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LEE AND WHITE METHOD with subsequent conversion of prothrombin t

P: The coagulation time of whole blood is the
length of time required for a measured amount
of blood to clot under specific conditions.
PROTHROMBIN TIME
>screening for extrinsic coag, course of oral
anticoag therapy, least sensitive to deficiency of
factor II
P: The calcium in whole blood is bound by
sodium citrate, thus preventing coagulation.
Tissue thromboplastin, to which calcium has
been added, mixed with plasma and clotting
time is noted.
ACTIVATED PARTIAL THROMBOPLASTIN TIME
>screening for intrinsic coag, inhibitors, monitor
heparin therapy
P: The calcium in whole blood is bound by
sodium citrate, thus preventing coagulation.
The plasma, after centrifugation, which contains
all intrinsic factors except calcium and platelets.
Calcium as an activator and phospholipid
substitute for platelets are added to plasma.
Clotting time is measured.
PLASMA RECALCIFCATION TIME (PLASMA
CLOTTING TIME)
>intrinsic coag, deficiency of platelets not
detected
P: Calcium chloride (substitute of calcium bound
by anticoag) is added to platelet-poor plasma
and clotting time is determined.
thrombin and fibrinogen to fibrin. Deficiencies
in Factor V, X, and prothrombin may be
detected.
REPTILASE TIME
>reptilase found in venom of Bothrops atrox
snake, test for functional fibrinogen when
thrombin time is prolonged due to heparin
>Dysfibrinogenemia- prolonged thrombin and
reptilase time
P: When reptilase is added to plasma, it acts by
releasing fibrinopeptide A from the fibrinogen
molecule. The resultant monomers polymerize
end to end, forming a clot.
THROMBIN TIME
>availability of functional fibrinogen, sensitive
detecting heparin inhibition, normal prolonged-
newborn, multiple myeloma.
P: A measured amount of thrombin is added to
plasma. Length of time to form a clot is measure
and recorded as thrombin time.
PREKALLIKREIN (FLETCHER FACTOR)
SCREENING TEST
P: Patients with Fletcher factor deficiency will
have a prolonged APTT. If the plasma + APTT
reagent mixture is incubated for 10 mis (instead
of routine 3 or 5 mins), the prolonged APTT will
be shortened, or almost normal, if the def is
due to Fletcher factor.
FACTOR XIII SCREENING TEST

STYPVEN TIME (RUSSEL’S VIPER VENOM TIME) > When factor XIII is present, the fibrin clot
formed is INSOLUBLE in 5 M Urea and 1%
>deficiency of prothrombin, fibrinogen, factor V
monochloroacetic acid when left standing for
and X, factor VII not detected
24 hrs. Generally, a level of 1% of factor XIII is
P: Russel viper venom is a thromboplastin like sufficient to make the clot insoluble to urea
substance that activates Factor X. This reagent
P: The patient’s plasma is clotted by the
is added to plasma, together w platelets and
addition of calcium chloride. Urea is added to
calcium chloride, activating coagulation process,
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the clot. If Factor XIII is absent in the patient’s
plasma, the clot is dissolved in less than 24 hrs.
FACTOR IDENTIFICATION (PT AND APTT
SUBSTITUTION TEST)
P: An APTT or PT is performed on the patient’s
plasma diluted 1:1 with:
1.) Adsorbed plasma (Factors V, VIII, XI, and
XII)
2.) Aged serum (Factors VII, IX, X, XI and
XII)
3.) Sodium chloride, 0.85%
4.) Normal control plasma
The patients undiluted plasma is also tested in
the same manner. Specific coag may be
detected by noting which reagent corrects the
APTT or PT.
QUANTITATIVE FIBRINOGEN
P: An excess amount of thrombin is added to
specimen of diluted plasma and the clotting
time is noted. Concentration of fibrinogen in
the unknown sample by comparing results with
clotting times of standard reference using
known amounts of fibrinogen. The higher the
clotting time, the lower the fibrinogen
concentration.
FACTOR V (II, VII, X) ASSAY
PT is performed on factor V deficient substrates
containing varying dilutions of patient’s plasma.
The amount of correction by the patient’s
plasma correlates with its factor activity level
and is compared to the results of PT performed
on varying dilutions of an assayed reference
plasma. The factor V content of the patient’s
plasma is expressed as the percentage of
normal.
