Blood Exercises

PHY-ANA LAB BLOOD

EXERCISE

S

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

of the plasma

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:

– 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

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

• 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+