Download - Blood Exercises
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PHY-ANA LAB BLOOD
EXERCISE
S
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COMPLETE BLOOD COUNT
• Hematocrit
• Hemoglobin
• Differential white blood cell
• Red blood cell
• White blood cell
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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)
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HEMATOCRIT
• One of the simplest, most accurate, & valuable tests
• Detecting cases of anemia
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HEMATOCRIT
• Specimen
– Fresh capillary blood (with heparin)
• Adam’s Microhematocrit Method
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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
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HEMATOCRIT
• Capillary tube with
seal towards the
outside
• Balance tubes in
the centrifuge
• Securely screw the
cover of the
centrifuge
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HEMATOCRIT
• When rotation has stopped,
remove tube
• Take note of the appearance
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HEMATOCRIT
• Capillary tube
with seal
toward the
center
• Align upper
portion of the
seal with the
black line
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HEMATOCRIT
• Rotate the
whole assembly
so that the pin
stops (100
mark)
• Rotate the
upper disk to
move the curve
line with the top
of the plasma
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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
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HEMATOCRIT
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HEMOGLOBIN
• Red-pigmented protein
• Transports oxygen and
carbon dioxide
• Measured as
oxyhemoglobin– Indirectly measured by
converting to compounds
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HEMOGLOBIN
• Acid Hematin
Method
– Yellowish brown
solution is compared
to the color standard
in the comparator
block
– Darker the color =
higher Hgb content
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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)
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DIFFERENTIAL WBC COUNT
• Examination of a thin smear determining the
percentages of WBC types
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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
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DIFFERENTIAL WBC COUNT
• Blood smear
preparation
– Most important step
• Two-Slide or Wedge
Method
– Simplest
– Most popular
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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
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DIFFERENTIAL WBC COUNT
• Good smear:
– Thick to thin
– Occupy 2/3 or ¾
– Smooth and even
surface
– Free from ridges,
waves, holes
– Margin-free
– Feathery edge
• Factors that affect:
– Angle of the spreader
slide
• Greater angle: thicker &
shorter
– Size of the blood drop
– Speed of spreading
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DIFFERENTIAL WBC COUNT
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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
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DIFFERENTIAL WBC COUNT
• Staining of Blood
Smears
– Dip Method (Rapid)
• Quick method
• Modified Wright-Giemsa
buffered in methanol at
pH 6.8
• Tightly sealed
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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
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DIFFERENTIAL WBC COUNT
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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
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DIFFERENTIAL WBC COUNT
• Method of Differential Counting
– Battlement
• Count 100 white blood cells while
differentiating
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DIFFERENTIAL WBC COUNT
• Method of Differential Counting
– Battlement
• Count 100 white blood cells while
differentiating
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DIFFERENTIAL WBC COUNT
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HEMACYTOMETER
• Counting chamber
• WBC pipette
• RBC pipette
• Accessory devices
– Suction device
– Thick cover slip
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COUNTING CHAMBER
• Heavy, colorless glass
• 3 parallel platforms
separated by moats
– Central: 0.1 mm lower
than lateral
• Transverse groove
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COUNTING CHAMBER
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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
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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
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DILUTED BLOOD
PREPARATION
• 0.5 mark: blood
• Diluting fluid
– 11 WBC, 101
RBC
– Constant
rotation
• Over aspirate
or presence of
bubbles: repeat
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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
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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
– Stem 0.0 to 1.0
– Bulb 1.0 to 11
– Stem volume is 10x the
bulb volume: 10 units
RBC
PIPETTE
WBC
PIPETTE
Upper mark 101 11
Bore Smaller Bigger
Bead Red White
Dilution 1:200 1:20
Size of bulb Bigger Smaller
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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
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COUNTING CELLS
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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
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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
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BLOOD GROUPS
• Antigens
• Antibodies
• Agglutination
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ABO BLOOD GROUPING
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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
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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
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COAGULATION TIME
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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
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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
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CAPILLARY RESISTANCE
TEST
• Assesses the fragility
of capillary walls
• Hemorrhagic
tendency
– Thrombocytopenic
purpura
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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
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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
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CAPILLARY RESISTANCE
TEST
• Interpretation of results
Number of petechiae Grade
0-10 1+
11-20 2+
21-50 3+
51 and above 4+