Clinical Coagulation Laboratory
A division of Duke University Regional Referral Laboratory Services
Beware of falling CLOTS!
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Mechanical clot detection using the ST4 |
Organon Teknika MDA 180 Analyzer |
Chromogenic assays on the ACL 300+ |
COAGULATION TEST DESCRIPTIONS
The coagulation tests available from the Clinical Coagulation Laboratory are breifly reviewed in this section.
Emphasis is placed on sampling conditions specific for a given test and clinical conditions detected by this
test. Current normal ranges are included for some tests especially if the normal range is unlikely to be
affected by changes in reagents. However, NORMAL RANGES ARE SUBJECT TO CHANGE; therefore, the normal range
for a given test should be verified by checking the current computer report or calling the Clinical Coagulation
Laboratory before decisions regarding patient management are made.
The Coagulation Laboratory offers a variety of assays designed to aid the physician in diagnosing and managing
patients with coagulopathies. Many of these coagulation assays have both a functional (activity) and quanitative
(antigenic) method for measuring pecific coagulation proteins. The Clinical Coagulation Laboratory recommends
the functional assays as the initial method of choice. These functional assays more closely mirror the physiological
action of these proteins, while antigenic assays do not assure normal function.
PLEASE CALL 919-684-6366 FOR TEST CPT CODE
ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Platelet poor plasma is incubated with a platelet membrane substitute (phospholipid) and a factor XII activator, and
then is recalcified. The clotting time is measured. All procoagulant factors except VII and XIII are measured by
this assay. The normal range can be obtained with each lot of reagent (approximately once a year). The current normal
ranges can be obtained from the computer generated test result report or by calling the Clinical Coagulation Laboratory.
The aPTT will prolong when: 1) one of the aPTT based clotting factors decreases to less than about 30-40% of normal (exact
cuttoffs differ between factors and with different aPTT reagents); 2) an inhibitor to a specific clotting factor is
present; or 3) a nonspecific inhibitor of the intrinsic pathway (heparin or lupus anticoagulant) is present. A normal
aPTT does not rule out the existence of a possible clinically significant mild clotting factor deficiency since even a
slight prolongation of the clooting time which corrects with mix suggests less than about 30-40% factor activity.
ACTIVATED PROTEIN C RESISTANCE ASSAY (APC) aPTT-BASED
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
This aPTT based assay is a screen for the factor V (FV) Leiden mutation ( R 509 Q) that has been implicated in thrombotic
events. Activated protein C (APC) has the ability to recognize and degrade its physiologic substrates, FV and factor VIII
(FVIII), and can be monitored by an aPTT assay. The FV Leiden mutation prevents APC from binding with FV, thus preventing
the APC from degrading FV. This assay is performed in two parts: first, a baseline aPTT (without added APC) is performed;
next, the aPTT is performed with APC added. The natural activity of APC is to degrade FV, which can be evidenced by a
prolongation of the aPTT. The presence of the FV Leiden mutation prevents APC from degrading FV, therefore no prolongation
of the aPTT is seen. Two ratios are then calculated: first, the ratio of the aPTT without APC to the aPTT with APC is
calculated; next, the ratio of the patient is compared to the ratio of the normal control (normalized ratio). A normalized
ratio of <0.86 is considered to be resistant to APC.
ACTIVATED PROTEIN C RESISTANCE ASSAY (APC) PT-BASED
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
This assay is a PT-based assay that can be used to detect activated protein C (APC) resistance in patients on anticoagulant
therapy (heparin or warfarin) or with lupus anticoagulants. This assay is also independent of the baseline concentration
of factor V in the patient sample. Serial dilutions of patient plasma are prepared and mixed with factor V deficient plasma.
These plasma samples are then clotted by sequential addition of tissue factor (thromboplastin) and either Ca2+ or Ca2+ with
APC. The presence of APC will cause the clotting time to prolong in normal individuals, but will have much less of an effect
on patients who are APC resistant. The differences in the clotting time (with and without APC) for the different dilutions
of the patient samples are plotted and compared to normal reference standards. APC resistance is determined by comparing
the prolongation of the patient sample with the normal reference sample at comparable sample dilutions.
ACTIVATED PROTEIN C RESISTANCE ASSAY (FACTOR V LEIDEN)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, citrated whole blood (refrigerated or room temp)
In over 90% of cases, activate protein C (APC) resistance is due to a single point mutation in the gene for
factor V (G--->A) at position 506), resulting in replacement of the arginine residue at position 506 with a
glutamine. The resultant molecule, referred to as Factor V Leiden, is less effficiently degraded by APC than
the normal factor V, which results in the APC resistance observed in the plasma based assays. It is recom
mended that DNA based analyses be performed on all patients with abnormal plasma-based APC resistant assays
in order to confirm the diagnosis as well as to determine the genotype of the affected individual. These
results are important in guiding clinical decisions making for the patient with APC resistance.
This assay utilizes genomic DNA isolated from the buffy coat to determine the genotype of the patient. A
portion off the factor V gene is amplified by polymerase chain reaction (PCR), following which the DNA is
then restricted with the restriction enzyme Mnll. The DNA is then analyzed by agarose gel electrophoreses
and compared to restricted samples from a normal individual and an individual who is homozygous for the
Factor V Leiden mutation.
ALPHA 2 PLASMIN INHIBITOR LEVELS (Alpha-2-Antiplasmin)
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Patient plasma is incubated with human plasmin, then residual plasmin activity is measured by a reaction with
a specific chromogenic substrate. The amount of free chromogenic material is inversely proportional to the
amount of functional alpha-2-plasmin inhibitor in the plasma. reduced plasma levels of alpha-2-plasmin inhibitor
may occur in DIC, liver disease, or during thrombolytic therapy. Rarely, a congenital deficiency may exist that
results in a bleeding disorder.
ANTICARDIOLIPIN ANTIBODIES (IgG, IgM, IgA)
TAT: 2/Week
Requirements: 2 tube-2cc/tube, frozen serum
Antibodies to cardiolipin and other negatively charged phospholipids occur most commonly in patients with systemic lupus
erythematosus or SLE like disorders, but may also be seen in patients with other autoimmune diseases, cerebral infarctions,
and recurrent deep venous thrombosis. The term anti-cardiolipin syndrome refers to a group of patients exhibiting the
following symptoms: high titers of anticardiolipin antibodies (ACA), thrombosis, thrombocytopenia, and recurrent fetal loss.
The role played by ACA in thrombotic events is speculative. It has been proposed that ACA may facilitate thrombosis by cross
reacting with phospholipid antigens present on endothelial cell membranes. This antibody binding prevents the release of
arachidonic acid; thus reducing the production of prostacyclin with increased platelet aggregation. ACA may also inhibit
prekalikrein or plasminogen activator causing a decrease in fibrin clearance. The ACA response is composed of antibodies
bearing different isotypes (IgG, IgM, IgA). There is no statistical association between any single isotype with thrombosis,
fetal loss, or thrombocytopenia. However, the presence of high titers of IgG, ACA in the majority of patients with these
symptoms suggest that this finding may be the most important in determining susceptibility to thrombotic complications.
ANTITHROMBIN III FUNCTIONAL ASSAY (Activity Assay)
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Dilute patient plasma is mixed with a known amount of thrombin in the presence of excess heparin and then a specific
thrombin chromogenic substrate is added. Residual thrombin not bound to ATIII reacts with the chromogenic substrate.
The amount of functional ATIII inversely related to the amount of chromogenic substrate hydrolyzed by the residual
thrombin. Some patients with recurrent venous or arterial thrombosis, have congenitally reduced or dysfunctional ATIII
molecules. Extensive thromboses, protein wasting nephropathies or enteropathies, heparin, DIC, liver disease, or
extra-corporeal circulation have all been implicated as causes of an acquired deficiency of ATIII.
