CLINICAL COAGULATION SECTION
HANDBOOK FOR PATHOLOGY RESIDENTS

INTRODUCTION

This section is intended to provide practical information on use and interpretation of laboratory tests and assays in coagulation. Appendix A contains the recommendations and guidelines of the National Committee for Clinical Laboratory Standards on important parameters in specimen preparation. This information is timeless and too often overlooked or assumed to be well controlled on all samples. However, one must be absolutely sure that the specimen is acceptable or all of the data collected will be in doubt. Special sample collection procedures for certain tests are described under the heading below entitled "Special Considerations". The pathologist responsible for test interpretation should confer with the lab supervisor often to assure proper specimen acquisition and handling.

 

SCREENING TESTS

Prothrombin Time (PT):

For defects of deficits in Factors II, V, VII, and X and severe dys- or hypofibrinogenemia; includes monitoring of long term anticoagulant therapy affecting these factors, such as coumadin.

Activated Partial Thromboplastin Time (aPTT):

Primarily for defects or deficits in Factors VIII, IX, XI, and contact activation components, but also sensitive to all other clotting factors except VII; frequently used to monitor heparin therapy.

Reagents for PT or aPTT:

These two tests differ in the way coagulation is initiated, basically to discriminate between the classical extrinsic and intrinsic pathways schematized in the "clotting cascade". The reagent used in the PT is called a full thromboplastin because it leads almost directly to generation of the activated prothrombinase complex for thrombin production. The commercial thromboplastins are prepared from solvent extraction of rabbit or other animal brain, which provides a crude source of tissue factor for activating Factor VII, a source of procoagulant membranes for the lipid-dependent steps of activation of Factor X and prothrombin conversion, and Ca2+ is added for reversing the citrate anticoagulant. The available commercial preparations produce slightly different normal ranges and differ in extent of prolongation of clotting times in Coumadin-treated patients. When the laboratory changes brands of thromboplastin or when a new lot of the same product is obtained, the normal range of PTs must be reestablished.

For the aPTT, a partial thromboplastin reagent is used which provides procoagulant membranes but not tissue factor or Ca2+. Natural source material is either soybean oil or animal brain extracted for the cephalin lipid fraction, which does not include much protein. In addition, the commercial aPTT reagents contain an agent to initiate the contact activation system, usually ellagic acid or micronized silica (Kaolin was the old favorite, but is a particulate suspension of clay and settles out too easily in use). A contact initiator reagent was not included in the original, unactivated PTT which depended solely on the glass of the test tube to initiate contact activation in plasma. Variability in activity among the available commercial preparations is considerable. After an optimal incubation time of test plasma with the partial thromboplastin reagent, Ca2+ is added separately to permit thrombin generation based on the activation of all preceding steps in the cascade. Thus a normal test result requires an intact intrinsic and common clotting cascade pathway.

 

PT or aPTT Mix:

When an abnormal PT or aPTT is obtained, it indicates that there is either a factor deficiency or an inhibitor of coagulation present. A simple method to check is to try to shorten the clotting time by mixing the test plasma with an equal volume of normal plasma. A low factor level in the test plasma will be brought up to at least half of normal levels in this mix. The clotting time should then be in the normal range unless an inhibitor is present, because a 50% level of any one clotting factors will still produce a normal clotting time. The types of inhibitors that can lead to abnormal clotting times include an antibody directed at a specific clotting factor, a lupus-type anticoagulant, fibrin(ogen) degradation products (FDP), or heparin. Some Factor VIII antibodies will be missed unless the mix of plasmas is incubated at 37ºC for 1 - 2 hours. A partial correction of the clotting time (shortened after the mix, but not within normal range of clotting times) is consistent with the presence of a non-specific inhibitor like heparin -- not an antibody.

 

 

Thrombin Clotting Time (TCT):

This test is primarily used for detection of direct inhibitors of thrombin or of fibrin polymerization. It is simply the clotting of undiluted citrated patient plasma by exogenous thrombin. The TCT is particularly sensitive to heparin, FDPs, and hypo- or dysfibrinogenemia. Thus this screening test is useful in evaluating a prolonged PT or aPTT by discriminating between a problem in thrombin generation (normal TCT) versus inhibition of thrombin activity (abnormal TCT). However, few general practice physicians recognize its utility, so it is often the clinical pathologist who suggests the test to decide if factor assays are the next logical diagnostic step in the workup of a bleeding patient. The TCT is also used in monitoring the extent of the lytic state in thrombolytic therapy (since it is sensitive to fibrin[ogen] degradation products). At PCMH, the TCT is performed routinely on cardiac post-op samples to monitor heparin reversal (see below, Activated Coagulation Time); an abnormal TCT result is followed by repeat testing after adsorption of heparin from the test plasma (Heparsorb) to see if there has been inadequate heparin reversal in the OR, which could explain other abnormal coag test results post-op. A version of this test employing a snake venom enzyme (Reptilase) instead of thrombin to initiate clotting can also be used to determine whether a prolonged TCT or other test is due to heparin, since the Reptilase time would be normal in the presence of heparin and yet sensitive to fibrinogen concentration and the presence of degradation products.

 

 

Bleeding Time:

This test measures the time it takes to form a primary hemostatic plug to arrest hemorrhage from a standard skin incision under controlled conditions. A normal response requires proper platelet count and function and vasoconstriction; single factor clotting deficiencies do not usually produce an abnormal test result (even a severe hemophiliac can produce a normal bleeding time result but will have a very copious flow from the incision and possible re-bleeding afterwards). However, because of uncontrolled parameters such as skin vascularity and thickness, the result across a variety of patients is only qualitative, not quantitative. An abnormal bleeding time result in an otherwise normal patient may indicate a potentially dangerous hemorrhagic tendency if subjected to significant trauma or surgery and should be followed up with further specific tests and assays. The more common disorders causing a prolonged bleeding time are von Willebrand's disease and drug-induced platelet dysfunction. In many normals, aspirin ingestion within 24 hours may result in a prolonged bleeding time without a real significant risk of hemorrhage. A platelet count is essential to interpretation of the test result. If the platelet count in below 100,000/µL, the bleeding time will be prolonged even if platelet function is normal. Appendix B contains figures showing the relationship of bleeding time to platelet count or function and a flow diagram of test interpretation. Recent re-evaluations of the bleeding time test in well-defined patient populations have shown in general poor correlation with actual risk of hemorrage, so its usefulness is in some doubt.

