Tests

Test Catalog
Test Name: ADAMTS-13 Activity (Service Code 5745)
CPT Code: 85397
Sample Requirements: Two 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less, ship on dry ice.
Max Turnaround: Samples are batch tested, usually twice weekly.
Methodology: Synthetic fluorescence resonsance energy transfer (FRET).
Availability: Contact Lab for availability.
Additional Information: This assay is based on quantifying the cleavage of a small fragment of von Willebrand Factor by the ADAMTS-13 protease. The cleavage of this synthetic substrate is detected by reading the fluorescence that results when the substrate is cleaved.
Test Catalog
Test Name: ADAMTS-13 Inhibitor (Service Code 5954)
CPT Code: 85335
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: Samples are batch tested, usually twice weekly.
Methodology: Mixing studies with normal pool using a synthetic Fluorescence Resonance Energy Transfer (FRET) labeled substrate.
Availability: Contact Lab for availability.
Additional Information: Functional assay to determine autoantibodies against ADAMTS-13 which is considered to be highly specific for TTP. Low plasma levels of ADAMTS-13 activity (<10%) and the presence of high titer inhibitor may be associated with poor prognosis.
Test Catalog
Test Name: Adenine Nucleotides (Service Code 5665)
CPT Code: 82030
Sample Requirements: Two 3.5 mL blue top (3.2% sodium citrate) tubes at room temperature.
Storage And Shipping: Maintain specimen as whole blood at room temperature and transport to the laboratory immediately. Must be received in laboratory within 3 hours of collection. Do not chill, platelets are activated at low temperatures.
Max Turnaround: Samples are batch tested, usually once or twice monthly.
Methodology: Luminescence
Availability: Contact Lab for availability.
Additional Information: An increase in the ATP/ADP ratio is consistent with a delta storage pool disorder.
Test Catalog
Test Name: Anticardiolipin IgA (Service Code 542I)
CPT Code: 86147
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: Batch tested once weekly
Methodology: Enzyme-linked immunoabsorbant assay (ELISA)
Availability: Contact Lab for availability
Additional Information: Evaluate patients with prolonged PTT and thrombotic tendency, including patients with systemic lupus erythematosus, recurrent spontaneous abortion of early pregnancy, and suspected antiphospholipid antibody syndrome.
Test Catalog
Test Name: Antiphosphatidylserine Antibodies (IgG, IgM) (Service Code 547X)
CPT Code: 86148 x 2
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: Batch tested once weekly
Methodology: Enzyme-linked immunoabsorbant assay (ELISA)
Availability: Contact Lab for availability
Additional Information: BACKGROUND

Antiphospholipid antibodies are a group of very heterogenous autoantibodies with reactivity against various negatively charged phospholipids, including phosphatidylserine, cardiolipin and others. The clinical importance of antiphospholipids is their strong association with recurrent venous and arterial thrombosis, thrombocytopenia and fetal abortion. These antibodies can be detected by coagulation assays or by solid phase ELISA, where currently the most common antigen in clinical use is the phospholipid cardiolipin.

Phosphatidylserine, unlike cardiolipin is found in the membranes of platelets and endothelial cells and participates in the coagulation cascade. Phosphatidylserine comprises 10-15% of the total phospholipds in plasma membranes, and is normally located in the interior of the lipid bilayer. Upon cell activation, phosphatidylserine is redistributed to the external surface where it may bin B2-glycoprotein I or other protein cofactor, leading to clot formation. For this reason, phosphatidylserine is a more physiologically relevant anionic phospholipid than cardiolipin, which has not been shown involved in coagulation. In addition, when aPS antibodies are injected into a mouse model, an experimental antiphospholipid syndrome is induced, confirming their pathogenic role.

aPS ASSAY

Anti-phosphatidylserine testing uses an indirect ELISA format to detect antibodies in human serum or plasma. Since autoimmune anti-phospholipid antibodies require a cofactor (B2-glycoprotein I) for optimal binding, a source of cofactor is included for increased detection of clinically relevant antibodies. The concentration of both IgG and IgM type phosphatidylserine antibodies are determined and reported out separately. The clinical specificity of the aPS assay is comparable to the specificity of the anticardiolipin (aCL) assay.

The heterogeneity of antiphospholipid antibodies complicates the laboratory diagnosis. Patients with elevated levels of antibodies to both cardiolipin and phosphatidylserine are more likely to have clinical complications than those positive to only one. Studies have shown that antiphosphatidylserine antibodies are prevalent in autoimmune populations and correlate with clinical manifestations of antiphospholipid antibody syndrome.

The heterogenity of antiphospholipd antibodies complicates the laboratory diagnosis. Patients with elevated levels of antibodies to both cardiolipin and phosphatidylserine are more likely to have clinical complications than those positive to only one. Studies have shown that antiphosphatidylserine antibodies are prevalent in autoimmune populations and correlate with clinical manifestations of antiphospholipid antibody syndrome.

CLINICAL APPLICATION

The anti-phosphatidylserine (aPS) assay, when run in concert with aCL and Lupus Anticoagulant assays provide a more complete profile for establishing a diagnosis of antiphospholipid antibody syndrome and for assessing the risk of thrombotic complications in SLE patients. It can offer valuable information in the following clinical situations:
  • patients diagnosed with autoimmune disease
  • patients with neurologic disease (cerebral ischemia, strokes, TIA’s, etc.)
  • patients with vascular disease
  • onset of myocardial infarcts
  • recurrent fetal loss
Test Catalog
Test Name: Antiphospholipid Antibodies (IgG, IgM) (Service Code 5651)
CPT Code: 86147 x 2
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: Batch tested once weekly
Methodology: Enzyme-linked immunoabsorbant assay (ELISA)
Availability: Contacted Lab for availability
Additional Information: Used to evaluate patients with prolonged PTT and thrombotic tendency, including patients with systemic lupus erythematosus, recurrent spontaneous abortion of early pregnancy, and suspected antiphospholipid antibody syndrome.
Test Catalog
Test Name: Antiplasmin Activity (Service Code 536A)
CPT Code: 85410
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: Batch tested once weekly
Methodology: Chromogenic substrate assay
Availability: Contact Lab for availability
Additional Information: Measure the amount of the major inhibitor of plasmin(ogen) in the fibrinolytic system.
Test Catalog
Test Name: Antithrombin III Antigen (Service Code 537H)
CPT Code: 85301
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 21 days
Methodology: Radial Immunodiffusion (RID)
Availability: Batch tested, once weekly.  Contact Lab for availability.
Additional Information: Must receive specimen in laboratory before day of testing.

Antithrombin III antigen measures total ATIII, functional and immunoreactive. It is ordered in conjunction with ATIII Activity to diagnose a dysproteinemia.
Test Catalog
Test Name: Cryoprecipitate QC (Service Code 547B)
CPT Code: 85240, 85384
Sample Requirements: One bag of cryoprecipitate, in compliance with your standard operating procedure. Criteria for Rejection: Broken bag
Storage And Shipping: Freeze at -70°C or less; ship on dry ice.
Max Turnaround: 3 days
Methodology: Clotting/Clauss method
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Test includes: Factor VIII:C, Fibrinogen

Evaluate whether blood products meet the standards of the blood banking industry.
Test Catalog
Test Name: Factor Assays (Service Code 547Y)
CPT Code: 85210, 85220, 85230, 85260, 85250, 85270, 85280
Sample Requirements: Two 3.5 mL blue top (sodium citrate) tube. (Min. 2 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder, ship on dry ice.
Max Turnaround: 1 day (excludes weekends and holidays)
Methodology: N/A
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Includes Factors II, V, IX, VII, X, XI, XII
Test Catalog
Test Name: ADAMTS-13 Autoantibody (Service Code 5913)
CPT Code: 83520
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: Samples are batch tested, usually every two weeks.
Methodology: Enzyme-linked immunoabsorbant assay (ELISA)
Availability: Contact Lab for availability.
Additional Information: Detects IgG antibody serologically. Less specific than the functional assay. Positive results have been observed in people without severe ADAMTS-13 deficiency.
Test Catalog
Test Name: Factor II (Service Code 536E)
CPT Code: 85210
Sample Requirements: One 3.5 mL blue top (3.2%sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 1 day (excludes weekends and holidays)
Methodology: Photometric; Factor deficient substrate plasma
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Quantity specific coagulation factor activity.
Test Catalog
Test Name: Factor IX (Service Code 536J)
CPT Code: 85250
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice.
Max Turnaround: 1 day
Methodology: Photometric; Factor deficient substrate plasma
Availability: Monday
Tuesday
Wednesday
Thursday
Friday

STAT testing available for off-hours.
Additional Information: Detect specific coagulation factor IX activity.
Test Catalog
Test Name: Factor IX Inhibitor (Service Code 537U)
CPT Code: 85335
Sample Requirements: Two 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 7 days
Methodology: Nijmegen-modified Bethesda assay
Availability: Batch tested twice weekly.
Additional Information: Specimen must be received before day of testing.

Used to evaluate patients with inhibitors to Factor IX. Recommend prior Factor IX Activity testing.  The Factor IX activity must be <25% (0.25 U/mL) to distinguish inhibitor activity.
Test Catalog
Test Name: Factor V (Service Code 536F)
CPT Code: 81479
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice.
Max Turnaround: 1 day
Methodology: Photometric; Factor deficient substrate plasma
Availability: Monday
Tuesday
Wednesday
Thursday
Friday

STAT testing available for off-hours.
Additional Information: Detect specific coagulation factor activity.
Test Catalog
Test Name: Factor V Cambridge (Service Code 557I)
CPT Code: 81479
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 2 mL) Should be received within 3 days of collection.
Storage And Shipping: Room temperature as whole blood
Max Turnaround: 21 days Batch Tested
Methodology: PCR-based assay that amplifies a 228 bp fragment of the factor V genomic DNA. Amplified fragment is restriction enzyme digested and fragments are separated by agarose gel electrophoresis.
Availability: Contact lab for Availability
Additional Information: Test performed every other Monday; results available Friday.

Background

Venous thromboembolism has been associated with molecular defects in several hemostatic components. Inherited resistance to activated protein C (APC) is a cause of familial thrombophilia and is the most common genetic risk factor for venous thrombosis. In more than 90% of cases, APC resistance is linked to a single point mutation in the factor V gene (Leiden mutation). In 5-10% of patients with deep vein thrombosis and APC resistance and no Leiden mutation it can be due to pregnancy, lupus anticoagulant activity, high factor VIII levels (“acquired” APC resistance) or another genetic mutation. In 1998, a new factor V mutation associated with APC resistance and thrombosis was identified. This mutation results in a replacement of Arg at position 306 to a Thr of the factor V gene.

Methods

A polymerase chain reaction (PCR) based assay amplifies a 228 bp fragment of the factor V genomic DNA containing the putative APC cleavage site. The amplified fragment is then restriction endonuclease digested using the BstN I. Digest fragments are separated by agarose gel electrophoresis to allow detection and discrimination of homozygosity or heterozygosity for the point mutation in the factor V gene. The frequency of this mutation is relatively rare.

Clinical Significance

It is clear from the literature and our own investigations that the frequency of this mutation is relatively rare. Testing for the presence of this gene should not be used for screening and should be done under the following circumstances:
  • Family and patient history of thrombosis
  • Abnormal APC resistance test and negative for the Leiden mutation
  • Exclusion of acquired APC resistance; negative testing for lupus anticoagulant, patient is not pregnant and normal levels of factor VIII.
The interaction of this genetic defect with other thrombotic risk factors is unknown. The assumption is there is accumulative risk if other acquired or genetic risk factors are present.
Test Catalog
Test Name: Factor V HR-2 Haplotype (Service Code 5591)
CPT Code: 81400
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 2 mL). Should be received within 3 days of collection.
Storage And Shipping: Room temperature as whole blood
Max Turnaround: 21 days (Batch tested)
Methodology: PCR-based assay that amplifies an 828 bp fragment of the factor V genomic DNA. Amplified fragment is restriction enzyme digested and fragments are separated by agarose gel electrophoreseis.
Availability: Contact Lab for availability
Additional Information: Test performed every other Monday; results available Tuesday.

BACKGROUND

Venous thromboembolism has been associated with molecular defects in several hemostatic components. The most frequent genetic risk factor for deep venous thrombosis (DVT) is a poor anticoagulant response to APC. Resistance to APC (APCr) is found in ~23% of our local unselected patient population with venous thrombosis. The majority of these patients have APCr due to the presence of the Factor V Leiden mutation. Researchers have actively studied the factor V gene to determine the cause of APCr in the 5-10% of patients that do not carry the Leiden mutation. Recently, evidence has emerged that shows that Factor V genetic components other than the Leiden mutation contribute to the APCr phenotype and are associated with increased thrombotic risk.