ANTITHROMBIN III ANTIGEN (Immunologic Assay)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Patient plasma is introduced into agar containing antibody to ATIII and zones of precipitation are measured. The
diameter of the zone is directly proportional to the concentration of ATIII. A normal antigenic level does not assure
adequate function of the protein; therefore, a functional level is usually the Preferred assay. The antigen level is
most often obtained to assess for a qualitative abnormality of the protein.
AUTOMATED BLOOD COUNT
TAT: Daily
Requirements: 1 tube-5cc/tube Lavender (EDTA) Vacutainer
The Automated Blood count consists of Hgb, Hct, RBC, MCV, MCH, MCHC, PLT, WBC and auto differential lymphocyte (1,2), monocyte (1,2), neutrophil (1,2), eosinophil (2),
basophil (2), percentages. The Automated blood counts are used to evaluate anemia, inflamatory diseases, infections and leukemia. The ABC can also provide
information about the state of hydration of a patient and other red cell and platelet abnormalities may also be detected.
Note: three part diff (1), five part diff (2)
AUTOMATED BLOOD COUNT WITH DIFFERENTIAL
TAT: Daily
Requirements: 1 tube-5cc/tube Lavender (EDTA) Vacutainer
The Automated Blood count consists of Hgb, Hct, RBC, MCV, MCH, MCHC, PLT, WBC and auto differential lymphocyte (1,2), monocyte (1,2), neutrophil (1,2), eosinophil (2),
basophil (2), percentages. The Automated blood counts are used to evaluate anemia, inflamatory diseases, infections and leukemia. The ABC can also provide
information about the state of hydration of a patient and other red cell and platelet abnormalities may also be detected.
Note: three part diff (1), five part diff (2)
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Bernard Soulier |
Grey Platelet Syndrome |
PlateletsNormal Blood Film |
BLEEDING TIME
TAT: Daily
Requirements: Must be performed by DUMC technologist † († Local Customer only)
The bleeding time is performed with a commercial bleeding time device for standardization and reproducibility. Vessel
integrity, vWF activity and platelet function are assessed. Normal: 3-9.5 minutes. The bleeding time is the only
physiologic test of primary hemostasis; however, both its sensitivity and specificity are limited. The test is not a
good indicator of bleeding risk when used alone, but can help support a clinical impression. This test is available
only to local customers and patients referred to Duke University Medical Center.
D-DIMER: LATEX AGGLUTINATION ASSAY (D-dimer assay)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Plasma is mixed with latex beads coated with an antibody to the Fragment D-dimer domain of fibrin. The D-Dimer epitope
is formed by the cross-linking of fibrin monomer D-domains by factor XIIIa. Fragment D-dimer appears in the blood only
after plasma degrades cross-linked fibrin. The assay is a highly specific measurement of fibrin (not fibrinogen) degradation.
Normal plasma does not have detectable amounts of fragment D-dimer. Fragment D-dimer levels increase when fibrin is lysed by
thrombolytic therapy, in DIC, during acute thromboembolic episodes, or after recent trauma or surgery.
D-DIMER, TITERED: LATEX AGGLUTINATION ASSAY (D-dimer assay)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Plasma is serial diluted and mixed with latex beads coated with an antibody to the Fragment D-dimer domain of fibrin. The
D-Dimer epitope is formed by the cross-linking of fibrin monomer D-domains by factor XIIIa. Fragment D-dimer appears in the
blood only after plasma degrades cross-linked fibrin. The assay is a highly specific measurement of fibrin (not fibrinogen)
degradation. Normal plasma does not have detectable amounts of fragment D-dimer. The D-dimer, Titered test is only ordered
for patients with results >1.25 ug/mL on the latex agglutination screen. Fragment D-dimer levels increase when fibrin is
lysed by thrombolytic therapy, in DIC, during acute thromboembolic episodes, or after recent trauma or surgery.
D-DIMER (ELISA) ASSAY (D-dimer assay)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
The presence of XL-FDP in plasma was first demonstrated by immunoprecipitation and gel electrophoresis techniques.
Development of the monoclonal antibody, DD-3B6, which recognizes D-dimer and degradation fragments containing the
D-dimer epitope, has enabled measurement of XL-FDP by enzyme immunoassay. DD-3B6 is covalently bound to
microtiter wells. When exposed to plasma or standard, this capture antibody immobilizes fragments containing the
D-dimer epitope. The EIA plate is washed after the capture and a tag antibody (DD-1D2), conjugated to horseradish
peroxidase, is added and binds to immobilized XL-FDP fragments. Color is developed by addition of substrate
enabling the estimation of XL-FDP levels.
Plasmin digests XIIIa crosslinked fibrin generating a heterogeneous array of XL-FDP derivatives. D-dimer is the
smallest plasmin-resistant fragment and elevated levels indicate clot lysis. Elevated XL-FDP levels are seen in
many vascular conditions including deep vein thrombosis (DVT), pulmonary embolism (PE), disseminated intravascular
coagulation (DIC), and other thrombotic conditions. This EIA assay does not cross-react with fibrinogen or its
degradation products, enabling reliable assessment of plasma XL-FDP.
DILUTE RUSSELL VIPER VENOM SCREEN (dRVVT)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma.
The dilute Russell Viper venom time (dRVVT) is a test used primary to screen for antibodies to phospholipids (i.e., lupus
anticoagulants). Russell Viper venom activates factor X (FX) and factor V (FV), thus initiating the clotting Cascade without
requiring clotting factors proximal to FX (i.e., factors XII, XI, IX, VIII or VII). The dRVVT is a phospholipid dependent
clotting test similar to the aPTT. By diluting the phospholipid in the assay, the dRVVT becomes more sensitive to the presence
of a phospholipid antibody. A patient’s plasma is considered to be abnormal when the ratio of the dRVVT result on a mixture of
patient and normal plasma to the normal plasma alone equals or is greater than 1.2.
DILUTE RUSSELL VIPER VENOM MIX / CONFIRM
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma.
A dRVVT mixing study (patient plasma 1:1 normal plasma) may be used to differentiate between deficiencies of factors X, V, II
or fibrinogen and the presence of inhibitors (either a phospholipid antibody or a specific inhibitor to factors X, V, or II).
Unlike the aPTT, the dRVVT test is not influenced by deficiencies or antibodies to factors XII, XI, IX, or VIII. If an
inhibitor is suspected after performing the dRVVT and dRVVT mix, then a confirmation tests is performed to differentiate between
specific factor inhibitors and a phospholipid antibody. The confirmation test is performed by repeating the dRVVT screen in the
presence of excess plant phospholipid. If the inhibitor is specific for phospholipid, then the phospholipid spiked dRVVT should
shorten compared to the original dRVVT due to the binding of the anti-phospholipid inhibitor to the extra phospholipid added to
the assay. The dRVVT confirm test is considered to be positive for a lupus anticoagulant in samples for which the ratio of the
base line dRVVT confirm result equals or exceeds 1.2.
EUGLOBULIN CLOT LYSIS TIME
TAT: Daily
Requirements: 2 tubes-2cc/tube, frozen citrated plasma.
Patient plasma is acidified (with acetic acid) and the “euglobulin” fraction of protein is precipitated. This fraction contains
fibrinogen, plasminogen, plasminogen activators, and plasmin, if present. Inhibitors of fibrinolysis remain in the supernatant.
The precipitate is separated from the supernate, redissolved, and thrombin is added to form a clot. The clot is kept at 37°C and
observed for lysis. Normal: greater than 120 minutes. Rapid lysis indicates increased fibrinolytic activity. Short euglobulin
clot lysis time are seen in the DIC syndrome, liver disease, and primary fibrinogenolysis due to prostate cancer and other
malignancies. THE PATIENT SAMPLE MUST BE FROZEN.
