In medicine, when a result is normal, the laboratory confirms that the patient is free of disease, infection, malformation or not abnormal.  Coagulation results aid the clinician in determining if a patient is at a risk for thrombosis or bleeding. 

But coagulation is not a perfect science.  Coagulation result interpretation is based on the individual patient history relative to their clinical situation.  Using all this information, the question can then be asked, "Is this result really normal?"

Case 1
A patient comes in for a pre-operative work-up for a hip replacement. He has a history of bleeding so coagulation studies are performed.  Results are as follows:

• PT= 11.2 seconds (10.5-12.5 seconds)
• APTT= 34.8 seconds (26.2- 35.3 seconds)

The screen is normal, so the patient is brought to surgery.  The patient requires one unit of fresh frozen plasma due to excessive bleeding.  Were the PT and APTT really normal?

PT and APTT

The PT and APTT are screening tests that evaluate the intrinsic, extrinsic and common pathway factors.  A normal result should demonstrate that a patient has a factor level within the normal range (50-150%).  Patients can maintain hemostasis at factor levels as low as 30%. This means that test results should exceed the upper limit of the laboratories normal range, when a factor falls below 30%.¹

Some reagents are insensitive to certain factors and will give a normal PT or APTT result when the factor level is actually < 30%. The reagent is said to be insensitive to that particular factor and can result in a patient that will bleed.  It is important for laboratories to understand how sensitive their PT and APTT reagents are to determine if a normal screen really means the patient will not bleed.

Outcome

In Case 1, this particular reagent had a poor sensitivity to factor XI, resulting in the inability of the reagent to provide a prolonged APTT until the level of factor fell below 25%. This reagent will miss detecting a patient with a FXI deficiency.  It is important to understand how your reagent performs, as well as the clinician understanding the patient bleeding history and any other relevant history. 

In particular, patients of Ashkenaki Jewish decent can present with deficiencies of FXI.  This is an autosomal recessive disorder that can affect up to 8% of this population in both sexes; deficiency is most problematic during surgical procedures, causing excessive blood loss requiring replacement product.²

Case 2
A whole blood platelet aggregation test is performed on a cardiac patient. The agonists of ADP and AA (aracidonic acid) are performed.  The patient has a normal platelet count, hemoglobin and hematocrit. Both of the agonists of ADP and AA are normal.  The patient has a heart attack.

Platelet Aggregation

This African America patient was on dual antiplatelet therapy that included aspirin and clopidogrel. Acetylsalicylic acid or aspirin works by inhibiting cyclooxygenase-1 (COX)1 and thus inhibiting platelet function.³ As a result, aracidonic acid (AA) should show decreased or abnormal aggregation.  When testing with a low concentration of ADP, this pattern should also be abnormal in a patient on aspirin. 

While it has been demonstrated that aspirin has many clinical benefits, some patients are still at risks for a cardiovascular event.  These patients may have a resistance to aspirin and predisposition to atherothrombotic complications. Resistance may be due to a genetic single-nucleotide polymorphism of COX-1.³   

Clopidogrel is a second generation thienopyridine.⁴ This is a prodrug that requires activation via several cytochrome P450 enzymes, including CYP2C19.  It works by covalently and irreversibly binding to the P2Y12 receptor, the major receptor involved in ADP-induced aggregation of platelets.⁵ 

Clopidogrel is given to patients with aspirin to manage coronary artery disease, with acute coronary syndromes (ACS), and after percutaneous coronary interventions.  However, a significant number of patients remain at risk for MI, stent thrombosis and stroke due to insufficient inhibition of platelet aggregation.⁴

Outcome

Genetic polymorphisms in the CYP2C19 enzymes responsible for metabolism of the drug play an important role in individual variability to the anti-platelet effects of clopidogrel.⁵ Prevalence varies by ethnicity. The most common variants are carriers of the *2 and *3 alleles, resulting in reduced clopidogrel-induced platelet inhibition. Twenty-five percent of Caucasians carry one copy of *2 allele, 30% of Blacks and 40-50% of Asians.⁵

Studies have shown that almost 15% of patients are resistant.³ Several definitions of resistance to these drugs have been proposed; definitions of resistance vary from <10% decrease in ADP-induced aggregation by LTA to <20% decrease in aggregation.⁵

In this case, genetic testing may aid in determining resistance in this patient. Higher doses may be given that will allow platelet inhibition; however, it is important to not put a patient at a risk for bleeding.