METHODS

A polymerase chain reaction (PCR) based assay amplifies an 828 bp fragment of the factor V genomic DNA containing the R2 allele. The amplified fragment is then restriction endonuclease digested using Rsa I. Digest fragments are separated by agarose gel electrophoresis to allow detection and discrimination of homozygosity or heterozygosity for the polymorphism in the factor V gene.

CLINICAL SIGNIFICANCE

A haplotype (HR2) including nine polymorphisms in exon 13 is always found in subjects carrying the Factor V A4070G allele (R2 allele). The R2 allele was found to affect the APC anticoagulant response both in noncarriers of the Leiden mutation and in carriers, pointing to an association between R2 allele and APCr. Homozygosity for HR2 has been shown to increase thrombotic risk. In addition, combined heterozygosity of HR2 and Factor V Leiden confers a 3 to 4- fold increase in relative risk of thrombosis as compared with Leiden alone. The clinical significance of the HR2 haplotype in combination with other genetic factors such as the Prothrombin Gene Variant has not yet been determined. Testing for the presence of this haplotype should not be used for screening. It should be done on patients with a family or patient history of thrombosis under the following circumstances:
  • Markedly abnormal APC resistance test, heterozygosity for the Leiden mutation, and multiple thrombotic episodes
  • Abnormal APC resistance test and negative for the Leiden mutation
  • Exclusion of acquired APC resistance; negative testing for lupus anticoagulant, patient is not pregnant and normal levels of factor VIII.
REFERENCES
  1. Bernardi F., Faioni, EM, Custoldi, E, et al. A factor V component differing from factor V R506Q contributes to the activated protein C resistance phenotype. Blood 1997; 90; 1552-7.
  2. Faioni, EM, Franchi, F, Bucciarelli, P., et al. Co-inheritance of the HR2 haplotype in factor V confers an increased risk of venous thromboembolism to carriers of the factor V R506Q (factor V Leiden). Blood 1999; 94; 3062-6.
  3. Alhenc-Gelas, M, Nicaud, V, Gandrille, S, et al. The factor V gene A4090G mutation and risk of venous thrombosis. Thromb. Hemost. 1999; 81; 193-7.
  4. DeVisser, MC, Guasch, JF, Kamphuisen, PN, et al. The HR2 haplotype of factor V effects on factor V levels, normalized activated protein C sensitivity ratios and risk of venous thrombosis. Thromb. Hemost. 2000; 83; 577-83.
Test Catalog
Test Name: Factor V Leiden Mutation (R506Q) (Service Code 557F)
CPT Code: 81241
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 2 mL) Should be received within 3 days of collection.
Storage And Shipping: Room Temperature as whole blood.
Max Turnaround: 10 days; Batch tested once weekly.
Methodology: Invader assay from Hologic Inc. is a linear signal amplification system for directly detecting and quantifying gene-specific sequences in complex mixtures of nucleic acids. Employs FRET detection format for increased sensitivity.
Availability: Contact Lab for availability
Additional Information: BACKGROUND

Venous thromboembolism has been associated with molecular defects in several hemostatic components: protein C, protein S, and antithrombin III. However in over 90% of patients the cause remains obscure. Recently, resistance to activated protein C has been observed in 30-40% of patients with idiopathic thrombotic disease. This abnormality has been shown in the majority of APC resistant patients to be due to a single basepair mutation in the factor V gene (FV Leiden mutation) resulting in the amino acid substitution of Arg 506 to Gln. This mutation alters the APC cleavage site of factor V and produces a mutant molecule that cannot be properly inactivated by APC. This defect forms the molecular basis for the thrombotic events associated with APC resistance.

METHODS

Lymphocytes are processed to extract DNA. Hologic Inc.'s Invader assay is used to genotype the target DNA following PCR amplification. Briefly, two specific oligonucleotide probes and controls hybridize in tandem to a specific region of the gene encoding the mutation site. Cleavase III enzyme cleaves at a specific invasive structure generated within the probe oligonucleotides creating products. These sequential cleavage reactions produce 1-10 million fluorescein labeled products per target sequence hour. This results in the accumulation of a signal molecule only when the specific target DNA or control sequence is present. A fluorescent multiwell plate reader is used to quantitate signal amplification for interpretation of genotype.

CLINICAL SIGNIFICANCE

The high prevalence of the mutation in the normal population (6%) and its association with APC resistance and thrombosis suggests that it is worthwhile to screen thrombophilic patients for the mutated allele. It is recommended that the factor V mutation test be performed on APC resistant positive patients and it may offer valuable information in the following situations:
  • Venous thrombotic events
  • Recurrent TIAs or strokes, especially in young patients
  • Patients being considered for anticoagulant therapy
  • Investigation of familial thrombosis
  • Screening prior to oral contraceptive use
The factor V mutation test has considerable advantages over the APC resistance clot-based screening method, since it can be performed on patients receiving anticoagulant therapy and those with familial or acquired disorders of the intrinsic coagulation pathway. 

REFERENCES
  1. Bertina, R. et al. Mutation in blood coagulation factor V associated with resistance to activated Protein C. Nature 369:64-67; 1994.
  2. Zöller, B. and Dahlbäck, B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 343:1536-1538; 1994.
  3. Voorberg, J. et al. Association of idiopathic venous thromboembolism with single point-mutation at Arg506 of factor V. Lancet 343:1535-1536; 1994.
Test Catalog
Test Name: Factor VII (Service Code 536G)
CPT Code: 85230
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice.
Max Turnaround: 1 day
Methodology: Photometric; Factor deficient substrate plasma
Availability: Daily

STAT testing available off hours
Additional Information: Detect specific coagulation factor activity.
Test Catalog
Test Name: Factor VIII Inhibitor (Service Code 537T)
CPT Code: 85335
Sample Requirements: Two 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL frozen platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice.
Max Turnaround: 7 days (Batch tested twice weekly)
Methodology: CDC Modification: Heat-Inactivated Nijmegen Assay
Availability: Contact Lab for availability
Additional Information: Specimen must be received before day of testing.

Used to evaluate patients with inhibitors to Factor VIII:C (hemophilia or acquired).
Test Catalog
Test Name: Factor VIII:C (Service Code 5364)
CPT Code: 85240
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 1 day
Methodology: Photometric; Factor deficient substrate plasma
Availability: Daily

STAT testing available off hours
Additional Information: Detect coagulant Factor VIII deficiency; screen for anti-VIII.
Test Catalog
Test Name: Factor X (Service Code 536H)
CPT Code: 85260
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice.
Max Turnaround: 1 day
Methodology: Photometric; Factor deficient substrate plasma
Availability: Daily

STAT testing available off hours
Additional Information: Detect specific coagulation Factor X activity.
Test Catalog
Test Name: Factor X Chromogenic (Service Code 537I)
CPT Code: 85260
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 7 days - batch tested twice weekly
Methodology: Chromogenic
Availability: contact laboratory for availability
Additional Information: BACKGROUND

Lupus anticoagulants are phospholipid antibodies that interfere with phospholipid dependent clotting reactions in vitro, most frequently in aPTT and dRVV assays. Recently it has been shown that the prothrombin time (PT) is frequently prolonged in patients with lupus anticoagulants since thromboplastins also contain phospholipids. Oral anticoagulant therapy with warfarin is monitored by the PT; if patients with a baseline prolongation of their PT due to a LAC are treated with warfarin, a certain PT-INR level may not reflect the true level of anticoagulation. To prevent supratherapeutic or subtherapeutic anticoagulation, these patients must be individually monitored with a test that is insensitive to lupus anticoagulants.

The chromogenic factor X assay is the method of choice for oral anticoagulant monitoring in patients with lupus anticoagulants affecting the prothrombin time. A therapeutic range of factor X is established by correlation to the appropriate target INR range (2.0-3.5). This assay is insensitive to the effects of lupus anticoagulants and can provide monitoring for effective anticoagulant therapy and improved patient management.

METHODS

The chromogenic factor X assay indirectly measures the level of the vitamin K dependent factor X in human plasma. This is done in two stages. In the first stage factor X is activated in the presence of calcium to Factor Xa by the activator Russell’s Viper Venom (RVV). In the second stage the generated factor Xa hydrolyzes a chromogenic substrate resulting in a color change. The intensity of the color change is proportional to the factor X activity in the sample. This assay does not require a phospholipid membrane surface and is insensitive to the effects of a phospholipid antibody.

CLINICAL APPLICATION

The chromogenic factor X assay is an alternate approach for the management of oral anticoagulants in patients with lupus anticoagulants influencing the prothrombin time. An INR of 2.0 to 3.5 correlated to a chromogenic factor X level of 23% to 45% in patients with lupus anticoagulants. A supratherapeutic result is less than 23% and a subtherapeutic result is greater than 45%.

REFERENCES
  1. Moll, S. and Ortel, T.L. Monitoring warfarin therapy in patients with lupus anticoagulants. Ann. Int. Med., 1997; 127: 177-185.
  2. Ciavarella, S., et al., Multicenter evaluation of a new chromogenic factor X assay in plasma of patients on oral anticoagulants. Thromb. Res., 1980: 19: 493-502.
Test Catalog
Test Name: Factor XI Inhibitor (Service Code 5386)
CPT Code: 85335
Sample Requirements: Two 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL frozen platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice.
Max Turnaround: Batch tested twice weekly
Methodology: CDC Modification: Heat-Inactivated Nijmegen
Availability: Contact Lab for availability
Additional Information: Specimen must be received before day of testing.

Used to evaluate patients with an inhibitor to factor XI. If a prolonged APTT fails to correct after an incubated mix with normal plasma and factor analysis reveals a marked decrease in factor XI only, a specific factor XI inhibitor is suspected. A titer to quantitate the amount (strength) of the inhibitor, such as the Bethesda assay, is useful in patient management and to monitor the success in eradicating the inhibitor.
Test Catalog
Test Name: Factor XII (Service Code 536L)
CPT Code: 85280
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 1 day; Available as a STAT test
Methodology: Photometric; Factor deficient substrate plasma
Availability: Daily
Additional Information: Detect specific coagulation factor XII activity.
Test Catalog
Test Name: Factor XII Inhibitor (Service Code 5387)
CPT Code: 85335
Sample Requirements: Two 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL frozen platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice.
Max Turnaround: 7 days (Batch tested twice weekly)
Methodology: Nijmegen Assay
Availability: Contact Lab for availability.
Additional Information: Specimen must be received before day of testing.

Used to evaluate patients with an inhibitor to factor XII. The factor XII activity must be <25% (0.25 U/mL) to distinguish inhibitor activity. If a prolonged APTT fails to correct after an incubated mix with normal plasma and factor anaylsis reveals a marked decrease in factor XII only, a specific factor XII inhibitor is suspected. A titer to quantitate the amount (strength) of the inhibitor, such as the Bethesda Assay, is useful in patient management and to monitor the success in eradicating the inhibitor.
Test Catalog
Test Name: Factor XIII Activity - Quantitave (Service Code 6289)
CPT Code: 85290
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -20°C or colder; ship on dry ice.
Max Turnaround: Batch tested once weekly
Methodology: See below
Availability: Contact Lab for availability
Additional Information: The FXIII Activity test is an assay that quantitatively determines the patient’s FXIII functional ability. The previous FXIII Antigen method only measured the amount of FXIII protein present in the sample rather than the patient’s FXIII functional capabilities. Therefore, the newer FXIII Activity-Quantitative assay may be more useful than the discontinued antigen method. In addition, the new FXIII Activity-Quantitative test is more specific than qualitative assays for FXIII, such as clot lysis methods (FXIII Screen), since these latter clots lysis tests can only detect FXIII abnormalities below 1-2%.

The sample collection requirements for the FXIII Activity-Quantitative assay are exactly the same as the previous discontinued FXIII Antigen test. Please collect the patient’s sample using one 3.2% sodium citrate (blue-top) tube and centrifuge/remove plasma within 1 hour of collection. Please freeze plasma at -20°C or colder and send to ITxM Diagnostics Coagulation Laboratory frozen as usual. A minimum of 1.0 mL of frozen plasma is required for this new assay. Similar to the discontinued procedure, the new FXIII Activity-Quantitative samples will be tested in batches once weekly. Therefore, the turn-around-time may be up to two-weeks from receipt of the sample.
Test Catalog
Test Name: Factor XIII Screen - Quantitative (Service Code 536M)
CPT Code: 85291
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice.
Max Turnaround: 1 day (excludes weekends and holidays)
Methodology: MCA Lysis
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: A Factor XIII deficiency of less than 1-2% can be detected by this in vitro method.  Abnormal/Positive results occurs only if the FXIII deficiency is less than 1-2% by this in vitro method.
Test Catalog
Test Name: Fibrinogen (Service Code 5373)
CPT Code: 85384
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Modified thrombin time (Clauss) using preassayed fribinogen standards
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Identify congenital afibrinogenemia or hyprofibrinogenemia.
Test Catalog
Test Name: Fibrinogen Antigen (Service Code 536U)
CPT Code: 85385
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL frozen platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 11 days
Methodology: Radial Immunodiffusion Assay
Availability: Friday
Additional Information: Specimen must be received before day of testing.

