ROUTINE LABORATORY

EVALUATION of COAGULATION
REPORTER #8
TIMBASAL, FATIMA ALLIN
TUBAING, SHEENA MARIE
YBIO, AMIE
Routine Laboratory Evaluation of
Coagulation
REFERENCE RANGES
TEST FOR THE INTRINSIC AND COMMON
PATHWAYS
1. Lee and White Whole Blood Coagulation Time
2. Plasma Recalcification Time
3. Activated Clotting Time
4. Partial Thromboplastin Time
TEST FOR THE EXTRINSIC AND COMMOM
PATHWAYS
1. ProthrombinTime
2. Other tests
Routine Laboratory Evaluation of
Coagulation
Reference range
- in hemostasis are significantly
affected by patients in populations,
methodology, reagent systems, instrument
system and combination thereof.
- Consequently, every laboratory must
establish its own reference ranges.
Test for the Intrinsic and Common
Pathways
Lee and White Whole Blood Coagulation Time
- described the procedure for the whole blood
coagulation time that was a modification of the
numerous methods described on literature time.
L-W Test
- Base on the fact that when venous blood is put
into a glass tube, it will form a solid clot.
- The time required for this response is a measure
of the overall intrinsic and common pathways of
coagulation
Test for the Intrinsic and Common
Pathways
Plasma RecalcificationTime
- A modification of the L-W,
- using citrated plasma instead of whole blood.
- Glass or siliconized tubes
- Platelet-rich Plasma (PRP)
- Platelet-poor Plasma (PPP)
This test is based on the fact that except for calcium, normal
PRP contains all the components of the coagulation
mechanism necessary for generating a fibrin clot.
REFERENCE RANGE
PRP 100 150 seconds
ppp 130 240 seconds
Test for the Intrinsic and Common
Pathways
Plasma RecalcificationTime
- Disadvantage-
- 1. Are the difficulty in standardizing the number of
platelet in the PRP
- 2. Length of time necessary to perform the test, which
moreover is insensitive to moderate factor deficiencies.
- 3. Errors in collection technique can significantly affect
results.
It is important that the same size tube always be used for
testing and that the specimen be tilted uniformly.
Plasma Recalcification Time
The sensitivity of this test is somewhat improved by
diluting the plasma
This accomplishes three things:
1. It adjust the PRP closer to in vivo platelet count
2. It increases the sensitivity of the test system to
factor deficiencies.
3. It dilutes the natural inhibitors to coagulation that
are present
A normal control should run with each test.
ACTIVATED CLOTTING TIME TEST
Heparin-monitoring procedure
Uses diatomaceous earth (diatomite) as an activator
of the contact factor.
Requires to be kept warmed to a constant 37C by
taking a special incubator to the patients bedside.
The most reliable and rapid screen of the intrinsic and
common pathways.
ACTIVATED CLOTTING TIME TEST
PRINCIPLE
- Whole blood contains all the components
necessary to produce a clot when removed from the
veins and put into a glass tube.
- By adding an activator and keeping the blood at a
constant 37C.
REAGENTS
- 2 evacuated tubes containing 12mg of diatomite are
needed
ACTIVATED CLOTTING TIME TEST
PROCEDURE
1. Two tubes containing diatomite are brought to 37C in a
heat block at the patients bedside.
2.Using good venipuncture technique, at least 2 ml of blood is
drawn into a tube and discarded.
3.The tourniquet is removed, and the 1
st
tube of diatomite is
attached to the needle.
4. When blood starts to flow into the tube, the stopwatch is
started.
5. The tube is filled, mixed and placed in the heat block.
6. The procedure is repeated with the 2
nd
tube, and 2
nd
stopwatch is started.
7. After 60 seconds, the 1
st
tube is observed by tilting it at 5-
second interval until clot is formed, same procedure with test
tube 2.