 

 

Screening Procedures at PCMH:

At this hospital, the PT, aPTT, and mix studies are performed on an automated analyzer (currently MLA 1600) with reagents from Pacific Hemostasis. A normal range is established for each new lot of reagents by assaying 20 - 30 healthy donors in duplicate and taking the mean ±2 SD of the observed values after statistical review. This 'normal range' is used to decide whether a patient test result is 'normal' or 'abnormal', but should not be considered strictly reliable with results that are near the threshold. Daily quality control of the instrumentation and reagents is performed with standardized commercial plasma preparations (from the same vendor) with expected normal or abnormal values. If control test results are not within acceptable limits, the supervisor will be advised of the problem. Any corrective action taken must be documented and reviewed by the pathologist for CAP compliance. A backup procedure has been established for performance of PT and aPTT tests on the fibrometer.

The TCT is best performed on a fibrometer, but has been adapted to the automated analyzers. We deliberately use a very dilute thrombin solution, which produces long clotting times in normal samples, but is particularly sensitive to heparin. For samples collected during lytic therapy, standardized commercial control plasmas are run for QC. The Reptilase time is rarely requested and is now a send-out.

Bleeding time tests are performed at PCMH with the Simplate device (General Diagnostics). This test is hard to control due to technique variations among technologists. Skin tone, hirsutism, and patient anxiety can also affect results. Nevertheless, this is the only in vivo platelet function test currently available. The pathologist may be advised by the technologist of unsuitable conditions pertaining to an abnormal result, and in almost every case it is advisable to repeat the test if a significantly prolonged bleeding time is found in presurgical screening. Often the patient history data in the SUNQUEST computer system or on the test order form is not completely accurate about drug history, so both the patient and the requesting physician should be quizzed carefully about possible interfering substances affecting platelet function.

 

CLOTTING FACTOR ASSAYS

Factors II - XII:

Factors VIII, IX, XI, and XII are assayed by performing the aPTT on dilutions of patient plasma mixed with an equal volume of substrate plasma that is prepared by the manufacturer to be deficient specifically in the clotting factor of interest. Factors II, V, VII, and X are assayed similarly with the PT. This assay principle is based on the original method of identifying clotting factor deficiencies by mixing a test plasma with plasma from patients known to have a deficiency in an established clotting factor. If the test plasma did not correct the clotting time of the known factor-deficient plasma (and no inhibitor was present), then one could conclude that the test plasma was similarly deficient in that clotting factor. If the test plasma shortened the clotting time of all the known deficient plasmas, then a new factor deficiency was suspected. For many years, coagulation laboratories used substrate plasmas prepared from patients with established factor deficiencies. The diagnosis of rare hemophilias was limited by availability of these substrate plasmas. More recently, immunodepleted plasmas have been manufactured by using immobilized-antibodies to remove a particular clotting factor from normal plasma. This product presents a much-reduced risk of HIV or hepatitis infection to the coagulation technologist and is more readily available than naturally deficient plasmas.

The attempt to quantify the activity of clotting factors in plasma is based on the bioassay concept. A standardized commercial normal pool plasma is serially diluted and assayed with factor-deficient substrate plasma to construct a standard curve showing the dependence of the assay result on the concentration of the missing factor. A documented normal control and/or abnormal control plasma are similarly assayed, and the interpolated results are read from the standard curve. These results must agree with the factor levels stated on the control plasma vial to within ±15% or be repeated. Finally, the patient plasma is assayed in three or more serial dilutions and the results interpolated from the standard curve.

The bioassay method assumes that the control curves and the patient curves (log clotting time vs. log reciprocal dilution) are parallel to the standard curve (Log clotting time vs. log clotting factor concentration). A robust statistical test of this assumption is not yet in place with the current hardware; the coag supervisor or pathologist should review all data before releasing an averaged result. A few of the hematology specialists on staff at ECU/PCMH in other departments can also interpret results correctly over the phone or in person. Check with the lab supervisor or other residents' experience on how to handle emergency high demand situations.

 

Factors XIII:

At PCMH we have only a screening test for severe deficiency in Factor XIII (less than 1% of normal). The test consists of recalcifying citrated patient plasma to form a clot which is placed in 5M urea. During a 24 hour incubation at room temperature, the clot is checked to see if the fibrin cross-linking activity of Factor XIII prevented dissolution of the clot. An abnormal test result is dissolution in 24 hours or less. Though simple and insensitive, this test is adequate since it is thought that only a severe deficiency in F. XIII is related to bleeding.

 

 

Contact Activation Factors:

The contact activation system of plasma consists of proteins that have activity in the coagulation cascade but appear to play more important physiologic roles in other systems. Deficiencies in Prekallikrein (also called Fletcher Factor) or High Molecular Weight Kininogen (also called Fitzgerald Factor) can result in markedly prolonged aPTT clotting times, but bleeding is not usually associated with these disorders; this remark is also true for Factor XII deficiency. Mixing studies are performed at PCMH to identify a deficiency in Prekallikrein or H.M.W. Kininogen by mixing 1:1 the test plasma with a substrate plasma deficient in one of these factors. As discussed above, a corrected aPTT in the mix rules out a deficiency in the tested factor. If an abnormal result is obtained with the Fletcher Factor-deficient mix, the test should be repeated with a 10 minute preincubation with the aPTT reagent to see if the clotting time gets shorter (hallmark of Fletcher Factor).

