AVAILABLE TESTS AND CLINICAL INDICATIONS:

PLATELET FUNCTION SCREEN (PFA):

Used to detect platelet dysfunction induced by intrinsic platelet defects, von Willebrand disease or
exposure to platelet inhibiting agents such as acetylsalicylic acid (ASA) or medications containing ASA.
The closure time is prolonged by thrombocytopenia (platelet counts < 100,000) and anemia.

PROTHROMBIN TIME (PT):

The prothrombin time (PT) measures the integrity of the extrinsic and common pathways of coagulation
(factors VII, X, V and II). The PT can be used to: 1) monitor warfarin therapy, 2) screen for inherited and
acquired deficiencies of factors in the extrinsic (factor VII) and common pathway (factors V, X and II) and
3) screen for inhibitors to factors VII, V, X and II.

ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT or PTT):

The activated partial thromboplastin time (APTT) measures the integrity of the intrinsic and common
pathways of coagulation (factors XII, XI, IX, VIII, X, V, and II). The APTT can be used to: 1) monitor
heparin therapy, 2) screen for congenital and acquired deficiencies of factor VIII (hemophilia A), factor IX
(hemophilia B), Von Willebrand disease and other congenital or acquired deficiencies of the intrinsic and
common pathway factors, 4) screen for inhibitors to factors VIII, IX, XI, XII, X, and V, and II, and 5)
screen for the presence of a lupus anticoagulant. Heparin contamination must be excluded.

MIXING STUDY (APTT or PT):

Mixing studies are used to determine whether a prolonged PT or APTT is due to a factor deficiency or an
inhibitor. Mixing studies are based on two principles: 1) the inhibitor is present in excess, and if present,
it will inhibit normal and patient plasma, and 2) that 50% of any factor is enough to yield a normal test
result. Correction into the normal range indicates a factor deficiency. Lack of correction suggests the
presence of an inhibitor. Heparin contamination must be excluded. Mixing studies cannot be performed
when the PT or APTT is within 2 or 5 seconds of normal range, respectively.

THROMBIN TIME (TT):

The thrombin time (TT) screens for defects in the conversion of fibrinogen to fibrin. This test is a useful
screening test for abnormal fibrin formation, including: 1) the diagnosis of hereditary fibrinogen
deficiencies and dysfibrinogenemia, 2) DIC, 3) liver disease and 4) fibrinolysis. The thrombin time is
prolonged by abnormally high fibrin degradation products, low amounts of heparin, thrombolytic agents,
and direct thrombin inhibitors.

REPTILASE TIME (RT):

The reptilase time is an alternative screening test for abnormal fibrin formation and can be used to: 1)
identify heparin or a heparin-like inhibitor as a cause of a prolonged TT and 2) evaluation of patients with
quantitative or qualitative fibrinogen disorders. Unlike the thrombin time, the reptilase time is unaffected
by heparin.

EUGLOBULIN CLOT LYSIS TIME (SEND OUT TEST):

The euglobulin clot lysis time is a screening procedure for the measurement of fibrinolytic activity and
can detect either primary or secondary fibrinolysis. A result less than 2 hours indicates an increase in
fibrinolysis. Prolonged results indicate inadequate fibrinolysis.

DISSEMINATED INTRAVASCULAR COAGULATION SCREEN:

The most useful panel to screen for disseminated intravascular coagulation (DIC) includes D-dimer,
prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen and platelet count.
However, these tests are not specific for DIC.

FIBRINOGEN ACTIVITY (FACTOR I):

This test measures the concentration of functional fibrinogen and can be used to evaluate 1) disseminated
intravascular coagulation (DIC), 2) liver disease, 3) monitor individuals undergoing thrombotic therapy,
and 4) diagnose inherited and acquired coagulopathies (afibrinogenemia, hypofibrinogenemia, and
dysfibrinogenemia). Fibrinogen is an acute phase reactant and can be increased in inflammatory states,
pregnancy and malignancies. High concentrations of heparin or fibrin degradation products can result in
abnormal results.