FACTOR ASSAYS (FACTOR II, VI, VII, VIII, IX, X, XI, XII, PREKALLIKREN, HMW KININOGEN)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma (per factor requested)
Coagulation factor assays are functional measurements of the individual clotting proteins which are performed using the photo
optical methodology for clot detection. Factor assays are performed using either the PT or aPTT procedure, depending on the
pathway required. Factor II, VII, and X utilize the PT whereas Factors V, VIII, IX, and XII utilize the aPTT. To perform a
factor assay, the patient’s plasma is diluted with a plasma deficient in a single factor but with normal amounts of all other
clotting factors. This substrate plasma is obtained from a person congenitally deficient in the factor of interest or is produced
from normal plasma which has been immunodepleted of the specific factor. Mixing the patient plasma with this factor deficient
substrate plasma will correct all factor deficiencies in the patient’s plasma except for the factor being tested. The mixed plasma
is then serially diluted and tested using the appropriate PT or aPTT assay. The degree of correction in the clotting time is
proportional to the amount of factor present in the patient’s plasma. Specific factor assays are available for Factors I
(fibrinogen), II, V, VII, VIII, IX, X, XI, XII, high molecular weight kininogen, and
prekallilrein.
FACTOR V LEIDEN (ACTIVATED PROTEIN C RESISTANCE PCR ASSAY)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, citrated whole blood (refrigerated or room temp)
In over 90% of cases, activate protein C (APC) resistance is due to a single point mutation in the gene for
factor V (G--->A) at position 506), resulting in replacement of the arginine residue at position 506 with a
glutamine. The resultant molecule, referred to as Factor V Leiden, is less effficiently degraded by APC than
the normal factor V, which results in the APC resistance observed in the plasma based assays. It is recom
mended that DNA based analyses be performed on all patients with abnormal plasma-based APC resistant assays
in order to confirm the diagnosis as well as to determine the genotype of the affected individual. These
results are important in guiding clinical decisions making for the patient with APC resistance.
This assay utilizes genomic DNA isolated from the buffy coat to determine the genotype of the patient. A
portion off the factor V gene is amplified by polymerase chain reaction (PCR), following which the DNA is
then restricted with the restriction enzyme Mnll. The DNA is then analyzed by agarose gel electrophoreses
and compared to restricted samples from a normal individual and an individual who is homozygous for the
Factor V Leiden mutation.
FACTOR Xa (Chromogenic assay)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
The chromogenic Factor Xa (FXa) level is useful in determining factor X (FX) levels when a clotting based assay is not indicated,
or in measuring the anticoagulant effect of warfarin in patients that have a prolonged baseline prothrombin time assay due to a
specific factor deficiency/inhibitor or lupus anticoagulant. The method is based on a two-stage principle. First, FX is activated
in the presence of calcium to FXa by the FX activator in Russell's Viper venom. Second, the generated FXa hydrolyzes a specific
chromogenic substrate, liberating the chromophore group pNA. The generated FXa and the intensity of the color reaction are directly
proportional to the FX activity in the original sample. Heparin concentrations below 30 U/mL do not influence the assay.
FACTOR XIII SCREEN
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Plasma is recalcified and allowed to clot. 5M urea us then added to the clot. The clot will dissolve within 24 hours if less
than 1% Factor XIII is present. A normal result is > 24 hours without clot lysis.
FIBRIN(OGEN) DEGRADATION (SPLIT) PRODUCTS: Latex Agglutination (FSP/FDP)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
The plsam is mixed with latex beads coated with an antibody to either fibrinogen or the degradation products D and E of fibrinogen.
Agglutination occurs in the presence of either fibrinogen or fibrin degradation products. Dilutions of the plasma can yield semi
quantitative results. Normal: less than 5 mg/mL. The test does not distinguish between fibrinogen or fibrin degradation. False
positives occurif there is a high titer of a rheumatoid factor in the plasma. Increased fibrin(ogen) degradation products are seen
in DIC, in primary fibrinolytic syndromes, after lytic therapy, in severe liver disease, during acute thromboembolic episodes and after
injury or recent surgery.
FIBRINOGEN ASSAY (Functional assay)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Plasma fibrinogen levels can be measured by isolation methods such as heat precipitation, salt precipitation, or clotting with
thrombin and then weighing the clot or measuring its protein content. It can also be measured by immunological methods, by optical
density techniques, or by a modified thrombin clotting time. At Duke University Medical center, a modified Clauss fibrinogen
performed on the Organon Teknika MDA coagulometer is used. The concentration of fibrinogen is inversely proportional to the clotting
time when a known amount of thrombin (in excess) is added to dilute patient plasma. Therapeutic heparin levels do not modify the
laboratory results because of the excess thrombin. Low functional fibrinogen levels may be seen in DIC, during massive bleeding
associated with consumption of the protein, in liver disease, after lytic therapy, in the rare congenitally deficient patient
(afibrinogenemia or hypofibrinogenemia), or if the fibrinogen protein is present but does not function appropriately (i.e., dys-
fibrinogenemia). In newborns, the fibrinogen level may be slightly lower than normal due to the presence of fetal fibrinogen which
is not accurately assayed by this method.
FIBRINOGEN ANTIGEN ASSAY (Immunological assay)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Patient plasma is introduced into agar containing antibody to fibrinogen and zones of precipitation are measured by RID. The
diameter of the zone is directly proportional to the concentration of fibrinogen. A normal antigenic level does not assure adequate
function of the protein; therefore, a functional level is usually the preferred assay. The antigen level is most often obtained to
assess for a qualitative abnormality of the protein.
HEPARIN ASSAY (Anti-Xa assay) - Unfractionated Heparin
TAT: Daily
Requirements: 2 tubes-2cc/tube, frozen citrated plasma, plus 1 tube-1cc/tube of heparin sample
Special Requirements: Whenever possible, send a sample of the unfractionated Heparin the patient is receiving (optional)
A known amount of Factor Xa is added to the patient’s plasma in the presence of excess antithrombin III and then a Factor Xa specific
chromogenic substrate is added. Heparin in the patient’s sample combines with antithrombin III to inhibit Factor Xa. Residual Factor
Xa reacts with the chromogenic substrate and is measured and compared to the starting amount of Factor Xa. This assay is the standard
method to measure anticoagulant properties of heparin. The patient sample must be
CLEARLY MARKED AS TO WHETHER THE PATIENT IS RECEIVING PORCINE HEPARIN OR SOME OTHER HEPARIN PREPARATION AS A DIFFERENT STANDARD
CURVE IS USED FOR EACH OF THESE SPECIFIC DRUGS. The Therapeutic heparin range for treatment of thromboembolic events at Duke
University Medical Center has been extrapolated to correspond to 0.25-0.45 anti-FXa units. This range may change slightly as more
precise information becomes available.
HEPARIN ASSAY (Anti-Xa assay) - Low Molecular Weight Heparin
TAT: Daily
Requirements: 2 tubes-2cc/tube, frozen citrated plasma, plus 1 tube-1cc/tube of low molecular weight heparin sample
Special Requirements: Whenever possible, send a sample of Low Molecular Weight Heparin the patient is receiving (optional)
A known amount of Factor Xa is added to the patient's plasma in the presence of excess antithrombin III and then a Factor Xa specific
chromogenic substrate is added. Heparin in the patient's sample combines with antithrombin III to inhibit Factor Xa. Residual Factor
Xa reacts with the chromogenic substrate and is measured and compared to the starting amount of Factor Xa. This assay is the standard
method to measure anticoagulant properties of unfractioned heparins, low molecular weight heparins, and heparinoids. The patient sample must be
CLEARLY MARKED AS TO WHETHER THE PATIENT IS RECEIVING HEPARIN LOW MOLECULAR WEIGHT HEPARIN (LMWH) OR HEPARINOID AS A DIFFERENT STANDARD
CURVE IS USED FOR EACH OF THESE SPECIFIC DRUGS. Therapeutic ranges for LMWH is not yet clearly established and it may
differ from heparin.
