Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 42. Laboratory evaluation of hemostasis

George A. Fritsma

OUTLINE

Hemostasis Specimen Collection

Patient Management During Hemostasis Specimen Collection

Hemostasis Specimen Collection Tubes

Hemostasis Specimen Collection Protocol

Specimen Collection Using Syringes and Winged-Needle Sets

Selection of Needles for Hemostasis Specimens

Specimen Collection from Vascular Access Devices

Specimen Collection Using Capillary Puncture

Anticoagulants Used for Hemostasis Specimens

Hemostasis Specimen Management

Hemostasis Specimen Storage Temperature

Hemostasis Specimen Storage Time

Preparation of Hemostasis Specimens for Assay

Platelet Function Tests

Bleeding Time Test for Platelet Function

Platelet Aggregometry and Lumiaggregometry

Testing for Heparin-Induced Thrombocytopenia

Quantitative Measurement of Platelet Markers

Immunoassay for the Anti Platelet Factor 4 (Heparin-Induced Thrombocytopenia) Antibody

Assays for Platelet Activation Markers

Clot-Based Plasma Procoagulant Screens

Prothrombin Time

Partial Thromboplastin Time

Partial Thromboplastin Time Mixing Studies

Thrombin Clotting Time

Reptilase Time

Russell Viper Venom

Coagulation Factor Assays

Fibrinogen Assay

Single-Factor Assays Using the Partial Thromboplastin Time Test

Bethesda Titer for Anti Factor VIII Inhibitor

Single-Factor Assays Using the Prothrombin Time Test

Factor XIII Assay

Tests of Fibrinolysis

Quantitative D-Dimer Immunoassay

Fibrin Degradation Product Immunoassay

Plasminogen Chromogenic Substrate Assay

Tissue Plasminogen Activator Assay

Plasminogen Activator Inhibitor-1 Assay

Global Coagulation Assays

Objectives

After completion of this chapter, the reader will be able to:

1. Properly collect and transport hemostasis blood specimens.

2. Reject hemostasis blood specimens due to clots, short draws, or hemolysis.

3. Prepare hemostasis blood specimens for analysis.

4. Describe the principles of platelet aggregometry.

5. Apply appropriate platelet function tests in a variety of conditions and interpret their results.

6. Diagnose von Willebrand disease and monitor its treatment.

7. Analyze plasma markers of platelet activation platelet factor 4 and β-thromboglobulin.

8. Describe the principle of, appropriately select, and correctly interpret the results of clot-based coagulation screening tests, including activated clotting time, prothrombin time, partial thromboplastin time, and the thrombin clotting time.

9. Interpret clot-based screening test results collectively to reach presumptive diagnoses, and then recommend and perform confirmatory tests.

10. Perform partial thromboplastin time mixing studies to detect factor deficiencies, lupus anticoagulants, and specific factor inhibitors.

11. Describe the principle of, appropriately select, and correctly interpret coagulation factor assays.

12. Describe the principle of and correctly interpret Bethesda titers for coagulation factor inhibitors.

13. Describe the principle of, appropriately select, and correctly interpret tests of fibrinolysis, including assays for D-dimer, plasminogen, plasminogen activators, and plasminogen activator inhibitors.

14. Interpret global coagulation assay tracings: Thromboelastograph and ROTEM.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 54-year-old woman experienced a pulmonary embolism on September 26 and began oral anticoagulant therapy. Monthly PT values were collected to monitor therapy. From October through January, her INR was stable at 2.4, but on February 1, her INR was 1.3. The reduced INR was reported to her physician.

On questioning, the patient reported that there had been no change in her warfarin (Coumadin) dosage or in her diet. She recalled, however, that the phlebotomist had used a tube with a red-and-black stopper. She had thought this to be out of the ordinary and had remarked about it to the phlebotomist, who made no response. The medical laboratory practitioner who had performed the PT assay reexamined the blood specimen and saw that it was in a blue-topped tube.