Case 3
A 75-year-old female is sent to the ED as a result of a motor vehicle accident. Patient presents with multiple traumas requiring surgery. The patient is on a new oral anticoagulant, dabigatran, for prevention of stroke due to paroxysmal atrial fibrillation. The patient takes 150 mg twice daily. Results are as follows:

• PT = 12.4 seconds  (10.5-12.5 seconds)
• APTT = 38 seconds  (26.2- 35.3 seconds)
• Thrombin Time= 22 seconds (12-22 seconds)

Oral Direct Thrombin Inhibitors

Dabigatran is an oral, reversible thrombin inhibitor that does not require laboratory monitoring.  However, in several situations it is needed to know if a patient has a peak level and may be at a risk for bleeding. In a study, samples were spiked with dabigatran and tested with several reagents for PT, APTT, fibrinogen, antithrombin, and APCR to look at peak and trough concentrations. At peak concentration, all APTT results were prolonged; higher concentrations resulted in doubling the APTT in all reagents. PT reagents were much less affected with almost no samples above INR 1.2 at a peak concentration. If a patient on dabigatran has prolonged APTT (>90 seconds) and PT,  over-dosing or accumulation of dabigatran should be considered.⁶

However, high inter-individual variability has been demonstrated. Results may also be dependent on renal function since the drug is cleared by the kidneys. Body weight and gender, drugs, coagulation reagents and functionally determined concentrations may add to the variability.⁷

Outcome

Due to the normal coagulation results, it appears that the levels of dabigatran are at a trough level and this patient should not be at a risk for bleeding.  Understanding how coagulation tests perform with these new oral anticoagulants and understanding individual patient conditions all play a role in assessment of the drugs and their impact on hemostasis.

Case 4
A specimen is sent to the coagulation laboratory from labor and delivery for a fibrinogen level and thrombin time.  The laboratory performs a Clauss Fibrinogen test and uses a bovine thrombin reagent for the thrombin time.  Additional fibrinogen levels are sent to the laboratory. Results are as follows:

• Fibrinogen:  300 mg/dL         TT = 19 seconds (12-22 seconds)
• 30 minutes later:  270 mg/dL
• 30 minutes later:  260 mg/dL

Is this result normal?

Fibrinogen

Fibrinogen is a 340-Kd soluble glycoprotein synthesized in the liver. In the final step of coagulation, fibrinogen is converted to fibrin with the formation of  a fibrin clot.

Fibrinogen is an acute phase reactant, which means it is elevated in inflammation, stress, infection and pregnancy.

Normal levels of fibrinogen are in the range of 200-400 mg/dL. Increased levels of fibrinogen also have a strong association with an increased risk of artherosclerotic vascular disease, while low levels of fibrinogen most commonly results from acquired conditions, such as decreased synthesis in the liver and increased consumption in disseminated intravascular coagulation.⁸

During normal healthy pregnancy there are substantial changes in the hemostatic system.   Pregnant women are specifically at risk for developing DIC.  This can occur when there is injury to the uterus or a placental abruption (secondary to high levels of thromboplastin in the placenta and plasminogen activator in the uterus). As fibrinogen levels in late pregnancy are often double those of the non-pregnant female, a "normal" fibrinogen level may represent early DIC.⁹

Outcome

Normal fibrinogen levels in pregnant women can be an early indicator of DIC.  This test should be part of investigating the possibility of this consumptive coagulopathy.  A prolonged PT, APTT and elevated D-dimer with a decreased platelet count all support the possibility of DIC.

Coagulation testing is complicated in most situations. Evaluation of results are dependent on reagents, patient history, ethnicity and gender. Understanding how all of these conditions can impact results will enable clinicians to provide the best outcomes for their patients.

Donna Castellone is clinical science manager, Roche Diagnostics.

References for archive

1. Bowyer A, et al. The responsiveness of different APTT reagents to mild factor VIII, IX and XI deficiencies. Int J Lab Hem 33:154-8, 2011. 
2. Seligsohn U., Factor XI deficiency in humans. J Thromb Haemost., 1:84-7, 2009.
3. Pulcinelli, FM,  Pignatelli, P.,Celestini, A.,, Riondino, S., et al.,  Inhibition of platelet aggregation by aspirin progressively decreases in long-term treated patients, J Am Coll Cardiol. 2004, (6):979-984. 2004.
4. Shah,  SH, Buehler, B, Clopidogrel Dosing and CYP2C19  http://emedicine.medscape.com/article/1733280-overview#aw2aab6b3
5. Karon, B. Jaben, B. Platelet Function: Laboratory Methods for Evaluating Effectiveness of Anti-platelet Therapy, Clinical Laboratory News, 32:4. 2011.
6. Lindahl TL, Baghaei F, Blixter IF, Gustafsson KM, Stigendal L, Sten-Linder M, Strandberg K, Hillarp A., Effects of the oral, direct thrombin inhibitor dabigatran on five common coagulation assays. Thromb Haemost. :371-8 2011
7. Freyburger G, Macouillard G, Labrouche S, Sztark F. Coagulation parameters in patients receiving dabigatran etexilate or rivaroxaban: two observational studies in patients undergoing total hip or total knee replacement. Thromb Res. 127(5):457-65. 2011.
8. Singh N, Gandhi M. Altered Levels of Fibrinogen in Relation to the Pathophysiology of Recurrent Spontaneous Abortions . WebmedCentral OBSTETRICS AND GYNAECOLOGY 2011;2(6):WMC001964
9. Seligsohn, U,  DISSEMINATED INTRAVASCULAR COAGULATION, Chapter 126 In  Williams Hematology, Seventh Edition, Lichtman, M., Beutler, E., Kaushansky, K. et al., pg. 1677, 2001.