19

Section 1: Hematology
3 mI Bore of the stem
mm
A

101
0.5 1.0

Bead in the bulb

11
0.5 1.0

Bulb with a bead

pipettes). (A) RBC

and B: The diluting pipettes (blood
Figs. 11A
markings--(1) 0.5, (2) 1.0, and
(3) 101; and (B)
Pipette: it has three markings-(1)0.5,(2) 1.0, and
(3) 11.
WBC pipette: it has three
blood cell)
(RBC:red blood cell; WBC: white

Partsßfa Diluting Pipette

narrowstem has a capillary
bore and
The stem: The long equal
1/20 mm 1/5 mm 1/4 mm tip. It is divided into 10
a well-grounded conical etched
Fig. 10:The counting grid of improved Neubauer's ruling. The four has only two numbers
parts (graduations) but the
corner groupsof 16 squares (side = 0.25 mm) are used for counting [Link] stem, and 1.0 (or l) at
WBCS. The RBCS are counted in five groups of 16 smallest squares on it--0.5 in the middle
unction of stem and the bulb.
each (side = 0.05 mm). The grid also provides aconvenient scale Thebulb: The stem widens into a bulb which
contains
eggs. (RBCS:
for measuring the size of small objects like parasite RBC pipette, and white
red blood cells; WBCs: white blood cells) a free-rolling bead-red in the
in the WBCpipette. The bead helps in mixingthebleed
quick identification )
The central densely etched large square (l mm x1 mm), and the diluent and also helps in
called the RBCsquare, is divided into 25 medium sized aaglance.
squares, each of which has a side of l/5
mm.
Rábber tub and mouthpiece:
The bulb narrows again
is set off (separate) rom narrow, and soft
Eachof thesemedium squares into a short stem to which a-long,
(tram red in RBC
its neighbors by very closely placed double lines rubber tube bearing a mouthpiece (often
lines extend The
lines) or triple lines. These double or triple pipette, and white in WBC pipette) is
attached.
the 9 mm?
in all directions beyond the boundaries of rubber tube is 25-30 cm long to facilitate filling of the
around the
ruling, i.e. in between all the WBC squares pipette by gentle suction. It also allows the pipette to be
central RBC square.
1/5 mm), held horiZontally sothat one can comtortably watchthe
Each of the 25 medium squares (side = blood or diluting fluid entering the pipette.
or
bounded by double lines (which are 0.0l mm apart) Just beyond the bulb, the numbe 101 /s etched on the
l6 smallest squares
triple lines, is further divided into RBC, and 11on the WBC pipettes.
square has a side of
by single lines. Thus, each smallest
l/400mm².
1/5x 1/4= 1/20 mm, and an area of Principle Underlying the Use of Diluting Pipettes
Note: Since each group of 16 smallest squares
is demarcated from It is important to understand that the numbers marked on
the dimensions of the RBC
its neighbors by double or triple lines, larger than 1 mm the pipettes--0.5, 1.0, and l01 on the RBC pipette and 0.5,
central square) must be slightly
square (largest dimensions of the smallest 1.0, and1lon the WBC pipette-do not indicate absolute
X1 mm, as indeed they are [the exact or definite amounts (or yolumes)in terms ot so many
surface.
squares (1/400 mm)]etched on the chamber
cublc millimeters (or uL), amistake commonly made by
Study of the Diluting Pipettes the students)These figures only indicate relative volumes
(parts)/or relative volumes in relation to each other Thatis,
1. Red blood cell pipette half volume(lirom tip to mark 0.5),one volume (from the tip
2. White blood cell pipette. to mark l.0)in both pipettes; and 1l volumes (from tip to
Figures 1lA and Bshows the two glass capillary pipettes mark 1l above the bulb in WBC pipette), and 101 volumes
used for diluting the blood. Each pipette has a long narrow
stem (for measuringthe blood), which widens into a bulb (trom tip to mark 101 in RBC pipette). Of course, all these
(for diluting the blood/which in turn, leads to a short stem). volumes will have certain definite volumes in terms of cubic
20
Textbook of Practical Physiology
Gently suck on
the cdge of tlhe drop. is just above
millimeter, but we are not concerned with these but only the itstip within blo0d until it
the
relative volumes or parts in relation to each otherltcanbe mouthpiece
and draw
action
cannot fill
the pipette at this
seen that the capillary bore in VBCvinette is widerthanthat mark 0.5 (capillary (say 2
adequatesize
inRBC pipette, andtherefore, will hold moreblood though angle). drop should
be of
The blood small or ifthe tip is lifted
the volume of the stem inboth cases is 1.0 (one). Ifit is too
mmin
diameter).
enterthe
pipette along with
drop, air will skin, the bOre
out of the against the
Differences between Red Blood Cell Pipette and blood. If the
tip presses
blocked and
no blood will enter the
White Blood Cell Pipette get
the tip willifyou suck hard at the
mouthpiece.

rubhbe
removing the
The differences between RBC pipette and WBC pipette are stem even pipette (after
described in Table 3. Alternately, the
filled with
blood
(without
sucking/
tube) may be below the horizontal). and
end
its bulb
bylowering blood to flowdownthe. stem by gravity.
Note: Thoughthe dilution obtaned with the RBC pipette is 10 times
that obtained with WBC pipette, its bulb is not 10 times bigger. The allowing the drop and clean i
blood
reason is the much fner bore in the red cellpipette. pipettefrom the toward
Remove the
cotton swab by Wiping it
with a otherwise some
outer surface bore at the tip
touchthe
Filling the Pipette (Fig. 12) the tip. Do not
pulled out. time, bring the
Placea drop ofanticoagulated blood on a glass slide or blood willbe horizontal allthe
Keeping the pipette wiping the tin
get a fingerprick under aseptic conditions. stem to the exact mark 0.5 by
the couple of times till
the
Holding the mouthpiece of the pipette between your blood in
facing on your palm (or on apaper) a
lips and keeping the pipette (with its graduations place blood recedes to
the exact mark.
40° to the horizontal,
you) at an angle of about
will absorb a laroe
paper for this purpose as it
Note: Do not use filter should you try to blow out
the extra
Tabi3: Differences between RBC pipette and WBC pipette. amount of blood, and neither
RBCpipette WBC pipette blood.
Calibrations are 0.5 and 1.0 below Calibrations are 0.5 and 1.0 vertical,immerse its tip in
the bulb, and 101 above the bulb below the bulb, and 11above o Holding the pipette nearly watch glass, and suck the
the diluting fluid taken in a
it
diluent to the mark 11(WBC)or 101 (RBC).
As the fluid
DNe capillary bore is narrow, thus it Re capillary bore is wider, before it into the bulb
isaslowspeed pipette hence it is a fast-speed pipette is sucked up, the blood is swept
Bulb is larger and has ared bead Bulb is smaller and has a white of 10 volumes (WBC) or 100 volumes (RBC pipette).
bead The sucking up of diluting fluid should not be
The volume of the bulb is 10
done very quickly because blood being viscous, ifa
The volume of the bulb is 100 times
the volume contained in stem times the volume of the stem sufficient time is not allowed, a thick film of blood
(R8C: red blood cell; WBC: white blood cell) will remain sticking to the inside of the capillary
bore, thus introducing a significant error.
The dilution of blood should not be delayed
otherwise it is likely toclot in the stem.
Once the diluent has been taken to the appropriate
mark, keep the pipette horizontal so that the fluid
does not run out by gravity.
Donot place the pipette on the table, or delay the
mixing because it becomes impossible to dislodge
the cells from the walls of the bulb once they settle
down.
* Mixing the blood with the diluting fluid: Once
diluting fluid has been sucked up, remove the rubber the
tube. Holding the short stem above the
bulb between
your thumb and first two fingers, and
pressing the tip of
the pipette against the palm of the other
Fig. 12: Filling the pipette. toand fro for 3-4 minutes so that hand, rotate it
the blood and diluent
get thoroughly mixed.
 21
Section 1: Hematology
volume of the bulb
Red blood cell pipette: Since the
that 100 volumes
1S 100 (101 - 1.0= 100), it means
0.5 (half) part of
(or parts) of diluted blood contain
blood and 99.5 (100 - 0.5 = 99.5)
parts or volumes of
diluents.
the volume of the
White blood cell pipette: In this case,
is taken to the mark
bulb is 10(11-1=10). When blood
by diluent to the mark
0.5 (half partor volume) followed blood is now 10, which
11, the volume of the diluted
parts or volumes of
contains 0.5 part of blood and 9.5
dilution of 0.5 in 10 (half
the diluting fluid. This gives a
dilution factor being
ten), or l in 20 (one in 20), the
Similarly, if blood
20 (the blood will be diluted 20 times). diluted to mark
is taken tothe mark 1.0 followed by
Fig. 13: Charging a chamber. 11, the dilution noW would be l in l0.