1. What did the phlebotomist do?

2. What was the consequence of this action?

3. What else could cause an unexpectedly short PT?

Hemostasis specimen collection

Most hemostasis laboratory procedures are performed on venous whole blood collected by venipuncture and mixed 9:1 with a 3.2% solution of sodium citrate anticoagulant. The specimen is maintained as well-mixed whole blood for platelet function testing or centrifuged to provide platelet-poor plasma (PPP) for other procedures. Phlebotomists, patient care technicians, nurses, medical laboratory practitioners, and other health personnel who collect blood specimens must adhere closely to published protocols for specimen collection and management. The nursing or laboratory supervisor is responsible for the current validity of specimen collection and handling protocols and ensures that personnel employ approved techniques.1

Patient management during hemostasis specimen collection

Patients need not fast, but they should avoid vigorous activities and should rest quietly for 30 minutes prior to collection for hemostasis testing. Little additional preparation is necessary; however, there are numerous drugs that affect the outcomes of coagulation tests. For example, aspirin suppresses most platelet function, and Coumadin (warfarin) reduces the activities of factor II (prothrombin), factor VII, factor IX, factor X, protein C, protein S, and protein Z and prolongs the prothrombin time (PT) test. The phlebotomist should attempt to record all drugs the patient is currently taking, and patients should be instructed by their physicians to discontinue drugs that may interfere with coagulation test results before testing.

Phlebotomists may manage patients using standard protocols for identification, cleansing, tourniquet use, and venipuncture (Chapter 3). If there is a reason to anticipate excessive bleeding—for instance, if the patient has multiple bruises or mentions a tendency to bleed—the phlebotomist should extend the time for observing the venipuncture site from 1 to 5 minutes and should apply a pressure bandage before dismissing the patient.

Hemostasis specimen collection tubes

Most hemostasis specimens are collected in plastic blue-stopper (blue-top, blue-closure) sterile evacuated blood collection tubes containing a measured volume of 0.105 to 0.109 M (3.2%) buffered sodium citrate anticoagulant.2 Tubes of uncoated soda-lime glass are unsuitable because their negative surface charge activates platelets and plasma procoagulants. Siliconized (plastic-coated) glass tubes are available, but their use is waning because of concern for potential breakage, with consequent risk of exposure to bloodborne pathogens.3

Hemostasis specimen collection protocol

Laboratory directors typically prefer evacuated blood collection tube systems for hemostasis blood collections; however, many directors may require syringe collection and initial “discard tubes” in special circumstances. provides a list of collection errors. Table 42-1

• If the hemostasis specimen is part of a series of tubes to be filled from a single venipuncture site, it must be collected first or immediately after a nonadditive tube. The hemostasis tube may not immediately follow a tube that contains heparin (green stopper), ethylenediamine tetraacetic acid (EDTA, lavender stopper), sodium fluoride (gray stopper), or clot-promoting silica particles as contained in plastic red-topped or serum separator (gel) tubes. These additives may become transferred to the hemostasis specimen on the stopper needle and invalidate all hemostasis test results. Nonadditive tubes include red-topped glass tubes and clear-topped or red-and-gray marble-topped tubes. If nonadditive tubes are unavailable, the phlebotomist may use and discard a preliminary blue-topped tube.4 Some hemostasis laboratory directors specify that a nonadditive tube be collected and discarded prior to the hemostasis specimen when the specimen is intended for platelet function studies or specialized coagulation assays. Their purpose is to ensure the absence of tissue contaminants in the specimen.

• The ratio of whole blood to anticoagulant must be 9 parts blood to 1 part anticoagulant. Evacuated tubes are designed so that the negative internal pressure draws the correct volume of blood from the vein. Collection tube manufacturers indicate the allowable range of collection volume error in package inserts and provide a minimum volume line on each tube. In most cases, the volume of blood collected must be within 90% of the calibrated volume. A short draw—that is, a specimen with a smaller volume than the minimum specified by the manufacturer—generates erroneously prolonged clot-based coagulation test results because the excess anticoagulant relative to blood volume neutralizes test reagent calcium.5 Short-draw specimens are consistently discarded, and a fresh specimen is collected from the patient. Most plastic blue-topped tubes collect 2.7 mL of whole blood; the smaller the collection tube, the narrower the tolerance for short draws.