Fibrinogen antigen measures total plasma fibrinogen, functional and non-functional. Ordered in conjunction with fibrinogen activity to diagnose dysfibrinogenemia.
Test Catalog
Test Name: Fondaparinux (Arixtra) Level (Service Code 5762)
CPT Code: 85520
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Chromogenic/Inhibition of Xa
Availability: Monday
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Additional Information: Used to monitor Fondaparinux (Arixtra) levels in patients.
Test Catalog
Test Name: Glycoprotein Ia C807T (Service Code 5607)
CPT Code: 81479
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 2 mL)
Storage And Shipping: Room temperature as whole blood
Max Turnaround: 28 days
Methodology: PCR-based assay that amplifies a 231 bp fragment of the targeted region. Amplified fragment is restriction enzyme digested and fragments are separated by agarose gel electrophoresis.
Availability: Monday
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Additional Information: BACKGROUND

The GPIa/IIa complex mediates platelet adhesion to collagen. Although the GPIa C807T polymorphism does not change the amino acid sequence, it has been associated with the difference in the GPIa/IIa receptor levels on the platelet surface. Carriers of the 807T allele express higher levels of GPIa/IIa, while carriers of the 807C allele exhibit lower expression of the integrin. This polymorphism has been implicated to be associated with an increased risk of arterial thrombosis. Additional studies suggest this is a mild risk factor and is particularly important in synergism with known risk factors such as smoking, hypertension, diabetes or proteinuria, which may enhance its contribution to the overall cardiovascular risk.

METHODS

Lymphocytes are processed to extract DNA. Third Wave Technologies’ Invader assay is used to genotype the target DNA. Briefly, two specific oligonucleotide probes and controls hybridize in tandem to a specific region of the gene encoding the mutation site. Cleavase III enzyme cleaves at a specific invasive structure generated within the probe oligonucleotides creating products. These sequential cleavage reactions produce 1-10 million fluorescein labeled products per target sequence hour. This results in the accumulation of a signal molecule only when the specific target DNA or control sequence is present. A fluorescent multiwell plate reader is used to quantitate signal amplification for interpretation of genotype. Homozygosity for the C807T allele (T/T) is associated with increased arterial thrombotic risk.

CLINICAL SIGNIFICANCE

Platelets play an important part in arterial thrombosis therefore it is important to consider the role adhesion molecules of the platelet surface play in increasing arterial risk. Glycoprotein Ia/IIa is the major platelet collagen receptor and is responsible for platelet adherence to exposed vascular subendothelium. The single nucleotide polymorphism C807T allele is associated with increased collagen receptor levels and increased collagen induced platelet adhesion. Preliminary results suggest that the C807T variant of glycoprotein Ia may be a genetic risk factor for early-onset arterial thrombotic disease.

In general, screening for arterial thrombosis is best accomplished by evaluating for the traditional cardiovascular markers, such as diabetes mellitus, smoking, hypertension, and hypertriglyceridemia. The use of the glycoprotein Ia C807T assay may be of value in the following situations:
  1. Children, young individuals or pre-menopausal women with arterial thrombosis
  2. Any individual with arterial disease in the absence of atypical cardiovascular risk factors
REFERENCES
  1. Bussel, JB, Kunicki, TJ, Michelson, AD. Platelets: New understanding of platelet glycoproteins and their role in disease. Hematology (Am. Soc Hematol Educ Program; 222-240, 2000.
  2. Beer, JH, Pederiva, S, Poniggia, L. Genetics of platelet receptor single-nucleotide polymorphisms: clinical implications in thrombosis. Ann. Med. 32: 10-14, 2000.
  3. Reiner, AP, Kumar, PN, Schwartz, SM, et. al., Genetic variants of platelet glycoprotein receptors and risk of stroke in young women. Stroke 31: 1628-1633, 2000.
  4. Golanski, J, Golanski, R, Chizynski, K, et. al., Platelet hyperreactivity after coronary artery bypass grafting: the possible relevance to glycoprotein polymorphisms. A preliminary report. Platelets 12: 241-247, 2001.
  5. Casorelli, I, DeStefano, V, Leone, AM, et. al. The C807T/G873A polymorphism in the platelet glycoprotein Ia gene and the risk of acute coronary syndrome in the Italian population. Br. J. Haematol. 114: 150-154, 2001.
Test Catalog
Test Name: Heparin Level/Anti-Xa (Service Code 547L)
CPT Code: 85520
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.) Type of heparin for evaluation MUST be provided to the laboratory (e.g., Unfractionated, Lovenox, Fragmin, or Innohep).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Chromogenic/Inhibition of Xa
Availability: Monday
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Additional Information: BACKGROUND

Heparin, a naturally occurring polymeric mucopolysaccharide with anticoagulant and antithrombotic properties, is effective in the prevention and treatment of a variety of venous and arterial thromboembolic disorders. It is used as a prophylaxis against postoperative thrombosis, in conjunction with the initiation of oral anticoagulant therapy, in the treatment of unstable angina and acute myocardial infarction and to prevent clotting during cardiac bypass.

The principle inhibitor of thrombin, factor Xa and other coagulation serine proteases in plasma is antithrombin III. The rate of inhibition, under normal conditions, is slow, but can be increased several thousand-fold by heparin. This mechanism accounts for the anticoagulant effect of heparin. Low molecular weight therapeutic heparin preparations appear to catalyze the reaction between factor Xa and antithrombin III more readily than the reaction between thrombin and antithrombin III while standard heparin catalyzes both reactions equally. The factor Xa inhibition test is the most useful test for assaying the widest variety of therapeutic heparin preparations.

ANTI-Xa ASSAY

Laboratory monitoring of heparin therapy is desirable to ensure that an appropriate antithrombotic effect is obtained, while guarding against bleeding complications of an overdosage. Currently, the APTT is the most common test used to monitor heparin therapy, however, this method is not without drawbacks. Monitoring by APTT evaluates heparin’s overall activity throughout the entire coagulation system, and the results can be affected by components in plasma not related to heparin. An alternative approach is to assay for heparin by exploiting its catalysis by antithrombin III inhibition of coagulation enzymes, particularly factor Xa. In this method, factor Xa is present in excess and the residual factor Xa activity is inversely proportional to the heparin concentration. Unlike assays based on thrombin, this assay is based on factor Xa is sensitive to both standard and low molecular weight (LMW) heparins.

CLINICAL SIGNIFICANCE

The aim of therapy is to produce truly stabilized levels of circulating heparin capable of maintaining an effective hypocoagulability, and avoiding unnecessary and dangerous overdosage. There are several clinical situations in which the specific measurement of heparin levels using the anti-factor Xa method may be necessary. Monitoring of heparin is difficult by conventional methods when the baseline APTT is prolonged as seen in patients with lupus anticoagulant and deficiencies of factor XII (Hageman factor), prekallikrein (Fletcher factor) and high molecular weight kininogen (Fitzgerald factor). A quantitative anti-Xa heparin assay makes heparin monitoring possible in these clinical situations. In pregnant women requiring anticoagulation, heparin is the drug of choice because it does not cross the placenta or produce untoward effects in the fetus or newborn when administered to the mother. The anti-Xa heparin assay can provide accurate monitoring to ensure therapeutic levels are maintained since the long term use of high doses of heparin is associated with osteoporosis. Heparin monitoring using the anti-Xa assay may also be clinically warranted in cases of suspected heparin resistance and in the use of LMW heaprins and heparinoids. Monitoring of LMW heparins can only be determined by anti-factor Xa activity since these agents do not produce a prolongation of the APTT at therapeutic levels.
Test Catalog
Test Name: Heparin PF4 (Platelet Factor 4) Platelet Antibody (Service Code 550B)
CPT Code: 86022
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 3 days
Methodology: Enzyme-linked immunoabsorbant assay (ELISA)
Availability: Monday
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Additional Information: BACKGROUND

Heparin-Induced Thrombocytopenia (HIT) can be a devastating syndrome that manifests itself by mild to severe thormbocytopenia and is accompanied by a relatively high risk of associated thrombosis. This syndrome occurs in two general forms. HIT Type I is a non-immune mediated benign form characterized by a slight decrease in platelet count occurring immediately after heparin or within the first 5 days of heparin therapy. The patient is often times asymptomatic.

HIT type II is a more serious, immune-mediated form characterized by a delayed onset (3-15 days) after initiation of heparin therapy. Patients often present with mild to severe thrombocytopenia (<100,000 platelet x 109/L) and may develop complicated thromboses. Due to the seriousness of this condition it is essential to identify the patient with HIT and discontinue heparin.

HIT occurs when heparin triggers the formation of antibodies directed at a heparin-platelet factor 4 complex. This complex binds to platelet Fc receptors resulting in platelet activation, platelet aggregation, platelet granule release and the formation of procoagulant microparticles that accelerate thrombin generation.

METHODS

The identification of platelet factor 4 (PF4) as the target component, allowed the design of an ELISA assay for the diagnosis of heparin induced thrombocytopenia. This assay incorporates the use of coated wells with PF4 complexed with heparin. Prediluted plasma containing heparin-PF4 antibodies will bind to the wells if present. A second conjugated affinity-purified anti-human IgG, M, A will bind to the complex and the intensity of observed coloration is directly proportional to the antibodies present in the test sample.

Most of the diagnostic methods for type II HIT are based on the use of platelet aggregometry tests or serotonin release assay (SRA). The platelet aggregation method requires careful selection of control platelets and lacks sensitivity and specificity. The SRA, considered by many to be the gold standard, also requires carefully selected control platelets, radio-isotopes and is difficult to perform. The heparin-PF4 ELISA assay has similar sensitivity to SRA and is reproducible and relatively easy to perform. Studies have shown that heparin-PF4 ELISA yields false negative results in approximately 10% of patients tested with clinically evident HIT.

CLINICAL SIGNIFICANCE

HIT carries high mortality and morbidity, the diagnosis of HIT is critical for the successful intervention and treatment of complications of heparin therapy. In addition the potential for developing HIT is high, since as many as two-thirds of all patients may be exposed to heparin at some point during their hospitalization. The incidence of HIT remains difficult to determine, however the majority of reports indicate that 5-10% of all patients exposed to heparin will develop an antibody to heparin. Laboratory procedures are useful in diagnosing HIT but cannot predict any thrombotic risk. It should be emphasized that a negative result in any assay does not rule out the presence of HIT. The heparin-platelet factor 4 induced antibody test by ELISA is recommended in the following situations:
  • Mild to severe thrombocytopenia post heparin therapy
  • Confirm clinical suspicion of HIT
  • Patients at risk for HIT due to previous heparin exposures
  • Patients undergoing cardiac procedures
Test Catalog
Test Name: Heparin Platelet Antibody Panel (Service Code 550C)
CPT Code: 86022 x 2
Sample Requirements: Refer to individual test components
Storage And Shipping: Refer to individual test components
Max Turnaround: 3 days
Methodology: Refer to individual test components
Availability: Monday
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Additional Information: Test includes: Heparin-Induced Platelet Antibody, Heparin PF4 Platelet Antibody
Test Catalog
Test Name: Heparin-Induced Platelet Antibody (Aggregation Method) (Service Code 535H)
CPT Code: 86022
Sample Requirements: Two 4.5 mL blue top (3.2% sodium citrate) tubes. (Min. 1 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Platelet aggregation
Availability: Monday
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Additional Information: Specimen must be received by 4:00pm.