 

 

Fibrinogen:

Fibrinogen is quantified in a somewhat different manner from factor assays. A series of dilutions of the test plasma is clotted directly with exogenous thrombin without the need for a substrate plasma. Unless interfering substances are present, the clotting time of diluted plasma treated directly with a high concentration of thrombin will be related strictly to fibrinogen concentration. The results are interpolated on a standard curve made with dilutions of a standardized commercial plasma which has been independently calibrated for fibrinogen protein content, or a purified fibrinogen solution. The standard curve is plotted in units of milligrams of fibrinogen per deciliter (mg%), rather than percent of normal. Dilution of the test plasma as required for this assay makes it less sensitive than the TCT to the presence of heparin, FDPs, or other inhibitors. The assay of fibrinogen can be artifactually low in samples from patients on lytic therapy unless an inhibitor of plasmin (such as aprotinin) is added to the blood collection tube. The inhibition of plasmin in the collected sample prevents further degradation of fibrinogen in vitro before the assay is performed. Specialized collection tubes for this purpose are commercially available but not in routine use at PCMH.

 

 

VON WILLEBRAND'S AND OTHER FACTOR ASSAYS

von Willebrand's Factor:

A prolonged bleeding time test result obtained in screening, or any report of family history positive for spontaneous mucosal bleeding is often followed up with an investigation of von Willebrand's Factor (vWF). A panel of three assays is performed at PCMH to reach or rule out a diagnosis of vWD; it is important that all three of these assays be run on the same plasma sample to evaluate the ratio of results. These tests are: a Factor VIII clotting activity assay (sometimes designated F. VIIIc), measurement of Factor VIII-related antigen levels (sometimes designated F. VIII: Rag) by immunoelectrophoresis, and an assay for ristocetin cofactor activity (sometimes designated F. VIII: vWF). The latter assay is performed by determining the rate of aggregation of normal platelets in dilutions of the test plasma treated with ristocetin. Commercial kits for this assay provide control plasmas, ristocetin, and fixed lyophilized platelets. The antibiotic ristocetin mediates binding of normal vWF to the platelet surface to promote agglutination (perceived as aggregation) of the platelet suspension. The concentration of vWF in the test plasma becomes rate-limiting for platelet agglutination under these conditions. The results obtained in the platelet aggregometer are not precise and should be treated as an estimate of the vWF activity, rather than an absolute determination. Another confirmatory qualitative test that can be employed is aggregation of the patient's platelet-rich plasma (if platelet count is normal) by dilutions of ristocetin. A more definitive classification of vWD is possible with analysis of vWF multimer size distribution in the test plasma. PCMH does not perform this analysis but it is available through send-out to referral centers in Chapel Hill or Miami Medical Center. See Appendix C for a review of vWD diagnosis and subclassifications.

 

 

Antithrombin III:

The Chemistry Section of PCMH Laboratories performs a chromogenic assay for antithrombin III (AT-III) activity on the Dupont ACA. This assay is usually ordered before a determination of the AT-III antigen level (send-out). Evaluation of the normal range for AT-III activity and antigen is critical to interpretation of patient results, because a partial deficiency in AT-III (< 75% of normal) may be associated with significant thrombotic risk (see McGann and Triplett, Lab Med 13:742-749, 1982). The determination of AT-III levels is important in the phenomenon of "heparin resistance" where it appears that a standard dose of heparin does not have a strong biological effect, or in D.I.C. when rescue with heparin is being contemplated.

 

 

Plasminogen and Antiplasmin:

Tissue plasminogen activator (tPA) and activator inhibitor (PAI) assays are available as send-outs, but plasma levels of these regulators may not relate directly to hyper or hypofibrinolysis. Plasminogen antigen levels and Ü2-antiplasmin activity assays can be performed on the Dupont ACA, but they are ordered so rarely that the cost of outdating kits precludes keeping any in inventory. This is the weakest area of diagnostic testing at PCMH and clinical coagulation in general. Suspicion of a defect in fibrinolysis should be confirmed through collaboration with experts at other medical centers (such as Dr. Charles Greenberg at Duke).

 

 

Protein C and Protein S:

Assays for Protein C and Protein S antigen and activity levels used to be evaluated in the Brody Outpatient Lab by chromogenic or ELISA techniques but are no longer available for fiscal reasons and are now send-outs. A critical value of Protein C antigen or activity predisposing to thrombotic risk has not yet been determined but may be ~60%. (see Miletich et al., NEJM 317:991-996, 1987). Of more interest is the recent discovery of a common Factor V mutation leading to resistance in degradation by activated Protein C (APC resistance; see Dahlback et al., NEJM 330:517, 1994). A test of APC resistance in the patient plasma is available through the send-out service through a few specialty laboratories. A genetic test via restriction fragment analysis is now available as a send-out to detect the presence of a mutation in the patient's Factor V molecule for the most common trait (Factor V Leiden), performed on nucleic acid recovered from WBC collected from an EDTA tube. Recently this genetic diagnosis has become more accepted and definitive for the diagnosis. Abnormal findings in these tests may explain up to half of the cases of unexpected familial thrombosis.

 

 

INHIBITORS

Bethesda Inhibitor Titer:

Antibodies to Factor VIII, factor IX, or other clotting proteins can arise in hemophiliacs receiving factor concentrate therapy or spontaneously in postpartum or autoimmune states. The presence of such an antibody is usually signaled by a markedly prolonged aPTT or PT which does not correct in the 1:1 test mix with normal plasma (see above). An estimate of the titer of the antibody can be obtained in standardized (Bethesda) Inhibitor units based on determination of the dilution of test plasma at which approximately 50% of the clotting factor activity in normal plasma is inhibited. This assay is performed by incubating a series of dilutions of the test plasma mixed 1:1 with normal plasma at 37ºC for 2 hours along with a control of normal plasma mixed with buffer. Single point determinations are then made of the factor level of interest, and the factor activity remaining in the mixes is compared to the factor activity in the control. The dilution of test plasma producing exactly a 50% decrease in factor activity relative to the control is said to have one Bethesda inhibitor unit per milliliter; the reciprocal of that dilution thus gives the titer of the antibody. An interpolation curve is used in the usual case when dilutions in this assay may not be optimal for every type of clotting factor antibody encountered; if the calculated titer is lower than expected from clinical or other data, a change in incubation time, temperature, or even pH may be attempted to better assess antibody strength.