HEPARIN ASSAY (Anti-Xa assay) - Heparinoid (danaparoid sodium)
TAT: Daily
Requirements: 2 tubes-2cc/tube, frozen citrated plasma, plus 1 tube-1cc/tube of heparinoid sample
Special Requirements: Whenever possible, send a sample of the Heparinoid (danaparoid sodium) the patient is receiving (optional)
A known amount of Factor Xa is added to the patient's plasma in the presence of excess antithrombin III and then a Factor Xa specific
chromogenic substrate is added. Heparinoid in the patient's sample combines with antithrombin III to inhibit Factor Xa. Residual Factor
Xa reacts with the chromogenic substrate and is measured and compared to the starting amount of Factor Xa. This assay is the standard
method to measure anticoagulant properties of unfractioned heparins, low molecular weight heparins, and heparinoids. The patient sample must be
CLEARLY MARKED AS TO WHETHER THE PATIENT IS RECEIVING HEPARIN LOW MOLECULAR WEIGHT HEPARIN (LMWH) OR HEPARINOID AS A DIFFERENT STANDARD
CURVE IS USED FOR EACH OF THESE SPECIFIC DRUGS. Therapeutic ranges for heparinoid is not yet clearly established and it may
differ from heparin.
HEXANGONAL II PHASE PHOSPHOLIPID ASSAY (STACLOT LA)
TAT: Daily
Requirements: 1 tubes-1cc/tube, frozen citrated plasma
This two part aPTT assay is used to screen for anntibodies to phospholipids (i.e. lupus anticoagulants) and confirm the presence
of a phospholipid antibody. Test plasma that is suspected to contain lupus anticoagulants (LAC) is incubated at 37 degrees C with and
without HPE (source of phospholipid). Next, an aPTT is performed on both samples (with and without HPE) using a LAC sensitive reagent.
If LAC were present in the test plasma, the antibodies would be neutralized by the HPE, resulting in a shortened clotting time versus
the sample without HPE. By comparing the difference between the two clotting times, the presence of LAC in the test plasma can be identified.
The Staclot LA is considered to be positive for the presence of a phospholipid antibody if the difference in the clotting times between the two
tubes is greater than + 8.0 seconds. The aPTT reagent in this assay contains a heparin inhibitor which makes the test system insensitive
to heparin levels up to 2.0 U/mL. The Staclot LA procedure also requires the addition of a normal test plasma to the test system to correct
a prolongation of the clotting time due to factor deficiencies that might be present. Due to the heterogeneity the phospholipid antibody, there
is no single test that is confirmatory for all phospholipid dependent antibodies. Therefore, a combination of procedures may be necessary to
confirm the presence of a phospholipid antibody.
HIGH MOLECULAR WEIGHT KININOGEN (HMWK) ASSAY (Fitzgerald Factor)
TAT: Daily
Requirements: 1 tubes-1cc/tube, frozen citrated plasma
The contact phase of the intinsic coagulation pathway includes prekallikrein (PK), high molecular weight kininogen (HMWK), factor XII (FXII) and
factor XI (FXI). These contact factors are thought to absorb negatively charged surfaces and initiate a series of reactions, ultimately leading
to activation of the intrinsic pathway. Kininogens are plasma proteins that release the vasoactive peptide bradykinin. HMWK is a cofactor for
FXIIa and kallikrein and normally circulates in plasma complexed with PK. Some HMWK is stored in the alpha granules. HMWK makes up only about
1/5 of the total plasma kininogen, with the remainder not playing a role in coagulation. The most common method for the evaluation of contact
activation is the aPTT. APTT reagents generally consist of a contact surface activator and a phospholipid source. To test for this deficiency,
patient plasma is first tested by performing an aPTT with an extended incubation time of 10 minutes. Correction of the aPTT with the prolonged
incubation suggests a PK deficiency, no correction suggests HMWK deficiency. Patient plasma is then mixed with a known HMWK deficient plasma and
incubated at 37 degrees C for 60 minutes. A plasma that is not HMWK deficient should correct the aPTT in less than 60 minutes - this prolonged
incubation will insure that the aPTT indeed does not correct.
INCUBATED MIX (APTT)
TAT: Daily
Requirements: 2 tubes-2cc/tube, frozen citrated plasma
The aPTT may be prolonged due to deficiency of one or more clotting factor or due to the presence of an inhibitor that interferes either
specifically or nonspecifically with the measurement of a coagulation factor. Either finding may be of clinical significance depending
on the type and level of factor or inhibitor present. An incubated mix should be ordered to investigate the cause of a prolonged aPTT.
This study is performed by mixing the patient’s plasma with an equal amount of pooled normal plasma and repeating the clotting test in
question immediately and after an established incubation period. At DUMC, the incubation period is 60 minutes at 37°C. Pooled normal
plasma is assayed concurrently and reported as a reference for both the 0 and 60 minute incubated mixes. This is important because the
aPTT tend to prolong upon incubation due to degradation of the labile clotting factors V and VIII. At DUMC, a result within 3 seconds
of the control mix for aPTT indicates full correction. Because only ~30-40% of an individual factor (exact cut-off differs between
factors and with different aPTT reagents) is necessary to yield a normal aPTT, a patient with a deficiency of a clotting factor should
correct completely when mixed with normal plasma. For example, if the plasma of a patient with a severe factor deficiency, e.g., <0.5%, is mixed with an equal volume of pooled normal plasma, which is assumed to have 100% of all factors, the deficient factor level in the final mix will be ~50%. This level is more than enough to yield a normal aPTT.
On the other hand, the aPTT of patients with inhibitors will remain abnormally long after the mix. In these cases, a 1:1 dilution of the
patient’s plasma is not sufficient to eliminate the full effect of the inhibitor on the aPTT. The most common inhibitors are non-specific
(not directed at a particular clotting factor). The LA interferes with the in vitro binding of clotting factors to phospholipids and is
usually manifested by an isolated prolongation of the aPTT. These antibodies are able to bind to their lipid targets quickly, even at room
temperature, thus interfering with the mix at both 0 and 60 minutes. A Lupus Anticoagulant Panel should be ordered to confirm the presence
of this type of inhibitor. A specific inhibitor refers to an antibody which is directed towards an individual clotting factor. Clotting
factor assays are required to define whether an inhibitor is specific or nonspecific. A specific inhibitor will inhibit one factor primarily
and all other factors will be near normal. An aPTT should be specified when ordering this mixing study.
INHIBITOR TITER (Bethesda titer)
TAT: Daily
Requirements: 2 tubes-2cc/tube, frozen citrated plasma
If a prolonged PT or aPTT fails to correct after mixing with normal plasma, then an inhibitor that interferes either specifically or non-
specifically with the measurement of a coagulation factor is present. A specific inhibitor is suspected if factor assays reveal a single
factor to be markedly decreased. Once a specific factor inhibitor (antibody) has been identified, a titer to quantitative the amount of
inhibitor present should be performed. Clinically, a low titer inhibitor may be more easily overwhelmed with clotting factor infusions and
therefore management of a patient will vary depending on the titer of the inhibitor present. In addition, inhibitor titers may be followed
in patients being treated with immunomodulating protocols to monitor the success of the protocol in eradicating the inhibitor.