For Red Blood Cell Counting

Alternately, remove the rubber tube. close the ninette
ends with thumband forefinger of yourright hand. and The red cells are counted in four corner groups and one
each of which has 16
shake it vigorously with a figure of eight motion. central group of medium squares,
Do not shake the pipette with an endwise motion as Smallest squares, i.e. in a total of 80 smallest squares.
1/20 mm = 1/400
this willforce the cells out of the bulb into the stem. Area of smallest square = 1/20 mm x
Charging thechamber: Once the blood and the diluent mm². volume
have been mixed well, "charge" the chamber (Fig. 13). Since the depth ofthe chamber is 1/1 0 mm, the
Charging the chamber requires patience, practice, of the smallest square = 1/400 x 1/10 = 1/4,000mm.
and understanding of how to correctly judge the size
of the drop, the angle at which the pipette should be For White Blood Cell (Total Leukocyte)Counting
held on the floorpiece, and the time needed for filling This countis done in the four corner groups of large squares,
(charging) the chamber. This is called the "speed of the each of whichhas 16 medium squares.
pipette" Obviously, it varieswith the size of thecapillary Area ofone medium=1/4 mmx 1/4 mm square =1/16 mm².
bore in the stem of the pipette. Volume of this square = 1/16 mm²x1/10 mm = 1/160 mm.
High-speed pipette: Since the bore of the WBC
pipette is wider, a drop willform more quickly at Focusingthe CountingGrid
its tip, and it will be larger, as compared to the RBC
pipette. This requires that this pipette should be held Bxamine the grid on each floorpiece, without the coverslip,
more horizontally say, at an angle of 10-20° and for under low and high nmagnifications. Rack the condenser up
a shorter time.
and down, closing/adjusting the diaphragm at the same
time. Find out the best combination of these two that shows
Slow-speed pipette: The bore of the RBC pipette
being narrow, it will take a longer time for a suitable he gia ines and squares clearly. When properly focused.
drop to form. It should, therefore, be held at a steeper the rulings (lines) appear as translucent darkish lines.
With low magnification of 100 times, one large square,
angle--say, 60-70. 1mm x 1 mm is visible in one field, i.e. a group of 16
It is for this reason that the students should first practice
charging a chamber with the RBC pipette and then with the medium squares (for WBC counting), or a groups of 25
medium squares (for RBC counting).
WBC pipette. 3 Examine the squares under high magnification.
Calculation of Dilution Obtained (Dilution Factor)
PROCEDURES
When blood is sucked up to the mark 0.5 (half part or
volume) and is followed by the diluting fluid, the blood 1. Assuming that the blood and the diluent have been
enters the bulb first and is followed by the diluent to the properly mixed, the next step is to charge the chamber.
mark 101 (RBC pipette), or mark l1(WBCpipette). The stem Place a coverslip on the chamber so that it spans the
in both pipettes contains only the diluent. Thus, the dilution floorpieces and the trenches around them-a process
of the blood occurs in the bulb only. called "centering" the coverslip.
22 Textbook ot Practical Physiology

2. Rollthe pipette once more between your palms to mix

the contents of the bulb. If this precaution is not taken,
the counts are boundto be unreliable.
3. Keeping your finger over the top of the pipette and
releasingit in acontrolled manner, allow the first two
cell-free
drops to drain by gravity. This fluid contains
diluting fluid in the stem which has not taken any part
in the dilutionof blood.
angle (depending
4. Hold the pipette at an appropriatewatching carefully,
on the speed of the pipette) and Then
allow a drop of diluted blood to formn at its tip. squares to which
the
floorpiece
quickly place the tip of the pipette on the
the
Arrows indicate
of cells.
of the coverslip. As the
in gentle contact with the edge coverslip, Fig. 14:
Counting

the fluid will cells belong.

surface of the drop touches the the adjacent
squares
form a uniform film. Lift counted with
run under it by capillarity and because they willbe counting a cell
twice (vo
floorpiece is covered with you will avoid horizontal and lef
the pipette as soon as the In this way, upper
diluted blood. cells lying on the lower and
one go and notin may omit lying on its
The chamber should be charged at vertical lines and count those
method is chosen,
it is best
of proper size. Try
parts. For this, one needs a drop right lines. But
whichever
chamber. cell counts).
to achieve an ideally-charged on the microscope to followit for all middle line is
Before placing the chamber bound by triple lines, the
for allowing the cells to 4. In squares square, and the same
stage, wait for 2-3 minutes considered the boundary of that
settle down.
ideally-charged chamber rules of counting apply. continuously "rack, the fine
Ideally-charged chamber: An cells,
diluted blood. If any blood 5. While counting the down so that cells sticking to the
is completely filled with adjustment up and
called "overcharging. If
flows into the trenches, it is floorpiece, or if underside of the coverslip
are not mnissed.
the fluid is insufficient to cover the pattern should be
followed for the entire
"undercharging" (air 6. Only one
there are air bubbles, it is called
bubbles are formed if the coverslip
or the floorpiece is counting.
dirty with grease or is
moist). Sources oferror in cell counting:
undercharging and overcharging: If there 1. Pipette error
Effects of chamber and
undercharging, wash the
is over or them, and recharge
2. Dilution error
coverslip in soap and water, dry
3. Chamber
the chamber. 4. Statistical
charged, move
5. Once the chamber has been properly minutes so that the 5. Field error.
itto the microscope. Wait for 2-3
cells settle [Link] cannot be started when the Three important sources of error: (1) pipette error, (2)
cells are moving and changing places due to currents chamber error, and (3) field error (see Experiment 1.4 for
in the fluid. details) can produce a variation of as much as 10-15%, or
Counting the cells: Focus the appropriate squares even more in the hands of a student. On the other hand,
under the required magnification and start counting hemoglobin and packed cell volume are easy to determine
as described here in Figure 14. and give enough information about the blood picture.
Rules ofcounting:
1. Care should be taken not to countthe same cells again. PRECAUTIONS
2. Count the cells ling withinasquare and those lying on
or touching its upper horizontal and left vertical line 1. The pipette should be clean and dry and the bead
and those cells on its right and lower linesare ignored should roll freely.
as they will be counted in the adjacent squares. This is 2. The pipette should not be lifted out of the blood drop
called "inverted Lpattern" Arrows indicate the squares while filling it with blood, otherwise air will enter it.
to which the cells belong.
3. Cells lying on or touching its lower horizontal and 3. The drawing up of diluent, after blood has been taken
right vertical lines are to be omitted from in the stem, should not be delayed, otherwise it will
that square clot in it.
 25
Section 1: Hematology

NormalValues Hayem's fluid, (RBC diluting fluid): The

ideal fluid
be isotonic and neither
Adults: tof dilutingthe bloodshould should
of red cells. It
Males: 3.2(4.5-6) million per mm'of blood. Cause hemolysis nAF ererattor also
of RBCs and
Females: 4.7(4.0-5.0)million per mm°of blood. have afixative to preserve the shape
Newboms: 6-8 million per mm'of blood. prevent their autolysis.
Methods of Counting Composition of Hayem's fluid:
1. Manual Sodium chleride fNaCI): 0.50 g
2, Automated Sodiumsulfate (Na So,): 2.50 g
Mercuric chloride (Hg CL,0.25 g
Manual Method Distillet water: To0 mL isotonicity
Sodium chloride andsodium sulfate provide
Principle suspended in diluted blood
So that the red cells remain
The blood is diluted 200timesin ared cell pipette and the without changing their shape and size
anticoagulant, and as a
cells are counted in the counting chamber. 3 Sodium sulfate also acts as an
prevent rouleaUx
Knowing the dilution employed, their number in Lxative to preserve their shape and to
undiluted blood can easily be calculated. formation (piling togetherof red cells).
Mercuric chloride acts as an antifungal and antimi
and growth of
Apparatus crobial agent, prevents contamination
1. RBC pipette: Consult Experiment 1.3 and Figures 15 microorganisms.
and 16 for its description. It should beclean and dry Note:
Hayem's
alternative to
and the bead should roll freely. O Dacie's solution: This diluting fluid is an
solution. It is simple to prepare and keep for a long time.
It
2. Improved Neubauer chamber with coverslip: These trisodium citrate, 1.0 mL of 37% formalin
should be clean and dust free. contains 3.13 g of
(commercial formaldehyde), and distilled water to 100 mL.
3. Microscope with LP and HP objectives and 10x O Normal saline: 0.9% sodium chloride solution can be used
if
eyepiece. Hayem's or Dacie's fluids are not available. However, the red cells
4. Disposable blood lancet/pricking needle: have to be counted within an hour or so of filling the pipette.
Sterile cotton/gauze swabs. Also the RBCS are likely to form rouleaux. Further, a stock solution
70% alcohol/methylated spirit. of normal saline cannot be kept for this purpose.

Procedure
* Consult Experiment 1.2 for obtaining a sample of
03 mark blcod capillary blood,Experiment 1.3 on filling apipette with
blood, diluting it, and charging a counting chamber.
05 Place about 2 mL of Hayem's fluid in a watch glass.
Examine the chamber with the coverslip "centered"
on it, under low magnification. Adjust the illumi
101mark RBC diluing fuid nation and focus the central l mm square (RBC
square on the counting grid) containing 25 groups
of 16 smallest squares each. All these squares will be
visible in one field. Do not change the focus or the
field. Admitting too much light is a common cause
Fig. 15: RBC pipette. of the inability to see the grid lines and squares
clearly.
Bore of the stem Move the chamber to your worktable for charging it
with diluted blood (It can be charged while on the
stage, but it is more convenient to charge it on the
0.5 1.0 101
table).
Filling the pipette with blood and diluting it: Get
Bead in the bulb a fingerprick. Wipe the first two drops of blood and
fill the pipette from a fresh drop of blood up to the
Fig. 16: The RBC pipette. It has 3markings-0.5, 1.0, and 101. mark 0.5. Suck Hayem's fluid to the mark 101 and
26 Textbook of Practical Physiology
others, the
chamber has to be
scanty at
places and
mix the contents of the bulb for 3-4 minutes as washed, dried,
and
recharged.
the left upn
described earlier. carefully and bring
Move the chamber
squares in the field
Charging the chamber: Observing all the pre corner block of l6 smallest
squares above and to
smallest
cautions, fill the chamber with diluted blood. Since (There are no
view
the RBC pipette is a slow-speed pipette, it will need its left).
the andB)
to be kept at an angle of 70-80° while charging
Counting (Figs.
17and 18A
chamber. Move the chamber to the microscope and Rules for 1.3
maybe o
Experiment

once again to see the central 1 mm d i s c u s s e d in

600-700 RBCs)
focus the grid Note: As be lin
over. Wait is not to be Counted
square with the red cells distributed all
WBC(may
Anoccasional granular but it
down because grayish and
for 3-4 minutes for the cells to settle appearing
moving and donein 80)smallest squares,
they cannot be counted when they are
cells.
with the red
the fluid. will have been
changing their positions due to currents in The counting
each.
again showing of 16 squares
During this time drawa diagram once i.e. in 5 blocks
groups of 16 square
the RBC square. Then draw fivepositions--the four Observations and Results
each, showing their relative entering under high
power (Fig. 19)
central group for the RBC square blocks of 16
Corner groups and one RBC in in each of the 5
Add up the number of cells between anv
of morethan 20
your counts.
to high magnification difference
Countingthe cells: Switch over smallest squares. A
distribution of cells. If they indicates uneven
distribution.
check the
(HP lens) and 2blocks
distributed, i.e. bunched
at some
are unevenly

WBC
WBG
0.2 mm
Ruled RBC
area counting
R
RRE

WBC
WBC 0.2 mm

Fig. 17: Rules of countingcells.