 

 

Lupus Anticoagulant:

Several autoimmune disorders, including but not restricted to systemic lupus erythematosus, may show the presence of a coagulation inhibitor that appears to be directed against procoagulant phospholipid (part of the procoagulant membranes described earlier). Some infections may transiently also produce the inhibitor with less clinical severity (see reference A). The effect of the inhibitor in the test tube is to prolong the aPTT or PT. Paradoxically, the patient may have a thrombotic process. The nature of the inhibitor varies from patient to patient; however, it is generally thought that the effect of the inhibitor in vitro is to bind to the partial thromboplastin phospholipid added in the PT or aPTT reagent. The amount of phospholipid used in the PT and aPTT tests is a critical determinant of the sensitivity of those tests to lupus anticoagulants (reference D). Tests with lower concentrations of procoagulant phospholipid tend to be more sensitive to the presence of lupus anticoagulants (D). An independent test also related to lupus anticoagulants is the demonstration of antibodies to the lipid cardiolipin (this panel is available through the send-out service). How these antibodies correlate with the anticoagulant producing a prolonged aPTT is not fully understood since in some patients the activities are dissociable (please see Appendix E for further details).

At PCMH, we used to employ routinely a qualitative test called the Platelet Neutralization Procedure (PNP), which consists of an addition of frozen-thawed platelets to the aPTT to absorb out or bypass the lupus anticoagulant. If the aPTT does not shorten by 10 seconds or more with this addition, then the test result is considered to be negative for a lupus-type anticoagulant. Obviously, this procedure will not be meaningful if the patient's aPTT is not at least 10 seconds above the upper limit of the normal range before addition of the platelet suspension. False positives may occur with oral anticoagulated plasma and plasma with a factor V inhibitor (reference E). Poor sensitivity of the aPTT reagent to lupus anticoagulants will often obscure identification of weak inhibitors. An anticardiolipin panel may be requested by the ordering physician if a lupus anticoagulant is suspected but the aPTT is near normal (see Lo et al., Am J Hematology 30:213-220, 1989) .

A more sensitive test for the presence of lupus anticoagulants is now available in the form of the dilute Russell's viper venom test (dRVVT), also known as the Stypven time (reference B). Dilute Russell's viper venom has an enzyme that activates factor X directly. This bypasses the need for any plasma proteins except factor V, factor X, prothrombin, and fibrinogen in order to form a clot (reference C). In this test, dilute Russell's viper venom is added to the patient's plasma along with dilute phospholipid. The mixture is incubated at 37 degrees C for 30 seconds. Calcium chloride is then added and the clotting time is measured. If the clotting time is prolonged, it may be due to a lupus anticoagulant or a factor deficiency in the common pathway (F. X, II, V, or fibrinogen); a mixing study is then performed by repeating the test after mixing the test plasma 1:1 with normal plasma to restore nominal factor levels if deficient. If the mix also produces a prolonged clotting time (judged by ratio with clotting time of the normal plasma alone with the Stypven reagent), then the patient plasma is tested again with a modified Stypven reagent that contains a high level of procoagulant lipids to bypass lupus anticoagulants. If this produces a normal clotting time then the diagnosis of a lupus anticoagulant is confirmed (reference C).

 

 

Fibrin Degradation Products (FDP):

The action of plasmin (the main effector enzyme of fibrinolysis) on fibrin clots and on soluble fibrinogen produces polypeptide fragments that inhibit the activity of thrombin. These fragments, collectively called FDP, can prolong the aPTT, PT, and TCT of patients on fibrinolytic therapy or in acute disseminated intravascular coagulation syndrome (DIC). At PCMH we have a calibrated quantitative assay for FDP on the Dupont ACA. The DuPont version of this test is performed on completely clotted serum generated in special collection tubes (black top) to avoid interference in the assay by fibrinogen. For patient samples containing heparin, as is often the case, a snake venom enzyme preparation called Atroxin must be added to coagulate the plasma and thereby remove all of the unbound fibrinogen from solution. Because of the frequent lack of accurate drug history on patients (especially in regard to heparin therapy), the pathologist may need to confirm a finding of elevated FDP by treating the sample with Atroxin to remove residual unclotted fibrinogen. The D-dimer assay is similar in that it gives a semiquantitative measurement of cross-linked fibrin strand fragments resulting from plasmin digestion of preformed clots; split products from native unclotted fibrinogen are NOT picked up in the D-dimer assay, only in the FDP assay. Recent advances in technology for precise quantitation of D-dimers has elevated the value of this test over that of the FDP assay for monitoring DIC and for detecting early thrombotic processes (pre- myocardial infarct), but PCMH has yet to implement the newer assay methodology.

 

MONITORING OF ANTICOAGULANT THERAPY

Coumadin - PT:

Long term anticoagulant therapy with Vitamin K antagonists (coumadin, dicumarol, warfarin) results in reduced activity of Factors II, VII, IX, X, and Protein C & S due to inhibition of the posttranslational carboxylation of glutamic acid residues. Both the PT and the aPTT are affected, but it is widely held that prolongation of the PT is the better monitor for titrating the dose of drug for best effect, due to its sensitivity to Factor VII. This factor is the shortest lived in circulation and therefore the first factor to decrease appreciably at he initiation of coumadin therapy. The PT is thus more sensitive than the aPTT would be to the early changes. The ordering physician may wish to know the clotting time obtained for the normal PT control on that shift or the mean of the PT normal range in order to calculate a ratio with the patient's PT. However, the preferred method of evaluating prolongation of PTs is the International Normalized Ratio (INR), for specific regimens of anticoagulant therapy (see Koepke and Triplett, Arch Pathol Lab Med 109:800-801, 1985). The calculation of INR values comes from the following formula:

INR formula

*Where the ISI equals the standardization index value for the particular thromboplastin in use versus the Manchester standard.


With the newer instrumentation (MLA 1600) there are simple computer algorithms to convert PT clotting times into INR units; both are reported out. Most therapeutic modalities with coumadin have a target INR of 2.0 to 4.0.