D-DIMER QUANTIFICATION:
Fibrinolysis is mediated by plasmin, which degrades fibrin clots into D-dimers and fibrin degradation
products (FDP). Plasmin can also degrade intact fibrinogen, generating FDP that are detected in FDP
assays. Elevated D-dimers may be seen in DIC, thromboembolism, systemic fibrinolytic states and liver
disease due to decreased clearance. D-dimers can also be elevated postoperatively, with significant
bleeding, hemodialysis, eclampsia, sickle cell crisis and other conditions. Cancer patients often have
positive D-dimers and FDP, usually representing low-grade, chronic DIC. Serial D-dimers may be used to
monitor resolution of a known thromboembolism.

HEPARIN-PF4 IgG ANTIBODIES (HIT):

Immune-mediated heparin induced thrombocytopenia (HIT type II) is due to an IgG antibody that
recognizes heparin bound to platelet factor 4 (PF4). A strong-positive ELISA result with positive
confirmatory testing in a high probability patient is strongly suggestive of heparin induced
thrombocytopenia (HIT). However, it has been reported that a high incidence of anti-heparin PF4
antibodies is observed in the plasma of patients who have undergone cardiopulmonary bypass. This does
not necessarily mean HIT. Therefore, results should be used in conjunction with clinical findings, platelet
counts and other laboratory tests. In patients with HIT, the most common clinical complication is
thrombosis (30-40% incidence) despite thrombocytopenia, and non-heparin anticoagulation is typically
required. Although the sensitivity of this testing is high (>90%), false negatives can occur. If there is a
strong clinical suspicion of HIT and test results are negative, repeat testing within a few days may be
useful since antibody titer can increase rapidly over time.

HEPARIN LEVEL- Xa INHIBITION FOR LMWH (chromogenic): The heparin anti-factor Xa assay is used
to monitor heparin therapy. It can be used to monitor standard, unfractionated heparin when the APTT
cannot be used, such as in patients with lupus anticoagulant. The anti-Xa assay is the only test that works
for low molecular weight heparin such as Lovenox or Fragmin , or the pentasaccharide fondaparinux.

HYPERCOAGULATION PANEL:
This panel tests for the presence of a hereditary or acquired deficiency of the more common factors
predisposing patients to thrombosis. It includes ATIII activity, fibrinogen activity, Protein C activity,
Protein S activity, APC resistance ratio, Factor V Leiden (PCR) and prothrombin gene mutation (PCR).
This panel requires 2 lavender tubes and 2 blue tubes. One lavender tube must be kept unspun and at
room temperature. One lavender tube must be kept on ice. The blue top tubes must be spun until they are
platelet free (<10,000) and the plasma frozen in appropriately labeled tubes.

PT and PTT
HEPARIN NEUTRALIZATION (performed if APTT is prolonged)
ANTI-THROMBIN III ACTIVITY
PROTEIN C ACTIVITY
PROTEIN S ACTIVITY
APC RESISTANCE RATIO
HOMOCYSTEINE
DNA-PROTHROMBIN MUTATION
FACTOR V LEIDEN
FIBRINOGEN

HEPARIN NEUTRALIZATION:
Heparin contamination of specimens can cause an unexpectedly prolonged APTT. Heparinase
(Hepzyme) can be used to determine if the APTT prolongation is due to heparin (UFH and LMWH). In
addition, heparinase can be used to remove heparin from samples to perform coagulation tests that
demonstrate heparin interference.

ANTITHROMBIN III ACTIVITY:

A deficiency of antithrombin III (AT III), a natural anticoagulant protein, leads to a hypercoagulable state
with an increased risk for venous thrombosis. Acquired AT III deficiencies are more common than
hereditary deficiencies. An acquired AT III deficiency has been described in DIC, septic shock, nephrotic
syndrome, liver disease, in L-asparaginase treatments and with heparin therapy or contamination.

