Instrumentation and quality control in DISADVANTAGE:
hemostasis
3 WAYS OF DETECTING FIBRIN CLOT
FORMATION
1. VISUAL DETECTION
2. ELECTROMECHANICAL DETECTION
3. PHOTO-OPTICAL DETECTION
VISUAL DETECTION
PRINCIPLE: manual visualization of clot
 To know if there is a clot what we do is
to tilt the tube
 Solid gel formation as clot
 SUBJECTIVE
 It is important We evaluate accurately
for the sample to form a clot
Disadvantages:
 Lack of accuracy: temp., pippettor,
timing
 Time consuming
 Less precise and reproducibility
Tubes should be always 37degree
ELECTROMECHANICAL DETECTION
Principle: fibrin is detected by 2 wire loops as
electrode which is incorporated to an
electromechanical instrument
Sensitive to low fibrinogen levels:
 With 2 different wire loops acts as
electrode
 1st loop- stationary
 2nd loop-moving
 Stop-form clot already
 SEMI-AUTOMATED
 Sensitivity- able to test smallest
concentration of analyte
 Sensitive: <50mg/dL
 Interval- 0.5 seconds
 Specimen carry over-if u will use it for
next patient if u not able to clean
properly the loops it will be
contaminated
Photo-Optical Detection
Principle: depends on the increase light
scattering meaning as u form the clot there will
be increase scattering of light
E.g.:
 Electra 750 and 750A
 FP910 Coaganalyzer
 Coag-A-Mate X2XC
 Ortho Koagulab16S
 ACL
Source of error:
 Transmittance is affected by: icteric
sample, lipemia, hemolysis, low
Fibrinogen, high heparin, protein ppt
 time interval=0.1
 guard interval- before your machine
measure your clotting time it will not
start to aspiration of specimen it will
start timing when reagent and
specimen mixed
Variables affecting coagulation testing and QC
•Instrument: temp(37), light source, detector,
timer, reagent delivery
•Specimen: collection, anticoagulant, delays,
storage
•Reagents and Controls: shipping, storage,
recon, contamination, deterioration, lot
changes
Reagent quality Control
•PT reagent
•APTT uses insoluble activators
•*reagent compatibility
•*cost
•*reproducibility
•*sensitivity
•*purposes
Considerations of control material used
 To monitor precision
 To determine SD
 Run:
We run control material not to create
normal values rather to check the
accuracy and precision of the test and
check machine if it is in good condition
We run control material= every after 20
test
In here Philippines we control testing
before running test
Proficiency Testing
 Inter laboratory comparison of the test
result obtained to assess lab
performance
 Reference laboratory sent a notes
sample to participating laboratory
 Reference laboratory will send
unknown sample and test it for specific
parameter and test it with your
laboratory and give them the result so
result must same as them
 To check if laboratory employees good
quality control practices result must
close to their result
 Example: RITM
Laboratory Evaluation of Secondary Hemostasis
•Extrinsic and Common
 End point of extrinsic will end the
activation of common pathway
•Intrinsic and Common
Example:
EX=abnormal
IN= normal
 So external is the problem
 If both are abnormal the problem si
common pathway
In secondary hemostasis the end point
is CLOT
WE MEASURED HERE IS THE TIME IT
TAKES FOR SPECIMEN TO FORM A CLOT
Extrinsic and Common
1. One stage Prothrombin Time (PT)
 Once vessels are damage the tissue
surrounding vessels release tissue
factor or THROMBOPLASTIN (CF3) that
will activate CF7
 CF7 form complex with thromboplastin
with the help of Ca+ forming EXTRINSIC
TENASE COMPLEX activate common
pathway
 7a + ca+ +thromboplastin
Test of choice: ORAL ANTICOAGULANT , suffer
stroke
Reagent: Simplastin (THROMBOPLASTIN +
CALCL2)
 Factor 3 is not with your plasma it is
within your tissue so you need tissue
factor
 Thromboplastin (from plants, animals
and humans)
 PPP + SIMPLASTIN
 Let simplastin activate extrinsic tenase
 WHO = recommended sensitivity is
equal to 1
 ISI( international sensitivity Index)
 ISI is 1= sensitive
 Higher 1= less sensitive
NV: 11-14 seconds form CLOT
INR: international normalize ratio= TO IDENTIFY
