HEMATOLOGY 2: LABORATORY TESTS

EXERCISE 2 BLOOD SMEAR PREPARATION (WEDGE FILM)

Place the lateral side of the clean side of the slide
against the slide with blood to form 25-45
With FEATHERY EDGE

EXERCISE 3 STAINING OF BLOOD FILM

Fix blood smear in METHANOL for 30sec to 1minute
air dry
Cover entire smear with WRIGHTS STAIN (count the
drops) Stand for 1minute
Add equal number of drops of BUFFER SOLUTION
Mix stain and buffer solution until a METALLIC SHEEN
appears on the surface of the mixture stand for 3 to
5 minutes
Using a spouted bottle of WATER, Flush the stains
Blot-dry the smear Air dry
Examine under OIL IMMERSION OBJECTIVE
Clean slide with XYLENE after to remove the immersion
oil
EXERCISE 4A PLATELET COUNT: DIRECT
(REES AND ECKER METHOD)

Red blood cell pipette

Rees & Ecker diluting fluid

Petri dish

Filter paper

Hemocytometer
Fill RCPipette w/ blood up to MARK 0.5

Suck diluting fluid to MARK 101

DILUTING FLUID: Rees and Ecker Diluting fluid Color
Blue Platelets are stained with blue

DF does not lyse Red Cell RBC can still be seen

DILUTION:

WBC Pipet -1:20

FORMULA:

WBC squares are used -4 WHITE CELL SQUARES

PLT CT = (# of plt x dl factor x depth factor)

/ area factor

(# of plt x 200 x 10)/ 4

Clinical Significance:

To quantify platelets

NORMAL REFERENCE VALUE:

150-400x109/L or 7fL

If below THROMBOCYTOPENIA

Bleeding tendencies

If above THROMBOCYTOSIS

Thrombotic tendencies

EXERCISE 4B PLATELET COUNT:

UNOPETTE SYSTEM
DILUTING FLUID: AMMONIUM OXALATE

Can lyse red cells

AUTOMATIC DILUTION = 1:100
RBC Pipet is always used
FORMULA:

RBC SQUARES use all 25 tertiary square for

counting; minimum of 10 tertiary squares

PLT CT = # of plt x area factor x dl factor x

depth factor

# of plt x 1 x 100 x 10

# of plt x 1000
Clinical Significance: To quantify platelets
EXERCISE 4C PLATELET COUNT: INDIRECT
(FONIOS METHOD)

Blood sample

Slides

Wrights or Giemsa Stain

Wedge smear Air Dry Stain
Examine blood smear under OIO
Observe 5 to 10 fields while counting calculate
FORMULA:

PLT CT = average # of plt x 20,000

Clinical Significance:

To quantify platelets
EXERCISE 5A BLEEDING TIME
(DUKES METHOD)

Lancet

Filter paper

Cotton balls

70% Alcohol

Stopwatch
Disinfect area to be punctured with 70% Alcohol dry
make a firm stab to produce a STANDARD 3MM
DEEP WOUND

DEPTH IS 2-3x2.5MM
Dont wipe the puncture area
Record time wait for 30 SECONDS to lapse and blot
the blood

without the paper to come in contact with the

skin
Repeat blotting every 30seconds until no more
blood adheres on the filter paper (END POINT)
CLINICAL SIGNIFICANCE:

Platelet Function Test Platelet adhesion test

Measuring 1o hemostasis (Platelet, BV, vWF, CF1,

CF4/Ca++) and Platelet number (Qualitative)

PROLONGED RESULT means defect in 1o

hemostasis
REFERENCE VALUE: 0 to 6 minutes

EXERCISE 5B BLEEDING TIME

(IVYS METHOD)

Standard Lancet

Blood Pressure Cuff

Filter paper

Stopwatch
Apply pressure cuff on one arm and inflate to 40mmHg
pressure
Other steps are the same with DUKES MTD
CLINICAL SIGNIFICANCE:

Platelet Function Test Platelet adhesion test

Measuring 1o hemostasis (Platelet, BV, vWF, CF1,

CF4/Ca++) and Platelet number (Qualitative)