PROTEIN C ACTIVITY:

Protein C is a natural anticoagulant protein that uses protein S as a cofactor to degrade factors V and
VIII. Deficiency of protein C leads to a hypercoagulable state with an increased risk for venous
thrombosis. Protein C levels are decreased by warfarin. The functional assay cannot be performed in
patients on hirudin or argatroban anticoagulation.

PROTEIN S ACTIVITY:
Protein S is a nonenzymatic cofactor required for the anticoagulant activity of protein C. Deficiency of
protein S leads to a hypercoagulable state with an increased risk for venous thrombosis. Protein S levels
are decreased by estrogen, pregnancy, inflammation, infection, acute phase reactions, nephrotic
syndrome, and warfarin. The functional assay cannot be performed in patients on hirudin or argatroban
anticoagulation.

ACTIVATED PROTEIN C (APC) RESISTANCE:

The activated form of protein C (APC) degrades Factor Va and Factor VIIIa and provides physiologic antithrombotic activity. APC resistance is the inability if protein C to cleave factors V and/or factor VIII.
Resistance to activated protein C is a condition which leads to a hypercoagulable state with an increased
risk of venous thrombosis. The majority of cases (85-90%) are explained by a mutation in the Factor V
gene (FVL).

HOMOCYSTEINE LEVELS:
Homocysteine is a thiol containing amino acid formed by demethylation of methionine. It is metabolized
by remethylation to methionine or by transsulfuration to cysteine. Elevated homocysteine levels may
occur as a result of inherited disorders which alter enzyme activity in the transsulfuration and
remethylation pathways (eg, MTHFR gene mutation). Alternatively, nutritional deficiencies of essential
cofactors of the enzymes including cobalamin (vitamin B12), folate or pyridoxine (vitamin B6), can results
in blockade of homocysteine pathways. Plasma homocysteine may be used as a graded risk factor for
cardiovascular disease (<11 mmol/L: desirable; 11-14 mmol/L: intermediate; 15-29 mmol/L: high and >29
mmol/L: very high).

DNA-PROTHROMBIN G20210A MUTATION:

Prothrombin G20210A mutation is a common hereditary predisposition to venous thrombosis. A
polymerase chain reaction (PCR)-based assay is used to determine the presence of this specific mutation
at nucleotide position 20210 in the prothrombin gene.

Factor V LEIDEN MUTATION (FVL):

Individuals with factor V Leiden have a mutation at one of the arginine cleavage sites in factor V, such that
factor V resists degradation by activated protein C. This is the most prevalent hereditary predisposition to
venous thrombosis. A polymerase chain reaction (PCR)-based assay is used to determine the presence of
this specific mutation.

FIBRINOGEN (FACTOR I):

This test measures the concentration of functional fibrinogen and can be used to evaluate 1) disseminated
intravascular coagulation (DIC), 2) liver disease, 3) monitor individuals undergoing thrombotic therapy,
and 4) diagnose inherited and acquired coagulopathies (afibrinogenemia, hypofibrinogenemia, and
dysfibrinogenemia). Fibrinogen is an acute phase reactant and can be increased in inflammatory states,
pregnancy and malignancies. High concentrations of heparin or fibrin degradation products can result in
abnormal results.

PLASMINOGEN (SEND OUT TEST):

Plasminogen is the precursor of plasmin, which lysis fibrin clots. Hereditary plasmin deficiency is rare
and it may predispose to venous thrombosis. The test is intended for the quantitative measure of
plasminogen levels and may be considered in patients with familial venous thrombosis and no evidence
for a more common hypercoagulable state or to monitor thrombolytic treatments.