• When specimens are collected using winged-needle butterfly sets, the phlebotomist must compensate for the internal volume of the tubing, which is usually 12 inches long and contains approximately 0.5 mL of air. The phlebotomist must first collect and discard a nonadditive tube or an identical blue-topped tube. This step ensures that the needle set tubing is filled with fresh patient blood before the hemostasis specimen is collected.6

• Clotted specimens are useless for hemostasis testing, even if the clot is small. A few seconds after collection, the phlebotomist must gently invert the specimen at least five times to mix the blood with the anticoagulant and prevent clot formation. If possible, the medical laboratory practitioner must visually examine for clots just before centrifugation and testing. Many coagulometers are equipped to detect the presence of clots. Clotted specimens are discarded, and a new specimen is collected from the patient.

• Excessive specimen agitation causes hemolysis (RBC rupture), procoagulant activation, and platelet activation. The phlebotomist must never shake the tube. The test results from visibly hemolyzed specimens are unreliable, and the specimen must be recollected.7

• Excess needle manipulation may promote the release of procoagulant substances from the skin and connective tissue, which contaminate the specimen and cause clotting factor activation. Consequently, test results from specimens collected during a traumatic venipuncture may be falsely shortened and unreliable.8

• During blood collection, the phlebotomist must remove the tourniquet within 1 minute of its application to avoid blood stasis.9 Stasis is a condition in which venous flow is slowed. Stasis results in the local accumulation of coagulation factor VIII and von Willebrand factor (VWF), which may result in false shortening of clot-based coagulation test results.

TABLE 42-1

Hemostasis Specimen Collection Errors That Require Collection of a New Specimen

Error

Comments

Short draw

Whole-blood volume less than 90% of required volume or less than manufacturer specified minimum.

Clot in specimen

Each specimen must be visually inspected prior to centrifugation; the presence of even a small clot requires that the specimen be recollected.

Visible hemolysis

Hemolysis, pink or red plasma indicates in vitro activation of platelets and coagulation. Results are unreliable.

Lipemia or icterus

Optical instruments may not measure clots in cloudy or highly colored specimens, especially chromogenic substrate methods. The practitioner must employ a mechanical instrument.

Prolonged tourniquet application

Stasis elevates the concentration of von Willebrand factor and factor VIII; falsely decreases fibrinolytic parameters; and falsely shortens clot-based test results.

Specimen storage at 1° C to 6° C

Storage at refrigerator temperatures causes precipitation of large von Willebrand factor multimers, activation of coagulation factor VII, and destroys platelet integrity.

Specimen storage at more than 25° C

Storage at above standard room temperature causes coagulation factors V and VIII to deteriorate.

Specimen collection using syringes and winged-needle sets

Managers of many hemostasis specialty laboratories insist that specimens from patients with difficult venous access and patients whose veins are small, fragile, or scarred by repeated venipunctures be collected by syringe. Additionally, in an effort to reduce the activation of platelets and coagulation, specimens for specialized tests such as platelet aggregometry are collected by syringe. Many hemostasis laboratories employ medical laboratory practitioners and phlebotomists who are specially trained in specimen collection to ensure the integrity of the specimen. The use of syringes presents additional needle-stick risk to the phlebotomist, so careful training and handling are essential.10

The phlebotomist selects sterile syringes of 20 mL capacity or less with nonthreaded Luer-slip hubs. The phlebotomist assembles syringes, a winged needle set (), a tubing clamp, and standard venipuncture materials. The phlebotomist then uses the following protocol: Figure 42-1

1. Use standard patient identification and standard blood specimen management precautions (Chapter 3).

2. Most syringes are delivered with the plunger withdrawn about 1 mm from the end of the barrel. Move the plunger outward and inward within the barrel. Expel all air from the barrel and affix the needle set to the Luer-slip hub.

3. Cleanse the venipuncture site, affix the tourniquet, and insert the winged needle. Immobilize the needle set by loosely taping the tube to the arm about 2 inches from the needle.