The most commonly encountered clotting factor inhibitor is that to FVIII. Inhibitor titer assays, such as the Bethesda assay, have been
specifically developed to monitor FVIII inhibitors; however, a modified Bethesda titer is used by the clinical coagulation laboratory for
all specific factor inhibitors. A Bethesda unit is defined as the reciprocal of the dilution of patient plasma that destroys half the factor
VIII procoagulant activity in an equal mixture of normal and patient plasma incubated at 37°C for 2 hours. The time and temperature must be
defined in the assay because of the observed variation of inhibitory effect based on these variables.
Methylenetetrahydrofolate Reductase Polymorphism (MTHFR)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, citrated whole blood (refrigerated or room temp)
Mild hyperhomocysteinemia is an established risk factor for venous thromboembolic disease as well as cardiovascular disease. Methylenetetrahydrofolate reductase (MTHFR) is a regulatory enzyme in folate-dependent homocysteine remethylation, catalyzing the reduction of 5, 10-methylenetrahydrofolate to 5-methytetrahydrofolate. A common polymorphism in the MTHFR gene (CèT at nucleotide position 677, resulting in substitution of valine for alanine) associated with a thermolabile enzyme and decreased specific MTHFR activity. The prevalence of the homozygous form ranges from 8% to 18% in various populations, but is very uncommon in individuals of African descent.
Clinically, homozygosity for this MTHFR polymorphism is associated with an increased risk for venous thromboembolism as well as premature vascular disease. It is possible to modulate this risk, however, by supplementing dietary folate and/or pyridoxine, which is associated with decreased homocysteine levels. Consequently, patients with elevated plasma homocysteine levels should be evaluated for the thermolabile MTHFR polymorphism, since this information is useful in determining an individual patient's therapeutic regimen as well as for genetic counseling of the patient and their family members.
PLASMINOGEN ANTIGEN (Immunologic assay)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Plasma is added to agar containing antibody to plasminogen and assayed by RID. Zones of precipitation are measured and the diameter of the
zone is directly proportional to the concentration of plasminogen. The presence of a normal amount of antigen does not exclude a qualitative
defects of the protein resulting in poor function of the protein; therefore a functional level is usually the preferred assay. The antigen
level is most often obtained to assess for a quantitative abnormality of the protein.
PLASMINOGEN FUNCTIONAL ASSAY (Activity assay)
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Plasma is incubated with streptokinase and then a plasma specific chromogenic substrate is added. The amount of color generated is directly proportional to the concentration of plasminogen in the
plasma. Plasminogen levels are reduced in liver disease, DIC syndrome, thrombolytic therapy and in a rare congenital deficiency of plasminogen that results in recurrent thrombosis.
PLATELET ANTIBODY - DIRECT
TAT: 24 Hours
Requirements: 2 tubes-5cc/tube Lavender EDTA vacutainer tubes, If the platelet count is below 50,000 x 103/ul, additional blood may be required.
The platelet antibody assay is a quantitative radioactive measurement of platelet bound IgG antibody. This antibody is detected by first incubating washed platelets with radiolabeled monoclonal
anti-human IgG antibody, then removing all unbound radiolabeled antibody and finally counting the radiolabeled anti-IgG that is bound to the platelets. Normal range of platelet bound IgG
is <1500 molecules IgG/platelet.
PLATELET ANTIBODY - INDIRECT
TAT: Weekly
Requirements: 1-10cc red top vacutainer tube.
The platelet antibody assay is a quantitative radioactive measurement of antibody present in the plasma of thrombocytopenic patients. This antibidy is detected by first incubating patient's
serum/plasma with four normal group O washed platelet samples and then a radiolabeled monoclonal anti-human IgG antibody. The unbound radiolabeled antibody is removed and the
radiolabeled anti-IgG bound to the patient's antibody and the donor platelets is counted and calculated. Normal range of plasma/serum IgG binding to normal platelets is <1000 molecules of IgG/platelet. Four group O donors are used to determine if the antibody has a specificity to one or more donors or is it a pan reactive antibody reacting with all four group O donors. The highest value of the four donors is reported.
PLATELET ANTIBODY - DRUG INDUCED
TAT: 24 Hours
Requirements: 1 tube-5cc/tube red top vacutainer tube is allowed to clot and the serum separated from the red cell clot.
Plasma from a 5cc lavender EDTA vacutainer tube may also be used.
Special Requirement: The suspected drug in the form the patient is presently taking should also be sent with the serum/plasma sample.
Many drugs have been reported to cause or suspected to cause thrombocytopenia. This assay was developed to determine if a identified drug was responsible. The assay involves the
incubation of normal platelets from a group O donor with patient serum or plasma and the suspected drug. The platelets are then washed and incubated with 125 I anti-IgG. After incubation,
the unbound radiolabeled antibody is removed and the radiolabeled antibody bound to the platelets is counted and calculated. The calculated molecules of IgG/platelet for the patient's
plasma/serum are compared with and without the drug and to normal plasam/serum versus the patient's plasma/serum.
PLATELET FACTOR 4 ASSAY (ELISA)
TAT: 1/WEEK
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Heparin-induced Thrombocytopenia (HIT) is an immune-mediated disorder that may develop in ~3% of patients receiving heparin therapy. These patients develop an antibody to the heparin-PF4 complex that binds to platelets, resulting in platelet activation and clearance from the circulation. Characteristically, the platelet count begins to fall five to eight days after starting heparin, with a typical platelet count nadir of ~20,000-150,000/ml. In contrast to most drug-induced thrombocytopenias, however, as many as half of these patients will develop a thrombotic complication. Not infrequently, these complications may result in severe morbidity (e.g., amputation) and mortality.
Prompt and accurate diagnosis is essential for the appropriate management of these complicated patients, especially now that alternative therapeutic agents are available. Two types of assays are available to diagnose HIT: functional and antigenic assays. The functional assay used at Duke University Medical Center for "rapid" screening of patients suspected of HIT is the heparin induced platelet aggregation assay (HIPA). The HIPA uses platelet rich plasma and a three-point system (analyzing heparin at three concentrations, one low, one medium, and one high), by using selected donors with "reactive" platelets, the sensitivity of the HIPA has been reported to be ~60-80% with a specificity of ~80-100%.
The antigenic method currently available at Duke University Medical Center is the Platelet Factor 4 ELISA. This ELISA assay is designed to detect antibodies specific for conformational changes that develop in PF4 when it is complexes with heparin or other polyanionic compounds. Studies that have compared the PF4/heparin ELISA with the 14C-serotonin release assay found that the sensitivity of the ELISA was ~87-90% with a specificity of ~92-98%. However, the ELISA assay also detected antibodies in 8% of patients who were negative by the 14C-serotonin release assay. The sensitivity for the PF4 ELISA kit is lower than what is published in the literature, which may reflect differences between research laboratory testing vs. use of a commercial kit. Nevertheless, the combination of these two tests provides a combined sensitivity (>90%) and specificity (90-100%) comparable to the serotonin release assay.
PLATELET SPECIFIC ANTIGEN - PLA1
TAT: 24 Hours
Requirements: 1-5cc lavender EDTA vacutainer tube. If the platelet count is below 20,000, up to six 5cc lavender tubes may be requested.
This test determines the presence or absence of the PLA1 antigen on platelets. Ninety-seven (97) percent of the population has PLA1 positive platelets. Patients with PLA1 negative platelets
may develop anti-PLA1 antibodies when exposed to the PLA1 antigen. PLA1 Antigen Positive Range is >4000 molecules IgG per platelet. Normal range for PLA1 sntibody is <1000 molecules IgG/platelet.
