Abnormal Screening Results

Abnormal Screening Results

Causes of Abnormal Screening Results

In many cases, a clinician must deal with an extended PT or aPTT in a patient who is not receiving anticoagulant therapy. Often, the key to identifying the cause of this laboratory finding is knowledge of the patient's clinical history. These tests are commonly ordered as part of the diagnostic work-up of a patient with a history of bleeding. This clinical situation is significantly different from the patient with no history of bleeding who is tested prior to surgery to rule out possible coagulation defects. The aPTT, or the more sensitive aPTT-LA, can be ordered as part of an antiphospholipid syndrome work-up of a patient with a history of thrombosis or recurrent miscarriage. Three distinct test algorithms should be employed for these three very different clinical situations.

In order to choose an optimal diagnostic algorithm, one should understand the many potential causes of an abnormal screening test. In the absence of prescribed anticoagulant therapy, prolongation of these tests generally can be attributed to five common causes: (1) specimen collection and transport issues, (2) medication, (3) pathologic conditions, (4) factor inhibitors, and (5) mixing studies: distinguishing factor deficiency from inhibitors.

Specimen Collection and Transport Issues

  • Traumatic Collection. Needles larger than 23 gauge should be used for phlebotomy to avoid mechanical disruption of platelets and red blood cells. The venipuncture should be clean (without trauma) to avoid hemolysis and contamination of the specimen with procoagulant factors. Prolonged application of the tourniquet (more than 1 minute) should be avoided because it can reduce venous circulation and result in production of coagulation factors that can affect test results.
  • Collection in Glass Tubes. Collection containers must be composed of nonreactive or nonwettable materials such as siliconized glass or plastic. Tubes made of borosilicate, soda lime, or soft glass cannot be used, as these materials may activate the contact pathway of coagulation.
  • Wrong Citrate Concentration. LabCorp provides blue-top collection tubes containing 3.2% (0.109M) buffered sodium citrate for coagulation sample collection. Reference intervals have been established with these tubes. Tubes with alternate citrate concentrations should not be used.1
  • Inadequate Tube Filling. Evacuated collection tubes must be filled to completion to ensure the proper blood to anticoagulant ratio is achieved.2 Sample tubes <90% filled will be rejected.
  • Heparin Contamination. Heparin contamination can occur when the sample is drawn inappropriately from arterial or central venous line. Blood for coagulation testing should never be collected from heparinized lines. When blood is drawn through an indwelling catheter, the line should be flushed with 5 mL of saline and the first 5 mL of blood discarded. Despite efforts to flush heparin from these lines, heparin contamination can still occur. When heparin contamination is suspected, a thrombin time can be performed (015230 Thrombin Time). A heparin level that will cause only a slight prolongation of the aPTT will cause a thrombin time >150 seconds.
  • Other Contamination. When multiple tubes are collected, collect sterile tubes and nonadditive [red-top] tubes prior to citrate [blue-top] tubes. Any tube containing an anticoagulant (ie, EDTA [lavender-top], heparin [green-top], or oxalate [gray-top]) should be collected after the blue-top tube is collected. Gel-barrier tubes and serum tubes with clot initiators should also be collected after the citrate tubes.
  • Inadequate Mixing. It is critical that the collected sample be mixed immediately by gentle inversion at least six times to ensure adequate mixing of the anticoagulant with the sample. Inadequate mixing can lead to clot formation, depleting coagulation factors and resulting in erroneous results.
  • Platelet Contamination. Clotting tests for LA are especially sensitive to contamination with platelet components.3 Specimens destined for LA testing should be collected by a method designed for the production of platelet-poor plasma (ie, platelet count <10,000/mL). Double centrifugation of the plasma should be performed.4

Frozen Gel Packs. To ensure specimen integrity during warm weather, follow these Instructions for Use of frozen gel packs and specimen lockboxes.

Medication

  • Warfarin. Oral anticoagulants decrease the activity of factors II, VII, IX, and X. The PT is generally more sensitive to the effects of warfarin therapy than the aPTT, but both can become extended.5
  • Heparin. The PT is usually less affected by heparin than the aPTT and can sometimes be normal.5
  • Thrombin Inhibitors. New anticoagulant medications, such as hirudin analogues and argatroban, can inhibit thrombin and extend both the PT and aPTT.5
  • Other Drugs. Drugs that can cause a prolonged screening test include hydroxyethyl starch, hematin, and suramin.6 Taularidine, an additive in some intravenous medications, can lengthen the aPTT.6

Pathologic Conditions

  • Vitamin K Deficiency. Extrinsic pathway (VII), intrinsic pathway (IX and X), and common pathway (II) factors can be affected. Both aPTT and PT results can be extended; however, factor VII has a relatively short half-life and can become depleted more quickly than other factors in vitamin K deficiency (or during oral anticoagulant therapy). For this reason, the PT is more sensitive to vitamin K deficiency than the aPTT.5
  • Consumptive Coagulopathy. Nearly all factors can become depleted in conditions associated with extensive thrombosis or in disseminated intravascular coagulation (DIC). The PT may become extended before the aPTT.5
  • Diminished Factor Production. Nearly all factors can become deficient in severe liver disease or in malnutrition. Due to the short half-life of factor VII, the PT will often be extended first while the aPTT will not become extended until the disease becomes more advanced.5
  • von Willebrand Disease. While von Willebrand factor (vWF) is not part of either the intrinsic or extrinsic pathways, vWF is the obligate carrier for factor VIII. Diminished factor VIII levels can produce extended aPTT results with normal PT.
  • Amyloidosis. This condition can produce an extended PT and/or aPTT due to diminished levels of factor X.6
  • Nephrotic Syndrome. Diminished levels of factors XI and XII associated with proteinuria can sometimes result in an extended aPTT with normal PT.5