4. Fill the syringe using a gentle, even pressure.

5. Place the syringe on a clean surface and clamp the tubing with a hemostat near the needle hub.

6. Remove the first syringe and discard to avoid tissue contamination of the hemostasis specimen. The phlebotomist may use this specimen for chemistry or other tests. Attach a second syringe for collection of the hemostasis specimen; release the clamp and fill the second syringe. Repeat if needed.

7. Replace the clamp, remove the needle set, and immediately activate the needle cover.

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FIGURE 42-1 A, Winged needle set and syringe for collecting special hemostasis specimens. The phlebotomist may use the option of drawing the desired volume of anticoagulant into the syringe prior to blood collection. B, Winged needle illustrating needle-covering safety interlock.

After seeing to the patient’s welfare, the phlebotomist cautiously transfers the blood specimen to sealed evacuated tubes by affixing a safety transfer device. The specimen is allowed to flow gently down the side of the tube. The specimen is not pushed forcibly into the tube, because agitation causes hemolysis and platelet activation. The phlebotomist must transfer the specimen within a few seconds of the time the syringe is filled, and the tube must be gently inverted at least five times. The specimen volume must be correct for the proper ratio of blood to sodium citrate.

Selection of needles for hemostasis specimens

Whether evacuated collection tubes or syringes are used, the bore of the needle should be sufficient to prevent hemolysis and activation of platelets and plasma procoagulants. If the overall specimen is 25 mL or less, a 20- or 21-gauge thin-walled needle is used (). For a larger specimen, a 19-gauge needle is required. A 23-gauge needle is acceptable for pediatric patients or patients whose veins are small, but the negative collection pressure must be reduced. All needles provide safety closures that either cover or blunt the needle immediately after completion of the venipuncture.Table 42-2

TABLE 42-2

Selection of Needles for Hemostasis Specimens

Application

Preferred Needle Gauge and Length

Adult with good veins, specimen ≤25 mL

20 or 21 gauge, thin-walled, 1.0 or 1.25 inches long

Adult with good veins, specimen ≥25 mL

19 gauge, 1.0 or 1.25 inches long

Child or adult with small, friable, or hardened veins

23 gauge, winged-needle set; apply minimal negative pressure

Transfer of blood from syringe to tube

19 gauge, slowly inject through tube closure

Syringe with winged-needle set

20, 21, or 23 gauge, thin-walled; use only for small, friable, or hardened veins or specialized coagulation testing

Specimen collection from vascular access devices

Blood specimens may be drawn from heparin or saline locks, ports in intravenous lines, peripherally inserted central catheters (PICC tubes), central venous catheters, or dialysis catheters. Vascular access device management requires strict adherence to protocol to ensure sterility, prevent emboli, and prevent damage to the device. Personnel must be trained and must recognize the signs of complications and take appropriate action. Institutional protocol may limit vascular access device blood collection to physicians and nurses. Before blood is collected for hemostasis testing, the line must be flushed with 5 mL of saline, and the first 5 mL of blood, or six times the volume of the tube, must be collected and discarded. The phlebotomist must not flush with heparin. Blood is collected into a syringe and transferred to an evacuated tube as described in the prior section on hemostasis specimen collection with syringes and winged needle sets.11

Specimen collection using capillary puncture

Several near-patient testing (point-of-care) coagulometers (Chapter 44) generate PT results from a specimen consisting of 10 to 50 μL of whole blood. These instruments are designed to test either anticoagulated venous whole blood or capillary (finger-stick) blood and represent a significant convenience to patients and to anticoagulation clinics.12 Many are designed for patient self-testing and pediatric or neonatal testing, and laboratory practitioners are often charged with training patients in proper capillary puncture technique.13

Capillary specimen punctures are made using sterile spring-loaded lancets designed to make a cut of standard depth and width, while avoiding injury (Chapter 3). The phlebotomist or patient selects and cleanses the middle or fourth (ring) finger and activates the device so that it produces a puncture that is just off-center of the fingertip and perpendicular to the fingerprint lines. After wiping away the first drop of blood, which is likely to be contaminated by tissue fluid, the phlebotomist places the collection device directly adjacent to the free-flowing blood and allows the device to fill. The phlebotomist wipes excess blood from the outside of the device and introduces it to the coagulometer to complete the assay. The phlebotomist then presses a gauze pad to the wound and instructs the patient to maintain pressure until bleeding ceases. The phlebotomist then provides a spot bandage to cover the wound.