 

 

Heparin - aPTT, Dupont ACA:

Heparin is a mixture of mucopolysaccharide polymers of varying size prepared from tissue extracts of porcine intestine or bovine lung. Synthetic heparin polymers (heparinoids) are also available through the PCMH Pharmacy, as well as semi-purified fractions of native heparin of specific size ranges (low molecular weight heparins). The anticoagulant effect of heparin is mediated through the naturally occurring inhibitor antithrombin III (AI-III) and heparin cofactor II. Heparin greatly increases the rate of reaction of AT-III with activated clotting enzymes, especially Factor Xa and thrombin. The aPTT is more sensitive than the PT to the effects of heparin, possibly because of inhibition of Factor IXa and contact activation factors or inhibition of thrombin feedback loops in the intrinsic clotting system. The extent to which the aPTT is prolonged by heparin depends a lot on the type of partial thromboplastin reagent employed. No standardization protocol is yet developed as in INR-units used for coumadin anticoagulation. Different regimens of heparin are used according to the degree of perceived risk for thrombosis in each hypercoagulable syndrome. The in vivo activity of heparin can vary widely among patients with similar diagnoses. Physicians generally follow "rules of thumb" in dosing heparin, such as prolonging the aPTT to 1 1/2 - 3x the upper limit of the normal range, depending on the heparin-sensitivity of the aPTT reagent.

If desired, a chemistry test can be performed on the Dupont ACA to measure the units of heparin activity in plasma. This assay is based on inhibition of the cleavage of a chromogenic substrate by Factor Xa in the presence of AT-III and the patient's plasma. Only a part of the biological effect of heparin is involved in this assay; inhibition of thrombin activity by heparin is not evaluated by this test. The low molecular weight fractions of heparin are enriched in anti-factor Xa activity relative to antithrombin activity, and in this assay they will appear to have a strong biological effect. Whether or not the in vivo anticoagulant effect is mediated mostly through anti-Xa or antithrombin activity is still debatable. Thus, the use of this assay alone in monitoring heparin therapy would not be advised. Also, we have found that protamine interferes in this assay, so it is of little use in monitoring heparin reversal in the cardiac OR.

 

 

Heparin & the Activated Coagulation Time (ACT):

The ACT is a simple test performed at the patient's bedside (during dialysis) or in the operating room on whole blood to estimate in vivo levels of heparin. It is performed by introducing a measured amount of freshly drawn blood into a glass tube containing diatomaceous earth (manual method) or a cartridge containing kaolin or other contact-activating material (automated method). The test is surprisingly insensitive to hemodilution and partial defects in clotting factors or platelet function, but lysed platelets may artifactually shorten the clotting time (see Bode and Eick, Am J Clin Path 91:430-434, 1989). Currently at PCMH two different mechanical devices are used to perform this test. The PCMH Pathology Laboratory is responsible for monitoring of heparin during open-heart surgery and dialysis procedures even though performed by others at ancillary sites; we have two specialists involved in maintaining quality control and training in these other sites. Generally, a baseline ACT is obtained before cardiopulmonary bypass (CPB) circulation or dialysis is begun and 5 minutes after the heparin loading dose is given. A line drawn between these points on a graph of heparin dose versus ACT is used as a rough dose-response curve to calculate how much more heparin is needed to reach a target ACT (usually 6 - 8 minutes) or how much protamine to give to neutralize the heparin at the end of CPB based on the assumption that 1 mg protamine will neutralize 100 units of heparin.

At the close of each open-heart procedure at PCMH, it is routine to send a citrated plasma sample for testing with the TCT, which is more sensitive than the ACT to residual heparin. If the TCT is prolonged (greater than 30 seconds), a cationic agent (HeparsorbR) is added to remove the anionic heparin molecules in the plasma, and the TCT is performed again. If the TCT is now returned to the normal range, then there is an indication of potentially significant levels of residual heparin in the patient. A prolonged TCT after Heparsorb treatment should be followed up with tests for other antithrombin substances, such as FDPs, or a fibrinogen assay.

 

 

PLATELET FUNCTION TESTING

Screening:

The bleeding time is a somewhat useful screening test for vWD and for platelet dysfunction; however, an accurate patient history can be just as valuable or better. Other information is also helpful before progressing to more specific tests of platelet status (such as drug history and recent transfusion record). The blood smear may reveal abnormal platelet size or appearance. The Coulter Counter in the Hematology Lab section gives not only a platelet count, but also a graphic and statistical analysis of platelet volume distribution. However, it is not reported out unless ordered by the physician. These tests may indicate a disorder in platelet production. A clot retraction test can be useful as a screening test for Glanzmann's Thrombasthenia, but it is not sensitive to all adhesion and aggregation abnormalities. With normal platelets, clot retraction is evident in 30 - 60 minutes after blood collection in glass tube. Severe bleeding in the presence of a normal platelet count and a normal vWD workup is a likely indication of a platelet function defect.

 

 

Aggregation Panel:

The patient's current medications should be examined for possible interference with platelet testing prior to scheduling of this panel. Ideally, the patient should be free of all drugs for ten days and be fasting prior to blood drawing. Ingestion of two beers or one mixed drink the night before by the patient (or by the technologist doing the testing) can also interfere with aggregation tests of platelets. Blood is drawn preferably through a 19 gauge butterfly needle into Ware's anticoagulant of citrate plus citric acid. One or two normal donors are also drawn as positive controls. Platelet-rich plasma (PRP) is obtained by low speed centrifugation and immediately tested in a turbidimetric device called the platelet aggregometer. To each aliquot of PRP is added a small volume of an agent known to produce aggregation of normal platelets by one or more of the known pathways of platelet activation. At PCMH, the panel consists of (final concentration):

  • 500 or 250g/mL arachidonic acid
  • 1 or 0.5 x 10-4 M epinephrine
  • 0.19 or 0.1 mg/mL collagen
  • 10 x 10-6 M adenosine diphosphate (ADP)
  • 4 x 10-6 M ADP
  • 2 x 10-6 M ADP
  • three concentrations of ristocetin (1.5, 1.0, and 0.5 mg/mL).

