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