The key to accurate PT measurement is a free-flowing puncture. Often it is necessary for the phlebotomist to warm the patient’s hand to increase blood flow to the fingertips. Blood collection device distributors provide dry, disposable warming devices for this purpose. The phlebotomist avoids squeezing (“milking”) the finger, because this renders the blood specimen inaccurate by raising the concentration of tissue fluid relative to blood cells.14

Anticoagulants used for hemostasis specimens

Sodium citrate (primary hemostasis anticoagulant)

The anticoagulant used for hemostasis testing is buffered 3.2% (0.105 to 0.109 M) sodium citrate, Na3C6H5O7 ⋅ 2H2O, molecular weight 294.1 Daltons. Sodium citrate binds calcium ions to prevent coagulation, and the buffer stabilizes specimen pH as long as the tube stopper remains in place.15

The anticoagulant solution is mixed with blood to produce a 9:1 ratio: 9 parts whole blood to 1 part anticoagulant. In most cases, 0.3 mL of anticoagulant is mixed with 2.7 mL of whole blood, which are the volumes in the most commonly used evacuated plastic collection tubes, but any volumes are valid, provided that the 9:1 ratio is maintained. The ratio yields a final citrate concentration of 10.5 to 10.9 mM of anticoagulant in whole blood.16 Some laboratory practitioners prepare specimen tubes locally for special hemostasis testing.

Adjustment of sodium citrate volume for elevated hematocrits

The 9:1 blood-to-anticoagulant ratio is effective, provided the patient’s hematocrit is 55% or less. In polycythemia, the decrease in plasma volume relative to whole blood unacceptably raises the anticoagulant-to-plasma ratio, which causes falsely prolonged results for clot-based coagulation tests. The phlebotomist must provide tubes with relatively reduced anticoagulant volumes for collection of blood from a patient whose hematocrit is known to be 55% or higher. The amount of anticoagulant needed may be computed for a 5 mL total specimen volume by using the graph in or the following formula, which is valid for any total volume: Figure 42-2

Image

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FIGURE 42-2 Graph for computing the volume of anticoagulant in a 5.0 mL specimen when the patient’s hematocrit is 55% or greater. Source: (From Ingram GIC, Brozovic M, Slater NGP: Bleeding disorders, investigations, and management, ed 2, Oxford, 1982, Blackwell, pp. 244-245.)

where C is the volume of sodium citrate in milliliters, V is volume of whole blood-sodium citrate solution in milliliters, and H is the hematocrit in percent.

For example, to collect 3 mL of blood and anticoagulant mixture from a patient who has a hematocrit of 65%, calculate the volume of sodium citrate as follows:

Image

Image

Image

Remove the stopper from the blue closure collection tube, pipette and discard 0.11 mL from the 0.3 mL of anticoagulant, leaving 0.19 mL. Collect blood in a syringe and transfer 2.81 (2.8) mL of blood to the tube, replace the stopper, and immediately mix by gently inverting four times. Alternatively, the laboratory practitioner can prepare for collection of 10 mL of blood and anticoagulant solution in a 12 mL centrifuge tube as follows:

Image

Image

In this instance, 0.64 mL of sodium citrate is pipetted into the tube, and 9.36 (9.4) mL of whole blood is transferred from the collection syringe. There is no evidence suggesting a need for increasing the volume of anticoagulant for specimens from patients with anemia, even when the hematocrit is less than 20%.