ACTIVATED CLOTTING TIME TEST
REFERRENCE RANGE
- 75 to 120 seconds
The target range during heparin therapy is
- 140 to 185 seconds
INTERPRETATION
- Prolongation of the ACT is indicative of one or more
factor defects in the intrinsic or common pathways or the
presence of circulating anticoagulant such as heparin.
PARTIAL THROMBOPLASTIN TIME
Hemophiliacs have a prolong bleeding time.
This test is more sensitive to abnormalities in the early stages
than were previous test in intrinsic system.
An important refinement of the PTT was the addition of
negatively charged activators to the system, resulting in
significantly shorter clotting time.
Modification is APTT
(Activated Partial ThromboplastinTime)
- test of choice to screen for factor deficiencies of the
common and intrinsic pathways and also for monitoring
heparin therapy.
Activated Partial Thromboplastin Time
PRINCIPLE
- The APTT measures all factors except VII and XII.
Maximum activation of the contact factor is accomplished
by addition of the activator. Phospholipid is supplied to
substitute for platelet factor 3 (PF3).
- APTT is essentially same as a recalcification time of
plasma.
REAGENTS
1. Phospolipid with activators
2. 0.025 M CaCl
2
(as recommended by the manufacturer)
Activated Partial Thromboplastin Time
CONTROLS
Commercial lyophilized controls are available in midrange,
and extended ranges.
It is recommended that a normal control and at least on
abnormal control can be used.
In-house preparation of pooled/frozen plasma may be
used as controls.
Each laboratory must specify when controls are to be
tested, what the satisfactory control limits are, and, if
duplicates are run, how closely the values should agree.
Activated Partial Thromboplastin Time
PROCEDURE
1. Platelet-poor plasma (0.1ml) is added to 0.1mL of APTT
reagent and incubated at 37C for the period of time
specified by the reagent manufacturer ( approximately 3
to 5mins)
2. After incubation, 0.1mL of warmed CaCl2 is added, and
the time for clotting to occur is recorded.
REFERRENCE RANGE
The ranges differ according to the reagent, method and
instrument used. May extend from lower limit of 20
seconds to an upper limit of 45 seconds
Activated Partial Thromboplastin Time
INTERPRETATION
A prolonged APTT in the absence of heparin use
indicates a factor deficiency, an acquired circulating
anticoagulant such as lupus inhibitor, or an antibody to a
specific factor such as factor VII.
Most commercial reagents will demonstrate a prolong
APTT if any factor measured by the APTT is less than
40% to 50% of normal.
The APTT is also known as:
Kaolin Cephalin Clotting Time (KCCT)
Partial Thromboplastin Time with Kaolin
(PTTK).
Prothrombin Time
Is a blood test that measures the
time it takes for the liquid portion
(plasma) of your blood to clot.
A prothrombin time test can be used
to check for bleeding problems. PT
is also used to check whether
medicine to prevent blood clots is
working.
TEST FOR THE EXTRINSIC AND COMMON
PATHWAYS
PROTHROMBIN TIME
PRINCIPLE
The PT reference interval varies from site to
site depending on the patient population, type of
thromboplastin used, type of instrument used, and pH and
purity of the reagent diluent. Each center must establish its
own range for each new lot of reagents or at least once a
year.
This may be done by testing a sample of at least 20
specimens from healthy donors of both sexes spanning the
adult age range over several days and computing the 95%
confidence interval of the results. A typical PT reference
interval is 12.6 to 14.6 seconds.
PROTHROMBIN TIME
REAGENTS
1. Thromboplastin/CaCl
2
(PT reagent)
2. Controls (discussed in APTT) are needed.
PROCEDURE
1. Aliqouts of control and patient plasma are warmed
according to method being used.
2. The PT thromboplastin reagent is warmed by
incubating it at 37C for 3 to 5 mins. , and 0.2mL of PT
reagent is added to 0.1mL of plasma (patient or
control)
3. The clotting time is recorded.
PROTHROMBIN TIME
REFERENCE RANGE
Normal values differ with the reagent and method.