Factor Deficiencies

 

Factor

Deficiency

Cases per Million Individuals*

Screening Tests

Specific Factor

Level Test N°

Comments

*More information about clinical aspects and testing for individual factor deficiencies can be found under the individual test descriptions.

vWF

>100

von Willebrand Profile

084715

aPTT can sometimes be extended

XII

 

aPTT

086322

It should be noted that factor XII deficiency is quite common but is not associated with bleeding

VIII

60-100

aPTT

086264

Affects males predominantly

IX

10-20

aPTT

086298

Affects males predominantly

XI

1

aPTT

086314

 

Fibrinogen

1

aPTT, PT

001610

 

II

<0.5

aPTT, PT

086231

 

V

<0.5

aPTT, PT

086249

 

VII

<0.5

PT

800599

 

X

<0.5

aPTT, PT

086306

 

XIII

<0.5

 

086330

Both PT and aPTT will be normal. Must test directly

Factor Inhibitors

  • Specific Factor Inhibitors. Antibodies to specific factors can prolong screening tests and are frequently associated with bleeding. The most commonly observed specific factor inhibitors affect factors of the intrinsic pathway and produce an extended aPTT with a normal PT. Alternatively, some patients spontaneously develop autoantibodies that rapidly clear their own coagulation factors. By far the most common of these specific autoantibodies are to the factor VIII molecule. Acquired antifactor VIII antibodies often occur in older patients and in pregnancy and can produce significant hemorrhage. Inhibitors to other factors are quite rare.5
  • Lupus Anticoagulants (LA). These nonspecific antibodies extend the clotting time of phospholipid-dependent clotting assays such as the aPTT.3,7 Unlike specific factor antibodies, LA are usually associated with venous thrombosis, pulmonary embolism, arterial thrombosis, and recurrent fetal loss.8 Lupus anticoagulants do not specifically inhibit individual coagulation factors; rather, they neutralize anionic phospholipid-protein complexes that are involved in the coagulation process. Testing for lupus anticoagulants and the antiphospholipid syndrome that is associated with these antibodies is described in more detail in the APS section of this appendix.
  • Factor VIII Inhibitor Profile, Comprehensive (117157)
  • Lupus Anticoagulant With Reflex (117892)
  • Lupus Anticoagulant Comprehensive (117054)

Mixing Studies: Distinguishing Factor Deficiency From Inhibitors

A mixing study is used to study the cause of a prolonged screening test. This study can determine if the cause is a deficiency of one or more factors or an inhibitor.7 In a mixing study, platelet-free, normal plasma that is replete with all coagulation factors (near 100% activity for each) is mixed with the patient's sample. For example, in a 1:1 mix, one part patient sample is mixed with one part normal plasma, and the mixture is tested. In this case, the lowest possible concentration for any individual factor in the mixture would be approximately 50% (in the case of a patient with a factor concentration of zero and the normal pool has an activity of 100%). The mixture is tested using the same test system that produced the extended screening result. Some inhibitors are time- and/or temperature-dependent. In these cases, the sample that is tested immediately after mixing will correct, while results after the sample has been incubated for one hour at 37° will not correct. All plasma samples that correct upon immediate mix are retested after an incubated mix to rule out time- and/or temperature-dependent inhibitors.

Three different types of results can be observed in mixing studies:

1. No Correction for Immediate Mix

  • When the addition of normal plasma fails to correct the clotting time into the normal range, the cause of the abnormal test is likely an inhibitor.
  • Addition of normal plasma to the reaction mixture serves to dilute inhibitors, but typically does not completely neutralize their effects.
  • The most common cause of inhibition that is observed in the clinical laboratory is caused by inhibitor-type, anticoagulants that have been given therapeutically or have contaminated the sample at collection (ie, from a heparin line).
  • Extended PT values observed in patients on oral anticoagulants typically correct in a mixing study because these therapeutics are not coagulation factor inhibitors. They work by reducing vitamin K-dependent factor levels.
  • When therapeutic factor inhibitors have been ruled out, further studies can be performed to differentiate specific factor inhibitors from lupus anticoagulants. (See Lupus Anticoagulant section.)

2. Correction for Immediate Mix − No Correction After Incubation

  • This result is consistent with the presence of a time- and/or temperature-dependent inhibitor.
  • Historically, it was felt that lupus anticoagulants were exclusively immediate-type inhibitors. Subsequent studies have shown that as many as 30% of lupus anticoagulants are time- and temperature-dependent.7 Specific inhibitors, particularly those to factor VIII, can also be time- and temperature-dependent.

3. Correction for Immediate Mix − Correction After Incubation

  • This result rules out the presence of immediate inhibitors as well as time- and/or temperature-dependent inhibitors.
  • The normal plasma supplies the deficient factor at a concentration high enough to allow normal clotting. In the absence of inhibitors, a sample with at least 50% of all factors will produce a normal clotting time for both the PT and aPTT tests.

Note: Mixing studies performed on samples with minimally prolonged screening tests (ie, less than eight seconds) will often produce confusing results. The addition of normal plasma can sometimes dilute weak inhibitors out. This causes the mixing study to correct, a result that is more consistent with a factor deficiency.

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