PLATELET NEUTRALIZATION PROCEDURE (PNP)
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
This two part aPTT based assay is used to screen for antibodies to phospholipids (i.e. lupus anticoagulants) and to confirm the presence of
a phospholipid antibody. The first part of the Platelet Neutralization Procedure (PNP) is a phospholipid dependent clotting test similar to
the traditional aPTT. An added reagent to the aPTT activates factor X (FX) and factor V (FV), initiating the clotting cascade without
requiring factors proximal to FX (i.e., factors XII, XI, IX, VIII or VII). The aPTT reagent used in this part of the assay contains a dilute
phospholipid concentration, making this part particularly sensitive to the presence of a phospholipid. The presence of a phospholipid antibody
will bind with the available phospholipid, thus prolonging this phase of the assay. The second part of the PNP is a confirmatory assay in which
an excess of phospholipid in the form of platelet lysate is added to the aPTT assay. If the inhibitor is specific for phospholipid, the clotting
time of the aPTT assay should shorten as compared to the original aPTT due to the excess of added phospholipid. The PNP is considered to be
positive for the presence of a phospholipid antibody if the difference in clotting times of part one minus part two is greater than +8.0 seconds.
Due to the heterogeneity of the phospholipid antibody, there is no single test that is confirmatory for all phospholipid dependent antibodies.
Therefore, a combination of procedures may be necessary to confirm the presence of a phospholipid anti-body.
PNH SCREEN
TAT: 24 Hours
Requirements: 1 tube-5cc/tube Lavender EDTA vacutainer tube.
Patients with unexplained hemolytic anemia, thrombosis, or aplastic anemia may be diagnosed with PNH. Identifying the patient with PNH helps in the treatment of the hemolytic anemia
and limits the transfusions needed. Aplastic anemia has been shown to develop into PNH in about 5% of the cases. Patients experiencing thrombosis and diagnosed with PNH will get
anti-thrombotic therapy to prevent thrombosis, especially of the liver. Results are reported as percent lysis. A result of 2% or more is positive for PNH. Further testing may be done in
the research laboratory to determine the percentages of each population of red cells usually seen in PNH patients.
PREKALLIKREIN (PK) ASSAY (Fletcher factor)
TAT: Daily
Requirements: 1 tubes-1cc/tube, frozen citrated plasma
The contact phase of the intinsic coagulation pathway includes prekallikrein (PK), high molecular weight kininogen (HMWK), factor XII (FXII) and
factor XI (FXI). These contact factors are thought to absorb negatively charged surfaces and initiate a series of reactions, ultimately leading
to activation of the intrinsic pathway. Prekallikrein is structurally similar to FXI and is activated to kallikrein by FXIIa in the presence of
HMWK and a negatively charged surface. About 1/4 of PK circulates freely with the remainder bound to HMWK. The most common method for the
evaluation of contact activation is the aPTT. APTT reagents generally consist of a contact surface activator and a phospholipid source. To test for this deficiency,
patient plasma is first tested by performing an aPTT with an extended incubation time of 10 minutes. Correction of the aPTT with the prolonged
incubation suggests a PK deficiency, no correction suggests HMWK deficiency. Patient plasma is then mixed with a known PK deficient plasma and
incubated at 37 degrees C for 60 minutes. A plasma that is not PK deficient should correct the aPTT in less than 60 minutes - this prolonged
incubation will insure that the aPTT indeed does not correct.
PROTEIN C ANTIGEN (Immunologic assay)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Patient plasma is introduced into agar containing antibody to protein C and the plasma is electrophoresed across the agar forming “rockets” at zones
of precipitation. These peaks are compared to normal plasma precipitant peaks. Dysfunctional protein C molecules have been reported to cause
thrombosis. A normal antigen level does not assure normal function, therefore a functional level is usually the preferred assay. The antigen level
is most often obtained to assess for a quantitative abnormality of the protein.
PROTEIN C FUNCTIONAL ASSAY (Clottable activity assay)
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Currently the laboratory offers two types of functional assays for Protein C. The routine screening assay is based on protein C’s ability to degrade
factors V and VIII and in monitored by an aPTT. A protein C activator is added to the patient’s plasma. The amount of protein C present is proport
ional to the prolongation of the aPTT. The aPTT prolongs as the activated protein C in the patient’s plasma inactivates factors V and VIII on the
reagent plasma. This aPTT based assay is a more physiologic evaluation of protein C’s ability to recognize and degrade its physiologic substrates,
factors V and VIII, instead of a chromogenic substrate.
PROTEIN C FUNCTIONAL ASSAY (Chromogenic activity assay)
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
The chromogenic assay is still available by special laboratory order for patients previously diagnosed using this assay or for special evaluation of
a dysfunctional protein C molecule. The chromogenic assay is performed using a specific protein C chromogenic substrate. The amount of protein C
present is proportional to the amount of chromogenic substrate hydrolyzed. Congenital absence of protein C (Homozygous deficiency) produces neonatal
purpura fulminans, a rapidly fatal syndrome unless treated with infusions of protein C. Heterozygotes produce reduced amounts of protein C which may
promote a thrombotic tendency. Protein C is a vitamin K dependent protein. A normal antigen level does not assure normal function, therefore a
functional level is usually the preferred assay. The chromogenic protein C level is most often obtained to further assess patients for a dysfunctional
abnormality of the protein, but the clot-based protein C assay is usually the preferred assay.
PROTEIN S Antigen (ELISA)-Free Protein S level)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
In the absence of protein S, protein C functions less well as an anticoagulant. In plasma, protein S circulates both free (~40%) and in a complex with
C4b binding protein (~60%). Only the free form of protein S can function as an anticoagulant. Protein S may be low secondary to a congenital deficiency,
an acquired decrease in total protein, or a shift of the free protein to the bound form during an acute phase response with evaluated C4b binding protein.
A congenital deficiency in protein S may lead to a recurrent thrombotic tendency. The functional protein S level is now performed as the screening assay
for patients suspected for being protein S deficient.
The internal wall of a plastic microwell is precoated with the first monoclonal antibody to free protein S. The second anti-free protein S monoclonal antibody that is coupled with peroxidase is added to the precoated microwell at the same time as the plasma whose free protein S is to be determined. The free protein S of the plasma being tested is simultaneously captured on the one hand by the first monoclonal antibody that is precoated on the microwell wall, and on the other by the second monoclonal antibody-
peroxidase conjugate , i.e., the "sandwich" is formed in a one-step reaction. Next, the bound peroxidase is revealed by its activity in a predetermined time on the substrate ortho-phenylenediamine in the presence of hydrogen peroxide. After stopping the reaction with a strong acid, the intensity of the color produced bears a direct relationship with the free protein S concentration initially present in the plasma sample.
PROTEIN S Antigen (Immunologic assay-Free Protein S level)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
In the absence of protein S, protein C functions less well as an anticoagulant. In plasma, protein S circulates both free (~40%) and in a complex with
C4b binding protein (~60%). Only the free form of protein S can function as an anticoagulant. Protein S may be low secondary to a congenital deficiency,
an acquired decrease in total protein, or a shift of the free protein to the bound form during an acute phase response with evaluated C4b binding protein.
A congenital deficiency in protein S may lead to a recurrent thrombotic tendency. The functional protein S level is now performed as the screening assay
for patients suspected for being protein S deficient. Free protein S is separated from the bound protein S in plasma by a polyethylene glycol precipitation
procedure. The protein S antigen is then determined by a Laurell rocket technique with the amount of free protein S present proportional to the height of
the antigen/antibody precipitin rocket compared to normal.
PROTEIN S Antigen (ELISA)-Total Protein S level)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
In the absence of protein S, protein C functions less well as an anticoagulant. In plasma, protein S circulates both free (~40%) and in a complex with
C4b binding protein (~60%). Only the free form of protein S can function as an anticoagulant. Protein S may be low secondary to a congenital deficiency,
an acquired decrease in total protein, or a shift of the free protein to the bound form during an acute phase response with evaluated C4b binding protein.