- 10 to 12 seconds (photo-optical systems)
- 12 to 14 seconds (manual methods)
INTERPRETATION
Prothrombin times may be reported in several ways;
1. Patient time (in seconds) with reference range.
2. Patient time with control time (in seconds) with
reference range.
3. Prothrombin ratio
(PT of the patient mean of reference range x 100)
Prothrombin Time Quality Control
The medical laboratory scientist tests normal and
prolonged control PPP specimens at the beginning
of each 8-hour shift or with each change of reagent.
Although lyophilized control PPPs are commercially
available, the laboratory manager may choose to
collect and pool PPP specimens from designated
subjects to make home-brew controls. In this case,
the specimens must be collected and managed
using the same tubes, anticoagulant, and protocol
that are used for patient plasma specimen collection.
The samples are pooled, tested, and aliquoted.
Regardless of whether commercial or locally
prepared controls are used, the control is tested
alongside patient specimens using the same
protocol as for patient PPP testing
The normal control result should be within the
reference interval and the prolonged control result
should be within the therapeutic range for warfarin. If
the control results fall within the stated limits in the
laboratory protocol, the test results are considered
valid.
If the results fall outside the control limits, the
reagents, control, and equipment are checked; the
problem is corrected; and the control and patient
specimens are retested. The operator records all the
actions taken. Control results are recorded and
analyzed at regular intervals to determine the long-
term validity of results
Use of Prothrombin Time as a Diagnostic Assay
The PT is performed diagnostically when any
coagulopathy is suspected. Acquired multiple
deficiencies, such as disseminated intravascular
coagulation (DIC), liver disease, and vitamin K
deficiency, all affect factor VII activity and are
detected through prolonged PT results. The PT is
particularly sensitive to liver disease, which
causes factor VII levels to become rapidly
diminished
Reporting of Prothrombin Time Results and
the International Normalized Ratio
The medical laboratory scientist reports PT results
to the nearest tenth of a second along with the PT
reference interval. If the PT assay is performed in
duplicate, the results are averaged, and the
average is reported.
In view of the inherent variations among
thromboplastin reagents, most laboratories report
the international normalized ratio (INR) for patients
with a stable anticoagulation response using the
following formula.
Factors That Interfere with the Validity of
Prothrombin Time (PT) Results
Limitations of Prothrombin Time
Anticoagulant volume must be adjusted when the
hematocrit is greater than 55% to avoid false
prolongation of the results.
Specimens must be inverted five times
immediately after collection to ensure good
anticoagulation, but the mixing must be gentle.
Clotted and visibly hemolyzed specimens are
rejected, because they give unreliable results.
Plasma lipemia or icterus may affect the results
obtained with optical instrumentation.
Heparin may prolong the PT. If the patient is
receiving therapeutic heparin, it should be noted
on the order and commented on when the results
are reported. The laboratory manager selects
thromboplastin reagents that are maximally
sensitive to oral anticoagulant therapy and
insensitive to heparin. Many reagent
manufacturers incorporate Polybrene into their
thromboplastin reagent to neutralize heparin.
The medical laboratory scientist may detect
unexpected heparin by using the TCT test, which
is described subsequently
Other Tests for Extrinsic and Common
Pathway
1. Stypven time
2. Prothrombin-proconvertin time
OTHER TESTS
STYPVEN TIME
- Utilizes the powerful coagulant properties of Russells
viper venom, obtained from the snake Vipera ruselli.
- This venom is capable of bypassing the action of factor
VII and directly activating factor X to Xa.
OTHER TESTS.
Prothrombin-proconvertin time
- Based on the earlier observations that minor deficiencies
can be more pronounced when the plasma is diluted.
- Today, the test has little value as on overall screen for
extrinsic system because of its insensitivity to factor V
and fibrinogen
- Also, with the current use of sodium citrate as the
anticoagulant of choice, the instability of factor V in no
longer a great problem.
END