Other anticoagulants used for hemostasis specimens

EDTA-anticoagulated specimens are not used for coagulation testing because calcium ion chelation by EDTA is irreversible, interfering with coagulation assays.17 Calcium ion chelation with citrate, on the other hand, is reversed with the addition of calcium. EDTA is the anticoagulant used in collecting specimens for complete blood counts, including platelet counts. EDTA may be required for specimens used formoleculardiagnostic testing, such as testing for factor V Leiden mutation or the prothrombin G20210A mutation. Likewise, acid citrate dextrose (ACD, yellow stopper) and dipotassium EDTA (K2EDTA) with gel (white stopper) tubes may be used for molecular diagnosis, as specified by institutional protocol. Heparinized specimens have never been validated for use in plasma coagulation testing but may be necessary in cases of platelet satellitosis (satellitism) as a substitute for specimens collected in EDTA or sodium citrate. Citrate theophylline adenosine dipyridamole (CTAD, blue stopper) tubes are used to halt in vitro platelet or coagulation activation for specialty assays such as those for the platelet activation markers platelet factor 4 (PF4) and platelet surface membrane P-selectin (measured by flow cytometry) or the coagulation activation markers prothrombin fragment 1+ 2 and thrombin-antithrombin complex.

Hemostasis specimen management

Hemostasis specimen storage temperature

Sodium citrate-anticoagulated whole blood specimens are placed in a rack and allowed to stand in a vertical position with the stopper intact and uppermost. The pH remains constant as long as the specimen is sealed. Specimens are maintained at 18° C to 24° C (ambient temperature), never at refrigerator temperatures (). Storage at 1° C to 6° C activates factor VII, destroys platelet activity through uncontrolled activation, and causes the cryoprecipitation of large VWF multimers.Table 42-31819 Also, specimens should never be stored at temperatures greater than 24° C because heat causes deterioration of coagulation factors V and VIII.

TABLE 42-3

Hemostasis Specimen Storage Times and Temperatures

Application

Temperature

Time

PT with no unfractionated heparin present in specimen

18–24° C

24 hours

PTT with no unfractionated heparin present in specimen

18–24° C

4 hours

PTT for monitoring unfractionated heparin therapy

18–24° C

Separate within 1 hour, test within 4 hours

PT when unfractionated heparin is present in specimen

18–24° C

Separate within 1 hour, test within 4 hours

Factor assays

18–24° C

4 hours

Optical platelet aggregometry using platelet-rich plasma

18–24° C

Wait 30 min after centrifugation, test within 4 hours of collection

Whole-blood aggregometry

18–24° C

Test within 3 hours of collection

Storage in household freezer

−20° C

2 weeks

Storage for 6 months

−70° C

6 months

PT, Prothrombin time; PTT, partial thromboplastin time.

Hemostasis specimen storage time

Specimens collected for PT testing may be held at 18° C to 24° C and tested within 24 hours of the time of collection. Specimens collected for partial thromboplastin time (PTT) testing also may be held at 18° C to 24° C, but they must be tested within 4 hours of the time of collection, provided that the specimen does not contain unfractionated heparin anticoagulant. If a patient is getting unfractionated heparin therapy, specimens for PTT testing must be centrifuged within 1 hour of the time of collection, and the plasma, which should be PPP, must be tested within 4 hours of the time of collection.20

Preparation of hemostasis specimens for assay

Whole-blood specimens used for platelet aggregometry

Blood for whole-blood platelet aggregometry or lumiaggregometry must be collected with 3.2% sodium citrate and held at 18° C to 24° C until testing. Chilling destroys platelet activity. Aggregometry should be started immediately and must be completed within 4 hours of specimen collection. The practitioner mixes the specimen by gentle inversion, checks for clots just before testing, and rejects specimens with clots. Most specimens for whole-blood aggregometry are mixed 1:1 with normal saline before testing, although if the platelet count is less than 100,000/μL the specimen is tested undiluted.21

Platelet-rich plasma specimens used for platelet aggregometry

Light-transmittance (optical) platelet aggregometers are designed to test platelet-rich plasma (PRP), plasma with a platelet count of 200,000 to 300,000/μL. Sodium citrate–anticoagulated blood is first checked visually for clots and then centrifuged at 50 xg for 30 minutes with the stopper in place to maintain the pH. The supernatant PRP is transferred by a plastic pipette to a clean plastic tube, and the tube is sealed and stored at 18° C to 24° C (ambient temperature) until the test is begun. PRP-based light-transmittance aggregometry is initiated no less than 30 minutes after the specimen is centrifuged and completed within 4 hours of the time of collection. To produce sufficient PRP, the original specimen must measure 9 to 12 mL of whole blood. Light-transmittance aggregometry is unreliable when the patient’s whole-blood platelet count is less than 100,000/μL.

