PHY-ANA LAB

BLOOD
EXERCISES
 COMPLETE BLOOD COUNT

• Hematocrit
• Hemoglobin
• Differential white blood cell
• Red blood cell
• White blood cell
 HEMATOCRIT

• % of formed cells in whole blood

– 99% RBCs and 1% WBCs and platelets

• Estimate if RBCs are adequate

• Greek hemato “blood” and crit “to

judge”

• Aka packed cell volume (PCV), Hct

or erythrocyte volume fraction (EVF)
 HEMATOCRIT
• One of the simplest, most accurate, & valuable tests
• Detecting cases of anemia
 HEMATOCRIT

• Specimen
– Fresh capillary blood (with heparin)
• Adam’s Microhematocrit Method
 HEMATOCRIT

1. Blood  ¾ of the
capillary tube
2. Sealing clay (3 mm)
3. Centrifuge 10,000
rpm for 4-5 minutes
4. Level of packed RBC
using
microhematocrit
reader
 HEMATOCRIT

• Capillary tube with

seal towards the
outside
• Balance tubes in
the centrifuge
• Securely screw the
cover of the
centrifuge
 HEMATOCRIT

• When rotation has stopped,

remove tube
• Take note of the appearance
 HEMATOCRIT

• Capillary tube
with seal
toward the
center
• Align upper
portion of the
seal with the
black line
 HEMATOCRIT

• Rotate the
whole assembly
so that the pin
stops (100
mark)
• Rotate the
upper disk to
move the curve
line with the top
 HEMATOCRIT

• Rotate the entire assembly

until the curved line is lined
up with the boundary
between packed RBC and
plasma

• Read the % packed cells at

the right
HEMATOCRIT
 HEMOGLOBIN

• Red-pigmented protein
• Transports oxygen and
carbon dioxide
• Measured as
oxyhemoglobin
– Indirectly measured by
converting to compounds
 HEMOGLOBIN

• Acid Hematin
Method
– Yellowish brown
solution is compared
to the color standard
in the comparator
block
– Darker the color =
higher Hgb content
 HEMOGLOBIN
1. 0.1N HCl  2 mark of Sahli’s tube
2. Aspirate 0.02 mL blood using Sahli’s pipette
3. Expel the blood sample to the tube
4. Rinse the pipette with dist. water 3x  add to the
mixture  Stand 10 mins
5. Add dist. water drop by drop (mix with stirring rod)
until color matches with the block
6. Reading  lower meniscus
7. Report gm% or gm/dL or gm/100mL (CU) and in
gm/L (SI)
 DIFFERENTIAL WBC COUNT

• Examination of a thin smear determining the

percentages of WBC types
 DIFFERENTIAL WBC COUNT
• Specimen
– EDTA blood
– Within 2-3 hours of
collection
– Within the mark of the
tube

• Avoid
– Old specimen
– Excessive amount of
anticoagulant to specimen
 DIFFERENTIAL WBC COUNT

• Blood smear
preparation
– Most important step
• Two-Slide or Wedge
Method
– Simplest
– Most popular
 DIFFERENTIAL WBC COUNT

1. Drop of blood from mixed

sample on a clean glass slide
2. Spreader slide at an angle of
about 30-45o
3. Allow blood to spread evenly;
Control thickness of smear
4. Air dry
– Do not blow dry: RBC artifact
 DIFFERENTIAL WBC COUNT

• Good smear: • Factors that affect:

– Thick to thin – Angle of the spreader
– Occupy 2/3 or ¾ slide
– Smooth and even • Greater angle: thicker &
shorter
surface
– Size of the blood drop
– Free from ridges,
waves, holes – Speed of spreading
– Margin-free
– Feathery edge
DIFFERENTIAL WBC COUNT
 DIFFERENTIAL WBC COUNT

• Staining of Blood Smears

– Methanol: fixative (30s)
– Eosin: acidic dye (6s)
• Stains Hgb & leukocytes
– Methylene blue: basic dye
(4s)
• Nucleoproteins, nucleic acids
– Buffer solution (pH 7.2) for
45s
 DIFFERENTIAL WBC COUNT

• Staining of Blood
Smears
– Dip Method (Rapid)
• Quick method
• Modified Wright-Giemsa
buffered in methanol at
pH 6.8
• Tightly sealed
 DIFFERENTIAL WBC COUNT

• Blood Smears
– RBC: pink to salmon
– Nucleus: dark blue to purple
– Neutrophils: lavender to lilac
– Basophils: dark blue to black
– Eosinophils: red to orange
– Area between cells: colorless,
clean and free of precipitates
DIFFERENTIAL WBC COUNT
 DIFFERENTIAL WBC COUNT

• Smear Examination
1. LPO
• Assess overall quality
• Rapid detection of large
abnormal cells
• Not overlapping or too scanty
2. Shift to OIO
 DIFFERENTIAL WBC COUNT

• Method of Differential Counting

– Battlement
• Count 100 white blood cells while
differentiating
 DIFFERENTIAL WBC COUNT