The listed agents bind to receptors on the platelet surface or enter the cell to activate biochemical pathways leading to release of the platelet's storage granule contents. The release reaction is associated with aggregation of platelets in clumps large enough to cause a measurable increase in light transmittance in the aggregometer. The lack of a complete wave of aggregation usually indicates a defect in the release reaction, or, more rarely, a receptor defect. Fibrinogen is a necessary plasma cofactor in these aggregation studies. Aggregation due to ristocetin is more appropriately termed agglutination since it does not require signal transduction and the release reaction. Plasma vWF is necessary for aggregation of the patient's platelets with ristocetin. The low dose of ristocetin (0.5 mg/mL) will not produce aggregation of normal platelets; only vWD type IIB or platelet-type vWD will show a response. In actuality, the aggregation of patient PRP with ristocetin is more a test of vWF function, rather than platelet function.

Spontaneous aggregation of platelets may occur in some patients, especially those with reduced surface charge on the platelet membrane or with a hypercoagulability syndrome. The test may take up to 30 minutes incubation of patient PRP in the aggregometer (with no additions) to show a positive response, and thus should be run after the rest of the reactions have been completed.

Anomalies seen in aggregation tracings include: no response at all, only a partial or reversible change of 20 - 30% across the chart paper, or a slow rate of aggregation. Interpretation of these results requires comparison with controls and published patterns for known platelet disorders (see Appendix D). Hereditary dysfunction of platelets can be the result of a deficiency in certain membrane proteins (Bernard-Soulier Syndrome, Glanzmann's Thrombasthenia) or a dysfunctional response mechanism (Storage Pool Disease, Gray Platelet Syndrome, etc.). Acquired platelet dysfunctions are associated with certain drugs (especially those containing aspirin), autoimmune disorders (ITP, SLE), myeloproliferative disorders, and other pathological conditions. Congenital platelet abnormalities are rare, so the pathologist must be conservative in applying a definitive diagnosis on the basis of platelet aggregation studies. Appropriate interpretations require adequate history from the clinician. All findings should be repeated and possibly followed up with special testing in research laboratories and/or electron microscopy facility. For example, flow cytometry phenotyping of platelet may be used to reveal specific membrane receptor defects such as in Bernard-Soulier platelets.

Recently a new aggregometer (Chronolog LumiAggregometer) was purchased by PCMH that will also measure the platelet release reaction by means of the luminescence produced when ATP released from platelet granules energizes the luciferin-luciferase reagents added in at the end of the aggregation reaction. This procedure is still in the testing phase but will soon become the mainstay for diagnostic platelet aggregation studies as well as for the demonstration of HIT (see next section). Release of granule contents from platelets during activation is a surer test of platelet functionality than aggregation patterns alone, and this new capability will add significantly to our ability to detect acquired or congenital platelet defects.

 

Heparin-Induced Aggregation:

A small number of patients receiving heparin infusions or exposed to heparin in blood vessel catheters will develop an antibody to heparin with thrombotic consquences. Heparin normally binds to PF4 released from platelet alpha granules; but if the PF4 has already bound back to the platelet, then this antibody to heparin will also become associated with the platelet surface. The Fab portion of the antibody binds to the heparin/PF4 complex, but the Fc portion is free to bind to the platelet Fc receptor. The binding of the antibody to the Fc receptor on the platelet sends an intracellular signal that serves to activate the platelet, thereby inducing systemic aggregation and expression of procoagulant activity. When the patient has clinically significant numbers of antibodies, s/he experiences a sudden drop in platelet count with thromboses and possibly hemorrhage or DIC. The consequences can be rapidly fatal due to the widespread development of arterial occlusions. Unfortunately, there is no predictive test to use to screen patients for antibodies to heparin before heparin is given. When the physician (usually a surgeon or internist) suspects a heparin-induced thrombocytopenia (HIT), the lab can perform a test of the ability of the patient's plasma to aggregate normal platelets in the presence of heparin. This test should be interpreted very cautiously, because a negative result does not rule out a heparin-platelet interaction in vivo (the test is not very sensitive for HIT), but a positive result signals the need to avoid further use of heparin in that patient (the test is fairly specific for HIT if run with proper controls). Depending on the last time we obtained a positive result, we may or may not have a positive control.

The test is based on methodology utilized 20 years ago to investigate adverse reactions of patients receiving heparin, but is best performed with a release assay (see below) instead of aggregation studies (see next). Our current method employs a very high (1000 U/mL), high (100 U/mL), low (10 U/mL), and zero dose of heparin in normal donor and patient plasma added to normal. The normal plasma should not produce aggregation of normal platelets at the high or low dose of heparin. If the patient has developed an antibody directed by heparin against normal platelets, then the low dose of heparin in the patient plasma should produce an aggregation wave after 5 - 10 minutes incubation in the PRP. The high dose of heparin should be non-responding due to saturating the antibody in suspension and preventing cross-linking. The zero dose of heparin in the patient's plasma may produce aggregation if a significant amount of heparin is in the blood at the time of sample collection (hence the reason for ensuring that the patient has been off of heparin for at least 24 hours before the specimen is collected). If all three doses of heparin in the patient's plasma produce aggregation, then a heparin-independent effect may be the cause of thrombocytopenia in the patient (such as with ITP). A negative result at all heparin doses does not completely exclude a heparin-induced antibody to platelets; a more sensitive test such as the lumiaggregometry assay described below would detect the release reaction, rather than aggregation, to rule out false negatives more effectively (see Sheridan et al., Blood 67:27-30, 1986).

The gold standard of platelet release assays has traditionally been the C-serotonin release assay in which the test platelets are preloaded with radioactive serotonin before challenge with the patient's plasma containing heparin. This assay is 99% sensitive and specific for HIT. Needless to say, this method is impractical in most laboratories, including the one at PCMH, due to the use of radioactive material.

The next best platelet release assay, which does not entail the use of radioisotopes is the ATP release luminescence assay. The method employs the firefly enzyme luciferase which uses ATP in its rate-limiting step to phosphorylate firefly luciferin. The phosphorylated luciferin is luminescent and can be detected using a lumiaggregometer (reference C). Thus, ATP secretion (via luminescence) and turbidity of the sample (via spectrophotometry) are simulataneously measured. The sensitivity of this assay is higher than that of the conventional turbidometric assay, but testing of the lumiaggregometer in our laboratory is still underway as of the writing of this manual. The other advantage of this test is that it is not affected by other conditions which might increase the turbidity of the solution (i.e. lipemia) (reference G). Refer to the coag-specialist at the PCMH bench for the latest procedural data.

