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Coagulation

Coagulation laboratory tests

Protein C assays


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

Indications
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● Necrotic skin in newborns days 1-3 of life (purpura fulminas neonatorum, can also test parents also for heterozygosity); evaluation of cause of venous thromboembolism (recommended to use chromogenic protein C assays initially)
Non-indications: screening before oral contraceptives or oral anticoagulants (discontinue for 10 days, or test family members)

General
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● Deficiencies are either quantitative (type I - reduced amount of normal protein) or qualitative (type II - normal amount of defective protein, Thromb Haemost 1984;51:1)
● Assays are either functional (measure protein activity) or antigenic (immunoassays that measure quantity, not function)
● Perform functional assay first - if decreased, perform antigenic assay; must exclude acquired causes (Arch Pathol Lab Med 2002;126:1337)
● Low values should be confirmed on a new specimen
● Assays should be performed with platelet poor plasma, using sodium citrate collection tubes
● Functional assays are clot-based or chromogenic

Clot based functional assays
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● Detect all known type I and II variants; patientís protein C is activated by Southern Copperhead venom (Agkistrodon contortrix contrortrix), which degrades synthetic substrate, factor Va or factor VIIIa with clot based PTT assay; the prolongation of clotting time is proportional to the amount of factor activity
● PT based assay or amidolytic assays are affected by lupus anticoagulants (raises protein C result), elevations of factor VIII > 200% (decreases the result), acute phase reactions or factor V Leiden mutation (decrease the result); cannot perform on patients taking hirudin or argatroban

Chromogenic functional assays
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● Not affected by lupus anticoagulants, factor VIII levels, factor V Leiden or other coagulation abnormalities that interfere with clot-based functional assays; may not detect qualitative deficiencies detected by clot-based assays; patientís protein C is activated by snake venom, which cleaves a synthetic substrate, which releases a chromogenic that is measured spectrophotometrically

Other assays
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Antigenic assays: either ELISA, electroimmunoassay (Laurell rocket method) or radioimmunoassay; variable levels, so use 3 standard deviations as cutoff
ELISA: uses antibody to protein C immobilized to microtiter place; add plasma; add secondary anti-protein C antibody coupled to an enzyme for colorimetric detection; use standard curve to determine plasma protein C
Laurell rocket antigenic assay: agarose gel has antibody to protein C; plasma samples are put into wells and electrophoresed; antigen-antibody complexes precipitate during electrophoresis, and height of precipitin arc is proportional to plasma protein C, which is compared to standard curve using pooled normal plasma; may be unable to detect protein C levels < 5%
Radioimmunoassay: similar to ELISA but uses single, radiolabeled antibody

Interpretation
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● Values falsely increased by bivalirudin, lepirudin, argatroban and fondaparinux (Arch Pathol Lab Med 2004;128:1142), lowered by warfarin (must discontinue for 10 days prior to testing)
Reference range: 70-140% of normal; newborns levels are 20-30% of adult values; usually rise to near adult levels by age 6 months, but may remain below adult normal levels until age 10 years

Acquired causes of altered levels
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Acquired causes of low Protein C levels: more common than hereditary deficiencies - clot formation, surgery, liver disease, warfarin (should be discontinued at least 10-30 days prior to testing), DIC, vitamin K deficiency, vitamin K antagonist therapy and L-asparaginase therapy; repeat protein C test once these conditions are no longer present
Acquired causes of increased Protein C (may mask protein C deficiency): ischemic heart disease, pregnancy, postmenopausal women, hormone replacement therapy and oral contraceptives

End of Coagulation > Coagulation laboratory tests > Protein C assays


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