Figs. 18A and B: (A) Microscopic víew of a charged chamber

showing even distribution of red cells. Agroup of
squares is shown in the middle: (B) Rules of counting:16 smallest
cells lying within asquare and those lying on Count the
or touching its upper
horizontal and left vertical line cells
horizontal and right vertical lines are tolying
be
or touching its lower
Counted in the adjacent squares. Arrows omitted as they will be
which the red cells belong. indicate the squares to
Fig. 19: Red blood cells in the RBC
square.
 27
Section 1: Hematology
Calculation of dilutionobtained (dilution factor), the cells in each,
O Taking repeated samples of blood, cOunting
Refer Experiment [Link] that the dilution with this and taking their mean value can eliminate pipette error. But this
pipette is I in 200 as blood is taken to mark 0.5. has hardly any practical value.
Final volume attained (100 parts)
lhus, the dilution=
Volume of blood taken (0.5 part) Normal Red Cell Count
factor is
Express your result as..... million/mm
Calculation of volumeof fluid examined. We know Ihe average cell counts and their ranges are:
the count in 80 smallest squares which have a volume Males =5.0 million/mm³(4.75-6.0 million/mm)
(space) of 1/50 mm., We can also know the cell count Females =4.5 million/mm (4.0-5.5 million/mm).
inl,spíallest square which has a volume (space) of
1/4.000 mm. Precautions
talculation of redcell count
described for getting a finger
Let Nbe the number of cells in 1/50 mm of diluted 1. Observe all precautions pipette with blood
blood. prick (Experiment l.2), flling the
chamber (Experiment
Cells in lmm° of diluted blood =Nx 50 and diluent, and charging the
Dilution employed was = 1in 200 1.3).
piece of cloth. Once
Therefore, number of cells inl mm³ of undiluted blood 2. Clean the chamber with a lint-free
cleaned, do not touch the central
part. Any oils left
will be Nx 50x 200 = Nx10,.000. ) or the chamber are
Thus, adding, 4zeros in front ofN willgive the RBC count from your fingers on the coverslip
per l mm of undiluted blood. -fatal to cell counting. and
wash, clean, dry,
3. Ifthere is over or undercharging,
refillthe chamber.
Example while counting
4. Continuously rack the fine adjustment
Number of cells in 80 smallest squares = 480 the cells.
These cells are present in 1/50 mm of diluted blood. from the stock bottle as it
5. Do not draw diluents directly
Dilution employed is = l in 200 cells.
Number of cells in lmm² of undiluted blood willbe: may contaminate the solution with
recharge the chamber if the
6. Discard the samnple and
= 480x 50 x 200 distribution is not uniformn.
= 480 x 10,000
7. Follow the rules of counting to avoid the counting
= 480,0000, i.e. 4.8 million/mm?. twice.
Sources of Error Physioclinical Significance
As mentioned in Experiment 1.3, despite all precautions in There are number of physiological and pathological
the procedures the degree of error with this methodis said conditions where RBC count can be altered.
to be about + 15%.Thus, with a count of 5.0 million/mm³, 1. Decrease RBCcount
the technical error of a single count comes to about + 0.75 Paysiological causes:
million/mm². And in the hands of a student, this error can Pregnancy
be as high as 20%. The following factors are responsible for RBC count is lower in children than adults
the error: Females have lower RBC count than males.
$ Pipette error: It may be due to inaccuracy of gradua Pathological-Anemia_
tions, the method of filling with blood and diluent, or Hemodilution, e.g. excess ADH secretion as in
mixing. posterior pituitary tumors.)
3 Chamber error: The counting grid and the depth of the 2. Increasein RBC count
chamber may not be accurate, or the process of charging Phy_iological causes:
it may introduce an error. High altitude
* Field error: The distribution of cells on the grid may. Newborns
not be uniform. Hemoconcentration, e.g. excessive-sweatmg.
Pathological:
Notes:
Hemoconcentration, e.g_diarrhea, vomiting
O To be of any value, the test must be carried out as meticulously
as possible. Chronic hypoxia, e.g, congenital heart disease,
O Repeated counts of a charged chamber do not give significantly chronic obstructive pulmonary disease
better results. Polycythemia vera.
 31
Sectlon 1: Hamatology
their
STAGES OFLEUKOPOIESIS (FLOWCHART 2) are not visible under the light microscope due to
usual dyes. The
Smallsize and poor staining with the
Normal count: 4,000-11,000 cells/mm' of blood. agranulocytes include-monocytes and lymphocytes.
The count after birth may be as high as 18,000-20,000/
mmt, the normal levels being reached in afew years. In the CHIEF FUNCTIONS OF LEUKOCYTES
adults, about 55-75% of the WBCs are granulocytes, while
in voung children, lymphocytes dominate. The count may $ immunity
The chief function of leukocytes is to provide constitute
he high in some physiologicalconditions such as heavy (protection)against these invaders and
thus
by preserving
exercise, stress, etc.
an important mechanism of survivalimmune system
health and fending off disease. The
are in a constant
TYPES OF LEUKOCYTES Consists of task-specific cells that
readiness-like the branches of
state of vigilance and
TheWBCs, unlike red cells contain nuclei butno hemnoglobin. invaders as "foreign"
armed forces. They recognize the
combat at the site of
Depending on the presence or absence of clearly visible and to the body and engage them in
conspicuous, chemical-filled granules (vesicles) in their invasion.
cvtoplasm (that are made visible by staining), they are 3 The tissue macrophages (that develop from blood
grouped into two typesgranular and agranular. ) "sentinels"), and neutrophils
monocytes and act as the
transported by
Granulocytes: Therè arethreetypes of granulocytes that (they act as the "infantry" and are
most quickly
can be recognized under the compound microscope blood to the site of invasion), respond
according to the coloration of their cytoplasmic and destroy the invaders by phagocytosing then (both
active amoeboid
these cells move through the tissues by
granules--neutrophils, eosinophils (eosin loving), and inflamed area).
basophils (basic loving). movements and are attracted to the
antimicrobial
Agranulocytes: In contrastto granulocytes whose Thus, these two types ofWBCs along with
nuclei are lobed, the nuclei of agranulocytes are proteins (interferons-alpha, beta, and gamma, and
not lobed but appear as a single mass. Although the complement system and natural killer lymphocytes,
cytoplasm contains chemical-filled granules, these form the second ine of defense).

Flowchart 2: Stages of leukopoiesis.

Multipotential hematopoietic stem cell

(Hemocytoblast)

Common myeloid progenitor Common lymphoid progenitor

Erythrocyte Mast
cell Myeloblast Natural killer cell
(Large granular
Small lymphocyte
Iymphocyte)
Tlymphocyte Blymphocyte
Megakaryocyte
Basophil Neutrophil Eosinophil Monocyte
Thrombocytes
Plasma cell

Macrophage
32 Textbook of Practical Physiology
settle for
3-4 and then
minutes,

cells to n i c r o s c o p e . Ir
Allow the to the
PRINCIPLE the chamber identify the
WRO
chrefully transfer
adjustmentagain
and try to
appear as rouna
the fine m a g n l f i c a t i o n :he
leukocytes
Asample of blood is diluted with a diluting tuid which Under low dots, with
around th
a halo
destroys the red cells aund stains the nucleiofthe leukocyts. shiny
(refracile),
darkish
nuclei, which
have been
stain
Ihe cells are then counted in acounting chanber and heir These "dots"
represent the
c y t o p l a s m is
not stained.
number in undlted blood reported as leukocytes/mm' violet. The
by gentian which have varying sizes
dust particles "haloe
confuse with opaque, with no
APPARATUS Note: Do not angular. They are usuallyyellow in color.
often brown, black
or
and shapes,
They may be
around them. do not keonR
Microscope: cells or counting them,
that the cell
Counting chamber with a heavy coverslip Important:
When
examining
"rack" the
m i c r o s c o p e so
you will
way,
Bloodlancet or pricking needle fxed focus but
continuously
of focus. In this
come into and
go out or confue
Sterile cotton or gauzeswabs u n d e r s u r f a c e of
the cOverslip,
and the lines the
stickingto
70% alcohol. miss cells WBCs is simils.
bulb, and WBCs. counting the

WBCpipettes (Fig.
20): White bead in dust particles for
The procedure for
dry cells:
markings 0.5, 1.0, and
l1. Two such clean and Counting the
employed for
21).
red cells (Fig. lens.
are required. to that low power low power.
pipettes with free-rolling beads diluting the blood. cells under
O Count the squares under total
is used for the WBCS in 16 each, i.e., in a
Turk's fluid: This fluid count square
(hemolyzes RBCs without O You may cells in the 4 groups of 16
Glacial acetic acid = 3 mL O Count the
for entering the
affecting WBCs). stains the
of 64 squares.
squares in your workbook
solution) = 1.0 mL (it ODraw
appropriate
Gentian violet (1% COunts.