Platelet-poor plasma required for clot-based testing

Clot-based plasma coagulation tests require PPP-plasma with a platelet count of less than 10,000/μL.22 Sodium citrate-anticoagulated whole blood is centrifuged at 1500 xg for 15 minutes in a swinging bucketcentrifuge to produce supernatant PPP. Alternatively, the angle-head StatSpin Express 2 (Iris Sample Processing, Inc., Westwood, MA) generates 4400 xg and can produce PPP within 3 minutes. Both make it possible for automated coagulometers to sample from the supernatant plasma of the primary blood collection tube. The advantage of the slower swinging bucket centrifuge head is that it produces a straight, level plasma-blood cell interface, whereas angle-head centrifuge heads cause platelets to adhere to the side of the tube. If the “angle-spun” tube is allowed to stand, the adherent platelets drift back into the plasma and release granule contents. Each hemostasis laboratory manager establishes the correct centrifugation speed and times for the local laboratory. Centrifugation must yield PPP from specimens with high initial platelet counts.

In the special hemostasis laboratory the manager may choose a double-spin approach. The primary tube is centrifuged using a swinging bucket centrifuge, and the plasma is transferred to a secondary plastic tube, which is labeled and centrifuged again. The double-spin approach may be used to produce PPP with a plasma platelet count of less than 5000/μL, which some laboratory directors prefer for lupus anticoagulant (LA) testing and for preparation of frozen plasma.

The presence of greater than 10,000 platelets/μL in plasma affects clot-based test results. Platelets are likely to become activated in vitro and release the membrane phospholipid phosphatidylserine, which triggers plasma coagulation and neutralizes LA if present, interfering with LA testing. Platelets also secrete fibrinogen, factors V and VIII, and VWF (Chapter 13). These may desensitize PT and PTT assays and interfere with clot-based coagulation assays. In addition, platelets release platelet factor 4 (PF4), a protein that binds and neutralizes therapeutic heparin in vitro, falsely shortening the PTT and interfering with heparin management.

The hemostasis laboratory manager arranges to perform plasma platelet counts on coagulation plasmas at regular intervals to ensure that they are consistently platelet poor. Many managers select 10 to 12 specimens from each centrifuge every 6 months, perform plasma platelet counts, and document that their samples remain appropriately platelet poor, even if the initial platelet count is elevated.

Laboratory practitioners inspect hemostasis plasmas for hemolysis (red), lipemia (cloudy, milky), and icterus (golden yellow from bilirubin). Visible hemolysis implies platelet or coagulation pathway activation. Visibly hemolyzed specimens are rejected, and new specimens must be obtained. Lipemia and icterus may affect the end-point results of optical coagulation instruments. The hemostasis laboratory manager may choose to maintain a separate mechanical end-point coagulometer to substitute for the optical instrument if the specimen is too cloudy for optical determinations. Conversely, some optical instruments detect and compensate for lipemia and icterus via spectrophotometric analysis.23

Specimen storage

Specimens for PT assay only may be held uncentrifuged at 18° C to 24° C for up to 24 hours, provided the tubes remain closed. Likewise, specimens for PTT measurement may be held uncentrifuged for up to 4 hours. However, specimens from patients receiving unfractionated heparin collected for PTT heparin monitoring must be centrifuged, and the supernatant PPP must be sampled or transferred within 1 hour to avoid false shortening of the PTT as platelet granule PF4 neutralizes the heparin.