 

NORMAL RANGES

The reporting of results from coagulation tests generally does not include an error of the estimate or a confidence interval. In the case of screening tests, there is not even a scaling factor to apply to interpretation of data. Thus, each coagulation laboratory must exert a considerable effort to put each patient's value in some sort of context that will better alert the physician to a result indicating potential or existing problems. This is the purpose for establishing ranges of values from 20 - 30 normal donors to be used as a reference for interpretation of tests performed on patient samples. The normal range is usually reported as the population arithmetic mean value ± 2 standard deviations. By statistical definitions, this range includes about 95% of the values likely to be encountered in the normal population. However, it is also true to state that the range may include values that would be abnormal in a few patients that usually have low clotting times, and may exclude values from a few donors who would be considered normal in the geriatric or infant populations. Therefore, the limits of the normal range should not be taken as absolute discriminators. Typical normal ranges established at PCMH for screening tests, and factor normal ranges taken from literature supplied by reagent vendors, are given below. The current normal ranges can be found in the Coagulation Laboratory Procedure Manual.

 

 

 

 

 

 

 

TEST

LOW RANGE LIMIT

HIGH RANGE LIMIT

PT

11.0 seconds

12.6 seconds

aPTT

26 seconds

40 seconds

TCT

13.1 seconds

20.1 seconds

Bleeding Time

2.5 minutes

9.5 minutes

Factor Assays (II-XII)

50% of normal plasma

150% of normal plasma

AT III Activity

84% of normal plasma

123% of normal plasma

AT III Antigen

80% of normal plasma

120% of normal plasma

Protein C Antigen

60% of normal plasma

130% of normal plasma

Fibrinogen

186 mg/dL

386 mg/dL

Test results markedly different from the normal range may constitute a "panic value" which requires immediate verification and then rapid communication of the finding to the attending physician or charge nurse. Panic value thresholds are set arbitrarily to include only the most extraordinary results; this is not intended to preclude rapid communication of test results not meeting panic value criteria but still of critical importance in a particular patient. The pathologist will need to advise the Coagulation Lab in advance of the need for drawing attention to results under these circumstances.

The established panic values at PCMH are:

PT

Greater than 30 seconds

aPTT

Greater than 100 seconds

Bleeding Time

Greater than 20 minutes

Fibrinogen

Less than 100 mg/dL

 

 

 

 

 

SPECIAL CONSIDERATIONS

Scheduling of Tests:

Screening tests and fibrinogen assays are performed on all three shifts at PCMH. If trained personnel are available on second and third shift at PCMH, factor VIII or IX assays can be performed (not all Hematology lab technologists are trained to do Factor assays). All other special testing, including nonemergency Factor assays, platelet aggregation panels, inhibitor studies, and antigen determinations is performed only on the first shift and must be scheduled in advance with the shift supervisor. Assays run on the Dupont ACA (FDP, Heparin levels, AT III activity) are available on all shifts in the Chemistry Lab Section.

 

 

Turnaround Times:

Many of the communications between the Coagulation Lab Section and ordering physicians have to do with demands for timely release of test results and data. Average turnaround time for "STAT" screening tests on the automated analyzer or fibrometer is less than 30 minutes from the time the sample was received at the front desk. However, during the hours of 5 - 8 AM every day, presurgical and prerounds screening requests hit a peak that can cause delays of up to an hour or more. In addition, approximately half of the screening tests ordered during the day are labeled "STAT", which further slows down processing of regular orders.

Factor assays on the MLA-1600 require analysis of standards and controls as well as patient dilutions, so the typical turnaround time is 2 hours from receipt of sample. F.VIII:vWF Antigen determinations take 2 - 3 days before final analysis. Platelet aggregation panels take 2 - 3 hours to perform and require a pathologist's review before release of results. A Bethesda Inhibitor Titer similarly requires 3 - 4 hours to perform, and a pathologist must review the results to write an interpretation. Results on send-out tests can often take over a week to come back.

The pathologist may have to spend a considerable amount of time defending the lab from unreasonable demands of turnaround time in the face of personnel shortages, equipment failure and malfunction, or other intractable delays. The Lab personnel need this defense since they have enough to do already to produce the data and quality control. The pathologist or resident should perform the communicator role if possible to buffer the Lab from unreasonable requests and constant inquiries for test results which are frequently already reported in the SUNQUEST lab computer system, or awaiting review and interpretation, or are in process.

 

 

Tests Requiring Special Sampling:

The PT, aPTT, Factor Assays, TCT, and related clotting assays can be performed on citrated plasma from blood collected into Vacutainer blue top tubes. Assays of Factor XI, XII, or the contact activation system screening tests require avoidance of glass surfaces (even the siliconized glass walls of a Vacutainer tube), so blood should be drawn into plastic syringes containing 3.8% trisodium citrate. Platelet aggregation samples also require drawing of blood into plastic syringes with a special anticoagulant (Ware's) instead of Vacutainers. It is advisable, but not required to use plastic syringes in collecting blood for the vWF assay. Qualitative or quantitative FDP assays are performed on defibrinated serum and must be collected in special Vacutainer tubes marked "FDP assay" (D-dimer tests are done on standard bluetop citrate tubes). Whenever possible, the patient requiring special coagulation testing should be brought to the Lab phlebotomy facility for blood drawing by the Coagulation Lab Staff. The Bleeding Time Test can be performed at the bedside; the ACT is no longer performed at bedside by lab personnel.

 

Names and Numbers:

  • The Pathologist-in-charge of Coagulation is Gregory Gagnon, M.D., phone 816-5016, beeper 757-7686
  • The Scientific Director is Arthur P. Bode, Ph.D., phone 816-5021, beeper 757-5802.
  • The clinical consultants at PCMH are:
    • Charles L. Knupp, M.D., Dept. of Medicine, phone 816-2560, beeper 757-5572.
    • Darla Liles, M.D., Dept. of Medicine, phone 816-3326.
  • Other faculty available for consultation include:
    • Ruth Ann Henriksen, Ph.D., Dept. of Medicine, phone 816-3155.