nuclei of leukocytes).
Distilled waer = 100 mL. OBSERVATIONS A N D RESULTS

unless the RBCS

impossible to count WBCs
Note: t would be acid. (This concentra
is achieved by acetic Calculations (Fig. 22)
were first destroyed. This membranes of WBCS). The dye gentian (10 parts)/
cell volume achieved
tion does not affect the without staining their =final
only the nuclei of leukocytes Dilutionfactor
violet stains
(0.5 parts) = 20
cytoplasm. pipette and for charging original volume taken
Experiment 1.3 on filling the Volume of fluid:
Go through
the chamber.
WBC squares -4x 1x l = 4 mm?
Area of4
-0.l mm
Depth of the chamber 4>x0.l=0.4 microliter
WBCsquares =
Volume offluidinthe 4
PROCEDURE

the
in a watch glass. Place Calculation of TLC:
Take l mL of Turk's fluidmicroscope stage. Adjust the of WBCs in 4 WBC
squares
counting chamber on the 16WBC Let N be the total number undiluted blood = Nx
illuminationand focus the
right upper group of Total [Link] WBCs in l microliter of
in one field. 50
squares. You will see all
the squares dilution factor (20)/O.4 = Nx
aseptic precautions,get a fingerprick,
Observing all the
ofblood, and let a good-sized
discard the first two drops
pipette: Dip the tip of the
drop to form. Filling the mark -WBC
pipette in the edge of the
drop, draw blood to the WBG
contents
the markll. Mix the
0.5 and suck Turk's fluid to
minutes.
of the bulb thoroughly for 3-4
Charging the chamber: Discard the first two drops of IR: ER!
chamber on both
fluid from the pipette and charge the over-charged nor R
sides. The chamber should neither be
under-charged. R R

1.0 11 1WBC WBC

0.5

Bead in the bulb

Fig. 20: The WBC pipette. It has 3 markings-0.5, 1.0, and 11. Fig. 21: Counting of cells.
 33
Section 1: Hematology
the diluted blood starts
lor 30-40 minutes, After that,.
receding due todrying and the count decreases. The
not be delayed.
COunting of the cells should, therefore,

PHYSIOCLINICAL SIGNIFICANCE

Conditions that increase TLC:

Physiological causes: About 95% of
the people have a
3 Physiologicalleukocytosis
TLCwithin the normal range.
or tissue injury) has no
(i.e. in the absence of infection
decrease or absence
clinical significance. There is no feature of
(eosinopenia) which is a
of eosinophils Physiological leukocytosis
leukocytosis due to infection.
from the marginal pool
is due to mobilization of WBCs leukocytosis). It is seen
("Shift"
or bone marrow reserve
Fig. 22: VWhite blood cells in WBC square in the,following conditions:
be as high as
Normal infants: The count may level within
but it returns to normal
Sources and Degrees of Error 18-20,000/mm3

The sources of errors are the same as described for RBOC -2 years. leukocy
counting and include: Pipette error, chamber error, field Food intake and digestion ("digestivereturns to
which
error, and experimental error (page 27). tosis"): There is a mild increase
The degree of error which may be 30% or more in RBC normal within an hour or so.
counting is much less in TLC(about 5-10%) because of the Physical exercise
low dilution employed (1 in 20) in this case. The error can Mental stress
be further reduced if counting is done on both platforms Pregnancy: The count may be quite high especially
of the counting chamber. That the error in TLC counting is during the first pregnancy.
tissue
much less important than that in the RBC count is obvious Parturition: The high TLC is possibly due to
hemorrhage.
from the following example: In a TLCof 8,000/mm, even an injury, pain, physical stress, and
error of 20% will give a count of 9,600/mm³ which is again Extremes of temperatures: Exposure to sun, or to
count.
well within the normal range. very low temperature can increase the WBC
Pathological causes: Arise in TLC in disease is seen in:
leukemia,
Note: In case when the leukocyte count is very high,as in Acute infection with pyogenic (pus forming)
increased. For this purpose the RBC pipette
the dilution has to be bacteria: The infection (due to cÍcci bacteria
which the blood is sucked up to mark 1 and diluted
can be used in
100 times. Further calculation is done accordingly. Streptococcus, Staphylococcus) may be: (a) Localized,
such as boils, abscess, tonsillitis, appendicitis, etc.
PRECAUTIONS (b) Generalized such as in septicemia and pyemia,
bronchitis, pneumonia, peritonitis, meningitis, etc.
Observe all precautions described for getting a finger Myocardial infarction: The rise in TLC is due to
counting
prick, filling the pipette, and charging the tissue injury is not seen immediately after a heart
chamber. attack but only after 4-5 days.
$ Keep all the equipment ready before geting a prick. Acute hemorrhage: Maximum response occurs in
the Turk's fluid, give
$ When mixing the blood with 8-10 hours, the count returning to normal in 5-6
sufficient time for complete hemolysis of red
cells.
centrifuged days.
However, ensure that the leukocytes are not Burns: Maximum response occurs in 5-15 hours, the
avoided by
toward the ends of the pipette which can be count returning to normal in 2-3 days.
keeping the pipette horizontal while mixing the contents Amebic hepatitis.
of the bulb.
Malignancies: High counts are seen in half the
Continuously "rack" the microscope while identifying
cases; secondary infection enhances the count.
and counting the cells for reasons described earlier.
$ Though the condition of a charged chamber may remain
Surgical operations: Apostoperative rise is seen
in all cases.
stable for 80-90 minutes, the count is usually stable
34 Textbook of Practical Physiology
volume a
pipette, the
the RBC can gi
20. In stem, which
Conditions that decrease total leukocyte count: the volume
of the
of leukC
the count
3 Physiological causes: Adecrease in TLC under normal or 1in
200. Since
requires much
less
physiological conditions is unusual and rare. Exposure mm³,the
blood millions/r
conditions and in count which
is in
to extreme cold, even under arctic red cell
to only of the be
spite of acclimatization, may reduce the count What is the
function

slightly below the 4,000/mm' level. Q.4. purposes:

serves three
Pathological causes: Leukopenia is due to
disease, The bead blood with th
mixing the
where TLC is abnormally low is never beneficial
to the 1. It aids
identifying the
pipet
in
2. It helpswhether
body. In fact, it may endanger the life of the
patient.
the pipette isdr
The condition is almost always due to a
decrease in 3. It tells freely without
by various the bead rolls
neutrophils (neutropenia) and may be caused infections the bulb which
would happen
certain
drugs ysed in treatment, radiation, or of W
the other uses
as described here: Q.5. What are
Typhoid
Infection with nonpyogenic organi[Link] be used for
The WBC pipette can in cases
fevers, and sometimes in
aaparatyphoid less forcounting RBCs
also be u
'infection like malaria.
counting platelets. It can
Viral infections: Influenza, mumps, smallp0x, and bacteria.
acquired immunodeficiency syndrome (AIDS).
Drugs: Chloramphenicol, sulfonamides, aspirin, 0.6. What is the composition of
penicillins, cyclosporins, phenytoin, etc. Cytotoxic function of each constituent?
drugs used in treating malignanciesmay also cause The diluting fluid for TLC cont
leukopenia by depressing the bone marrow (other gentian violet, and distilled wate
blood cells may also decrease). red cells without affecting the W
Repeated exposures to X-rays and radium: These The dye stains the nucleiof leuko
are usedas radiotherapy incancers, and cause bone
marrow depression. [Link] is meant by the term "
Chemical poisons that depress bone marrow: acid in the Turk's fluid be glacia
Arsenic, initrophenol, antimony, and others. The termglacial means pure aceti
Malnutrition: Deficiency of vitamin B,, and folate, can give the typical "shine" (halo)
12
general malnutrition, starvation, extreme weakness the WBCs due to
swelling of
and debility. from dust particles which are nucl
opa
Hypoplasia and aplasia: Partial or complete (t is called glacial
depression of bone marrow, i.e. failure of stem the
because durir
may occur as a result of cells, appearance of a glacier at on
factors. autoimmunity, and other Q.8. Why are
the red cells not
Preleukemic stage of leukemias leukocytes?
leukopenia. may show The red cells are not
by the seen bec
acid (they would,
QUESTIONS leukocytes). The remnants otherw
Q.1. How will you visible-the so-called ghostof cell
red
RBC pipette? differentiate a WBC pipette Q.9. Can any
See page 20. from a cells? other agent be u
[Link] do the three No. Any weak
How do you get
See text ab a markings on
dilution of the pipette long time to lysehemolytic agent
46
Textbook of Practical Physiology
DETERMINATION OF
BLOODSMEAR AND
1.7: EXAMINATION OFDIFFERENTIAL
APERIPHERALLEUKOCYTE COUNT

