
sample for WBCT
starting immediately
after collection

NORMAL: a solid clot Discussion

is retained upon  The activated clotting time test uses 2mL of whole blood
inversion of the tube placed in a gray stoppered BD vacutainer tube containing
at 20 to 30 minutes diatomaceous earth.(Blood should be drawn using two-
(Grade 0, no way syringe technique)
coagulopathy)
- The procedure is carried out at 37°Cand the tube
tilted after the first minute, and thereafter at 5-
10second intervals until the clot forms
- Using this procedure coagulation is normal complete
in less than 101seconds
 Poor venipuncture technique causing hemolysis or tissue
thromboplastin to mix with the blood, shortens the
clotting time
 Bubbles entering the syringe when the blood sample is
being obtained increase the rate of coagulation.
Unnecessary agitation of the blood shortens the
coagulation time
Always tilt the tube in the same direction and at the same angle
so that the blood is moving in the same pathway up the side of
the tube each time
dane.
COAGULATION SCREENING PROCEDURES:
- The detection and diagnosis of a hemostatic
disorder should begin with a
• Physical examination
• Clinical history
• Drug/medication history
- Presence of the abnormality yields a clue as the
type of disorder:
• Liver disease
• Renal failure
• Cancer
• Vitamin K deficiency
• Alcoholism
• Certain disorders- secondarily to the
primary disease.
- Screening procedures:
• Vasculature
• Platelets
• Coagulation mechanism
• Fibrinolysis
- Comprehensive and careful clinical history-most
valuable screening test
- Abnormality in primary hemostasis may present
with easy bruisability and/or bleeding of the mucus
membrane.
- TEST:
• Bleeding time
• Platelet count
• Review of a peripheral blood smear
• Estimation of numbers
- The results of the bleeding time should be
correlated with the platelet count. When these test
results do not agree (e.g prolonged BT, normal
platelet count) von Willebrand’s disease or other
functional platelet disorders maybe suspected and
further appropriate testing (such as platelet
aggregation studies) may then be performed.
- Patients with factor deficiencies will usually exhibit
bleeding from larger blood vessels.
BLEEDING TIME
- Measures the ability of small blood vessels to
control free flow of blood after injury
- The duration of the bleeding time is measure of the
function of platelets as well as the integrity of the
blood vessel wall.
- It is a screening test for detecting disorders of
platelet function and von Willebrand’s disease and
is directly affected by the platelet count and the
ability of platelets to form a plug
- The thickness and vascularity of the skin and the
ability of the blood vessels to constrict and retract
may also affect test results.
- The coagulation method mechanism, however does
not influence the bleeding time unless there is a
severe deficiency present
- Prolonged bleeding times will result when the
platelet count is lower than 30,000 to 50,000/ul,
when the platelet are dysfunctional, in von
Willebrand’s disease, and following ingestion of
aspirin, aspirin-containing compounds, anti-
inflammatory drugs, anticoagulants, some
antibiotics and certain other drugs
- These medications should not be taken for a
minimum of 1 week prior to performance of this test.
Drug therapy is the most common cause of a
prolonged BT
- Historically, bleeding times were performed using
the DUKE method. In this procedure, the earlobe or
finger tip was punctured using a sterile blood lancet
- The wound was blotted every 30seconds until
bleeding ceased. This method was not precise or
very accurate
- The Ivy method introduced some standardization
into the procedure. A blood pressure cuff (inflated to
40mm Hg) was used to exert a uniform pressure on
the blood vessels
- Tow standardized punctures of the forearm were
made using disposable blood lancets. The wound
was blotted every 30 seconds until bleeding ceased
- This procedure was modified by Mielke Jr, who
introduced the, which utilized a template containing
a standardized slit in place of the disposable
lancets.
- A Bard-parker or similar disposable blade was
placed in a special handle such as that when the
template was placed on the forearm, the blade
would make an incision having a standardized
depth and length.
- Simplate contains a spring-loaded blade within a
plastic case (Simplastin II holds a double blade.
When activated, the blade spring forward from the
housing and makes a guillotine like cut in the skin
MATERIALS:
• 70% isopropyl alcohol
• Sphygmomanometer cuff
• Blood lancet
• Timer
• Cotton
• Filter paper
Procedure: (Duke’s Method)
1. Cleanse the earlobe or the 3rd or 4th finger with
70% isopropyl alcohol and allow it to dry
2. Make a relatively deep puncture with a sterile
blood lancet and start timing.
3. Blot the blood using filter paper every 30
seconds.
Note: do not allow the filter paper to touch the wound
as this will hasten the bleeding time
4. Stop timing as bleeding cease and record the
bleeding time
Normal value: 2-4minutes
IVY’S METHOD
1. Apply a sphygmomanometer cuff on the patient’s
upper arm, inflate it at 40mmHg. Maintain the
pressure during the entire procedure.
2. Cleanse an area on the valor surface of the
forearm with 70% alcohol and allow it to dry. The
selected area should be free from visible veins.
3. Make three successive punctures in the form of a
triangle to a depth of 2-3mm using a disposable
lancet. Start timing
 4. Blot the blood with filter paper at 30-second 1. Perform a kin puncture. Discard the
interval until the bleeding stops. first drop of blood
Record the time when the bleeding stops 2. Place a drop of blood on a clean
Normal value:1-7minutes glass slide. Start timing
3. Draw the tip of the lancet across the
COPLEY- LALITCH METHOD drop of blood at 30second interval
1. Cleanse the fingertip with alcohol and allow it to until fibrin threads cling to the finger
dry. tip
2. Make a puncture to a depth of 6mm. Start timing 4. Stop timing and record the clotting
3. Immense the punctured finger in sterile time
physiologic saline solution warmed at 37DC until Normal value: 2-4 minutes
the bleeding stops B. Capillary tube or Dale and Laidlaw’s
4. Record the bleeding time method:
Normal Value: < 3minutes 1. Make a skin puncture. Wipe of the
first drop of blood
CLOTTING TIME 2. Fill a non-heparinized capillary tube
- Measures the period required for blood to clot or ¼ with blood
solidify after it has been extracted from the body. 3. Start timing as soon as blood enters
Materials: the tube
• 70% isopropyl alcohol 4. Set the capillary tube aside in a
• Blood lancet horizontal position for two minutes
• Timer 5. Break off about 1cm of the capillary
• Cotton tube at 30 seconds interval until fibrin
• Capillary tube (blue) thread bridges the broken ends of
• Wasserman tube (3) the capillary tube
• Water bath 37DC 6. Record the coagulation time
• Syringe with needle Normal value: 2-4minutes
• Gum label
• Clean glass slide

WHOLE BLOOD or LEE AND WHITE METHOD

1. Label three tubes (1,2 and 3) place the clean dry
tubes in a 37DC water bath
2. Obtain 3mL venous blood in a plastic syringe,
preferably using the two-syringe method
3. Place 1mL of blood into each of the tube starting
with tube 1
4. Start timing as blood is delivered into tube 1. Put
the tubes back into the water bath
5. Gently tilt the tube 3 every 30 seconds until blood
solidifies handle tube 2 in the same way. Finally,
tilt tube 2 until blood forms a solid clot. Stop timing
and record the coagulation time
Normal value: 7-15minutes

Drop or Slide Method

1. Capillary blood methods
A. Drop or slide