Confirm the presence of clinically suspected heparin induced thrombocytopenia.
Test Catalog
Test Name: Hereditary Hemochromatosis HFE C282Y (Service Code 557T)
CPT Code: 81256
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 3 mL.)
Storage And Shipping: Room temperature as whole blood.
Max Turnaround: 7 days
Methodology: PCR based assay that amplifies a 290 bp fragment of the HFE genomic DNA. Amplified fragment is restriction enzyme digested and fragments are separated by agarose gel electrophoresis.
Availability: Friday
Additional Information: Hereditary hemochromatosis is a genetically determined disorder characterized by iron overload. The carrier frequency for this mutation may be as high as 1 in 15 among people of European descent. A point mutation in the HFE gene results ina Cys282Tyr substitution. This substitution has been identified as the major genetic defect for this autosomally recessive inherited disorder. Most affected individuals are homozygous for the mutant allele, although a few may be heterozygous with variable phenotypic expression. Molecular detection of the mutation can lead to early diagnosis and treatment to prevent the long-term sequalae of iron overload.
Test Catalog
Test Name: Hereditary Hemochromatosis HFE H63D (Service Code 558D)
CPT Code: 81256
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 3 mL.)
Storage And Shipping: Room temperature as whole blood
Max Turnaround: 7 days
Methodology: PCR based assay that amplifies a 211 bp fragment of the HFE genomic DNA. Amplified fragment is restriction enzyme digested and fragments are separated by agarose gel electrophoresis.
Availability: Friday
Additional Information: BACKGROUND
Hereditary Hemochromatosis is a genetic disorder characterized by iron overload that results in organ damage. It occurs with a frequency of 1 in 300 in populations of northern European descent. The HFE gene was identified in 1996 and subsequent studies have shown HFE as being the responsible gene for hereditary hemochromatosis. C282Y has been shown to be the main mutation responsible for hemochromatosis in all study populations and accounts for 80-90% of HH chromosomes. Recently the functional significance of another HFE mutation, H63D was demonstrated. This mutation has been shown to be associated with a milder form of HH and shows incomplete penetrance.

METHODS Patient lymphocytes are processed to extract DNA. Using 250ng of human DNA, a polymerase chain reaction is performed to amplify a 211 b.p. fragment of the gene encoding the HFE protein.
The 211 b.p. fragment includes the H63D coding site that has been associated with hereditary Hemochromatosis. The amplimer is restriction enzyme digested with the enzyme BclI. Enzyme digest fragments are resolved on a 2.5% agarose gel and visualized by ethidium bromide. The 211 b.p. product of the normal HFE allele yields fragments of 69 b.p. and 138 b.p. Digestion of the H63D mutant HFE allele remains uncut at 211 b.p. Repeat digestion is performed on all homozygous mutants for verification.

CLINICAL SIGNIFICANCE

The H63D substitution is considered a genetic variant that increases the risk of developing a milder form of hemochromatosis. It has been reported that H63D represents 39% of affected individuals that do not carry the C282Y mutation. Most affected individuals are homozygous for the mutation. Compound heterozygotes C282Y/H63D have been reported to account for 7% of the hemochromatosis subjects. Molecular detection of the mutation can lead to early diagnosis and effective treatment to prevent the long-term clinical consequences of iron overload. Molecular detection of the mutant HFE allele may be useful in the following situations:
  • Patients with abnormally high iron measurements
  • Distinguishing between hereditary hemochromatosis and other iron overload conditions
  • Preclinical detection of hereditary hemochromatosis in individuals with a family history of hemochromatosis
  • Patients previously shown to be heterozygous for C282Y
REFERENCES
  1. Mura, C., et al. HFE mutation analysis in the 711 hemochromatosis probands: Evidence for S65C implication in mild form of hemochromatosis. Blood 1999; 93:2502-5.
  2. Feder, J.N., et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet l996; 13: 399-408.
  3. Adams, p.c. et al. The relationship between iron overload, clinical symptoms, and age in 410 patients with genetic hemochromatosis. Hepatology l997; 25: 162-166. Bulaj, Z.J. et al. Clinical and biochemical abnormalities in people heterozygous for hemochromatosis. N Engl J Med l996; 335: 1799-1805.
Test Catalog
Test Name: Hexagonal Phospholipid Neutralization (Service Code 547A)
CPT Code: 85730, 85732
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Photometric
Availability: Monday
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Additional Information: Background

Antiphospholipid antibodies (including the so-called lupus anticoagulant) have been associated with an increased risk of recurrent thromboses, fetal loss, and thrombocytopenia. These antibodies are heterogeneous, with differing patterns of test results from patient to patient. Although the lupus anticoagulant (LAC) may occur in lupus patients, the majority of patients with LAC’s do not have lupus. The occurrence of the LAC in an individual may be idiopathic, associated with a variety of autoimmune diseases, infections, malignancies, or drug-induced.

LAC’s are usually detected because they inhibit phospholipid dependent coagulation tests, such as the APTT, PT, and dilute RVV. They presumably do this by binding to phospholipids, not to individual coagulation factors. Due to the heterogeneity of composition and potency of LAC’s, there is no single test that is able to detect all positive individuals. Although there are several tests available for the detection of LAC’s, there are no accepted diagnostic criteria.

In 1991, an international committee proposed guidelines that may be used to identify the presence of LAC’s. These include:
  1. Prolongation of a phospholipid dependent clotting test
  2. Inability of normal plasma to correct the defect
  3. Correction of the defect by addition of exogenous lipids
Other properties such as the demonstration of antiphospholipid immunoglobulin and that the inhibition observed is not specific for any single clotting factor are helpful.

Hexagonal Phase Neutralization Assay: Rauch, Janoff, and their colleagues have been able to obtain antiphospholipid antibodies from human hybridomas which were produced from patients with lupus. These monoclonal antibodies were able to bind to hexagonal (II) phase phosphatidylethanolamine (HPE), but not lamellar (bilayer) phosphatidylethanolamine (PE). Additionally, HPE, but not PE, could induce the formation of monoclonal antibodies in mice that had LAC activity, and in many cases were able to cross react with cardiolipin as well. Furthermore, mice immunized with a mixture of cardiolipin and the plasma protein apoliproprotein H were induced to form monoclonal antibodies that cross reacted with HPE. These results suggested that in vivo LAC’s and anticardiolipin antibodies may either be induced by, and/or react with damaged cellular membranes. The hexagonal phase neutralization assay is based on the findings that HPE will bind to lupus anticoagulants, thus inhibiting their actions. The test features a dilute APTT reagent with decreased lipids that is highly sensitive to LAC’s. The patient plasma is mixed with an equal volume of normal plasma. If the clotting time with the addition of HPE is 8 or more seconds less than the mix without HPE, then the test is considered positive for LAC. This test is an important addition to our LAC profile, because it may be used for patients on oral anticoagulants or heparin.

Clinical Applications

The lupus anticoagulant profile may offer valuable information in the following clinical situations:
  • Recurrent spontaneous abortions
  • Arterial and venous thromboembolic events
  • An unexplained prolonged APTT
  • Recurrent TIA’s or strokes, especially in young patients
Test Catalog
Test Name: HMW Kininogen (Fitzgerald Factor) (Service Code 536N)
CPT Code: 85293
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 10 days
Methodology: Photometric; Factor deficient substrate plasma
Availability: Monday
Additional Information: Availability is variable (dependent on deficient plasma availability).

Investigate the causes of prolonged APTT; identify HMW Kininogen deficiency (Fitzgerald Factor deficit).
Test Catalog
Test Name: Homocysteine, Quantitative (Service Code 558)
CPT Code: 83090
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma immediately after collection or, as an alternative, place on wet ice until separation. Freeze at -70°C or less; ship on dry ice.
Max Turnaround: 7 days
Methodology: Chemical modification with competitive agglutination
Availability: Wednesday
Additional Information: Screening test to quantitate homocysteine; aid in assessment of thrombotic risk, cardiovascular disease, and the development of atherosclerosis.
Test Catalog
Test Name: Lupus Anticoagulant (LAC) Panel (Service Code 547T)
CPT Code: 85610, 85730 x 2, 85732 x 2, 85613, 85705, 85670, 86147 x 2, 85240
Sample Requirements: Three 4.5 mL blue top (3.2% sodium citrate) tubes. (Min. 3 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Refer to individual test components
Availability: Monday
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Additional Information: Test includes: PT; APTT; APTT Mix; dRVV; Thrombin Time; Reptilase Time (if needed; will invoke additional charge); Tissue Thromboplastin Inhibition (TTI); Factor VIII:C; Hexagonal Lipid Neutralization; Anticardiolipin Antibodies (IgG, IgM); Anti-VIII (if needed; will invoke additional charge), and Physician Interpretation with clinical recommendations if warranted.

LAC Panel includes screening and confirmatory tests to detect the presence of phopholipid antibodies that may be associated with thrombosis. Normal test results do not necessarily rule out a lupus anticoagulant.
Test Catalog
Test Name: Lupus Anticoagulant (LAC) Panel II (Service Code 5774)
CPT Code: 85240, 85610, 85613, 85670, 85705, 85730 x 2, 85732 x 2, 86146 x 3, 86147 x 2
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Refer to individual test components.
Availability: Monday
Tuesday
Wednesday
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Friday
Additional Information: Test includes: PT; APTT; APTT Mix; dRVV; Thrombin Time; Reptilase Time (if needed; will invoke additional charge); Tissue Thromboplastin Inhibition (TTI); Factor VIII:C; Hexagonal Lipid Neutralization; Anticardiolipin Antibodies (IgG, IgM); Beta 2 Glycoprotein I Antibodies (IgG, IgM, IgA); and Physician Interpretation with clinical recommendations if warranted.

LAC Panel II includes screening and confirmatory tests to detect the presence of phopholipid antibodies that may be associated with thrombosis. Normal test results do not necessarily rule out a lupus anticoagulant.
Test Catalog
Test Name: Lupus Anticoagulant (LAC) Screen (Service Code 547S)
CPT Code: 85610, 85730, 85732, 85613, 85705, 85670
Sample Requirements: Two 4.5 mL blue top (3.2% sodium citrate) tubes. (Min. 2.5 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice
Max Turnaround: 2 days
Methodology: Refer to individual test components
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Test includes: PT; APTT; APTT Mix; dRVV; Thrombin Time; Reptilase Time (if needed; will invoke addtional charge); Tissue Thromboplastin Inhibition (TTI); and Interpretation.

Basic screen to detect the presence of phopholipid antibodies that may be associated with thrombosis. Normal test results do not necessarily rule out a lupus anticoagulant.
Test Catalog
Test Name: MTHFR Gene Variant A1298C (Service Code 557Y)
CPT Code: 81291
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 3 mL.)
Storage And Shipping: Room temperature as whole blood
Max Turnaround: 7 days
Methodology: Invader assay from Third Wave Technologies is a linear signal amplification system for directly detecting and quantifying gene-specific sequences in complex mixtures of nucleic acids. Employs FRET detection format for increased sensitivity.
Availability: Friday
Additional Information: BACKGROUND

Total blood levels of homocysteine (tHcy) have been shown to depend on both environmental and genetic factors, and to be associated with the risk of developing thrombosis (venous and arterial), coronary disease, and cerebral vascular disease. Studies have shown that homozygosity for the common C677T mutation in the MTHFR gene is a genetic risk factor for hyperhomocysteinemia and has been implicated in both neural tube defects and vascular disease. Although many studies have examined the effects of the C677T polymorphism on homocysteine levels, the variability in results, particularly in heterozygous individuals, suggested the presence of a second variant in the population. A second genetic variant has been characterized MTHFR (A1298C) and this assay is designed to detect this variant of MTHFR which results in reduced activity of the enzyme.

METHODS

Lymphocytes are processed to extract DNA. Third Wave Technologies’ Invader assay is used to genotype the target DNA. Briefly, two specific oligonucleotide probes and controls hybridize in tandem to a specific region of the gene encoding the mutation site. Cleavase III enzyme cleaves at a specific invasive structure generated within the probe oligonucleotides creating products. These sequential cleavage reactions produce 1-10 million fluorescein labeled products per target sequence hour. This results in the accumulation of a signal molecule only when the specific target DNA or control sequence is present. A fluorescent multiwell plate reader is used to quantitate signal amplification for interpretation of genotype.