APPARATUS

Student Objectives 1. Microscope

Aftercompleting this experiment, the student should be able to: glass slides
clean
1. Describe the relevance and special importance of preparing 2. Five to six
and staining a blood smear and doing the differential leukocyte 3. Sterile lancet
gauze swabs
count.
4. Cotton and
2. Name the components of Leishman's stain and explain the 5. 70% alcohol
function of each.
describe 6. Glass dropper
3. Prepare satisfactory blood films, fix and stain them, and 7. Leishman stain
the features of a well-stained flm. buffered
indicate the of distilled water (or water, if
4. ldentify different blood cells in a film, and 8. A wash bottle
identifying features of each type of leukocyte. available)
and basophils
5. Differentiate between neutrophils, eosinophils, 9. Fluff-free blotting
paper
lancet/pricking needle.
and between a large lymphocyte and a monocyte.
express your results in their Disposable, sterile blood
6. Carry out the differential count and 10. touch the surfaces of.
percentages and absolute numbers. Important: Once cleaned,do not
7. Describe the functions of each type of leukocyte. glass slides and
coverslips.
8. List the conditions in which their
numbers increase and simplification of Romanows
Leishman stain: This stain is a
decrease. one of the simplest and mo
group of stains. It is probably purpOses
pretise methods of staining blood for diagnostic
INTRODUCTION dye-eosinate of methylene blue
It contains a compound
dissolwedin acetone-free methyl alcohol.
Many hematological and other disorders can be diagn0sed Aain:Itisan acidicdye (negatively charged)and stains
bya carefulexamination ofastainedblood film. AphysICIan
may order adifferential leukocyte count (DLC) (always
basic (positive) particles-granules of eosinophils
(TLC)] to detect infection pink color.
along with total leukocytes count apd red blood cells (RBCs), a
orinflammation, determine the effects ofpossible posons
DLC is
iethylene blue: It is a basic dye (positively charged)
by chemicals, drugs, chemotherapy, radiation, etc.
diseases like leukemia, or to and stains acidic (negatively charged) granules n
also done to monitor blood
detect allergic and parasitic infections. The determination of the cytoplasm, nuclei of leukocytes, especially the
granules of basophils, a blue-violet color.
each type of whiteblood cell (WBC) helps in diagnosing the 3Acetone-free and water-free absolute methy
condition becauseaparticular type may show an increase
or decrease.
alcohol: The methyl alcohol is a fixative and must be
free from acetone and water. It serves two functions:
PRINCIPLE i. It [Link] blood smear ta the glass slide. The
Ablood film is stained with Leishman stain and scanned
alcohol precipitates the plasma proteins, which
under oil-immersion, from one end to the other. As each then act as a "glue" which attaches (fixes) the
WBCis encountered, it is identified until 200 leukocytes blood cells to the slide so that they are not washed
have beenexamined. The percentage distribution of each away during staining.
type of WBC is then calculated. Knowing the TLC and the ii. The alcohol preserves the morphology and
differential count, it is easy to determine the number of each Chemical status of the cells)
type of cell per cubic millimeter. The alcohol must be free from acetone because
Note: The special importance of a stained blood smear is that, acetone being a very strong lipid solvent, wil,
unlike any other routine blood test, the smear can be retained and if present, cause crenation, shrinkage, or even
preserved as a permanent original record. The slide can be taken destruction of cell membranes. This will make
out and reassessed whenever required after days, weeks, months or
even years. The slide can also be conveniently sent to specialists for the identification of the cells difficult. (Ifacetone
their opinion in doubtful cases. ispresent, the stain deteriorates quickly).
The stained smears can also provide information about The alcohol must be free from water since the
morphology and count of red cells and platelets, the
Hb) status, besides detecting the presence of and hemoglobin latter may result in rouleaux formation and even
malaria). various parasites (e.g.
hemolysis. The water may even wash away the
blood film from the slide.
 47
Section 1: Hematology
to the slide
PROCEDURE
Do not apply the blood drop at the finger amount of
placed on the table. One cannot see the
Cleaning the Slides blood placed on the slide.
the blood drop to
Prepare acid-dichromate solution by mixing l part of 4. Place the slide flat on the table. with
nor the skin of the
concentrated sulfuric or nitric acid with 9 parts of 2-3% the right side (neither your fingers, of the slide).
surface
potassium dichromate solution. Subject's finger should touch the
with your thumb and
Wash the slides and coverslips with soap and water and 3. Support the left end of the slide long edges
hngers of your left hand. Now graspbetween the
rinse in running water. Then soak them overnight in the thumb
acid-dichromate solution. Follow this with awash in of asecond slide, the "spreader"
that its free left end
running water, then in distilled water. and fingers of your right hand, so an angle of about
extends downward and to the left at
* Dip the slides in 90-95% alcohol and dry with a clean,
lint-free cloth. 40° to the horizontal.
slide,
6. Place the narrow edge of the spreader on the first
Another method is to use agood detergent for overnight the blood drop. Pull
soak in place of acid-dichromate solution. at an angle of 40°, just in front of touches the front of
The acid-dichromate solution is best kept in 1-2 L, wide the spreader back gently so that it little from
mouth jar. After washing the used slides, they are put the blood drop. Hold it there, (or move it a
junction
in this jar. The slides can then be treated as described side to side) till the blood, moving along the
capillarity, almost reaches the ends
of the two slides by side,
above. last 2 mm on each
Anticoagulated blood obtained from a student volunteer, of the spreader, except the its width. (If
thus distributing the blood evenly across
or spare blood obtained from the clinical laboratory may the blood drop is too big, you may start to spread the
be provided tothe students to avoid skin pricks at this smear before the whole of the blood spreads along the
time (a drop of blood can be put on the slide without slide).
touching it). Students may also se their own blood hand, and
7. Steady the first slide with your left
from skin pricks. maintaining a light but even pressure and 40° angle
1. Get a fingerprick under aseptic conditions. Discard (step 2, Figs. 25A to D), move the spreader forward to
the first 2 drops and allow a good drop to form. the left in a single, smooth, fairly fast gliding motion,
Holding a coverslip by its edges between your thumb puling the blood behind it in the form of athin smear.
and finger, touch its center to the blood drop,thus The smear should be spread in about half a second.
forming a bead. Any hesitation willresult in striations in the film.
2. Invert and carefully drop the coverslip (along with 8. Make as many trialsas possible to get acceptable films,
the blood drop under it) in the center of a glass slide. keeping in mind the features of an ideal blood smear,
Do not press. The blood drop will spread into a thick as described here. Dry the film by waving the slide in
film by the weight of the coverslip. the air (do not try to blot-dry the film).
3. Examine the preparation under low and high
magnifications and record your observations. Fixingand Staining of Blood Films
While supravital staining is employed for living cells, the
Preparationof a Blood Film (Blood Smear) staining of blood films involves dead cells. Fixation is the
(Figs. 25A to D) process that makes the blood film and its cells adhere to
Blood films can be made from anticoagulated or fingerprick the glass slide. It also preserves the shape andchemistry of
blood. blood cells as near living cells as possible. Staining is the
1. Place three or four slides on a white sheet of paper on process that stains (colors) the nuclei and cytoplasm of the
your work-table, the surface of which should be even cells. Both these purposes are achieved by the Leishman
and smooth. stain.
2. Allowa medium-sized drop of blood to form on the 1. Fixing the blood films: Place the slides, smear side up,
fingertip. on a "staining rack" assembled over a sink (twoglass
3. Steady the pricked finger of your partner with your rods placed across the sink, with the ends fitted into
left hand. Lift a slide from the table, holding it along short pieces of rubber tubing). Ensure that they are
horizontal.
its long edges. Then touch its center, about l cm from
the narrow end, to the blood drop. (If anticoagulated 2. Pour 8-10 drops of the stain on each unfixed slide by
blood is being used place a drop of blood in a similar dripping it from adrop bottle or use a dropper. This
position with a dropper). amount of stain usually covers the entire surface
 Textbook of Practical Physiology

Step 1
Step 2

Spreader Direction of movement

of spreader
Blood drop
Blood running along full
width of the spreader

45°

Step 1: Obtain a small drop of blood; Step 2: Spreader drawn

narrow edge of spreader placed at to touch the blood drop, backwards so as
anangle of 45° in front of the blood drop bood
the full width of spreader runs along
capillary action automatically by

An ideal blood
smear
D
Step 3: Spreader is
smoothly to the othermoved
end
slowly and
Figs. 25Ato D: (A) angle of 45° maintaining the
touches the blood. Method of spreading a blood
Step 2: Spreader is film. Step 1: The spreader is
movement of the objective
blood smear.
pushed
over it; (C) Steps in forward to spread the film; placed in front of the blood drop and
preparation of a blood smear; (B)(D)The appearance ofa well-prepared pulled back till t
Schematic film, showing ie
representation of steps in preparation
oo
 49
Section 1: Hematology

and "stands up" from the edges of the slides without (cells lying on top of each other like a pile of coins) and the
running off. Note the time. Observe if any leukocytes
number of cells in a rouleaux,
possible. Do they show any
1 Al}lowthe stain to remain undisturbed for 1-2 minutes, are seen, and their types if platelets-in clumps,
as advised. During this time, watch the stain carefully, ameboid movement? Do vou see any
may see fibrin
especially during hot weather, and see that it does not or showing disintegration? After atime you
become syrupy (thick) due to evaporation of alcohol. threads when clotting starts. naked
diffuse light, with
If the stain dries, it will precipitate on the blood film Examine the slides against
smear? Does it appear thick,
and appear as round, blue granules. eye. What is the color of the
any striations-longitudinal
This can be prevented by pouring more stain on the thin, or granular? Are there vacant places in the film?
any
slides as required. Or transverse? Are there
the middle two-thirds of the
4 Staining the blood flm: After the fixing time is over, Is it uniformly distributed in
straight and about l cm
add an equal number of drops of distilled water (or Slide? Is its head--the starting point,
about 2 mm from the long sides
buffered water, if available) to the stain. If the water Irom the end? Are its edges
answer all these questions.
is carefully dripped from a drop bottle or a dropper, oftheslide? Is there a tail? Try to
high magnification.
the entire mixture will stand up from the edges of the Examine the slides under low and
various cells and compare
slides (due to surface tension) without spilling over. Describe what you see about the
5. Mix the stain and water by gently blowing at different with what you saw in the drop preparation.
places on the slides through a dropper, without
scratching the smear. A glossy greenish layer (scum) Features of an ldeal Blood Smear (Fig.26)
sOon appears on the surface of the diluted stain. Allow middle two-thirds
1. The blood film should occupy the
the diluted stain to remain on the slide for 6-8 minutes, margin of about
(about 5 cm) of the slide, with a clear
or as advised. 2mm on either side.
6. Flush off the diluted stain in a gentle stream of distilled 2. It should be tongue-shaped, i.e. broad at the head
water for about 30 seconds and leave the slides on the (starting point), and taper toward
the other end, but
rack for about a minute with the last wash of water without any "tails"
throughout,
covering them. Drain the slides and put themn in an 3. should be translucent, uniformly thick
(longitudinal or
inclined position against a support, stained sides with no vacant areas, striations
facing downward (to prevent dust particles settling on transverse) or "granular" areas.
them) to drain and dry. The under sides of the slides 4. It should be neither very thick nor very thin (this can
may be blotted with filter paper. be learned only with practice). Athin film looks faintly
pink against a white surface, while a thick smear
Examination of Peripheral Blood Smear appears red. An ideal film appears "buff" colored.
Note the degree of separation of cells. Do they lie in a single 5. The red cells, as seen under the microscope, should
layer or in two, three or more layers? The red
cells are non lie separately from each other, just touching here and
there, but without any crowding, or rouleaux formation.
nucleated, flat biconcave disks, round, oval or pear-shaped,
thínner in the center and appear as colorless, or pale pink Note: A thick blood flm results from taking too large a drop of
structures. (When stained with Leishman stain, they appear blood, a faster movement, and a smaller angle of the spreader.
formation
dullorange-pink). Note if there is any rouleaux

-Vacoules

[Link] ideal 2. A film too long, too wide 3. A film too thick 4. Afilm which has been spread
blood smear grossly irregular in thickness with an irregular edged spreader;
made on a greasy slide it also shows long tails

Fig. 26: Blood films made on glass slides.