A congenital deficiency in protein S may lead to a recurrent thrombotic tendency. The functional protein S level is now performed as the screening assay
for patients suspected for being protein S deficient.
The internal wall of a plastic microwell is precoated with the first monoclonal antibody to total protein S. The second anti-total protein S monoclonal antibody that is coupled with peroxidase is added to the precoated microwell at the same time as the plasma whose free protein S is to be determined. The total protein S of the plasma being tested is simultaneously captured on the one hand by the first monoclonal antibody that is precoated on the microwell wall, and on the other by the second monoclonal antibody-
peroxidase conjugate , i.e., the "sandwich" is formed in a one-step reaction. Next, the bound peroxidase is revealed by its activity in a predetermined time on the substrate ortho-phenylenediamine in the presence of hydrogen peroxide. After stopping the reaction with a strong acid, the intensity of the color produced bears a direct relationship with the total protein S concentration initially present in the plasma sample.
PROTEIN S Antigen (Immunologic assay-Total Protein S level)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
In the absence of protein S, protein C functions less well as an anticoagulant. In plasma, protein S circulates both free (~40%) and in a complex with
C4B binding protein (~60%); the combination of these two fractions equal the Total Protein S Antigen. Only the free form of protein S can function as an
anticoagulant. Protein S may be low secondary to a congenital deficiency, an acquired decrease in total protein, or a shift of the free protein to the
bound form during an acute phase response with evaluated C4b binding protein. A congenital deficiency in protein S may lead to a recurrent thrombotic
tendency. The functional protein S level is now performed as the screening assay for patients suspected for being protein S deficient. Free protein S is
separated from the bound protein S in plasma by a polyethylene glycol precipitation procedure (see Protein S Antigen - Free). The total protein S assay
employs the same methodology as the free protein S, without the precipitation procedure with polyethylene glycol. The total protein S antigen is then
determined by a Laurell rocket technique with the amount of total protein S present proportional to the height of the antigen/antibody precipitin rocket
compared to normal.
PROTEIN S FUNCTIONAL (Activity Assay)
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
A new protein S functional assay is now available and has replaced the free protein S antigen level as the screening test for identification of protein S
deficient patients. The functional level reflects the free protein S component in the plasma as protein S bound to C4b binding protein is not active as
a protein C cofactor. The actual units of the functional level and the free antigen level differ, therefore direct comparison between values is not
applicable. Both the total and free protein S antigen assays are available for further evaluation of patients found to have a low functional level and
for follow-up of patients previously identified by the antigen levels. Acquired decreases in protein S activity may be seen in vitamin K deficiency,
liver disease, warfarin therapy, or with evaluation of C4b binding protein (an acute phase reactant protein) which results in a decrease of free,
functional protein S. A congenital deficiency in protein S may lead to a recurrent thrombotic tendency. The protein S activity assay is an aPTT based
assay designed to evaluate the enhancement of the anticoagulant activity of protein C. This enhancement is reflected by the prolongation of the clotting
time (aPTT) of a system enriched with factor Va which is a physiological substrate for activated protein C.
PROTHROMBIN (PT) ANTIGEN
TAT: 1/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Diluted plasma is electrophoresed into a gel containing antibody to Prothrombin antigen utilizing the Laurell rocket immunoassay technique . A standard
curve is prepared by plotting the height of the rockets formed with known quantities of Prothrombin in normal plasma and comparing to this normal curve
the height of the rocket formed using the patient’s plasma. This assay is designed to detect the presence of Prothrombin protein but cannot differentiate
between functional and non-functional protein. A normal antigen level does not assure normal function, therefore a functional level is usually the preferred
assay. The antigen level is most often obtained to assess for a quantitative abnormality of the protein. The assay is used to help diagnose Prothrombinemia
and other disorders of prothrombin consumption. (bovine factor inhibitors).
Prothrombin (G20210A) Gene Polymorphism (PTG G20210A)
TAT: 1/Week
Requirements: 1 tube-1cc/tube, citrated whole blood (refrigerated or room temp)
Prothrombin is the precursor of the serine protease thrombin, a key enzyme in the processes of hemostasis and thrombosis. Decreased levels of prothrombin are associated with an increased risk for hemorrhage, whereas elevated prothrombin levels have been associated with an increased risk for venous thrombosis.
Recently a genetic polymorphism located in the 3'-untranslated region of the prothrombin gene has been described. The polymorphism has been associated with increased prothrombin levels as well as an increased risk for venous thrombosis. The polymorphism is due to a single base pair substitution at position 20210 of a guanine (G) for an adenine (A) nucleotide. The mechanism whereby this polymorphism results in increased levels of prothrombin is unknown.
The prevalence of this polymorphism ranges from 1% to 3% in Caucasian populations, but is very uncommon in individuals from Asian or African descent. In contrast, several studies have reported that between 5% to 18% of patients with venous thrombosis have this polymorphism. Furthermore, this polymorphism is not infrequently encountered in patients with an additional hypercoagulable state, especially factor V Leiden and/or thermolabile polymorphism in the methylenetetrahydrofolate reductase (MTHFR).
PROTHROMBIN-PROCONVERTIN ASSAY (P+P)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
The Prothrombin-Proconvertin (P+P) Assay is used to monitor patients on oral anticoagulant therapy, and to screen for deficiencies in the extrinsic
coagulation pathway. There is a population of patients on oral anticoagulant therapy (warfarin) that present with a prolonged baseline Prothrombin
Time (PT) and International Normalized Ratio (INR) due to inhibitors (such as lupus anticoagulants and bovine thrombin inhibitors) or factor
deficiencies in the extrinsic and/or common pathways that prevent accurately monitoring the level of anticoagulation by traditional methods
(INR). This assay in conjunction with a chromogenic factor X assay will allow the physician to accurately monitor the anticoagulation status of
these patients with prolonged baseline PT assays.
PROTHROMBIN TIME (PT)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Thromboplastin, a mixture of tissue factor and phospholipids, and calcium are added to plasma and the time to clot formation is determined. The clotting
time is dependent upon the level of factor VII and of the common pathway factors II, V, X and fibrinogen. The normal range for a PT changes with each new
lot of reagent (approximately once a year). The current normal range can be obtained from the computer generated test result report or by calling the
Clinical Coagulation laboratory. The prothrombin time is a useful screening test of the extrinsic or initiator pathway of coagulation. It is also used to
monitor anticoagulation therapy with warfarin (Coumadin). The prothrombin time may be abnormal in patients with liver disease and vitamin K deficiency.
The PT is only a screening test of coagulation and the PT will not begin to prolong until one of the PT based clotting factor decreases to less than about
30-40% of normal (Exact cutoffs differ between factors and with different PT reagents). Therefore, a normal PT does not always correspond to normal levels
of clotting factors, and even a slight prolongation of the clotting time suggests a factor level of less than about 30-40%.
PROTHROMBIN (PT) MIXING STUDY
TAT: Daily
Requirements: 2 tubes-2cc/tube, frozen citrated plasma
The PT may be prolonged due to deficiency of one or more clotting factor or due to the presence of an inhibitor that interferes either specifically or
nonspecifically with the measurement of a coagulation factor. Either finding may be of clinical significance depending on the type and level of factor
or inhibitor present. An Prothrombin (PT) mixing study should be ordered to investigate the cause of a prolonged PT. This study is performed by mixing
the patient’s plasma with an equal amount of pooled normal plasma and repeating the clotting test (PT). Pooled normal plasma is assayed concurrently and
reported as a reference for the PT mix. At DUMC, a result within 1 second of the control mix for PT indicates full correction. Because only ~30-40% of
an individual factor (exact cut-off differs between factors and with different PT reagents) is necessary to yield a normal PT, a patient with a deficiency
of a clotting factor should correct completely when mixed with normal plasma. For example, if the plasma of a patient with a severe factor deficiency,
e.g., <0.5%, is mixed with an equal volume of pooled normal plasma, which is assumed to have 100% of all factors, the deficient factor level in the final mix will be ~50%. This level is more than enough to yield a normal PT.