CLINICAL SIGNIFICANCE

A second common polymorphism in the gene encoding Methylenetetrahydrofolate Reductase (MTHFR) (A to C transition at nucleotide position 1298) generates a heat-labile enzyme with reduced activity. Neither the heterozygous or homozygous state of this mutation is associated with higher plasma homocysteine levels or a lower plasma folate concentration. However, there appears to be an interaction between the two common mutations. Combined heterozygosity for the MTHFR mutations was associated with reduced MTHFR specific activity, higher homocysteine, and decreased plasma folate levels. Studies have also suggested that combined heterozygosity may be a risk factor for increased neural tube defects and vascular disease. The clinical significance of heterozygosity for MTHFR (A1298C) in combination with other genetic factors such as the Factor V Leiden and Prothrombin Gene Variant has not yet been determined. It is recommended that the Methylenetetrahydrofolate Reductase Variant (A1298C) genetic assay be used in the following situations:
  • Patients who are heterozygous for MTHFR C677T and have a history of thrombosis
  • Investigations of familial thrombosis, vascular disease and neural tube defects.
  • Venous or arterial events
REFERENCES
  1. van der Put, et al. 1998. A second common mutation in the methylenetetrahydrofolate reductase gene: An additional risk factor for neural tube defects? Am. J. Hum. Genet. 62: 1044-1051.
  2. Weisberg, I. et al. 1998. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol. Genet. Metabol. 64: 169-172.
Test Catalog
Test Name: MTHFR Gene Variant C677T (Service Code 557X)
CPT Code: 81291
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 3 mL.)
Storage And Shipping: Room temperature as whole blood.
Max Turnaround: 7 days
Methodology: Invader assay from Third Wave Technologies is a linear signal amplification system for directly detecting and quantifying gene-specific sequences in complex mixtures of nucleic acids. Employs FRET detection format for increased sensitivity.
Availability: Friday
Additional Information: BACKGROUND

A deficiency in the methylenetetrahydrofolate reductase (MTHFR) enzyme, as well as deficiencies in folate, vitamin B12 and B6 can result in elevated homocysteine levels in the blood. Moderate homocysteinemia has been associated with thrombosis (venous and arterial), coronary disease and cerebrovascular disease. Homocysteine is believed to induce endothelial cell damage and promote platelet adhesion. In vitro, it has been shown to activate clotting factors on the surface of endothelial cells and inhibit the activation of protein C. Additionally, it has been reported that patients who have both the Factor V Leiden mutation as well as homocysteinemia, have an increased risk of thrombosis, greater than having either defect alone. This assay is designed to detect a genetic variant of the MTHFR enzyme (C677T) which results in reduced activity of the enzyme.

METHODS

Lymphocytes are processed to extract DNA. Third Wave Technologies’ Invader assay is used to genotype the target DNA. Briefly, two specific oligonucleotide probes and controls hybridize in tandem to a specific region of the gene encoding the mutation site. Cleavase III enzyme cleaves at a specific invasive structure generated within the probe oligonucleotides creating products. These sequential cleavage reactions produce 1-10 million fluorescein labeled products per target sequence hour. This results in the accumulation of a signal molecule only when the specific target DNA or control sequence is present. A fluorescent multiwell plate reader is used to quantitate signal amplification for interpretation of genotype.

CLINICAL SIGNIFICANCE

A common polymorphism in the gene encoding the Methylenetetrahydrofolate Reductase (MTHFR) gene (C to T transition at nucleotide position 677) generates a heat-labile enzyme with reduced activity. In the homozygous state, this point mutation leads to elevated plasma homocysteine levels associated with an increased risk of venous and/or arterial thrombosis. The clinical significance of a heterozygotic state for this variant, which can occur in 50% of unselected individuals, has not yet been determined. The risk of thrombosis is increased in combination with other genetic factors such as the Factor V Leiden mutation. It is recommended that the Methylenetetrahydrofolate Reductase Variant (C677T) genetic assay be used in the following situations:
  • Venous thrombotic events
  • Patients who are known carriers of the Factor V Leiden mutation or the Prothrombin Variant
  • Investigation of familial thrombosis

REFERENCES
  1. Heijer, M. et al. l996. Hyperhomocysteinaemia as a risk factor for deep-vein thrombosis. New Engl. Jrnl. Med. 334: 759-762.
  2. Mandel, H. et al. l996. Coexistence of hereditary homocystinuria and factor V Leiden - effect on thrombosis. New Engl. Jrnl. Med. 334 : 763-768.
  3. Prosst, P. et al. l995. A Candidate genetic risk factor for vascular disease: methylenetetrahydrofolate reductase. Nature Genetics 10: 111-113.
Test Catalog
Test Name: PAI-1 (Plasminogen Activator Inhibitor) 4G/5G Polymorphism (Service Code 5620)
CPT Code: 81400
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 2 mL.)
Storage And Shipping: Room temperature as whole blood.
Max Turnaround: 28 days
Methodology: PCR based assay that amplifies a fragment of the genomic DNA. Amplified fragments are detected directly by agarose gel electrophoresis.
Availability: Monday
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Additional Information: Availability is variable.

PAI-1 is a primary regulator of the firinolytic system. A single guanosine insertion/deletion (4G/5G) in the promoter region affects an individual's predisposition for thrombosis. Subjects with 4G/4G genotype have increased plasma PAI-1 concentrations and this increased activity has been associated with increased risk for venous thrombosis and myocardial infarction.
Test Catalog
Test Name: PAI-1 (Plasminogen Activator Inhibitor-1) Activity (Service Code 547P)
CPT Code: 85415
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked immunoabsorbant assay (ELISA)
Availability: Monday
Tuesday
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Additional Information: Availability is variable.

Quantitates the activity of PAI-1 in the plasma. An increased plasma level of PAI is an important reason for impaired fibrinolytic function and may be associated with thrombotic disease. Increased levels have been found in pregnancy and sepsis.
Test Catalog
Test Name: PAI-1 (Plasminogen Activator Inhibitor-1) Antigen (Service Code 5391)
CPT Code: 85415
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked immunoabsorbant assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable.

Quantitates the amount of human plasminogen activator inhibitor-1 antigen in the plasma. The clinical utility of the assay is to detect disorders of the fibrinolytic system.
Test Catalog
Test Name: PL A1/A2 Polymorphism (Service Code 5621)
CPT Code: 81400
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 2 mL.)
Storage And Shipping: Room temperature as whole blood
Max Turnaround: 28 days
Methodology: PCR based assay that amplifies a fragment of the genomic DNA. Amplified fragments are restriction enzyme digested prior to separation by agarose gel electrophoresis.
Availability:

Monday
Tuesday
Wednesday
Thursday
Friday

Additional Information: Availability is variable.

Platelet membrane Glycoprotein IIb/IIIa, a receptor for fibrinogen and von Willebrand's factor, plays a major role in platelet function. An association of the PLA2 allele with increased platelet aggregability in vitro has been described and several studies have linked this marker to cardiovascular disease.
Test Catalog
Test Name: Plasminogen Activity (Service Code 537A)
CPT Code: 85420
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 7 days
Methodology: Chromogenic substrate
Availability: Tuesday
Additional Information: Determine plasminogen and plasmin activity in plasma; study of dysplasminogens; monitor thrombolytic therapy; evaluate DIC.
Test Catalog
Test Name: Plasminogen Antigen (Service Code 537L)
CPT Code: 85421
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 11 days
Methodology: Radial Immunodiffusion (RID)
Availability: Friday
Additional Information: Must receive specimen before day of testing.

BACKGROUND

The type 1 plasminogen activator inhibitor (PAI-1) is a primary regulator of the fibrinolytic system in vivo. PAI-1 binds to tissue plasminogen activator and inhibits plasminogen activation, which decreases fibrinolysis. A single guanosine insertion/deletion (4G or 5G) 675 base pairs from the start site of the genes promoter region affects an individual’s predisposition for thrombosis. Studies have shown a correlation between PAI-1 levels in plasma and the 4G/5G polymorphism. Subjects with the 4G/4G genotype have plasma PAI-1 concentrations that are 25% higher than those with the 5G/5G genotype. Increased PAI activity may be associated with increased risk for venous thrombosis and myocardial infarction. The prevalence of the 4G/4G genotype in the normal population is 26%.

METHODS

Lymphocytes are processed to extract DNA. Third Wave Technologies’ Invader assay is used to genotype the target DNA. Briefly, two specific oligonucleotide probes and controls hybridize in tandem to a specific region of the gene encoding the mutation site. Cleavase III enzyme cleaves at a specific invasive structure generated within the probe oligonucleotides creating products. These sequential cleavage reactions produce 1-10 million fluorescein labeled products per target sequence hour. This results in the accumulation of a signal molecule only when the specific target DNA or control sequence is present. A fluorescent multiwell plate reader is used to quantitate signal amplification for interpretation of genotype. Homozygosity for the 4G polymorphism is associated with increased thrombotic risk.

CLINICAL SIGNIFICANCE

In population studies, a family history of ischemic coronary events is a major predictor of coronary artery disease (1). In addition, several twin studies have shown a strong genetic component in the pathogenesis of cardiovascular ischemia (2). These findings support the hypothesis that genetic factors play a significant role in MI and vascular risk factors (3). Impaired fibrinolysis is related to raised plasma levels of PAI and has been documented in subjects who develop MI. Recently increased PAI plasma levels have been shown to be related to a single base pair guanine deletion/insertion (4G/5G) polymorphism (4). Subjects with the 4G homozygous genotype have plasma PAI concentrations that are 25% higher than those with the 5G homozygous genotype. There is substantial evidence reported in the literature to support that carrier status of the 4G polymorphism with increased PAI levels is an independent risk factor for coronary events. It is recommended that the PAI 4G/5G genetic assay be used in the following situations:
  • Patients presenting with family histories of early heart disease and stroke
  • Patients with coronary heart disease
  • Patients undergoing vascular surgery
  • Pregnant women with past complications during pregnancy.
  • Select cases of patients with family history of venous thrombosis

REFERENCES
  1. Margaglione, M.; Cappucci, G., Colaizzo, D., Giuliani, N., Vecchione, G., Grandone, E., Pennelli, O.; DiMinno, G., The PAI-1 Gene Locus 4G/5G Polymorphism Is Associated with a Family History of Coronary Artery Disease. Arterios.Thromb. Biol, 18: 152-156, 1998.
  2. Glueck, G.J., Phillips, H., Cemaron, D., et.al., and The 4G/4G polymorphism of the hypofibrinolytic plasminogen activator inhibitor type 1 gene: an independent risk factor for serious pregnancy complications. Metabolism 49: 845-852, 2000.
  3. Anvari, A., Schuster, E., Gottsauner-Wolf, M., et.al., PAI-1 4G/5G polymorphism and sudden cardiac death in patients with coronary artery disease. Thromb. Res. 103: 103-107, 2001.
  4. Francis, C.F. Plasminogen Activator Inhibitor-1 Levels and Polymorphisms. Association with Venous Thromboembolism. Arch. Pathol Lab Med. 126: 1401-1404, 2002
Test Catalog
Test Name: Platelet Aggregation Panel (Service Code 5358)
CPT Code: 85576 x 7
Sample Requirements: Four 4.5 mL blue top (3.2% sodium citrate) tubes at room temperature
Storage And Shipping: Maintain specimen as whole blood at room temperature and transport to the laboratory immediately. Must be received in the ITxM Diagnostics laboratory within 3 hours of collection. Do not chill - platelets are activated at low temperatures. (rev. 4/14/16)
Max Turnaround: 1 days
Methodology: Aggregometry with 7 agonists
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Sample must be received in laboratory by 4:00pm.

Test includes: Platelet Aggregations with 7 agonists (3 concentrations of ADP – 20µM, 10µM, 5µM; Collagen; 2 concentrations of Ristocetin – 1.2mg, 0.3mg; and Arachidonic Acid) and Interpretation.

Evaluate platelet function; aid in diagnosis of von Willebrand disease, Glanzmann's disease, platelet storage pool disease, Bernard-Soulier syndrome, "gray platelet syndrome" and Raynaud's phenomenon.
Test Catalog
Test Name: Platelet Associated Autoantibody Assay for Platelet Antibodies (Service Code 5714)
CPT Code: 86023 x 3
Sample Requirements: Four 5 mL lavender top (EDTA) tubes. Min. 10 mL - dependent on platelet count (see below): Platelet count <20,000----->40 mL blood needed Platelet count 21,000-50,000----->30 mL blood needed Platelet count >51,000----->20 mL blood needed
Storage And Shipping: Maintain specimen as whole blood at room termperatue. Transport to the laboratory within 24 hours.
Max Turnaround: 8 days
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Tuesday
Additional Information: Specimen must be received before day of testing.

Test includes: Presence/absence of IgG, IgM, IgA antibodies to GP Ia/IIa; GP Ib/Ix; and GP IIb/IIIa.

Used to evaluate idiopathic (autoimmune) thrombocytopenic purpura.
Test Catalog
Test Name: Platelet Function Panel (Service Code 547Z)
CPT Code: 85576 x 5
Sample Requirements: Four 4.5 mL blue top (3.2% sodium citrate) tubes at room temperature.
Storage And Shipping: Maintain specimen as whole blood at room temperature and transport to the laboratory immediately. Must be received in the ITxM Diagnostics laboratory within 3 hours of collection. Do not chill - platelets are activated at low temperatures. (rev. 4/14/16)
Max Turnaround: 2 days
Methodology: Aggregometry with 5 agonists
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Sample must be received in laboratory by 4:00 pm.

Test includes: Platelet Aggregations with 5 agonists (3 concentrations of ADP – 20µM, 10µM, 5µM; Collagen; and Arachidonic Acid) and an Interpretation; may be added to a von Willebrand Profile without duplication of tests.

