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Hereditary bleeding disorders

Hereditary bleeding disorders - testing


Jeremy C. Parsons, M.D.

Last author update: 1 June 2012
Last staff update: 25 April 2022

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PubMed Search: Hereditary bleeding disorders testing

Jeremy C. Parsons, M.D.
Table of Contents
Factor VII assay | Factor VIII assay | Factor VIII inhibitor assay / Bethesda assay | Factor IX assay | Factor XI assay | Factor XIII assay | Protein C assays | Protein S assays | Prothrombin gene 20210A testing
Cite this page: Parsons JC. Hereditary bleeding disorders - testing. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/coagulationhereditarybleedingtesting.html. Accessed January 10th, 2025.
Factor VII assay
  • Usually a one stage, prothrombin based assay
  • Results distorted by cold activation of factor VII, by variable sensitivity of thromboplastins to activity of factor VII versus VIIa
  • Can also use a chromogenic assay (Haemostasis 1983;13:161)
Factor VIII assay
  • Assay is usually a PTT based clotting assay that measures factor VIII activity
  • Use severely deficient factor VIII plasma as substrate for one stage clotting assay, although there is tremendous interlaboratory variability
  • Can also use chromogenic assay, particularly when assessing B chain deleted recombinant Factor VIII (i.e., refacto)
  • Low levels: hemophilia, vWF disease with decreased vWF antigen levels, delay in transporting specimen to lab (Goldman: Cecil Medicine: Expert Consult, 23rd Edition, 2007)
    • Levels are not greatly decreased at birth or throughout childhood
    • Levels may increase during pregnancy
Factor VIII inhibitor assay / Bethesda assay
  • Methodology (Am J Clin Pathol 2009;131:552):
    • Prepare serial dilutions of patient plasma in citrated saline from 1:1 to 1:160 (or higher)
    • Mix each dilution with an equal volume of normal plasma containing a normal amount of coagulation factors, incubate for 2 hours, then perform factor VIII assay
    • Titer of inhibitor is dilution that inhibits 50% of factor VIII in assay
    • Can use porcine Factor VIII to assess cross reactivity and assist in therapeutic decisions
Factor IX assay
  • To detect congenital or acquired factor IX deficiency
  • Congenital factor IX deficiency (hemophilia B) is inherited as a sex linked recessive in 1 per 30,000 males
  • Acquired factor IX deficiency is seen in liver disease, vitamin K deficiency, Coumadin therapy
  • A linear, dose dependent, false decrease is caused by bivalirudin, lepirudin, argatroban and fondaparinux (Arch Pathol Lab Med 2004;128:1142, Goldman: Cecil Medicine: Expert Consult, 23rd Edition, 2007)
Factor XI assay
  • Determine by one step clotting activity assay, comparing dilutions of patient plasma to clotting times of dilutions of pooled plasma from normals
  • Use plasma from individuals with < 1% activity of immunodepleted normal plasma
  • Can also use chromogenic substrate after add inhibitors to factor XIIa and kallikrein
  • Levels may decrease during pregnancy (J Clin Pathol 1975;28:332)
Factor XIII assay
  • Indications:
    • Patients with familial bleeding disorder but normal PT and PTT and normal von Willebrand panel
    • Factor XIII deficiency causes delayed bleeding after clot formation due to deficient crosslinking of the fibrin clot
  • Screening assay:
    • Evaluates clot stability in 5M urea
    • Add calcium to patient plasma to make it clot, incubate for 30 minutes at 37°C, then place clot in 5M urea for 24 hours at room temperature
    • Normal patients have stable cots but patients with factor XIII deficiency of 1 - 2% of normal have clots that dissolve in urea
    • Screening assay does not detect heterozygotes
    • 2% acetic acid can also be used as a clot stability screening reagent but will only detect patients down to 4% deficiency
  • Quantitative assay:
    • Reference range is 70 - 140% of normal
    • Detects values of 50% of normal (heterozygous deficiencies)
    • Expensive and not readily available, factor XIII is activated by thrombin, attaches glycine ethyl ester to a peptide substrate, releasing ammonia detected by photometer
    • High serum ammonia levels falsely decrease the result
    • Newborns may have lower levels than adults
Protein C assays