• Method of Differential Counting

– Battlement
• Count 100 white blood cells while
differentiating
DIFFERENTIAL WBC COUNT
 HEMACYTOMETER

• Counting chamber
• WBC pipette
• RBC pipette
• Accessory devices
– Suction device
– Thick cover slip
 COUNTING CHAMBER

• Heavy, colorless glass

• 3 parallel platforms
separated by moats
– Central: 0.1 mm lower
than lateral
• Transverse groove
COUNTING CHAMBER
 COUNTING CHAMBER
• 1 Primary square
– 3x3 mm (9 sq. mm)
• 9 Secondary
squares
– 1x1 mm
– 4 corners: WBC
count
• 16 tertiary squares
• W1, W2, W3, W4
• 64 squares
 COUNTING CHAMBER
• Central secondary
square
– 25 tertiary squares
• 0.2 mm each
• 16 quaternary
squares
• Total number of
quaternary: 400
– RBC count
• 5 tertiary squares:
80 squares
 DILUTED BLOOD
PREPARATION
• 0.5 mark: blood
• Diluting fluid
– 11 WBC, 101
RBC
– Constant
rotation
• Over aspirate
or presence of
bubbles: repeat
 CHARGING
• Cover slip
– No dirt, thumb marks,
tissue strands
• Discard
– WBC 2-3 drops
– RBC 5-6 drops
• Angle of the pipette
(30-35)
• Stand for 5-10 minutes
 RBC AND WBC THOMA
PIPETTES
• RBC pipette
– Stem 0.0 to 1.0 contains
1 unit of volume
– Mixing chamber or bulb
0.1 to 101 holds 100
units of volume
• WBC pipette
RBC WBC
PIPETTE PIPETTE
– Stem 0.0 to 1.0 Upper mark 101 11
– Bulb 1.0 to 11 Bore Smaller Bigger

– Stem volume is 10x the Bead Red White

bulb volume: 10 units Dilution 1:200 1:20

Size of bulb Bigger Smaller
 STANDARD PATTERN OF
COUNTING
• Cells touching
any of the lines
on the top and
left borders are
included
• Cell difference
between 2
squares
– RBC 20 or less
– WBC 12 or less
COUNTING CELLS
 COMPUTATION
• RBC COUNT
– No. of cells/cumm = total number of cells counted
area X depth X dilution

= total number of cells counted

1/5 X 1/10 X 1/200

= cells counted X 10,000

Normal values: male: 4.5-6.0 M/cumm

female: 4.0-5.5 M/cumm
 COMPUTATION
• WBC COUNT
– No. of cells/cumm = total number of cells counted
area X depth X dilution

= total number of cells counted

4 X 1/10 X 1/20

= cells counted X 50
Normal value: 5,000-10,000/cumm
 BLOOD GROUPS

• Antigens
• Antibodies
• Agglutination
ABO BLOOD GROUPING
 BLEEDING TIME
• Duke’s Method
– Finger prick
• Allow blood to flow freely
– Start time: drop of blood
appears
– Blot with filter paper
• Do not touch the wound
– Stop time: when bleeding stops
– Normal: 1-3 minutes
 COAGULATION TIME

• Drop or Slide Method

– Prick
– Drop of blood on a slide
• Start: when in contact with the slide
– Tip of lancet every 30 second
interval
– Observe fibrin formation
• Stop timer
– Normal: 3-6 minutes
COAGULATION TIME
 HYPEREMIA OR
CONGESTION
• Note the skin color, blood vessel
condition, temperature of left index
finger
• Immerse in hot water (60C) for 5
minutes
• Note the changes and the
sensation felt
• Rubber band (5 minutes)  2nd
interphalangeal joint
• Note the changes and the
sensation felt
 HYPEREMIA OR
CONGESTION
• Hyperemia: active increase in
blood volume
– Dilation
– Physiological: blushing or during
exercise
– Reddish
• Congestion: passive increase in
volume of blood
– Impaired venous blood flow or
venous obstruction
– Reddish-blue (cyanosis)
– Always pathological
– Cardiac failure
 CAPILLARY RESISTANCE
TEST
• Assesses the fragility
of capillary walls

• Hemorrhagic
tendency
– Thrombocytopenic
purpura
 CAPILLARY RESISTANCE
TEST
• Thrombotic Thrombocytopenic Purpura
– (clots)-(low platelet number)-(purple bruises)
– Rare blood disorder
• Blood clots form in capillaries
• Uses up platelets
• Bleeding problems
 CAPILLARY RESISTANCE
TEST
• Tourniquet Test (Rumpel-Leede or Hess)
– Mark red spots on the arm
– Wrap the cuff of sphygmomanometer around
– Inflate to 100 mmHg (5 mins) or 50 mmHg (10
mins)
– Release pressure (15-20 mins elapse)
– Count the number of petechiae (ventral)
– Interpret results
 CAPILLARY RESISTANCE
TEST
• Interpretation of results

Number of petechiae Grade

0-10 1+
11-20 2+
21-50 3+
51 and above 4+