Evaluate platelet function; aid in diagnosis of Glanzmann's disease, platelet storage pool disease, Bernard-Soulier syndrome, "gray platelet syndrome" and Taynaud's phenomenon.
Test Catalog
Test Name: Prekallikrein (Fletcher Factor) (Service Code 536P)
CPT Code: 85292
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 7 days
Methodology: Photometric; Factor deficient substrate plasma
Availability: Monday
Additional Information: Availability is variable.

Investigate cause of prolonged APTT; identify prekallikrein deficiency (Fletcher factor deficit).
Test Catalog
Test Name: Protein C Activity (Service Code 535Q)
CPT Code: 85303
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 4 days
Methodology: Clotting End Point
Availability: Tuesday
Thursday
Friday
Additional Information: Background

One major and important regulatory system with both anticoagulant and fibrinolytic properties is the Protein C system. This system consists of a complex collection of plasma proteins that interact with vessels and platelets to inhibit coagulation. Protein C (PC) and Protein S (PS) are the central components of this system, which when either is significantly decreased, will predispose an individual to an increased risk for thromboembolic disease.

Both Protein C and S are vitamin K-dependent proteins primarily synthesized in the liver. Circulating Protein C is converted to an active protease, activated Protein C (APC), by the action of thrombin in the presence of the endothelial cell cofactor thrombomodulin. Upon activation, anticoagulant activity of PC is expressed only after the formation of a functional complex that consists of a phospholipid surface, factor V and free Protein S. In the procoagulant pathway, the Protein C/Protein S activated complex inhibits coagulation by inactivating factors Va and VIIIa rendering them nonfunctional.

Assays for Protein C Activity 

The functional assay for Protein C is based on the ability of Protein C in a patient’s plasma to prolong the clotting time of an APTT reaction reconstituted with Protein C deficient plasma. Thus, this assay tests for the activity of Protein C on its natural substrates (Factors Va and VIIIa). Patient samples that are shown to have a deficiency in biologic function that cannot be attributed to liver disease or anticoagulant therapy can be further characterized with an antigenic assay for confirmation of the defect. This functional assay replaces a chromogenic assay previously used to screen for Protein C deficiencies. The clotting endpoint functional assay more accurately reflects Protein C function in vivo.

Clinical Applications

Abnormal levels of Protein C and Protein S as well as antithrombin III can contribute to thromboembolic complications and must be considered when diagnosing patients with recurrent thrombotic complications and/or a family with a history of thrombosis. Hereditary hypercoagulable states cannot be distinguished by the patient’s clinical profile. They are best diagnosed with functional assays for Protein C, Protein S, antithrombin III and activated Protein C (APC) resistance. These assays are included in the thrombotic risk screen or can be ordered separately.

Protein C and Protein S functional assays can detect low protein levels and dysfunctional or inactivated molecules, thus are more informative than antigen assays. Functional assays will be used to screen for deficiencies of these proteins when activity studies are requested. The appropriate antigenic assay is recommended to confirm and further characterize a detected abnormality.
Test Catalog
Test Name: Protein C Antigen (Service Code 537M)
CPT Code: 85302
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 11 days
Methodology: Radial Immunodiffusion (RID)
Availability: Friday
Additional Information: Specimen must be received before day of testing.

Investigate patients with thromboses, especially venous thromboses in young adults; study patients with hypercoagulable state. Protein C antigen measures total protein C functional and immunoreactive. Order in conjunction with Protein C Activity to determine and type deficiency.
Test Catalog
Test Name: Protein S Activity (Service Code 537Q)
CPT Code: 85306
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 4 days
Methodology: Clotting end point; mechanical
Availability: Tuesday
Thursday
Friday
Additional Information: Background

One major and important regulatory system with both anticoagulant and fibrinolytic properties is the Protein C system. This system consists of a complex collection of plasma proteins that interact with vessels and platelets to inhibit coagulation. Protein C (PC) and Protein S (PS) are the central components of this system, which when either is significantly decreased, will predispose an individual to an increased risk for thromboembolic disease.

Both Protein C and S are vitamin K-dependent proteins primarily synthesized in the liver. Circulating Protein C is converted to an active protease, activated Protein C (APC), by the action of thrombin in the presence of the endothelial cell cofactor thrombomodulin. Upon activation, anticoagulant activity of PC is expressed only after the formation of a functional complex that consists of a phospholipid surface, factor V and free Protein S. In the procoagulant pathway, the Protein C/Protein S activated complex inhibits coagulation by inactivating factors Va and VIIIa rendering them nonfunctional.

Assays for Protein S Activity

The functional assay for Protein S is based on the ability of Protein S to serve as a cofactor for activated Protein C in a Factor V dependent clotting assay (reconstituted with Protein S deficient plasma). Cleavage of activated Factor V by activated Protein C with Protein S as a cofactor will prolong the clotting time.

This assay detects only free Protein S which is the active form of this molecule. The Protein S functional assay is a new addition to the testing repertoire for hypercoaguable states. If Protein S activity is deficient, and this deficiency cannot be attributed to liver disease or the use of anticoagulants, the patient’s plasma can be further evaluated for Protein S antigen levels for confirmation.

Clinical Applications

Abnormal levels of Protein C and Protein S as well as antithrombin III can contribute to thromboembolic complications and must be considered when diagnosing patients with recurrent thrombotic complications and/or a family with a history of thrombosis. Hereditary hypercoagulable states cannot be distinguished by the patient’s clinical profile. They are best diagnosed with functional assays for Protein C, Protein S, antithrombin III and activated Protein C (APC) resistance. These assays are included in the thrombotic risk screen or can be ordered separately.

Protein C and Protein S functional assays can detect low protein levels and dysfunctional or inactivated molecules, thus are more informative than antigen assays. Functional assays will be used to screen for deficiencies of these proteins when activity studies are requested. The appropriate antigenic assay is recommended to confirm and further characterize a detected abnormality.
Test Catalog
Test Name: Protein S Free Antigen (Service Code 5705)
CPT Code: 85306
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 14 days
Methodology: Immunoturbidimetric assay
Availability: Friday
Additional Information: Patients on oral anticoagulants may have decreased functional protein C/S values. For accurate testing results, the patient should be off anticoagulant therapy (coumadin) for two weeks.
Test Catalog
Test Name: Protein S Total Antigen (Service Code 536I)
CPT Code: 85305
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked Immunosorbent Assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable.

Patients on oral anticoagulants may have decreased functional protein C/S values. For accurate testing results, the patient should be off anticoagulant therapy for 2 weeks.
Test Catalog
Test Name: Prothrombin Fragment 1+2 (Service Code 5677)
CPT Code: Research Only (83520)
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable. Maximum turn around time is 21-28 days.

Used as an aid for diagnosis, monitoring and evaluating acquired or hereditary blood coagulation disorders, especially in the assessment of risk of thrombosis. It may also be used in monitoring anticoagulant therapy. Elevated levels of PT F 1+2 may be found in patients with thrombosis, pulmonary embolism, DIC, trauma, and sepsis. Levels of PT F 1+2 may also be elevated in patients with hereditary thrombophilia with Factor V Leiden or Protein C or Protein S deficiencies. Patients undergoing oral anticoaglulant therapy or heparin therapy may show significant decreases in PT F 1+2 levels.
Test Catalog
Test Name: Prothrombin Gene Variant (Service Code 557V)
CPT Code: 81240
Sample Requirements: One 7 mL yellow top (ACD) tube preferred. EDTA or citrate also acceptable. (Min. 2 mL.)
Storage And Shipping: Room temperature as whole blood.
Max Turnaround: 7 days
Methodology: Invader assay from Third Wave Technologies is a linear signal amplification system for directly detecting and quantifying gene-specific sequences in complex mixtures of nucleic acids. Employs FRET detection format for increased sensitivity.
Availability: Friday
Additional Information: Background

Venous thromboembolism is a significant medical problem for approximately 1 in 1000 people in the general population. The Factor V Leiden mutation is the most common genetic risk factor for venous thrombosis. Recently, a genetic point mutation in the prothrombin gene (G20210A) has been identified as an additional risk factor for venous thrombosis. The mutation in the prothrombin gene is located in the 3’ untranslated (noncoding) region of the gene and results in a G to A transition at position 20210.

Prothrombin is also known as Factor II. It is the precursor of thrombin, which late in the clotting cascade leads to the formation of fibrin. The prothrombin gene mutation (G20210A) leads to elevated levels of prothrombin in plasma and is associated with an increased risk of venous thrombosis. The frequency of this allele has been reported to be 1-2% in the general population. Among patients with venous thrombotic episodes or a family history of venous thrombotic episodes, the prevalence has been reported to be 6-20%. The role of the prothrombin gene variant in arterial disease is not yet known and is currently under investigation.

Methods

Patient lymphocytes are processed to extract DNA. Using 250 ng of human DNA, a polymerase chain reaction (PCR) is performed to amplify a 118 bp fragment of the gene encoding the prothrombin protein. The 118 bp fragment includes the G20210A transition site which has been associated with increased prothrombin levels and venous thrombosis. The amplimer is restriction enzyme digested with the enzyme Taq 1 to identify and enable genotypic analysis of the G20210A polymorphism. Enzyme digest fragments are resolved on a 3.0% agarose gel and visualized by ethidium bromide staining. The 118 bp product of the wild type allele yields fragments of 98 bp and 20bp. The mutant allele is not susceptible to digestion by Taq 1 and yields a 118 bp fragment.

Clinical Significance

The Prothrombin variant (G20210A) occurs in 1-2% of the population. It has been reported to cause a 2-3 fold increased risk of venous thrombosis in individuals carrying the mutant allele. Among venous thrombosis patients the frequency of the allele has been reported to be 6-20% depending on the population tested. The risk of venous thrombosis significantly increases in combination with other genetic factors such as the Factor V Leiden mutation or with acquired risk factors such as oral contraceptive use.

It is recommended that the Prothrombin Variant (G20210A) genetic assay be used in the following situations:
  • Venous thrombotic events
  • Recurrent TIA’s or strokes especially in young patients
  • Patients being considered for anticoagulant therapy
  • Investigation of familial thrombosis
References
  1. Poort SW, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3’ untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood l996; 88: 3698-703.
  2. Hillarp A, Zoller B, Svensson PJ, Dahlback B. The 20210A Allele of the Prothrombin gene is a common risk factor among Swedish outpatients with verified deep venous thrombosis. Thromb. Haemost. 1997; 78: 990-2.
Test Catalog
Test Name: PT (Prothrombin Time) (Service Code 5359)
CPT Code: 85610
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Photometric
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Evaluate extrinsic coagulation system; screen for congenital deficiencies of factors II, V, VII, X; deficiency of prothrombin; dysfibrinogenemia (complete); coumadin or warfarin effect; liver failure; disseminated intravascular coagulation (DIC); screen vitamin K deficiency.
Test Catalog
Test Name: PT (Prothrombin Time) Mix (Service Code 535P)
CPT Code: 85611
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Photometric
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Detect the presence/absence of an inhibitor.
Test Catalog
Test Name: Reptilase® Time (Service Code 537D)
CPT Code: 85635
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less, ship on dry ice.
Max Turnaround: 2 days
Methodology: Photometric
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Distinguish hypofibrinogenemias from heparin contamination and fibrin split products (FSPs).
Test Catalog
Test Name: Ristocetin CoFactor Activity (Service Code 537N)
CPT Code: 85245
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 6 days
Methodology: Immunoturbidometric
Availability: Monday
Wednesday
Friday
Additional Information: Evaluate von Willebrand factor activity. Differential diagnosis between hemophilia and von Willebrand's disease.
Test Catalog
Test Name: Russell Viper Venom Time, Dilute Ratio (Service Code 535B)
CPT Code: 85613
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Photometric
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Aids in the diagnosis of lupus anticoagulants by evaluating the presence of inhibition in the common pathway of coagulation.
Test Catalog
Test Name: Spontaneous Platelet Aggregations (Service Code 5648)
CPT Code: 85576
Sample Requirements: Four 4.5 mL blue top (3.2% sodium citrate) tubes at room temperature.
Storage And Shipping: Maintain specimen as whole blood at room termperature and transport to the laboratory immediately. Must be received in laboratory within 4 hours of collection. Do not chill; platelets are activated at low temperatures.
Max Turnaround: 2 days
Methodology: Aggregometry
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Detect hyperaggregability of platelets.
Test Catalog
Test Name: TAT (Thrombin-Antithrombin) Complex (Service Code 5676)
CPT Code: Research Only (83520)
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 1 days
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable. Maximum turn around time is 21-28 days.

