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)

Adams Microhematocrit Method

HEMATOCRIT
1. Blood 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.
2.
3.
4.

0.1N HCl 2 mark of Sahlis tube

Aspirate 0.02 mL blood using Sahlis pipette
Expel the blood sample to the tube
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:
Thick to thin
Occupy 2/3 or
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

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
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

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
Dukes 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+