Definition / general
  • 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:
    • Detect all known type I and II variants; patients 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:
    • 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; patients protein C is activated by snake venom, which cleaves a synthetic substrate, which releases a chromogenic that is measured spectrophotometrically
  • Other assays:
    • Antigenic assays: either ELISA, electroimmunoassay (Laurell rocket method) or radioimmunoassay; variable levels, sso use 3 standard deviations as cutoff
    • ELISA: uses antibody to protein C immobilized to microtiter place; add plasma; add secondary antiprotein 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

Etiology
  • 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

Diagnosis
  • 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)
  • Nonindications: screening before oral contraceptives or oral anticoagulants (discontinue for 10 days or test family members)

Interpretation
  • Values falsely increased by bivalirudin, lepirudin, argatroban and fondaparinux, lowered by warfarin (must discontinue for 10 days prior to testing) (Arch Pathol Lab Med 2004;128:1142)
  • 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
Protein S assays

Definition / general
  • Deficiencies are either quantitative (type I: reduced normal protein) or qualitative (type II: normal amount of defective protein)
  • Assays are either functional (measure protein activity) or antigenic (immunoassays that measure quantity, not function)
  • Gold standard to measure free protein S or APC cofactor activity of protein S is considered the polyclonal ELISA with or without polyethylene glycol precipitation, although this procedure has poor reproducibility
  • Perform functional assay first (detects all types of deficiencies)
  • Functional assays are clot based, cannot be performed in patients taking hirudin or argatroban
  • Antigenic assays measure free protein S (functionally active form) or total (bound plus free) protein S; usually 60% of protein S is bound to C4b binding protein
  • Free protein S levels in protein S deficient patients are very sensitive to timing, temperature and dilutional conditions of assays compared to normal individuals

Etiology
  • More common than hereditary deficiencies; clot formation, surgery, liver disease, warfarin (should be discontinued at least 10 days prior to testing), nephrotic syndrome, DIC, L-asparaginase therapy, any stimulus to acute phase response (increases C4b binding protein, decreases free protein S), newborns (12 - 60% of adult levels, rise to adult levels by 6 months), women (lower than men before menopause, while taking oral contraceptives, during pregnancy or with hormone replacement therapy), vitamin K antagonist drugs, vitamin K deficiency, elevated factor VIII levels (> 200%) in PTT based functional assays or thrombosis; also nephrotic syndrome, varicella infection and HIV infection
  • Classification of deficiencies: all have low functional protein S; I: also low free and total protein S; II / IIb: also normal free and total protein S; III / IIa: low free but normal total protein S

Laboratory
  • Methodology:
    • Clot based protein S method is based on the addition of activated protein C, which in the presence of protein S, accelerates the inhibition of thrombin activated factors VIII and V
    • The prolongation of clotting time is proportional to the amount of factor S activity
    • Interference may occur with elevated factor VIII (acute phase reactions or otherwise) (Thromb Res 1995;77:375)
    • Values falsely increased by bivalirudin, lepirudin, argatroban and fondaparinux, lupus anticoagulants (Arch Pathol Lab Med 2004;128:1142)
  • Reference ranges (nmol/L)
    • Each lab should establish its own, values in acute phase plasma are higher:
    • Total protein S: - 65% of value in pooled normal human plasma (289 - 397)
    • Free protein S: 71 - 115
    • C4 binding protein beta+: 228 - 310
    • Total C4 binding protein: 257 - 423
Prothrombin gene 20210A testing

Definition / general
  • Mutation in G to A transition at nucleotide 20210 in 3 untranslated portion of prothrombin gene, which introduces a new Hind III restriction site
  • Can identify heterozygotes and homozygotes
  • G20210A mutation in heterozygotes is associated with increased risk of first venous thromboembolic episode (Br J Haematol 2001;113:630)
  • Multiplexed arrays test for factor V Leiden, MTHFR C677T and other sequences
  • Specimen is whole blood

Laboratory
  • Usually PCR amplification of 3 untranslated region of prothrombin gene surrounding the 20210 polymorphism, then either gel electrophoresis, radioisotopic probing or restriction endonuclease digestion with Hind III to detect the nucleotide sequence
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