 Sectlon 1: Hematology 51

able 5: Appearance of white blood corpuscles in a stained blood film.

Cell type Diameter (ue Nucleus Cytoplasm Gytoplasmlc granules
Granulocytes
g Blue-violet violet pink
Neutrophils 10-14 aSlate-blue in color O Fine,closely-packed
(40-70%) (1.5-2 Xa RBC) 2-6lobes, connected by chromatin ONot seen separately
threadsk O Give ground-glass
appearance
o Seen clearlythrough cytoplasm) ODo not cover nucleus
Eosinophils 10-15 Blue-violet O Eosinophilic OLarge, coarse
(1-6%) Birobed, lobes connected by thick O Light pink-red O Uniform-sized
or thinchomatin band (spectacle O Granular OBrick-red to orange
shaped) O Seen separately
nucleus
O Seen clearly through cytoplasm O Do not cover

Basophils 10-15 Blue-violet OBasophilic OLarge, very coarse

(0-1%) Va Irregular shape, may be S-shaped, a Bluih O Variable-sized

rarely bilobed OGranular O Deep purple

O Not clealy seen, because overlaid O Seen separately
cover
with gr§nues DCompletely fill the cell and
the nucleus

Agranulocytes
12-20 aPale blue-violet O Abundant O No visible granules
Monocytes
(5-10%) (1.5-3 XaRBC) Large single o Frosty
a May be indentedtrorseshoe, QF Slate-blue
kigney-shapsd (can appear oval or O Amount may be larger
round, if seen from the side) than that of nucleus
Small 7-9 a Deep blue-violet Hardly visible O No visible granules
lymphocytes oSingle, large,round, almpstfitscef a Thin crescent of clear,
(20-40%) 2 Condensed, lumpy chromatin, gives light blue cytoplasm
"ink-spor"appearance
10-15 tDeep blue-violet Large, crescent of ONo visible granules
Large
lymphocytes Singl. large, round or oval,almost clear, light blue
(5-10%) fillscelt cytóplasm
May be central or eccentric Amount larger than in
small lymphocyte

1. Size: The size of a WBC assessed by comparing it 4. Cytoplasm-nucleus ratio: Note the amount of
with that of the surrounding red cells which have a cytoplasm in relation to the size of the cell and the
uniform size of 7.2-7.8 um. nucleus.
2. Nucleus: Note if the nucleus can be clearly seen
Important: Whenever there is a doubt about a cell, try to draw its
through the cytoplasm and whether it is single or diagram exactly as you see it its size (draw 2-3 RBCS alongside),
lobed. If single, note its location-central or eccentric, nucleus (single or lobed, and how they are connected to each
its shape-round, oval, or horseshoe or kidney other), and the cytoplasm--its granules, their size and color, etc. In
shaped. Study its chromatin and whether condensed most cases, this will make the identification possible.
and lumpy or open and reticular. If lobed, count their
number. Alsonote whether the lobes are connected by Differentiation between Various Leukocytes
chromatin filaments or wider bands (Figs. 27A to C). Note: A degenerating neutrophil is occasionally confused for a
3. Cytoplasm and cytoplasmic granules: The cytoplasm basophil. It is said that one should label a cell as basophil only when
may or may not show "visible" granules, or they may one is absolutely certain of its identification.
be very fine and not visible separately. Note the color
of the cytoplasm and the granules, whether neutral Caution: Occasionally, deep blue-violet, solid-Hooking granules
of precipitated stain may appear on a blood film, These uniformly
color (light violet-pink taking up both acid and basic round artifacts, spread all over the flm, should not be mistaken for
stains), or large and coarse-brick-red or red-orange cells, especially platelets.
(eosinophils) or deep blue-violet (basophils).
 69
Section 1: Hematology

antibodies,
TAble 8:Results of reaction of different blood group with Table 9: DIfferences between ABO and Rh
diferent antibody. ABO system antibodies Rh antibodies
Reaction with
The antibodies anti-A and anti-B Rh antibodies are of the lgG
cross the
Blood group Serum-A Serum-B Anti-serum Rh are of the larger lgM type. They type. They can easily
(antibody B) (antibodyA) (antibody anti-D) cannot cross the placenta. placenta.
The antigen-antibody
A These antibodies react best with
reactions ocCur best at body
the antigens at low temperatures
8 temperature. Hence, they are
of 5-20°C. They are, therefore, called"warm"antibodies.
AB called"cold" antibodies.
Rh incompatibility between
ABO incompatibility between may
a mother and her fetus rarely a mother and her fetus
Rh+ cause serious complications.
causes any problems.
Rh (Ug: immunoglobulin)
factor is so named
GeneticBasis of ABO System Rh antigen (or Rh factor) (Table 9). The Rh rhesus monkey
because this antigen was discovered in
the
The blood group (type) of a person is determined by two by Landsteiner andWiener in 1940. They injected red blood
genes, one on each of twO paired chromosomes. variety with red
cells ofrhesus monkey (the common Indian
These genes can be any one of three types--A, B, or O, but callosities) intorabbits. The rabbits immune system
only one type is present on each of the two chromosomes. ischial red cells, and
Th gene is functionless and does not produce Oantigen han reacted by forming antibodies against rhesus
therahhit' nlasmawas tested against humanied
on red cells, while Aand Bgenes produce strong antigens
of agglutination occurred in 80-85% of
individuals.
possible combinations
on red cells. Ihus, there are six Persons whose red cells contain this additional antigen
genes--AA, BB, AB, OA, OB, and 00-and each person is are called "Rh positive" (Rh +ve, Rh+)while those
who
one of thesesix genotypes. Aperson with genotype Ohas no lack this antigen are called "Rh negative" (Rh -ve, Rh-).
antigen on red cells and so the blood group is 0. A person There are several varieties of Rh antigen-C, D, E, C,
with genotype AA or OA is blood group A, while genotype d, and e-but the D antigen is the most common, and
BB or OB is blood group B. antigenically, the most potent. Therefore, Rh +ve persons are
alsocalled D +ve and Rh -ve are called D -ve. The antibody
Agglutinins of ABO System of D antigen is called anti-D antibody (anti-Rh antibody).
The antibodies in the plasma (they are gamma globulins) However, there are no naturally occurring antibodies
are the reciprocal of the Aand Bantigens. The agglutinin against Rh (D) antigen. The Rh (D) antigen is not present
cells. This
reacting with antigen A is called anti-A (or alpha, a) that in body fluids and tissues, but only on red
reacting with B antigen is called anti-B (or beta,
B). The antigen is "warm" antigen and can cross the placenta easily.
antigen O(Hantigen) has no corresponding agglutinins.
These antibodies are present without any specific red PRINCIPLE
cell antigenic stimulus. For example, they are absent in
The surfaces of red cell membrane contain a variety of
a newborn; the ABO antibodies start appearing in the
plasma by the age of 3-4 months due to cross-reactivity genetically determined antigens, called isoantigens or
agglutinogens, while the plasma contains antibodies
of ABO antigens present in naturally occurring bacteria,
viruses, pollen, etc. present in the environment (see Q1A (agglutinins). To determine the blood group of a person,
3for details). his/her red cells are made to react with commercially
Blood transfusion is a life-saving procedure in all cases available antisera containingknown agglutinins. The slide is
of severe loss of blood and in life-threatening anemias. then examined under the microscope to detect the presence
However, blood can only be given after blood grouping, or absence of clumping and hemolysis (agglutination) ofred
which is an essential requirement before blood is given cells, which occurs as aresult of antigen-antibody reaction.
toany individual. Blood grouping is also done to settle
paternity disputes and other medicolegal purposes. APPARATUS

Rh Factor 1. Microscope, "glass dropper with a long nozzle" sterile

In addition to antigens of ABO System, the red cells of blood lancet or needle, "sterile cotton/gauze swabs,
80-85% ofhumans also contain an additional antigen, called alcohol, 5 mL test tube, toothpicks.
70 Physiolooy
Textbook of Practical

2. Clean, dry microscope slides. (A special porrelain tile

with 12 depressions is available for this purpose and
may be used in place of glass slides).
saline
3. 1$sodiumcitrate in normal saline (or normal
alone).
anti-A antibodies
4. Anti-A serum:Contains monoclonal are also called
antibodies
(against human): these
The anti-A serum can
anti-A or alpha (a)agglutinins.
person with blood group B.
aBso be obtained from a
(see Q/A 6).
Anti-Bserum:Contains monoclonal anti-Bantibodies A
5. antibodies are also called
(against human); these serum can
agglutinins. The anti-B
anti-B or beta (B) blood group A.
person with
also be obtained from a
(see Q/A 6). monoclonal anti
serum: Contains
6. Anti-D (anti-Rh) antibodies
antibodies (against human). These
Rh (D) agglutinis.
are also called anti-D
not occur naturally
(see
against Rh factor do
Note: The antibodies
below) identifi
commercially. Foraquick
Theseantisera are available yellow, while the
Note: anti-B serum and anti-c
serum is tinted blue, B anti-B,
cation, the anti-A of serumanti-A,
colorless (Fig. 34). Figs. 35A and B: Marking
anti-D serum is Markth
with antisera and B).
the droppers provided "anti-D' (Figs. 35A
Caution: Do not interchange
corner of 3rdslide (for control).
of these3 slides "C"
bottles. right corners
(4th) "S" (for
only red cell
another slide
2. Mark will be added
PROCEDURE in saline, i.e. no antiserum
suspension

pencil, divide
three slides, on this slide).
center of slide "S"
1. Using a
glass-marking
drawn down the
middle
Place 8-10 drops of saline in the
halves byaline
each into two right sides as
3.
cell suspension: A suspension of
as "test sides" and Preparation of red
(the left sides will act
corner of lst slide
4.
saline should preferably be prepared and
sides"). Mark the left red cells in the
the "control
slide "anti-B" and
the left blood drops directly from
of 2nd used instead of adding
"anti-A" left comer
fingerpick to the antisera
for the following reasons
(Fig. 36): easy detection of
Dilution of blood permits
present. (Red cells
agglutination and hemolysis, if
large rouleaux and
in undiluted blood tend to form
disperse and may
masses. These may be difficult to
be mistaken for agglutination). agglutination
Plasma factors likely to interfere with
are eliminated.
conditions, and add
5. Get a fingerprick under aseptic the slide marked"S.
twodrops ofblood tothe saline on
dropper to
ANT Mix the saline and blood with aclean glass
use a toothpick
get a suspension of red cells. You may
for this purpose.
saline in a smal
Abetter method is to place 2 mL of
and allow
Fig. 34: Commercially available Anti-A, anti-B, and anti-D serum. (5 mL)test tube. Then get a finger pricked
 Section 1: Hematology 71