LUPUS ANTICOAGULANT PANEL AND ANTI-PHOSPHOLIPID ANTIBODIES:

Lupus anticoagulants (LAs) and antiphospholipid antibodies are a heterogeneous group of
autoantibodies of the IgG, IgM or IgA subtypes that are directed against negatively charged phospholipids
or complexes of phospholipids with proteins. The LAs prolong phospholipid-based in vitro clotting assays
(PT and/or APTT) and are not corrected by mixing patient plasma with normal plasma. The lupus
anticoagulant reflexive panel includes PT, APTT, APTT (or PT) mixing study, Russell's Viper Venom Test
and Staclot-LA. The lupus anticoagulant panel requires 3 blue top tubes. The tubes must be spun down
until they are platelet free (<10,000) and the plasma frozen in appropriately labeled tubes.

In contrast, antiphospholipid antibodies may not be associated with abnormalities in coagulation

screening tests (PT or APTT) and include anticardiolipin antibodies (ACAs), anti beta 2-glycoprotien I
antibodies (b2-GPI) and anti-phosphatidylserine antibodies. These autoantibodies are detected by
ELISA. The test requires a red top tube and are send out tests.

The presence of LA or antiphospholipid antibody is considered to be a significant risk factor in patients

with otherwise unexplained thrombosis and are often present in women who have recurrent fetal loss.

LUPUS ANTICOAGULANT REFLEXIVE PANEL

PT
PTT-LA
DILUTE RUSSELL VIPER ENOM TEST (DRVVT)
THROMBIN TIME (PERFORMED IF PTT IS PROLONGED
REPILASE TIME (PERFORMED IF TT IS PROLONGED)
PTT-HEPARIN NEUTRALIZED (PERFORMED IF PTT IS PROLONGED)
1:1 MIXING STUDIES (PERFORMED IF PTT OR DVVT ARE PROLONGED)

STACLOT LA

ANTIPHOSPHOLIPID ANTIBODIES (SEND OUT TEST)

ANTI CARDIOLIPIN ANTIBODIES, IgG AND IgM (ELISA)
ANTI- BETA 2-GLYCOPROTIEN I ANTIBODIES, IgG, IgM AND IGgA (ELISA)

SCREENING TESTS:
The PTT-LA and DRRV are screening tests used to detect the presence of a lupus anticoagulant (LA). If
these tests are negative, then no further testing is performed. If the tests are prolonged, thrombin time,
reptilase time, heparin neutralization, mixing studies and confirmatory assays are added.

THROMBIN TIME and HEPARIN NEUTRALIZATON:

If the PTT is prolonged, the thrombin time is added. If the thrombin time is normal (no evidence of
heparin), a PTT 1:1 mix will be added. If the thrombin time is prolonged, then reptilase time is added. If
the reptilase time is normal, PTT heparin neutralization is added. If heparin neutralization is prolonged,
then a PTT 1:1 mix will be added.

MIXING STUDY (1:1):

Mixing studies are used to determine whether a prolonged APTT or DRVVT is due to a factor deficiency or
an inhibitor. Mixing studies are based on two principles: 1) the inhibitor is present in excess, and if
present, it will inhibit normal and patient plasma, and 2) that 50% of any factor is enough to yield a
normal test result. Correction into the normal range indicates a factor deficiency. Lack of correction
suggests the presence of an inhibitor.

DILUTE RUSSELL VIPER VENOM TEST:

The dilute Russell Viper Venom Confirm is intended to confirm the presence of a lupus anticoagulant (LA)
in citrated plasma. Russell viper venom directly activates factor X and is not affected by contact factor
abnormalities or factor VIII deficiencies/antibodies. Persistence of the positive test must be demonstrated
on a separate blood sample collected at least 12 weeks apart. For negative results, consider repeat testing
if clinical suspicion remains high or test for anticardiolipin or beta-2 glycoprotein 1 antibodies is
recommended.