On the other hand, the PT of patients with inhibitors will remain abnormally long after the mix. In these cases, a 1:1 dilution of the patient’s plasma
is not sufficient to eliminate the full effect of the inhibitor on the PT. The most common inhibitors are non-specific (not directed at a particular
clotting factor). A specific inhibitor refers to an antibody which is directed towards an individual clotting factor. Clotting factor assays are required
to define whether an inhibitor is specific or nonspecific. A specific inhibitor will inhibit one factor primarily and all other factors will be near
normal. A PT should be specified when ordering this mixing study.
REPTILASE TIME
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Reptilase is an enzyme obtained from the venom of the Bothrops atrox snake which cleaves fibrinogen independently of thrombin and; unlike thrombin is not
inactivated by ATIII-heparin. A standard concentration of the reptilase reagent is added to plasma and the clotting time is measured Low fibrinogen,
abnormal fibrinogen, or elevated fibrin(ogen) degradation products will prolong the clotting time. This test is useful in determining if a long aPTT or
thrombin clotting time is due to heparin since the reptilase time will not be affected by heparin.
THROMBIN CLOTTING TIME (TCT)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
A standard concentration of human thrombin is added to plasma and the clotting time is measured. Low fibrinogen, abnormal fibrinogen, myeloma proteins,
antithrombins such as heparin, or fibrin(ogen) degradation products will prolong the thrombin clotting time. The clotting time is dependent upon the
concentration of thrombin in the assay. The TCT will vary between labs depending on what concentration of thrombin a lab elects to use in their assay.
The TCT is also used to monitor fibrinolytic therapy and to detect dysfibrinogenemia (abnormal fibrinogen) and heparin resistance. The TCT is more
sensitive than the aPTT for the detection of heparin. Exogenous thrombin is added to the patient’s plasma; therefore, deficiencies of clotting factors
(factors II-XII) will not prolong the TCT since they are not required to activate endogenous thrombin.
TISSUE THROMBOPLASTIN INIBITION TIME (TTIT)
TAT: Daily
Requirements: 1 tube-1cc/tube, frozen citrated plasma
The tissue thromboplastin inhibition time (TTIT) is a phospholipid dependent aPTT type assay utilizing a dilute thromboplastin reagent as the source of
phospholipid. This assay is used primarily as a screen for phospholipid antibodies (i.e. lupus anticoagulants). Patient plasma is incubated with a
dilute thromboplastin reagent, recalcified, and the clotting time determined. The same procedure is simultaneously performed using a pooled normal
plasma. If the ratio of the clotting time for the patient plasma as compared to the clotting time of the pooled normal plasma is greater than 1.2, it
is suggestive of a phospholipid antibody. This assay is not a part of the lupus anticoagulant panel and must be ordered separately.
von WILLEBRAND FACTOR ANTIGEN (vWF Ag)
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
Diluted plasma is electrophoresed into a gel containing antibody to vWF antigen utilizing the Laurell rocket immunoassay technique. A standard curve
is prepared by plotting the height of the rockets formed with known quantities of vWF in normal plasma and comparing to this normal curve the height of
the rocket formed using the patient’s plasma. This assay is designed to detect the presence of vWF protein but cannot differentiate between functional
and non-functional protein. The assay is used to help diagnose von Willebrand disease.
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von Willebrand Antigen (Laurel Rocket) |
von WILLEBRAND FACTOR ANTIGEN (vWF Ag)ELISA
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
von Willebrand Factor Antigen (vWF:Ag or Factor VIII related protein) by ELISA (enzyme linked immunosorbent assay) provides a quantitative measurement of this protein in citrated human plasma. von Willebrand factor antigen is a plasma protein found in circulation combined by non-covalent interactions with factor VIII, a procoagu- lant protein also known as the anti-hemophilic factor. These two proteins show distinct biochemical and functional properties as well as different antigenic determinants; their plasma levels may vary independently of each other. Deficiency of FVIII causes classic hemophilia while deficiency of vWF causes von Willebrand disease. Most of the vWF:Ag is synthesized and stored by endothelial cells while 15-20% is synthesized by megakaryo- cytes and stored in circulating platelets. A vWF:Ag unit has a molecular weight of about 250 kD and tends to polymerize in circulation, with multimers ranging in size from 850kD to as large as 15 x 108 D.
vWF:Ag plays a very important role in hemostasis; it protects FVIII from proteo- lytic cleavage in circulation and helps platelets to aggregate or to adhere to sites of vascular damage. The in vivo half-life of FVIII without vWF:Ag is shortened from 10-12 hours to a few minutes. These two mechanisms prevent bleeding. von Willebrand disease is characterized by an inherited deficiency of vWF. A decreased vWF activity in plasma can be the result of low concentrations (quantitative or type I defect) or a deficient function of vWF (qualitative or type II defect). von Willebrand disease is the most common inherited bleeding disorder characterized by easy bruising and prolonged bleeding from mucosal surfaces. The prevalence of von Willebrand disease has been esti- mated to be 1 – 3% of the general population. Approximately 80% of von Willebrand disease patients have a type I deficiency.
The laboratory diagnosis of von Willebrand disease may require both quantitative and qualitative (functional) determinations. Quantitative determinations are based on immunologic techniques such as radial immunodiffusion in gel and Laurell rocket immunoelectrophoresis. ELISA procedures applied to measure vWF:Ag are less labor intensive and are more objective, accurate and have more reproducible results.
von WILLEBRAND FACTOR ACTIVITY-RISTOCETIN COFACTOR (vWF Activity assay)
TAT: 2/Week
Requirements: 1 tube-1cc/tube, frozen citrated plasma
The Ristocetin cofactor assay is an indirect measure of vWF activity. Ristocetin is an antibiotic now used as a laboratory reagent because of its
ability to substitute ex vivo for the blood vessel wall during vWF mediated platelet adherence. Formaldehyde fixed platelets are mixed with ristocetin
forming Platelet - vWF - Ristocetin - vWF - Platelet bridges. This agglutination process is monitored by measuring changes in the light transmission
(similar to platelet aggregation studies). The time required for agglutination corresponds to the amount of the amount of vWF in the patient’s plasma.
von WILLEBRAND FACTOR MULTIMER PATTERN
TAT: 1/Month
Requirements: 2 tubes-2cc/tube, frozen citrated plasma
The 225-275,000 dalton von Willebrand protein subunit is capable of self-association to form multimers with molecular weights over 2 million daltons.
This ability to form multimers is required for full vWF activity. Defects in forming the vWF polymers result is a qualitative defect of vWF activity
with clinical manifestations that may differ depending on the type of defect present. The assay is performed by electrophoresing diluted plasma into
an agarose gel, and then reacting the gel with antibody to von Willebrand factor antigen. A normal multimeric pattern contains a full range of high
to low molecular weight forms of the von Willebrand factor protein. Type I vWF defect is characterized by low levels of antigen but normal multimeric
configuration (quantitative defect); type II variants of vWF are characterized by lack of intermediate and/or high molecular weight multimers
(qualitative defect with or without concurrent quantitative defects); and type III vWF pattern refers to the total lack of any multimers due to the
inability to make vWF protein.
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von Willebrand Multimer gel |
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Last Modified September, 2003
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