PROLONGED RESULT means defect in 1o

hemostasis
REFERENCE VALUE: 2 to 9 minutes
EXERCISE 6A COAGULATION TIME
(SLIDE OR DROP METHOD)

Lancet

Slide

Cotton balls

70% Alcohol
Whole blood clotting time method
Most commonly used method
PRINCIPLE: measures time required for drop of blood
placed on a glass surface to form fibrin strand as the
clotting blood is lifted using a lancet or any pointed
object
Ideal for pediatric patient
CLINICAL SIGNIFICANCE:

Whole blood clotting method

Test to evaluate the INTRINCIS (VIII, IX, XI, XII,

PK, HMWK) AND COMMON (X, V, II, I) PATHWAY
CLOTTINGFACTORS

Any abnormality or deficiency will have

PROLONGED TET RESULT
REFERENCE VALUE: 2 to 6 minutes

EXERCISE 6C COAGULATION TIME

(LEE AND WHITE)

5 ml test tubes, 3 pcs.

Syringe and Stopwatch

Start timing as soon as blood enters hub then add 1.5
ml blood to 3 glass test tubes.
After standing for 5 mins check for clot

Via titling at 45; if no clot yet tilt every 30 sec.

Always check TUBE#3 first

first part of blood that entered the syringe and

will be the first to clot
Check TUBE#2 30 sec after TUBE#3 clots check
TUBE#1 30 sec after TUBE#2 clots
Records result from last tube (END POINT)
PRINCIPLE:

blood normally forms a solid clot at a specific

time the placed on a glass tube (foreign surface
that activates contact factors)
CLINICAL SIGNIFICANCE:

Whole blood clotting method

Test to evaluate the INTRINCIS (VIII, IX, XI, XII,

PK, HMWK) AND COMMON (X, V, II, I) PATHWAY

Any abnormality or deficiency will have

PROLONGED TET RESULT
REFERENCE VALUE:

5 to 15 minutes (Steininger)

10 to 15 minutes (Manual)
EXERCISE 7A CLOT RETRACTION TIME
(HIRSHBOECK)

Lancet

Test tube

Castor oil

Sahli-Hellige pipette
PLATELET FUNCTION TEST
using a test tube add castor oil and place a small drop
of blood
allow to stand for 15 mins and wait for dimpling
phenomenon

when there is serum that comes out of clotted

blood
PRINCIPLE:

Allowing whole blood to clot

CLINICAL SIGNIFICANCE:

Measures entire function of platelets from

ADHESION TO RETRACTION
REFERENCE VALUE: 15 to 45 minutes

EXERCISE 7B CLOT RETRACTION VOLUME

(MCFARLANE METHOD)

Syringe

Conical tube

Glass rod

Water bath
Using a conical tube add 5ml of Whole Blood, place
wire loop and incubate
Incubate Whole Blood at 37C and check for clot
retraction at 24 hours
after 24 hours, remove the clotted blood and measure
the serum
FORMULA:

%CRV
=`mL of SERUM/mL of WHOLE BLOOD) x 100
CLINICAL SIGNIFICANCE:

To measure quantitative and qualitative platelet

defects, CF deficiency and

plasmin or fibrinolytic action affect clot

retraction
REFERENCE VALUE:

40 to 60% CRV
EXERCISE 8 PROTHROMBIN TIME
(QUICKS METHOD)

Materials for Venipuncture, Citrated Tube

Simplastin

Centrifuge

Stopwatch

Water bath
4.5mL of blood Citrated tube
Centrifuge at 3500g for 10 to 15 minutes PPP
Incubate 0.2mL of Simplastin & 0.1 mL PPP @ 37oC for
2 minutes
Simplastin + Plasma Gently tilt the tube (Start time)
GEL FORMATION (END POINT)
REAGENT USED:

PT REAGENT SIMPLASTIN (BRAND NAME):

composed of

THROMBOPLASTIN
derived from different animal sources
-rabbit brain, rabbit brain-lung,
combination or recombinant human
hence DIFFERENCE IN SENSITIVITY
WHO requires international sensitivity
index (ISI) of PT reagent to be near 1
(i.e. 1.1-1.2) if around 1.8/1.9
sensitivity of reagent is affected

CaCl2
PRINCIPLE: Citrated PPP is added with tissue extract or
thromboplastin with Ca++ to from a loss fibrin clot (GEL)
CLINICAL SIGNIFICANCE:

Test to evaluate the EXTRINSIC (VII, III)

COMMON (X, V, II, I) PATHWAY CLOTTING
FACTORS

Test of choice to SCREEN ORAL ANTICOAGULANT

THERAPHY

ORAL ANTICOAGULANTS (Vitamin K

Anticoagulants):

Warfarin & Coumadin

REFERENCE VALUE:

12 to 14 seconds or 11 to 14 seconds
EXERCISE 9 PARTIAL THROMBOPLASTIN TIME

PTT Reagent

0.025M Cacl2

Test tubes, 2pcs

Water bath

Citrated tubes

Materials for Blood Collection

4.5mL of blood in a Citrated tube Centrifuge @
3500g PPP
Tube #1: 0.1mL PTT reagent
Tube #2: 0.2mL CaCl2
Incubate @ water bath for 3 to 5 minutes Add
0.1mL of plasma in tubes
Gently tilt the tube GEL FORMATION (END POINT)
REAGENTS USED:

PTT REAGENT Two components:

ACTIVATORS: Kaolin, Celite, Micronized

Selica and Elagic Acid

PLATELET PHOSPHOLIPID SUBSTITUTE

0.025M CaCl2
CLINICAL SIGNIFICANCE:

Test to evaluate the INTRINCIS (VIII, IX, XI, XII,

PK, HMWK) AND COMMON (X, V, II, I) PATHWAY

Measures ALL CLOTTING FACTORS EXCEPT VII

AND XII

Test of choice for HEPARIN THERAPHY

MONITORING
REFERENCE VALUE:

20 to 45 seconds (Steininger)

35 to 45 seconds (Manual)

EXERCISE 12 PLASMA RECALCIFICATION TIME

Blood Collection Materials

Two Citrated Tubes

Centrifuge

0.025M CaCl2

Timer/Stopwatch

Water Bath (37oC)

Pipettes
5 to 6 mL of Citrated Blood (3mL of blood per tube)
Tube #1 Centrifuge at 100 to 200g and spin for 3
minutes PRP
Tube #2 Centrifuge at 2000 to 2500g and spin for 10
to 15 minutes PPP
PRP & PPP Clean test tubes & label
Incubate PRP, PPP and CaCl2 Reagent @ 37oC for 5
minutes
Transfer 0.2mL of CaCl2 into PRP and wait for CLOT
FORMATION (END POINT). Timing should be done as
soon as the reagent was mixed with plasma. DO THE
SAME FOR PPP
CLINICAL SIGNIFICANCE:

Test to evaluate the INTRINCIS (VIII, IX, XI, XII,

PK, HMWK) AND COMMON (X, V, II, I) PATHWAY
REFERENCE VALUE:

90 to250 seconds
EXERCISE 15 WHOLE BLOOD CLOT LYSIS TIME

Blood Collection Apparatus

Plain Glass Tube

Water Bath Set @ 37oC

3 to 5mL of Blood Transfer in Plain Tube
Allow to clot for 30 minutes
Incubate at 37oC for 48 hours
Observe the appearance of clot

The clot should remain intact after 48 hours of

incubation
CLINICAL SIGNIFICANCE:

To screen excessive clot lysis time

REFERENCE VALUE:

DISSOLUTION OF CLOT PRIOR TO 48 HOURS IS

INDICATIVE OF EXCESSIVE FIBRINOLYTIC
ACTIVITY

If clot has jagged boarder and serum has tinge or

excess red cell fall out =EXCESS IN PLASMIN OR
FIBRINOLYTIC ACTION

Serum must be color YELLOW

KTHMNTS