Lab Guide
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Test ID: FVIII CH
Factor VIII Chromogenic assay
Useful For

• This assay will be performed in situations where there is a clinical suspicion of hemophilia A diagnosis, but the 1-stage Factor VIII (FVIII) assay is normal
• For determination of FVIII activity in patients with hemophilia A receiving Emicizumab.
• For determination of FVIII inhibitor levels in patients receiving Emicizumab.
• Measurement of FVIII in patient with Lupus anticoagulant if LA insensitive APTT reagent is not available.
• FVIII assay in patient on direct thrombin inhibitor oral anticoagulants.
• For post infusion monitoring of modified, extended half-life recombinant concentrates, if chromogenic assay is recommended by the drug manufacturer.

Method name and description

Chromogenic factor VIII is a two-stage assay, the main rate-limiting step is the factor VIII level present in the test plasma. In stage one, factor VIII in the sample is activated by thrombin. Activated Factor VIII (FVIIIa) then accelerates the conversion of Factor X (FX) into Factor Xa (FXa) in the presence of activated Factor IX (IXa), Phospholipids (PL) and calcium ions.  In the second stage of this assay, the activated factor X cleaves a chromogenic substrate and the FXa activity is assessed by hydrolysis of a p-nitroanilide substrate specific to FXa.  The initial rate of release of p-nitroaniline (pNA) measured at 405nm is proportional to the FXa activity, thus to the FVIII activity in the sample.

Reporting name

FVIII Chromogenic

Clinical information

Endothelial cells of the liver are the major site of FVIII synthesis.

Factor  deficiency is often discovered when activated partial thromboplastin time is found to be prolonged. The deficiency causes bleeding disorder.

Aliases

NA

Specimen type / Specimen volume / Specimen container

Plasma Na Cit. Platelet-poor plasma.
Specimen Volume:2.7 ml.
Container/Tube: Light-blue top (citrate)

Collection instructions / Special Precautions / Timing of collection
  1. Samples collected in HMC facilities MUST reach the coagulation laboratory within 1 hour of collection.
  2. Samples collected in non-HMC facilities / external clients: MUST reach coagulation laboratory within 1 hour of collection, if not applicable, centrifuge the whole blood and carefully remove the plasma (by pipette not by decanting) and send it in a temperature-controlled environment (18- 25 ºC) within 2 hours of centrifugation. If the transportation of the plasma can’t be within a maximum of 2 hours, prepare platelet-poor plasma as follows:
  • Re-centrifuge the plasma again.
  • Remove the top portion of plasma leaving approximately 250 ul in the bottom to discard.
  • The double-centrifuged plasma should be aliquoted into labeled plastic tubes.
  • Freeze immediately at -20 ºC or below
  • Specimens must arrive frozen.
  1. If patient HCT is >55%, test will not be processed. The ordering Physician will be contacted to reorder the test and call the patient to go to Phlebotomy for recollection. Instruct the patient to inform the phlebotomy   about his high HCT% , the phlebotomy will contact the lab to request a coagulation tube after adjusting amount of anticoagulant.

 

Note:

Collection of blood for coagulation testing through intravenous lines that have been previously flushed with heparin should be avoided, if possible. If the blood must be drawn through an indwelling catheter, possible heparin contamination and specimen dilution must be considered. When obtaining specimens from indwelling lines that may contain heparin, the line should be flushed with 5 mL of saline, and the first 5 mL of blood or 6-times the line volume (dead space volume of the catheter) be drawn off and not used for coagulation testing. For those specimens collected from a normal saline lock (capped off venous port) twice the dead space volume of the catheter and extension set should be discarded.

Relevant clinical information to be provided

Patient/family history of bleeding  disorder. History of anticoagulant therapy.

Storage and transport instructions

Ambient

Specimen Rejection Criteria

Specimens with no label or missing required identification
Broken, leaking or contaminated specimen
Clotted samples 
Under-filled or overfilled sample tubes.
Wrong sample container sample received
Improper specimen transport temperature (e.g. like specimens which are  needed to be sent on ice)
Old specimen (test-dependent) 
Grossly Hemolyzed sample (test-dependent)                                          

Biological reference intervals and clinical decision values

 Reference range for age group > 18 years:70-150%. 

 For age group up to 18 years: reference range for FVIII clotting is used. 

0 Minutes-4 Days

0 Minutes-4 Days

4 Days-1 Months

1 Months-6 Months

6 Months-1 Year

1 Year-5 Years

5 Years-10 Years

10 Years-18 Years

50-178

50-178

50-154

67-141

70-213

83-170

75-163

80-166

  • FVIII activity is done by chromogenic assay.
  • Chromogenic Factor VIII activity generally correlates with the one-stage FVIII activity. However, no specific data are available for full term/premature neonates, infants, children, and adolescents.
  • Reference Interval: Factor levels are expressed as percent of blood plasma concentration. By definition, pooled plasma contains 100 %(1unit/ml) of factor VIII (clotting assay).
  • The reference range for age group up to 18 years is quoted from literature. The reference range values have not been verified /established by lab.
Factors affecting test performance and result interpretation

No interference up to:
Triglycerides  6.78 mmol/L
Hemoglobin  1.0  g/dL
Bilirubin 615.7  µmol/L

Turnaround time / Days and times test performed / Specimen retention time

For routine ordered test : 3 working days

For stat ordered test :

  • During the Laboratory working hours from 0700 hours to 1500 hours (Sunday to Thursday): TAT is Within 8hrs.
  • After duty hours, weekends and on holidays: TAT is within  12 hrs. with the following requirements:
  • Laboratory should  be notified by telephone about the sending  of a STAT sample.
  • The sample should  be hand-delivered to the central processing area and requesting  them to deliver it immediately to the hematology coagulation lab.

 Test is done : Daily.

Specimen retention time: NA