In times past there have been regularly-scheduled staff or residents conferences for review of clinical coagulation issues or special cases at PCMH; check with the chief resident in pathology to find out if any such conferences are offered during your lab rotation.

 

 

Recommendations for Hematology residents handling requests for special coagulation tests

General Recommendations

  1. Always get the patient's name, medical record #, unit/floor, and the beeper number of the attending or resident ordering the test.
  2. ALWAYS check the patient's laboratory data yourself on Sunquest to determine if the laboratory history matches the test ordered.
  3. If the patient's laboratory history does not match the test ordered, then call the clinician in order to get the clinical indications for the test (approaching it with the clinician as just being curious is usually a pretty nonconfrontational way to go).
  4. Important clinical history to obtain from the clinician includes the following:
    1. Laboratory data done elsewhere (especially if the test is ordered from the clinics)
    2. Family history of coagulopathy
    3. Current anticoagulant therapy
    4. Current bleeding or thromboses
    5. Other pertinent diseases in the patient such as hemoglobinopathies, splenomegaly, hepatomegaly, etc.
  5. Based on the patient's clinical and laboratory data, approve the test or suggest alternative testing which would better determine the patient's underlying coagulation disorder.
  6. Pearls (please feel free to add to these by sending Dr. Bode e-mail (bodea@mail.ecu.edu) for the next edition of the coag manual):
    1. Lupus anticoagulant tests are NOT a routine part of the workup for lupus.
    2. Transfusion of platelets in heparin-induced thrombocytopenia is contraindicated (some clinical residents are not aware of this fact).
    3. Results of mixing studies for prolonged PT or aPTT should be obtained before testing for inhibitors and/or factor deficiencies in the absence of active bleeding and a positive family history.
    4. IF YOU DON'T KNOW THE ANSWER TO THE QUESTION, ASK YOUR ATTENDING BEFORE GIVING OUT INCORRECT INFORMATION!!!!!(that's a diamond, not a pearl) A.P. Bode

 

 

Lupus anticoagulants

Typically, the patient presents with a prolonged aPTT or PT. The next step in the workup should be a PT or aPTT mixing study to rule out the effects of a possible factor deficiency. A Thrombin Clotting Time (TCT) may also be performed to rule out any lingering effects of heparin (A). The four criteria for the diagnosis of a lupus anticoagulant are:

  1. Prolonged phospholipid-dependent assay (i.e. PT or aPTT);
  2. Evidence of an inhibitor by PT or aPTT mixing studies;
  3. Evidence of the dependence of the inhibitor on phospholipid;
  4. Lack of specific inhibition by any one coagulation factor.

Remember that some inhibitors/lupus anticoagulants are time-dependent. So, if the PT or aPTT of the mixing study corrects, the sample must also be run again after incubation for 30 minutes to 1 hour. Up to 30% of inhibitors which did not prolong the mixing study immediately after mixing will prolong it after 30 minutes to 1 hour.

A relatively recent update in screening for and confirming the presence of lupus anticoagulants has recommended the use of at least two separate assays. Studies based on one concentration of phospholipid (i.e. PT and aPTT) should be screening tests only. The confirmatory test (i.e. dRVVT) should be related in methodology to the screening test that was abnormal. Assays for antiphospholipid and anticardiolipin antibodies are not confirmatory tests. There is no evidence that a positive confirmatory study in the presence of a negative screening test indicates a functioning lupus anticoagulant (D).

 

 

 

 

 

Specific Tests

  • Activated clotting time (ACT): You may occasionally get an inappropriate order for an ACT from the floor or the unit. Normally this test is performed during vascular and cardiovascular surgery as well as during dialysis. If the test is ordered outside of this arena, check with the clinical staff covering the patient as to the reason for the test (some may just be trying to pull a central line from the patient). If clinically indicated, encourage the clinicians to order an aPTT or another appropriate test instead of an ACT (the results will be quicker if they order a stat if none of the available clinical personnel knows how to do the ACT at the bedside). If the clinician still wants an ACT, a laboratory technologist will have to take a Hemotech ACT instrument to the floor/unit to perform the test (only certain technologists can perform the test, so check first to see if one is available). More details are available in the Clinical Pathology Call Handbook.
  • Dilute Russell's viper venom test: The venom used in this test can also activate factor IX under the appropriate conditions. Therefore, it is important to remember that an abnormal dRVVT may result in a patient with a factor IX deficient sample B hemophilia B/Christmas disease (a patient who does not have a lupus anticoagulant). Depending on the patient's clinical history, a shortened dRVVT may make you suspect the presence of a heparin-induced thrombocytopenia.
  • Anticardiolipin antibodies: Testing for this antibody, while not as good a predictor of thrombotic complications as the lupus anticoagulant tests, is a better predictor of identifying pregnancies at risk for intrauterine fetal demise (A). Find out if the patient is an obstetrical patient before informing the physician about in-house lupus anticoagulant tests.
  • Antiphospholipid antibodies: These are thought to be a subset of anticardiolipin antibodies with lupus anticoagulant activity (C). The antibody is purported to bind phospholipid complex with apolipoprotein H (beta 2 glycoprotein I). Eighty percent of patients with a lupus anticoagulant will have an antiphospholipid or anticardiolipin antibodies, whereas only 10 to 50 percent of patients with an anticardiolipin antibody will have a lupus anticoagulant (A).
  • Heparin-induced platelet aggregation study: Always check with the clinical staff taking care of the patient if this is the patient's first exposure to heparin, and if so, how long the patient has been exposed to heparin. Typically, heparin-induced thrombocytopenia will not develop until at least 7 to 14 days after the first exposure to heparin (typical primary immune response). If the patient has already been exposed to heparin and has developed an antibody (which may not have produced a significant thrombocytopenia at that time), then the next exposure may produce a heparin-induced thrombocytopenia within 2 to 4 days. It is also important to ask about clinical evidence of thromboses in the patient as it increases the pathologist's suspicion in the presence of a negative test. You might also want to ask about the use of coumadin in the patient, as coumadin has been known to precipitate deep venous thromboses in the presence of heparin-induced thrombocytopenia (F).