LUPUS ANTICOAGULANT SCREEN (STACLOT LA):

The Staclot LA test is used to detect lupus anticoagulants (LAs). The patients plasma is incubated with
and without hexagonal phase phosphatidylethanolamine (HPE). An APTT is performed on both tubes
using a lupus anticoagulant sensitive reagent. If LAs are present in the test plasma, it would be neutralized
by HPE, and this would result in the shortening of the clotting time. Heparin levels greater than 1 IU/ml
and thrombin inhibitors may lead to falsely positive results. Persistence of the positive test must be
demonstrated on a separate blood sample collected at least 12 weeks apart. For negative results, consider

repeat testing if clinical suspicion remains high or test for anticardiolipin or beta-2 glycoprotein 1
antibodies is recommended.

BETA-2 GLYCOPROTEIN I ANTIBODIES (SEND OUT TEST):

Beta-2 Glycoprotein I is a protein cofactor that has a high binding affinity for negatively-charged
phospholipids forming phospholipid-protein complexes. Anti-beta-2 Glycoprotein I antibodies are more
relevant for antiphospholipid antibodies with fewer false positive results than the anticardiolipin or
antiphosphatidylserine assay. Anti-beta-2 glycoprotein I antibodies are detected by ELISA.

ANTICARDIOLIPIN ANTIBODIES (SEND OUT TEST):

Anticardiolipin antibodies are antibodies directed against phospholipid-binding proteins and are detected
by ELISA.

FACTOR ACTIVITY:
The factor activity tests are used to determine the functional activity of factors in the extrinsic, intrinsic
and common pathways of coagulation.

PROTHROMBIN (FACTOR II) ACTIVITY (SEND OUT)

FACTOR V ACTIVITY (SEND OUT)
FACTOR VII ACTIVITY (SEND OUT)
FACTOR VIII ACTIVITY
FACTOR IX ACTIVITY
FACTOR X ACTIVITY (SEND OUT)
FACTOR XI ACTIVITY (SEND OUT)
FACTOR XII ACTIVITY (SEND OUT)

FACTOR XIII SCREEN (SEND OUT)

The clot solubility test is used to detect deficiencies or inhibitors to Factor XIII. Clots are suspended in 5
M urea which will disrupt hydrogen bonds. If Factor XIII level is <5%, then the clot will dissolve in 24
hours.

FACTOR INHIBITORS:
The inhibitor assays are used to determine the amount of immunoglobulin in a patient. Inhibitors occur
in four clinical situations: hemophilia, post partum, immunologic disorders (e.g. RA, SLE, UC, eg) and old
age without underlying disease. Factor inhibitor assays cannot be performed in patients with normal
factor activity levels. This panel requires 2 blue tubes. They must be spun down until they are platelet free
(<10,000) and the plasma frozen.

FACTOR VIII AND FACTOR IX INHIBITORS:

The amount of inhibitor present can be calculated in Bethesda units. One Bethesda unit is defined as the
amount that will inactivate 50% of the FVIII present.

VON WILLEBRAND PANEL:

This panel screens for von Willebrands Disease. It includes Factor VIII activity, von Willebrand factor
(vWF) activity (ristocetin cofactor assay) and von Willebrand antigen. This panel requires 2 blue tubes.
They must be spun down until they are platelet free (<10,000) and the plasma frozen.

FACTOR VIII ACTIVITY:

measures the activity of FVIII which is decreased in some forms of VWD.

VON WILLEBRAND ANTIGEN:

measures the quantity of von Willebrand factor (vWF) in patient plasma.

RISTOCETIN COFACTOR ASSAY (SEND OUT TEST):

measures the functional level of vWF protein in the plasma. The vWF activity is reduced in type 1 and 2
von Willebrand Disease and absent in type 3 vWD.

VON WILLEBRAND MULTIMER ANALYSIS (SEND OUT TEST):

Subclassification of von Willebrand Disease is based on the distribution of vWF multimers. vWF
multimers are detected using SDS-agarose gel electrophoresis with radiolabeled antibody detection or
transblotting and enzyme-linked antibody detection. Von Willebrand factor levels are influenced by ABO
blood type. The following conditions may elevate vWF and mask the diagnosis of von Willebrand Disease:
pregnancy, oral contraceptives, liver disease, inflammation, exercise and stress.