FACTOR VIII (VIII:C) (IX, XI, XII) ASSAY
P: APTT is performed on factor VIII deficient
substrates containing varying dilutions of
patient’s plasma. The amount of correction by
the patient’s plasma correlates with its factor
activity level and is compared to the results of
APTT performed on varying dilutions of an
assayed reference plasma. The factor VIII
content of the patient’s plasma is expressed as
the percentage of normal.
VONVILLEBRAND FACTOR ANTIGEN (vWF:Ag)
>specific antigenic determinant (vWF:Ag)
> hemophilia A: decreased factor VIII:C, normal
to increase (vWF:Ag)
> von Willebrand disease: decrease to normal
both
P: Plasma dilutions containing the vWF antigen
are incubated in microwells coated with rabbit
anti-vWF Ab. During incubation, the plasma
vWF Ag binds to the anti-vWF Ab. The anti vWF
peroxidase conjugate (rgnt 2) is added, which
binds to the free vWF Ag forming a “sandwich”.
The bound peroxidase acts on substrate
orthophenylenediamine in the presence of
H202 to produce a color change that is directly
proportional to the conc. of vWF Ag present in
the plasma.
RISTOCETIN COFACTOR ASSAY
>property of the plasma vWF, resp for in vito
platelet agglutination in the precense of
ristocetin. Decreased- assoc w/ vW syndrome
P: Patient’s plasma is added to a standardized
mixture of platelets and ristocetin reagent. The
degree of resultant platelet aggregation is
measured by platelet aggregometer. This result
is then compared to a curve prepared from a
normal reference plasma, and the % conc. of
ristocetin cofactor determined.
HEPARIN (ANTI-Xa) ASSAY
>heparin binds with anti-thrombin III-
immendiate anticoag effect; chromogenic
method for assaying the amount of heparin
present in plasma.
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P: Patient’s plasma(containing heparin) is
incubated with a measured amount of anti-
thrombin III and factor Xa. A complex of
heparin, anti thrombin III, and factor Xa is
formed. Substrate reagent is added to the
mixture and during incubation the remaining
factor Xa catalyzes the release of
paranitroanilide from the chromogenic
substrate. It is the measured by
spectrophotometer. Inversely proportional to
the amount of heparin. The O.D. of each plasma
is read from a reference curve to determine the
conc of heparin present.
FIBRINOGEN DEGRADATION PRODUCTS
>primary fibrinolysis, DIC with secondary
fibrinolysis, Thrombo-Wellcotest procedure
P: Whole blood is added to thrombin(ensure
clotting) and soya bean enzyme inhibitors
(prevent breakdown of fibrin). After complete
clotting, the patient’s serum is diluted and
mixed with latex particles coated with anti-FDP.
If FDP are present, agglutination will occur.
D-DIMER TEST FOR FIBRIN DEGRADATION
PRODUCTS
>Fibrinosticon procedure
P: A dilution of the patient’s plasma is mixed
with latex particles coated with monoclonal
antibodies to D-dimer. If FDP containing the D-
dimer are present, agglutination will occur.
F.S. TEST FOR SOLUBLE FIBRIN MONOMER
COMPLEXES
>presence of SFMC in the plasma indicated that
thrombin has been generated.
P: Citrated plasma is incubated with RBC coated
with fibrin monomers. The presence of soluble
fibrin monomer complexes in the plasma will
cause the red cells to agglutinate. This will not
occur in normal plasma.
ETHANOL GELATION TEST
>detect presence of fibrin monomers in plasma,
screening for DIC, distinguish from primary
fibrinolysis
P: During the process of DIC, the level of fibrin
monomer in the blood increases. Sodium
hydroxide is added to the plasma to increase
the pH to above 7.70. Ethyl alcohol, added to
plasma, will cause ppt of any fibrin monomers
which may be present.
PROTAMINE SULFATE
>detect fibrin monomers by causing the
formation of fibrin strands or gel like clots
(paracoagulation)
>normal plasma, no fibrin monomer
P: Patient and control plasma are mixed with
varying dilutions of protamine sulfate. Each
tube is incubated at room temp for 30 mins and
then observed for fibrin strand or gel formation.
The protamine sulfate causes gel formation of
fibrin monomers or early fibrin slit products
when they are present in plasma.
CLOT LYSIS
>for increased fibrinolysis, detect large amount
of fibrinolytic activity
P: One of the tubes in clotting time is left in
incubator at 37 C and inspected for 8, 24, and
48 hrs. Second tube is placed in refrigerator
right after clotting for control. If the incubated
tube become fluid in less than 48 hrs, confirm
by filtering. If the refrigerated clot is still intact,
it may assumed that the clot lysis has taken
place inside the incubator. If the refrigerated
clot has also disapperead, it may due to
deficiency rather than clot lysis for both tubes.