AB

showing aggluti
Fig. 37: Determination of blood groups (types)
clumping of red cells) and no agglutination
nation (hemolysis and between anti-D
(cells remain uniformly distributed). The reaction
Fig. 36: Preparation of red cell suspension. serum and red cells is not shown.

taken on
ablood drop to form. Now place the pricked fingertip toothpicks to transfer red cells to saline drops
on top of the test tube and invert it. Mix the blood the "control" sides of the three slides.
clumps
and saline by inverting the tube two or three times. "+":Agglutination RBC is massed together in
and loses their outline
Asuspension of red cells is now ready.
Washed red cellsuspension gives the best results. "_"; No agglutination RBCs remain separate and
The red cells are "washed" in saline bycentrifuging evenly distributed.
the diluted blood, removing the supernatant, and Important: Do this one at a time and then discard each
toothpick.
adding fresh saline to get a suspension of "washed"
red cells. 10. Wait for 8-10minutes then inspect the three antisera
6. Determination of blood group: Put one drop of anti-A red cell mixtures ("test" mixtures) and "control"
serum on the left half ("test side")of 1st slide (marked mixtures, first with the naked eye to see whether
agglutination (clumping and hemolysis of red cells)
anti-A),one drop of anti-B serum on the left half of 2nd
slide (marked anti-B), and one drop of anti-D serum has taken place or not. Then confirm under low
magnification microscope, comparing each "test
on the left half of 3rd slide (marked anti-D). mixture" with its corresponding "control mixture"
"control"
7. Put one drop each of normal saline on the
sides (right halves) ofthe three slides (i.e. areas marked
"C').
OBSERVATIONS AND RESULTS
slide
8. Add a drop each of red cell suspension (from the Itis essential that youshould be able to distinguish between
"S, or from the test tube of red cell suspension) on "agglutination" and "no agglutination" The features of
anti-A, one drop on anti-B and one drop on anti-D
each are as follows:
taken on
sera, and one drop each on the normal saline
the "control" sides of the three slides.
mixture on the
Agglutination
In this way, the red cells-saline 3 Ifagglutination occurs, it is usually visible to the
"control" sides of each slide will act as a control to
confirm agglutination or no agglutination on the naked eye. The hemolyzed red cells appear as isolated
corresponding test side (Fig. 37). (separate), dark-red masses (clumps) of different sizes
9. Mix the antisera and red cells, and saline and red cells and shapes.
There is brick-red coloring of the serum by the
on each slide by gently tilting it first one way and then
the other a few times. Take utmost care that the "test" hemoglobin (Hb) released from ruptured red cells.
mixtures and "control" mnixtures do not flow into each * Tilting or rocking the slide a few times, or blowing on it
other and get mixed up. The red cells and sera can also does not break or disperse the clumps.
be mixed by gently blowing on them. You may use three Under lichen planus (LP) objective, the clumps are
visible as dark masses and the outline of the red cells
separate toothpicks to transfer red cell suspension cannot be seen.
to the three antisera, and for mixing them, and three
 79
Sectlon 1: Hematology
recipient is
0.37. What is Bombay blood group? blood typing") the serum of the would be
This bloodtype is a rare phenomenon in which the H tested against red cells containing known antigens, i.e.
andO.
anticen is absent. Since there is no Hantigen, there is no ted cells from persons with blood types A, B, AB,
antigen Aor antigen Bon the red cells. However, the plasma If agglutination occurs with Aand AB red cells, the biood
ontains anti-A, anti-B, and anti-H antibodies. As a result, fne is Rifagclutination occurs with Band AB red celis, the
such aperson can receive blood only from a person having blood type is A;;ifagglutination
i occurs with A, B, and AB red
Bombay blood type. agglutination in
cells, the blood type is Oand if there is no
blood type there are
0.38. What is reverse blood typing? any RBCs, the bloodtype is AB. In AB
The blood grouping (typing) procedure (already described) noantibodies in the plasma (serum).
with blood typing
in which the red cells of a person (whose blood type is to The serum typing is done along
RBC antigens
be determined) are tested against anti-A and anti-B sera is as a precaution because in leukemias, the pseudomonas
in
called "blood typing" or "forward blood typing" may become considerably weak. Also,
In another but related procedure called "reverse infection, the RBCs become agglutinated by all antisera due,
blood typing" (also called "serum typing" or "backward probably, to unmasking of hidden antigens.

1.11: DETERMINATION OF BLEEDING TIME AND CLOTTING TIME

Student Objectives loss of blood is a result of a complex, natural,physiological

arrest of
response. The term is also used for surgical
After completing this practical, student should be able to: survival
bleeding Thus, hemostasis has a high degree of
1. Indicate the physioclinical importance of doing bleeding time interrelated steps:
(BT) and clotting time (CT). value. It involves the following four
2. Define hemostasis and describe how bleeding stops from a 1. Vasoconstriction (contraction of injured blood vessels)
fingerprick. 2. Platelet plug formation
3. Determine BT by Duke's Method and CT by capillary tube 3. Formation of a blood clot
method and give their normal values. 4. Fibrinolysis (dissolution of the clot).
4,Name the conditions in which BT and CT are increased.
5. Describe the physiological basis of bleeding disorders.
6. List the lotting factors and describe the steps in intrinsic and PHYSIOLOGICAL BASIS OF BLEEDING
extrinsic pathways of clotting. DISORDERS
7. Describe how clotting occurs in vivo and in vitro.
8. Define the terms thrombosis and embolism.
Excessive and prolonged bleeding with small injuries, or
9. Indicate how clotting can be prevented in vivo and in vitro. spontaneous bleeding may result from defects of:
Platelets
INTRODUCTION * Blood vessel walls
Bleeding after injury is a common experience for most of Coagulation of blood.
us. But bleeding stops automatically within a few minutes. Defects of Platelets and Vessel Walls
However, suspicion of a disease arises when there is
frequent and prolonged bleeding with minor injuries, such Defects of platelets and vessel walls typically cause
as during shaving, cutting of nails, or a fall on the knees. spontaneous bleeding from small vessels, or during cuts
In others, there may be spontaneous bleeding (without and bruises (e.g. pinpoint or petechial hemorrhages and
any apparent trauma) in the skin, gums, or into joints, and Purpuric lesions in the skin (blue-red patches)l., and
muscles, etc. It is in all such cases that various tests are bleeding in the gums.
carried out.
Defects of Clotting
HEMOSTASIS Defects of clotting are of two types:
1. Excessive bleeding into tissues (muscles, joints,
The term hemostasis (Greek hema = blood; stasis = halt) viscera, etc.) is usually due to injury to relatively large
reters to the process of stoppage of bleeding after blood vessels. The delay in the formation of a clot fails to
vessels are punctured, cut, or otherwise damaged. support the normal action of platelets in checking
Hemostasis which is ahomeostatic mechanism to prevent blood loss. Deficiency of cloting factors--inherited
80 Textbook of Practical Physiology