Used in the evaluation of patients for thrombotic events. Patients at risk of thrombosis and DIC may have elevated levels of TAT. Also, TAT is important in patients with multiple trauma, liver dysfunction, septicemia and preeclampsia. TAT may rise in patients with advanced stage malignancies. TAT may increase during the course of heparin and fibrinolysis therapy.
Test Catalog
Test Name: Thrombin Time (Service Code 537E)
CPT Code: 85670
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Photometric
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Determine severe hypofibrinogenemia and presence of heparin-like anticoagulants; diagnose and monitor DIC and fibrinolysis; monitor thrombolytic therapy. A reptilase time will be performed to evaluate fibrinogen when thrombin time is prolonged. The need to do a reptilase time will invoke an additional charge.
Test Catalog
Test Name: Thrombomodulin (Service Code 5975)
CPT Code: Research Only
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable. Maximum turn around time is 21-28 days.

Thrombomodulin is found on the endothelial surface and is known to bind thrombin and the thrombin-thrombomodulin complex is a strong activator of Protein C to Activated Protein C (APC). APC is a well-known protein involved in limiting the coagulation response and, thus, decreased thrombomodulin may lead to less APC and increased risk of thrombosis.
Test Catalog
Test Name: Thrombospondin (TSP-1) (Service Code 5974)
CPT Code: Research Only
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plama within one hour of collectin and freeze in polypropylene tube at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable. Maximum turn around time is 21-28 days.

TSP-1 is released from platelet granules during platelet activation . In association with von Willebrand Factor (vWF) and fibrinogen, TSP-1 contributes to clot formation. TSP-1 has been shown to inhibit angiogenesis and minimize new vessel growth.
Test Catalog
Test Name: Thrombotic Risk Screen (Service Code 547V)
CPT Code: 85260, 85300, 85303, 85306, 85307
Sample Requirements: One 4.5 mL blue top (3.2%sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 7 days
Methodology: Refer to individual test components.
Availability: Tuesday
Thursday
Friday
Additional Information: Test includes: APC Resistance; Antithrombin III Activity; Protein C Activity; Protein S Activity; Factor X; and a Physician Interpretation.

Screening tests for patients at risk of thrombosis from the more common congenital or acquired deficiencies. Refer to individual test components for additional information.
Test Catalog
Test Name: Tissue Factor Activity (Service Code 5982)
CPT Code: Research Only
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable. Maximum turn around time is 21-28 days.

Tissue Factor (TF) is also known as Coagulation Factor III or Thromboplastin and it is found on sub-endothelial cells. It plays a critical role in triggering blood coagulation by binding to Factor VIIa to form a Factor VIIa-TF complex. This complex then cleaves Factor X to FXa and Factor IS to FIXa to help initiate the coagulation cascade. The level of TF may be increased in malignancies, inflammation, atherosclerosis, sepsis, diabetes, sickle cell disease and acute coronary syndrome. During sepsis, increased activation of the coagulation mechanism may result from enhanced cytokine-mediated expression of TF on endothelial cells and monocytes. In atherosclerotic plaques, TF ruptures various pro-coagulant material including microparticles containing TF. TF may also pla a significant role in angiogenesis and metastasis.
Test Catalog
Test Name: Tissue Factor Pathway Inhibitor (Free) (Service Code 5981)
CPT Code: Research Only
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable. Maximum turn around time is 21-28 days.

TFPI inhibits the extrinsic coagulation pathway by forming a complex of TFPI-FXa-Tissue Factor (TF)-FVIIa. Most of TFPI is bound to the endothelial cell surface and two forms of TFPI are found in plasma (lipoprotein-associated TFPI and free TFPI). Only the free form is associated with any anticoagulant activity. TFPI can also be found in platelets and is released during platelet activation. TFPI is closely related to total and HDL-cholesterol levels. TFPI levels are higher in men than women, especially for the free form. TFPI is also released into ciruculation following heparin injections.
Test Catalog
Test Name: Tissue Factor Pathway Inhibitor (Total) (Service Code 6167)
CPT Code: Research Only
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable. Maximum turn around time is 21-28 days.

TFPI inhibits the extrinsic coagulation pathway by forming a complex of TFPI-FXa-Tissue Factor (TF)-FVIIa. Most of the TFPI is bound to the endothelial cell surface and two forms of TFPI are found in plams (lipoprotein-associated TFPI and free TFPI). Only the free form is associated with any anticoagulant activity. TFPI can also be found in platelets and is released during platelet activation. TFPI is closely related to total and HDL-cholesterol levels. TFPI levels are higher in men than women, especially for the free form. TFPI is also released into circulation following heparin injections.
Test Catalog
Test Name: tPA Antigen (Service Code 5388)
CPT Code: 85415
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 28 days
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Availability is variable.

Quantitates the amount of human tissue type plasminogen (tPA) antigen in the plasma. The clinical utility of the assay is tho detect disorders of the fibrinolytic system.
Test Catalog
Test Name: TTI (Tissue Thromboplastin Inhibition) (Service Code 535G)
CPT Code: 85705
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Photometric, prothrombin time performed with dilute thromboplastin
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Detect lupus-like anticoagulants. Prolonged prothrombin times invalidate the tissue thromboplastin inhibition test.
Test Catalog
Test Name: von Willebrand Profile (Service Code 5356)
CPT Code: 85730, 85732, 85240, 85245, 85246, 85576 x 4
Sample Requirements: 3-4.5mL blue top (3.2% sodium citrate) tubes whole blood maintained and shipped at Room Temperature (must be received within 4hrs of collection and by 4:00pm); AND 2-4.5mL blue top (3.2% sodium citrate) tubes: Separate plasma (min. 2.0mL platelet poor plasma) and freeze within 2hrs of collection and at -60 °C or colder; ship frozen plasma on dry ice
Storage And Shipping: Separate plasma and freeze at -60°C or less; ship on dry ice. NOTE - If client is unable to separate plasma, maintain as whole blood (total 5 tubes) and ship at room temperature. Room termperature tubes must be received in the laboratory within 4 hours of collection AND by 4:00pm.
Max Turnaround: 10 days
Methodology: Refer to individual test components
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: For availability, refer to individual test components.

Maximum turn around time is 10 days if multimers performed; otherwise, up to 5 days after setup.

Test includes: Factor VIII:C; Ristocetin CoFactor (RCoF activity); vW Antigen; APTT; APTT Mix; Ristocetin Induced Platelet Aggregations; Closure Time; vW Antigen Multimer Analysis (if needed, will invoke additional charge) and a Physician Interpretation with clinical recommendations if warranted.

Diagnosis and typing of von Willebrand's disease.
Test Catalog
Test Name: von Willebrand Screen (Service Code 536V0)
CPT Code: 85730, 85732, 85240, 85245, 85246
Sample Requirements: Two 4.5 mL blue top (3.2% sodium citrate) tubes. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 10 days
Methodology: Refer to individual test components
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Maximum turn around time is up to 10 days if multimers performed; otherwise up to 5 days after setup.

For availability, refer to individual test components.

Test includes: APTT; APTT Mix; Factor VIII:C; Ristocetin CoFactor Activity; von Willebrand Antigen; vW Antigen Multimers (if needed, will invoke additional charge); and a Physician Comment.

Screen provides diagnostic information and typing for von Willebrand's disease.
Test Catalog
Test Name: vW Ag Multimer Analysis (Service Code 536R)
CPT Code: 85247
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 10 days
Methodology: SDS-agarose electrophoresis
Availability: Tuesday
Additional Information: Specimens must be received before 9:00am on day of testing to be included.

Classify von Willebrand disease.
Test Catalog
Test Name: vW Antigen (Service Code 537F)
CPT Code: 85246
Sample Requirements: One 4.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 6 days
Methodology: Immunotrubidometric
Availability: Monday
Wednesday
Friday
Additional Information: Von Willebrand antigen measures total plasma vWF, functional and immunoreactive. Usually ordered as part of von Willebrand Profile. This test alone does not rule out von Willebrand disease. Useful in the differential diagnosis of hemophilia A and von willebrand disease.
Test Catalog
Test Name: Anticardiolipin Antibodies (IgG, IgM) (Service Code 537R)
CPT Code: 86147 x 2
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days (excludes weekends and holidays)
Methodology: Enzyme-linked immunoabsorbant assay (ELISA)
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Evaluate patients with prolonged PTT and thrombotic tendency, including patients with systemic lupus erythematosus, recurrent spontaneous abortion of early pregnancy, and suspected antiphospholipid antibody syndrome.
Test Catalog
Test Name: Antithrombin III Activity (Service Code 5365)
CPT Code: 85300
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 1 day
Methodology: Chromogenic substrate assay
Availability: Monday
Tuesday
Wednesday
Thursday
Friday

STAT testing available for off-hours.
Additional Information: Evaluate response to heparin, hypercoagulable, and fibrinolytic states; test for hereditary deficiency of antithrombin III. Antithrombin levels are of use in cases of suspected heparin failure, DIC, or personal or familial history of thromboembolic disease.
Test Catalog
Test Name: APC Resistance (Service Code 547C)
CPT Code: 85307
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 7 days
Methodology: Clotting End Point
Availability: Batch tested twice weekly.
Additional Information: Screening assay to determine the presence of the Factor V Leiden mutation.
Test Catalog
Test Name: APTT (Activated Partial Thromboplastin Time) (Service Code 5363)
CPT Code: 85730
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Photometric, clotting end point
Availability: Monday
Tuesday
Wednesday
Thursday
Friday

STAT Testing available for off hours
Additional Information: Useful screening test to detect factor deficiencies or inhibitors of the intrinsic pathway of coagulation. Also used to monitor heparin therapy. The APTT will prolong when: 1) there are deficiencies of factors II, V, VIII, IX, X, XI, and XII; 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.
Test Catalog
Test Name: APTT Mix (Activated Partial Thromboplastin Time Mix) (Service Code 535S)
CPT Code: 85732
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days
Methodology: Photometric, clotting end point
Availability: Monday
Tuesday
Wednesday
Thursday
Friday

STAT testing available for off hours
Additional Information: Plasma is incubated 1:1 with Normal Pool for 60 minutes at 37°C, to detect the presence/absence of an inhibitor. Incubated mixes are performed to increase inhibitor detection.  Recommend ordering APTT with APTT Mix.
Test Catalog
Test Name: Beta 2 Glycoprotein I Antibodies (IgG, IgM, IgA) (Service Code 5652)
CPT Code: 86146 x 3
Sample Requirements: One 3.5 mL blue top tube. (Minimum 1 mL plasma.)
Storage And Shipping: Separate plasma by centrifugation at 1500xg for 15 minutes. Freeze at -20°C or colder; ship on dry ice.
Max Turnaround: Batch tested twice weekly
Methodology: Enzyme-linked immunoabsorbant assay (ELISA)
Availability: Contact Lab for availability
Additional Information: Batch testing based upon test requests (Monday - Friday); usually performed 2-3 times per week

BACKGROUND

Antiphospholipid antibodies are a heterogenous group of autoantibodies associated with the clinical manifestations of APS. These antibodies frequently require the presence of a serum protein as cofactor for optimal in vitro binding activity forming phospholipid-protein complexes. Binding of B2GPI directly to a microtiter plate has been found to produce antigenic sites similar to those produced when bound to phospholipid. This assay minimizes the variability observed between classic antiphospholipid ELISAs (cardiolipin and phosphatidylserine bound to plastic, with exogenous source of cofactor). In addition, studies indicate anti-B2GPI antibodies are more specific for thrombosis than antibodies detected by anticardiolipin ELISAs. Specifically, it has been suggested by various research groups that anti-B2GPI may help to distinguish “infectious” from “autoimmune” antiphospholipid antibodies. Only autoimmune antiphospholipid antibodies are associated with thrombosis.

METHODS

Anti-B2GPI testing uses an ELISA format to detect antibodies in human plasma. The IgG type anti-B2GPI antibodies are determined since it is generally regarded as the most clinically significant isotype.

CLINICAL APPLICATION

Antiphospholipid antibodies are a heterogenous group of autoantibodies associated with the clinical manifestations of the antiphospholipid syndrome. One factor contributing to the heterogeneity is their immunologic reactivity to phospholipids or the phospholipid-cofactor complexes. The presence of antibodies to B2GPI has been regarded as more specific for thrombosis than anti-cardiolipin or anti-phosphatidylserine.