“If no lysis in both thubes, report no lysis after
48 hrs”
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EUGLOBULIN CLOT LYSIS TIME
>screening fibrinolytic activity, more sensitive
than clot lysis, euglobulin fraction contains
plasminogen, plasminogen activator, and
fibrinogen.
>normally, clot lysis does not occur in less than
1 hr. Clot lysis in less time is indicative of
abnormal fibrinolytic activity.
P: Addition of 1% acetic acid to diluted plasma
causes the euglobulin portion of the plasma to
precipitate. After removing the supernatant, the
euglobulins are dissolved in a buffer solution.
Thrombin is added in order to clot the
euglobulins. The clot is incubated at 37 C, time
of complete clot lysis is noted.
CIRCULATING ANTICOAGULANT (INHIBITORS)
>specific inhibitors: Abs that are directed
against specific coagulation factors, assoc. with
bleeding
>nonspecific inhibitors: lupus anticoags,
paraproteins, FDP; not directed to single coag
factor, Lupus anticoag directed against
phospholipids, not associated with bleeding
>2 most common: Factor VIII and lupus
inhibitor. Inhibitors are usually detected by
prolonged APTT or PT, wc is not corrected by
addition of normal plasma.
P: The test is performed in methods where
there is an abnormal result (APTT or PT). In the
presence of an inhibitor, there will be little to
no correction of the clotting time when the
patient’s plasma is mixed with normal plasma. If
a factor deficiency exists, since only 50% of
plasma factors are necessary for normal
coagulation, the clotting time will show
significant correction when mixed with normal
plasma.
PLATELET NEUTRALIZATION PROCEDURE
>detects lupus anticoag; increased amount of
phospholipid is added to the test system to
minimize the effect of the phospholipid-
dependent anticoag, which produces a
shortened clotting time.
P: The patient’s platelet poor plasma is mixed
with a suspension of ruptured platelets, APTT
rgnt and calcium chloride. Clotting tie is noted
and compared with (1) similar structure
substituting sodium chloride for platelets , and
(2) an APTT performed on the same plasma
sample. If a lupus inhibitor is present, the
effects of the anticoag will be decreased or
bypassed by the freeze-thawed platelets and
the clotting time will be shorter than the
clotting times of both the mixture containing
saline and original APTT.
DILUTE RUSSEL VIPER VENOM TEST
>major type of antiphospholipids of LA:
cardiolipin.
>LA has no anticoag effect in vivo; not assoc
with bleeding
P: DVVtest reagent – single vial (Russell’s vper
venom, calcium, limited conc of phospholipid.
RVV in the presence of of factor V, phospholipid
and calcium will activate factor X, and begin the
coagulation mechanism till thrombin formation.
When this reagent is added to plasma
containing a LA, some of the phospholipid will
be neutralized by LA, thus limiting the amount
of phospholipid available for coag thus
prolonging the clotting time.
INHIBITOR ASSAY
>Neutralizing inhibitors (Factor V, VIII, IX, XI,
and XIII, fibrinogen, and vWF) : inhibit fibrin
formation by acttacking the active sites of
enzymes or cofactors.
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>Non-neutralizing inhibitors (Abs to factor VIII,
and X, prothrombin and vWF) bing to the
NONactive sites of enzymes and form
complexes that are rapidly cleared by the body.
FACTOR VIII INHIBITOR ASSAY (BETHESDA
METHOD)
P: In the presence of an inhibitor, some or all
factor VIII activity in the normal pooled plasma
of the mixture (patient:NPP) will be inhibited.
Mixtures are incubated at 37 C for 2 hrs. Factor
VIII is measured in all tubes. Residual factor
(Factor VIII not destroyed or neutralized by
inhibitor) is determined by comparing the
difference between the factor activity of the
patient:NPP mixture with that of the NPP.
Convert. One Bethesda unit of inhibitor
inactivates 50% of the factor VIII activity
present in 1ml of normal plasma in two hrs at
37 C
ANTITHROMBIN III
>chromogenic assay
P: The test plasma containing anti-thrombin III
is diluted in the presence of heparin and
incubated with an excess amount of thrombin.
The antithrombin III present in the plasma will
neutralized the thrombin by forming an
antithrombin III-thrombin-heparin complex.
Upon addition of substrate, the remaining
thrombin will catalyze the release of
paranitroanilide from the substrate. The
amount of this will be measure
spectrophotometrically. Inversely proportional
to the amount of antithrombin III. The optical
density of each plasma is read from a reference
curve to determine the conc of antithrombin III
present.