vitamin K Apparatus
or in liver disease (hepatitis, cirrhosis, and
fingerprick.
for sterile
Bquipment
deficiency) are the usual causes. < Clean filter papers.
within
2. Thrombosis: It is clottingof blood that occurs
endothelium Stopwatch.
unbroken blood vessels. A roughened
or injury is the
due to arteriosclerosis, infection, Procedure
common cause.
1. free-flowing
fingerprick
under aseptic
to conditions
Get a deep blood. Start the stopwatch and note t
puncture of
the finger is
BLEEDING DISORDERS time. The time of known
inherited or acquired-the
Zero ime.7
every 30 seconds by
Bleeding disorders may be common. Disorders due to 2. Blot the
blood drops
the filter paper along
touchin
its
defects being more
[Link]
with
acquind than puncture site
defects are more common the the
platelet and vessel wall clotting pressing or squeezing
Coagulationdisorders that
are due todeficiencies of without
onward.
blood spots l bleeding stops, i.e. when there
factors. time when
3. Note the blood spot onthe filter paper.
no trace of This is the end
Encircle thi
TESTS FORHEMOSTASIS number it as well. point (Hp
spot and paper on the table and
keep the filter then
38). (Do not it).
on
1. Bleeding time (BT). press your
wound
spots and express your
BT). number of blood
2. Iy BT (hemostasis of Hess (tourniquet test). 4. Count the
Capillary fragility test seconds.
and
3. result in
minutes
4. Platelet count.
Normal BTËs 2-6 minutes. reliable in spite of the fao
5. Clotting tíme (CT).
time (CRT). The test is simple and quite
cannot be
controlled.
6. Clot retraction depth of the wound
that the
7. Clot lysistime (CLT).
(PT).
8. Prothrombin time include: Platelet aggregation
and
Precautions
Other tests: These consumption test,
for BT should be scrubbed wellss
The skin site chosen
prothrombin
adhesiveness tests, (TT),
thrombin time blood flow.
t h r o m b o p l a s t i n test,
partial alcohol to increase the be3
partial (PRT), activated and thepuncture should
plasma recalcification time clotting The skinshould be dry not squeezp
(APTT), Kaolin cephalin free-flowing blood. Do
thromboplastin time
assaying of clotting mm deep to give puncture site.
special tests including paper on the
time (KCCT), and Do not press the filter l0-12 minutes, ston
Ifbleeding continues for
more than
factors. the wound. Inform
sterile gauze on
the test and press a
bleeding.
BLEEDING TIME your teacher about the
puncture
interval between the skin
Bleedingtimeisthe time without pressure)
spontaneous, unassisted (i.e.
and vitro test of platelet
stoppage of bleeding. The BT
test is an in
function. It is determined by
two nethods:
60 sec
1. Duke method 30 sec
Tmin 30 sec
2. Ivy method.
tests that are used as a routine
Note, The BT and CT are two simple
before every minor andmajor surgery (e.g. tooth extraction), biopsy 2 min
anticoagulant therapy, whether
procedures, and before and during
or not there is a history of bleeding.
2 min 30 sec
Bleeding Time by"Duke" Method
(Fingertip; Earlobe)
Since the skin of thefingertip is quite thick in some persons, 3 min
asmall cut in the skin of the earlobe with the corner edge
ofa sterileblade gives better results. The earlobe method is
the original "Duke" method for BT. Fig. 38: Bleeding time.
 81
Section 1: Hematology
Another method is to geta fingerprick and dip the finger Comments: The BT test is an in vivo test of platelet function, and
i abeaker containing normal saline at 37°C. The blood a peripheral
"Ivy" method is probably the most reliable. However,platelets and
rons willbe seen falling to the bottom in a continuous examined for the number of
Dlo0d nim is always note that platelets are
etream. Note the tinme when bleeding stops. This method is neir morphology. The students should
and one is normally affected
notcommonly used. involved both in BT and CT tests
low platelet count
without the other. If the BT is prolonged due to
(thrombocytopenic purpura), the platelets that are available are
Bleeding Time by "lvy" Method sufficient to give a normal CT.
This methodis more reliable than the "Duke" method.
However, itrequires somepractice to apply the blood CLOTTING TIME
pressure (BP)cuft and maintain the pressure,
the entry of blood
Clotting time is the time interval between
oEasE0nge, and formation of
Procedure intothe glass capillary tube, methods:
1 Clean the skin over the front of the forearm with 70% fibrin threads. It is determined by two
tube method
alcohol. 1. Wright's capillary glass
2. Lee and White method.
2. Apply a BP cuff on the upper arm, raise the pressure
to40 mm Hg and maintain it there till the end of the Capillary Blood Clotting Time
experiment.
(Wright's Capillary GlassTube Method)
3. Clean the skin area once again. Grasp the underside
of the forearm tightly, make a 1-3 mm deep skin
puncture, about 5-6 cm below the cubital fossa. Note
Apparatus
Pricking sterile needle
the time. capillary tubes
4. Remove the blood every 30 seconds by absorbing * Chemically clean, 10-12 cm long, glass
mm.
with a uniform bore diameter of 1-2
it along the edges of a clean filter paper by gently $ Stopwatch.
touching the wound with it till the bleeding stops. This
is the end point. Procedure
end of the
Note: Instead of one prick, two lancet stabs may be given, 5 cm 1. Give a sterile fingerprick. Now dip one
apart, one after the other, and the BT noted in them separately. into the
capillary tube in the blood; the blood rises
enhanced
Normal BT with this method is up to 9 minutes. tube by capillary action (Fig. 39). This can be
by keeping its open end at a lower level.
Simplate Method 2. Note the time of prick. This is called as the Zero time.
Though the "Duke" and "Ivy, BT methods are fairly reliable, 3. Hold the capillary tube between the palms of your
by a hands to keep the blood near body temperature.
itis not possible to control the depth of thewound made
lancet or ablade. However, by careful standardization, it has 4. Gently break off 1 cm bits of glass tube fromn one endat
technique intervals of 30 seconds, and look for the formation of
become possible to do so. The most widely used fibrin threads between the broken ends. The endpoint
automated scalpel to control the
uses a "template" or an
mm deep and 9 is reached when fibrin threads span a gap of 5 mm
depth and length of the wound-usually l
mm long-and a BP cuff inflated to 40
mm Hgto distend between the broken ends ("fibrin thread formation").
the capillary bed of the forearm. Note the time (Fig. 40).
Normal BT is less than 7 ninutes. 5. The CT is taken as total time from the time of puncture
(zero time) till there is formation of fibrin thread.
Physioclinical Significance Normal CT is 3-8 minutes (at 37°C)
The BT is prolonged in purpura (platelet deficiency,
normal in
or vessel wall defects) while it is usually
hemophilia.
Lack of several clotting factors may prolong BT, though
it is especially prolonged by lack of platelets.
increased risk
Note: Although a BT of over 10 minutes has a slightly
becomes great when the BT exceeds 15 or 20
of bleeding, the risk
mìnutes. Fig. 39: Glass capillary tube filled with blood.
82 Physiology
Textbook of Practical
of the
onthe
on the size ofthe test tube. "Therefore, a high itselt,
evenThe CTdepends
dege
condition.
lass
standardlization is needed.

is more
reliable
rel than the capillary
This
method
offblood with
system)).htsThussue
admixture
Comments:

thereis no
CTmethod,
because t h r o m b o p l

(extrinsic
a s t i n

tissue system of blood

contains
clotting.
theoretically How
which intrinsic
only the
becausethe
CT Can
method tests nonspecific
in the intrinsic
this
metho is
of any of
the
factors
always means
system.
due to
deficiency

practice, a
prolonged

hour in
CT nearly
severe cases.
hemophi
actual exceed 1 methodIis less
may
which the CT
Another
method (drop
alarge
method):
drop of
This
bloodf accurapunateth
from askin
method. Place pin through the
the above
clean and dry glass
slide. Draw a
time when
fibrin threads adhere to dropevery
on a
and note the drop. The timeelapse the
the formation bewe
seconds, the blood
move with it out of slide and
of fible
and drop on the
placing the blood
CT.
threads is the
Fig. 40: Formation of fibrin thread. 2-4
minutes.
Normal CTis CT, two drops method for.
Duke's drop
Lee and White Test Tube Method Inthe original glass slide..The si,
placed on a
diameter are
4-5 mm intervals. The endpoint is s
Single Test Tube Method istilted at 30
second abseno
when the slide is
previousshape hel
arrangements than capillary tube ofchange in the
This method needs more
and reliable method for the
method but is more sensitive vertical.
determination of CT.
blood by a clean,
non-traumatic Physioclinical Significance
1. Draw 5 mL venous
venepuncture. Note the time
when blood starts to
this method involves bo
time. Transfer the The clotting of blood with
enter the syringe. This is the zero systems of clotting Th
the intrinsic and extrinsic
blood to a chemically clean and dry test tube. is injury tothe blood (coming in contact with glas
bath at 37°C, take it out
2. Holding the test tube in a water intrinsicpathway), and the injury to the tissues (extrins
The end point is when
at 30second intervals and tilt it.
blood. pathway).
the tube can be tilted without spilling the
Normal CT with this method is 5-10 minutes. ¢ The CT is prolonged in hemophilia and other clotie
disorders, because thrombin cannot normally be
Multiple Test Tube Method generated. Yet, the BT which reflects platelet plug
formation and vasoconstriction, independently of ca
The CT can be determined more accurately by using 3 test formation is normal.
tubes rather than l only.
1. Rinse 3 test tubes of 8 mm diameter with normal
saline, drain them and place them in a metal rack kept CLOT RETRACTION TIME
inwater at 37°C. Transfer 1.5 mL blood into each test
tube. Transfer the larger test tube containing clotted blood to a
2. Take out the first tube after l minute, tilt it to 45° and incubator at 30°C. Normally, the clot starts to shrink (retrad
return it to the rack. Repeat every 30seconds until in about 30 minutes (leaving behind straw-colored serum)
clotting occurs, i.e. where the test tube can be tilted becomes halfits size in 2-3 hours, and complete in 24 hous
without spilling the blood. Note the time. Note if there is any digestion of the clot or discolorationo
3. Repeat the tilting on the second test tube and note the serum.
time when clotting occurs (this happens a few seconds
later because tilting the tube hastens clotting). The Comments: Clot retraction (tightening or consolidation)
thírd tube acts as a control and acheck on the on the release of many factors from the platelets, and so depe
the
end depends on the platelet count. The fibrin-stabilizing factor CaU'
point in the second test tube.
more and more cross-linking bonds between
Note: Ifa siliconized test tube is used at the nearby tibrin
Their spicules also release contractile proteins--actin, myosin, and
(40-70 minutes) can be shown. same time, adelayed CT
thrombosthenin. The contraction (retraction) of the clottisactivated
NormalCT with this method is 5-10 by thrombin, and calcium ions
minutes. stored in thee endoplasmic reticulut
Golgi apparatus, and mitochondria.
 called tissue
fron the d
subcutaneous
factor ll)
is
vessels
released

(e.g. cells
ofskin,
-lipoprotein complex tissue,
theblood
phospholipid-l

specilic including platelets. he

etc). lt is d of all cells
membranes are
the
present on
is
surlaces

released
when cell damaged
or crush injury) activates
T'Ewhich
cut, prick,
(as by a The complex of TF + VIla
( F l o w c h a r t 4).
rperturbed

Vlla Xa
tactor Vl to activatefactor
Xto Xa. Factor combines
+ calcium
ions
calcium ions
toform the active enzyme
and
withfactor V
coagulation of
blood. Theeintrinsic and
Mechanism of
Flowchart 4: shown.
pathways for blood clotting are
extrinsic
Extrinsic system
Intrinsic system
Injury to tissue cells
or
Injury to blood cells outside blood vessels
with blood
cells in contact (Skin, subcutaneous
(Damaged endothelium, or tissue, etc)
Exposed collagen)
Tisssue
PPL phospholipid
HMWK
TEI (TPL;|I)
KALLIKREIN PPL
Vila VI
XIl Xlla
HMWK

XI- Xla
TPL
IX IXa PPL
Ca2*
VIlI VIlla
PPL
Ca2+
Thrombin Xa + Va + PPL Prothrombin
X
Ca? + Ca
24
activator (PTA)
(Prothrombinase)

XII
Prothrombin Thrombin
Ce2+

XIlla
Fibrinogen Fibrin
Cross linking
(Stabilization)

Stage 1:generation of
of prothrombin activator (PTA); Stage
thrombin fromn prothr 2: formation