Anti-B2GPI may provide a more complete profile for establishing a diagnosis of antiphospholipid antibody syndrome and for assessing the risk of thrombotic complications. It can offer valuable information in the following clinical situations:
  • patients diagnosed with autoimmune disease
  • follow-up testing of positive aCL to distinguish infectious from autoimmune antibodies (autoimmune associated with thrombosis)
  • testing of all clinically suspicious aCL negative patients
  • patients presenting with thrombosis
  • unresolved thrombocytopenia
REFERENCES
  1. Tsutsumi, A., Matsuura, E. Ichikawa K, et. al., “Antibodies to B2-Glycoprotein I and clinical manifestations in patients with systemic lupus erythematosus. Arthritis Rheum. 39: 1466-1474, 1996.
  2. Gomez-Pacheco, L., Villa, A.R., Drenkard, C., et. al., “Serum anti-beta2-glycoprotein-I and anticardiolipin antibodies during thrombosis in systemic lupus erythematosus patients.” Am. J. Med. 106: 4170423, 1999.
  3. Ichikawa, T., Tsutsumi, A., Matsuura, E., Koike, T. “Antiphospholipid syndrome”. Intern. Med. 38: 170-173, 1999.
Test Catalog
Test Name: Closure Time (Platelet Function Screen) (Service Code 551I)
CPT Code: 85576 x 2
Sample Requirements: Two 3.5 mL blue top (3.2% sodium citrate) tubes at room temperature
Storage And Shipping: Maintain specimen as whole blood at room termperature and transport to the laboratory immediately. Must be received in laboratory within 3 hours of collection. Do not chill, platelets are activated at low temperatures.
Max Turnaround: 1 days
Methodology: In vitro blood flow analysis. The process of platelet aggregation builds a platelet thrombus at the aperture thereby diminishing and finally arresting blood flow. The time from start of the test until the platelet plug occludes the aperture is reported as closure time.
Availability: Monday
Tuesday
Wednesday
Thursday
Friday

STAT testing available for off hours.
Additional Information: Sample must be received in laboratory by 4:00pm.

BACKGROUND

Platelet dysfunction may be acquired, inherited, or induced by platelet inhibiting agents. The most common causes of platelet dysfunction are related to uremia, von Willebrand’s disease (vWD), and exposure to agents such as acetyl salicylic acid (ASA, aspirin). Current methods to assess platelet function include bleeding time (BT) and aggregation studies.

A critical review of BT (1) concluded that the utility of BT is not enhanced by recent standardization attempts; that in individual patients there is no relationship between BT and platelet counts; that the BT is not a specific indicator of platelet function and that the BT is a poor indicator of bleeding risk. As a consequence many clinicians no longer use BT. The noted deficiencies of BT lead to the development of an in vitro device that globally measures platelet-related primary hemostasis. Closure time is sensitive to platelet adherence and aggregation abnormalities and therefore has increased sensitivity for von Willebrand’s screening when compared to bleeding time (2).

METHODS

The PFA-100 is an instrument and test cartridge system in which the process of platelet adhesion and aggregation following a vascular injury is simulated in vitro. This system allows for rapid evaluation of platelet function on samples of anticoagulated whole blood. Using biologically coated membranes (Collagen/Epinephrine [CEPI] and Collagen/Adenosine-5’-diphosphate [CADP]) and high shear rates generated under standardized flow conditions, platelet plug formation at the aperture can be evaluated. The time required to obtain full occlusion of the aperture is reported as the Closure Time (CT) in seconds.

CLINICAL APPLICATION

It is clinically important to assess platelet function as a potential cause of a bleeding diathesis. Closure times may be useful in the following:
  • Patients with a clinical suspicion of von Willebrand’s disease
  • Patients with a family history of bleeding or heavy bruising
  • Monitoring of ASA/NSAID use
  • Therapeutic response to DDAVP
  • Uremia
  • Screening for inherited platelet disorders
REFERENCES
  1. Rogers, RPC., Levin J., “A critical reappraisal of the bleeding time.” Semin. Thromb. Hemost. 1990; 16: 1-20.
  2. Cattaneo M., Federici, AB., et. al. “Evaluation of the PFA-100 system in the diagnosis and therapeutic monitoring of patients with von Willebrand’s disease.” Thromb. Haemost. 1999; 82: 35-9.
Test Catalog
Test Name: Collagen Binding Assay (Service Code 5922)
CPT Code: 85246
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -20 °C or colder; ship on dry ice.
Max Turnaround: Batch tested; contact lab for availability
Methodology: Enzyme-linked immunosorbent assay (ELISA)
Availability: Contact lab for availability
Additional Information: Availability is variable.

Quantitates the collagen binding capacity of vWF to collagen-coated microtiter wells. The CBA may correlate more closely with vWF function than vW antigen and/or vW Multimer testing.
Test Catalog
Test Name: Cryofibrinogen (Service Code 536B)
CPT Code: 82585
Sample Requirements: One 10 mL green top (heparin) tube OR two 4.0 mL. (Min. 2 mL plasma.)
Storage And Shipping: Maintain at room temperature (20°C to 25°C) and process within 2 hours. Incubate at 37°C for 10 minutes; mix well by gentle inversion, then spin at room temperature; freeze plasma.
Max Turnaround: Batch tested
Methodology: Clotting end point (Clauss)
Availability: Contact lab for availability
Additional Information: Availability and maximum turn around time are both variable.

Detect and quantitate cold precipitable fibrinogen.
Test Catalog
Test Name: D-Dimer Semi-Quantitative (Service Code 535A)
CPT Code: 85378
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -20°C or colder; ship on dry ice.
Max Turnaround: 2 days
Methodology: Latex particle assay
Availability: Monday
Tuesday
Wednesday
Thursday
Friday

STAT testing available off hours
Additional Information: Used to evaluate chronic or acute disseminated intravascular coagulation (DIC), acute myocardial infarction, and unstable angina. NOT SUITABLE AS EXCLUSION TEST TO RULE OUT DVT OR PE.
Test Catalog
Test Name: Dabigatran (Pradaxa) Level (Service Code 5997)
CPT Code: 85670
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 2 days (excludes weekends and holidays)
Methodology: Dilute Thrombin Time with Dabigatran calibrators
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Quantitative measurement of Pradaxa (Dabigatran), a Direct Thrombin Inhibitor (DTI), in citrated plasma as an aid in the management of patients receiving Dabigatran. Used to manage bleeding in cases of possible excess anticoagulant activity, patients with renal insufficiency, recurrent TIA/CVA symptoms despite therapeutic dose, and to ensure patient compliance.
Test Catalog
Test Name: DIC (Disseminated Intravascular Coagulation) Screen (Service Code 547W)
CPT Code: 85610 ,85611, 85730 ,85732, 85384, 85300, 85362, 85378, 85670
Sample Requirements: Four 3.5 mL blue top (3.2% sodium citrate) tubes. (Min. 3 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -20°C or colder; ship on dry ice.
Max Turnaround: 1 day (excludes weekends and holidays)
Methodology: See individual test
Availability: Monday
Tuesday
Wednesday
Thursday
Friday

STAT testing available with medical on call approval
Additional Information: Tests includes: PT; PT Mix; APTT; APTT Mix; Fibrinogen; Thrombin Time; Reptilase Time (if needed, will invoke additional charge); FDP (plasma); d-Dimer; Antithrombin III Activity; Physician Interpretation with clinical recommendations if warranted.

Diagnosis and follow-up of disseminated intravascular coagulation (DIC).
Test Catalog
Test Name: dRVVT (dilute Russell Viper Venom Time) (Service Code 535B)
CPT Code: 85613
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -20°C or colder; ship on dry ice.
Max Turnaround: 2 days (excludes weekends and holidays)
Methodology: Clotting
Availability: Monday
Tuesday
Wednesday
thursday
Friday
Additional Information: BACKGROUND

Antiphospholipid antibodies directed against anionic phospholipids or phospholipid-protein complexes that interfere with phospholipid dependent in vitro coagulation assays are known as lupus anticoagulants (LACs). LACs are considered to be a significant risk factor in patients with otherwise unexplained thrombosis and are often present in women who have recurrent miscarriages. LACs occur in various clinical conditions, especially autoimmune diseases, thrombocytopenia and neurological diseases. They may also appear as a result of adminis-tration of chlorpromazine, procainamide, thorazine and other medications.

Patients are referred for LAC work-up due to an abnormal screening test of hemostasis, usually a prolonged APTT or based on clinical symptoms. The latter requires sensitive reagents to detect the maximum number of patients with LAC. Besides the analytical variables associated with reagent sensitivity, the pre-analytical condition of the specimen and heterogeneity of antibodies make the diagnosis of a lupus anticoagulant difficult. Hence there is no single test that can be used to establish a diagnosis. Due to these difficulties guidelines have been set by the Scientific and Standardization Committee for the diagnosis of a lupus anticoagulant and are summarized as follows: 1) abnormal phospholipid dependent coagulation reaction (APTT, PT, dRVV or KCT), 2) evidence for the presence of an inhibitor e.g. mixing studies and , 3) evidence that the inhibitor is directed against a phospholipid and not a specific coagulation factor.

METHODS

Russell’s Viper Venom (dRVV) selectively activates factor X to Xa, with factor V and phospholipid, activates factor II to thrombin. By diluting the phospholipid and RVV, this assay becomes very sensitive to lupus anticoagulant. On addition of phospholipid, a relative correction or neutrali- zation of LAC results in a shorter clotting time. The new assay is a two step procedure that includes a dRVV screen and a confirmatory step. The result is reported as a ratio of Screen clotting time (seconds)/ Confirm clotting time (seconds) and results greater than 1.1 are positive for a lupus anticoagulant. The improved reagents contain polybrene to neutralize the effect of heparin. As a result, the dRVV ratio is a more specific test for the evaluation of LAC than APTT

CLINICAL SIGNIFICANCE

Lupus anticoagulants are frequent problems in clinical medicine for physicians in a wide variety of specialties. Studies have shown that LACs are associated with thrombosis in 25-30% of affected patients however the cause of the thromboses remains poorly defined. Lupus anticoagulants have also been associated with a variety of problems during pregnancy e.g. fetal growth retardation, early pre-eclampsia, chorea gravidarum and miscarriages. The dRVV ratio may offer valuable information in the following clinical situations:
  • An unexplained prolonged APTT
  • Arterial and Venous thromboembolic events
  • Recurrent spontaneous abortions
  • Recurrent TIA’s or strokes, especially in young patients
Test Catalog
Test Name: Euglobulin Lysis (Service Code 536C)
CPT Code: 85360
Sample Requirements: One 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 1 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or less; ship on dry ice.
Max Turnaround: 1 day (excludes weekends and holidays)
Methodology: Clot lysis
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Detect and evaluate pathological fibrinolytic activity.
Test Catalog
Test Name: Extended Lupus Anticoagulant (LAC) Panel
CPT Code: 85610, 85730 x 2, 85732 x 2, 85670, 85240, 85613, 85705, 86147 x 4, 86146 x 3, 86148 x 2
Sample Requirements: Two 3.5 mL blue top (sodium citrate) tube. (Min. 2 mL platelet poor plasma).
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder, ship on dry ice.
Max Turnaround: Batch tested
Methodology: N/A
Availability: Contact Lab for availability
Additional Information: Includes all test for Lupus Anticoagulant Panel I (Service Code 547T) and Antiphosphatidylserine (APS) Antibodies (Service Code 547X) (IgG/IgM), Antiphospholipid (APL) Antibodies (Service Code 5651) (IgG/IgM), Anticardiolipin (ACA) IgA (Service Code 542I) and Beta 2 Glycoprotein I (Service Code 5652) (IgG/IgM/IgA).
Test Catalog
Test Name: Coagulation Factor Profile (Service Code 537J)
CPT Code: 85210, 85220, 85230, 85240, 85250, 85260, 85270, 85280, 85610, 85611, 85730, 85732
Sample Requirements: Two 3.5mL blue top (3.2% sodium citrate) tubes. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice
Max Turnaround: 1 day (excludes weekends and holidays)
Methodology: N/A
Availability: Monday
Tuesday
Wednesday
Thursday
Friday
Additional Information: Includes Factors II, V, VII, VIII, IX, XI, X, XII, PT, PT Mix, APTT, APTT Mix
Test Catalog
Test Name: Factor Inhibitor (Service Code 535I)
CPT Code: 85335
Sample Requirements: Two 3.5 mL blue top (3.2% sodium citrate) tube. (Min. 2 mL platelet poor plasma.)
Storage And Shipping: Separate plasma within one hour of collection and freeze at -70°C or colder; ship on dry ice.
Max Turnaround: 7 days (Batch tested twice weekly)
Methodology: Heat-inactivate Nijmegen modified Bethesda
Availability: Contact Lab for availability
Additional Information: Specimen must be received before day of testing.
Please note Factor on requisition.
Recommend prior Factor activity testing.

Used to evaluate patients with an inhibitor to a Factor other than Factor VIII:C or Factor IX.  Client must specify which Factor is to be studied.  This Factor Activity must be <25% Normal (0.25 U/mL) to distinguish inhibitor activity.