PLASMINOGEN ASSAY
P: The patient’s plasma is incubated wit an
excess of streptokinase rgnt. There will be
plasminogen-streptokinase complex. When the
substrate is added to the plasma-streptokinase
mixture, the plasmin-like activity present will
release pNA from the substrate. Measured.
Directly proportional to the amount of
plasminogen present in the plasma. Conc of
plasminogen is read from a Reference curve.
PROTEIN C CHROMAGENIC METHOD
P: Plasma is incubated with the protein C
activator. Upon addition of substrate, activated
protein C will release pNA. Measured. Directly
proportional to the conc of Protein C. Reference
curve.
PROTEIN C CLOTTING ASSAY
P: The amount of protein C in plasma is related
to the degree of correction obtained when
diluted plasma is incubated with protein C
deficient plasma in the presence of protein C
activator. The activated protein C inhibits factor
V and VIII, thus prolonging APTT. The clotting
time obtained for each plasma dilution in
inversely proportional to the percentage of
protein C activity read by a calibration curve.
PROTEIN S (ELISA METHOD)
P: Plasma dilutions containing protein S antigen
are incubated in microwells coated with anti-
protein S antibody. During incubation, the
plasma protein S antigen binds to the anti-
protein S Ab. The anti-protein S peroxidase
conjugate (rgnt 2) is added which will bind to
the free antigenic determinants of protein S,
forming a “sandwich”. The bound enzyme
peroxidase acts on OPD, in the presence of
H2O2 to produce a color change that is directly
proportional to the conc of protein S Ag seen in
plasma.
FREE PROTEIN S
P: The protein S bound to C4b BP is removed
from the plasma by ppting with polyethylene
glycol. Following incubation with this chemical,
the supernatant plasma contains only free
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protein S, which is assayed using the same
procedure describes for total protein S.
PROTEIN S CLOTTING ASSAY
P: The percentage of functional protein S
present in plasma is determined by the degree
of prolongation of the clotting time obtained
when diluted plasma is incubated with protein S
deficint plasma in the presence of activated
protein C and factor Va. Free protein S in the
patient’s plasma acts as a cofactor of activated
protein C, thus enhancing anticoag effect of
protein C on factor Va. The clotting time
obtained for each plasma dilution is inversely
proportional to the percentage of protein S
activity.
BLEEDING TIME
>screening test for disorders of platelet function
and von Willebrand disease.
P: A blood pressure cuff is placed on the
patient’s arm above the elbow, inflated, and
maintained at a constant pressure throughout
the procedure. One/two standardized incisions
are made on the volar surface of the forearm.
The length of time required for the bleeding to
stop is recorded.
TOURNIQUET TEST (CAPILLARY FRAGILITY
TEST)
P: An inflated blood pressure cuff on the upper
arm is used to apply pressure on capillaries for 5
mins. The arm is examined for petechiae.
CLOT RETRACTION
>Retraction: serum is expressed from the clot,
and the clot becomes denser.
>abnormal: Glanzmann, thrombocytopenia,
paraproteinemias
P: Fresh whole blood is placed in a 37C water
bath and inspected at 1,2,3, and 24 hours, for
the presence of retracted clot.
PLATELET AGGREGATION
>ADP
P: PRP is placed in a test well of the platelet
aggregometer. An aggregating reagent is added
to PRP, at the same time, optical density is
measured. As the platelets in the plasma clump,
the plasma becomes more clear and light
transmittance through the specimen increases.
These changes in optical density are recorded
by the instrument in the form of a graph. The
aggregometer also calculates the slope of the
aggregation curve and the amount (%) of
platelet aggregation.
PLATELET ADHESIVENESS TEST
SALZMANN METHOD
P: A platelet count is performed on both
specimens of blood. The number of platelets
collected through the glass bead collecting
system, will be lower than the number obtained
in routine venipuncture. This is because
platelets have adhered to the column of the
glass beads due to their adhesive
characteristics. The result of this procedure are
expressed as the percentage of platelets
retained in the glass bead.
HEPARIN ASSOCIATED THROMBOCYTOPENIA
TEST (HATT TEST)
P: Patient’s plasma is incubated with PRP from a
normal control in the presence of heparin. The
degree of platelet aggregation observed is
measured by an aggregometer and compared to
aggregation obtained in the absence of heparin.
If the heparin induced antibody is present,
platelet aggregation will occur in the presence
of heparin.