PATIENT THAT UNDER ANTICOAGULANT
THERAPHY
INR=not taking anticoagulant
Higher= taking anticoagulant
(Patient’s PT/Mon mean of normal as
control)ISI
2. StypvenTime
  Uses snake venom
 From RUSSEL VIPER
 Venom here will assume the
function of FACTOR 7
 Venom will activate common
pathway
 Venom=acts as 7a
 To identify if problem is with
FACTOR 7 OR COMMON
PATHWAY
Example: PT=prolonged & ST=normal
=problem is extrinsic pathway or factor 7
If same prolonged problem will be common
pathway
•Determines problem in FVII or other factors in
common pathway
•NV
Intrinsic and Common
Clotting time is a measure of the ability
of the blood to clot and is NOT
influenced by the platelet functions
other than PF3
 Need phospholipids
 Need PPP
 Measure the only required for the
formation of traces of thrombin
sufficient enough to produce a visible
clot
1. Whole Blood Clotting Time Determination
Micro Methods
Principle: when blood exposed to glass surface
with negative charge they will form clot
Slide= 2-6 mins =clot
NV:
Capillary tube method (Dale and Laidlaw’s)=not
advisable /fibrin threads/break capillary tubes
NV:
Macro Method:
Lee and White: venipuncture negative charge
on test tube will form clot/ 10-15 mins forms
clot/manual visualizations of clot
NV:
2. Activated Clotting Time= same with lee and
white/add activators /75-120 SECONDS
Activator: 12 mg DIATOMITE
More reliable and faster result
NV:
UNDER HEPARIN= 140-185 seconds to form
clot
3. APTT=INTRINSIC/test patient taking
HEPARIN
 Need 9a + 8a + CALCIUM +
PLATELET PHOSPHOLIPID
 Because PPP has no platelet
Test of choice: HEPARIN
Activators:
Reagent: platelet substitute and activators=
kaolin, cellite, silica and ellagic acids
0.025 M CaCl2
NV:20-45s
Note: start timing after adding CA+
4.Plasma Recalcification Time
 More sensitive method than the
coagulation time of whole blood.-May
reveal abnormality which is not detect
able by the determination of the
clotting time of venous blood.-The
activated recalcification time makes use
of 0.025M CaCl2 as activator.
 Returning calcium
 Compare PPP (form clot faster) and PRP
NV
Indirect tests for Coagulation
1. PT consumption Test
-Sample:
-Reagent: SimplastinA
NV
2. Thromboplastin generation time
3. Two-stage Prothrombin and
ProconvertinTest(Owren and Aas)-It offers a
combine destimation of the level so
prothrombin and proconvertin
Tests for Fibrinogen
1. Thrombin Clotting time-add thrombin
Affected by: circulating anticoagulant and
affected by HEPARIN
2. Reptilase Time
-defects in fibrinogen
 Use snake venom ( BOTHROPS ATROX)
 Assume the function of thrombin
 Convert fibrinogen to fibrin
 To know if the problem is heparin or
fibrinogen
 Not affected by HEPARIN
Test for Factor XIII
•5M Urea Test/DUCKERT’S TEST
 Use to evaluate factor 13
 Sample will allow clot
 F13= stabilization of clot
 24 hours
MIXING STUDY
Plasma + blood
PX + fresh plasma Aged, serum, adsorbed
NOTE!!!
Fresh plasma=all clotting factors are present
Aged plasma= no 5 & 8
Serum plasma= 1, 5, 8, 13 (fibrinogen group)
Adsorbed (barium sulfate) =no 2, 7,9,10
EXTRINSIC= 7
INTRINSIC= 12,11,9,8
COMMON= 10,5,2,1,13
3-TISSUE
4-CALCIUM
Endpoint of pt and apt= clot
Lupus anticoagulant= inhibitor of
phospholipids/ fresh plasma is prolonged
•A. Screening Test:
•*presence of inhibitors
•*presence or absence of correction
B. PNT: platelet neutralization test
 use of freeze thawed platelets
 shortening of APTT
 Confirmed by: Anti-cardiolipin
Assay/ELISA
 Test to detect lupus anticoagulant
 PLASMA + PLATELETS
 LUPUS= NEUTRALIZE BY PLATELETS
C. Agarose Plasma Gel Technique:
 fresh plasma and CaCl2
 cloudy or opaque gel
 AGAROSE GEL WITH 2 WELLS (plasma +
calcium)
 Gel will become cloudy= indicates clot
 Clear=no clot /with inhibitor
D. Bethesda Inhibitor Assay:
 factor VIII inhibitors
 1 Bethesda Unit = 50% FVIII inhibited
  Add factor 8

E. Ristocetin Co-Factor:
 ristocetin& platelets
 NV: 50-150% activity
 Pag platelet di nagfunction after adding
this it will means presence of VWF
DISEASE