If the hemostasis test cannot be completed within the prescribed interval, the laboratory practitioner must immediately centrifuge the specimen. The supernatant PPP must be transferred by plastic pipette to a plastic freezer tube (non-siliconized glass materials are never used with plasma handling as it activates the coagulation cascade), sealed, and frozen and may be stored at –20° C for up to 2 weeks or at –70° C for up to 6 months. At the time the test is performed, the specimen must be thawed rapidly at 37° C, mixed well, and tested within 1 hour of the time it is removed from the freezer. If it cannot be tested immediately, the specimen may be stored at 1° C to 6° C for 2 hours after thawing. To avoid cryoprecipitation of VWF, specimens may not be frozen and thawed more than once.

Platelet function tests

Platelet function tests are designed to detect qualitative (functional) platelet abnormalities in patients who are experiencing the symptoms of mucocutaneous bleeding (Chapter 41). A platelet count is performed, and the blood film is reviewed before platelet function tests are begun, because thrombocytopenia is a common cause of hemorrhage (Chapter 40).24 Qualitative platelet abnormalities are suspected only when bleeding symptoms are present and the platelet count exceeds 50,000/μL.

Although hereditary platelet function disorders are rare, acquired defects are common.25 Acquired platelet defects are associated with liver disease, renal disease, myeloproliferative neoplasms, myelodysplastic syndromes, myeloma, uremia, autoimmune disorders, anemias, and drug therapy. Platelet morphology is often a clue; for instance, in Bernard-Soulier syndrome, the blood film reveals mild thrombocytopenia and large gray platelets (Figure 41-3). Similarly, the presence of large platelets on the blood film associated with elevated mean platelet volume often indicates rapid platelet turnover, such as what occurs in immune thrombocytopenic purpura or thrombotic thrombocytopenic purpura. Giant or dysplastic platelets are seen in myeloproliferative neoplasms, acute leukemia, and myelodysplastic syndromes.

Bleeding time test for platelet function

The bleeding time test was the original test of platelet function, although it is now largely replaced by near-patient analysis of platelet function using the PFA-100 (Siemens Healthcare Diagnostics, Inc., Deerfield, IL), the Multiplate (DiaPharma, West Chester, OH), or platelet aggregometry.26 To perform the test, the phlebotomist uses a lancet to make a small, controlled puncture wound and records the duration of bleeding, comparing the results with the universally accepted reference interval of 2 to 9 minutes. The bleeding time test was first described by Duke27 in 1912 and modified by Ivy28 in 1941. In 1978 some standardization was attempted. A blood pressure cuff was inflated to 40 mm Hg, a calibrated spring-loaded lancet (Surgicutt Bleeding Time Device; International Technidyne Corp., Edison, NJ) was triggered on the volar surface of the forearm a few inches distal to the antecubital crease, and the resulting wound was blotted every 30 seconds with filter paper until bleeding stopped.2930

A prolonged bleeding time could theoretically signal a functional platelet disorder such as von Willebrand disease (VWD) or a vascular disorder such as scurvy or vasculitis, and was a characteristic result of therapy with aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). Measurement of the bleeding time was often requested by surgeons at admission in an attempt to predict surgical bleeding, but a series of studies in the 1990s revealed that the test has inadequate predictive value. The bleeding time is affected by the nonplatelet variables of intracapillary pressure, skin thickness at the puncture site, and size and depth of the wound, all of which interfere with accurate interpretation of the test results. Owing to its poor predictive value for bleeding and its tendency to scar the forearm, use of the bleeding time assay has been discontinued at most institutions.

Platelet aggregometry and lumiaggregometry

Functional platelets adhere to subendothelial collagen, aggregate with one another, and secrete the contents of their α-granules and dense granules (Chapter 13). Normal adhesion requires intact platelet membranes and functional plasma VWF. Normal aggregation requires that platelet membranes and platelet activation pathways are intact, that the plasma fibrinogen concentration is normal, and that normal secretions are released from platelet granules. Platelet adhesion, aggregation, and secretion are assessed using in vitro platelet aggregometry.

An aggregometer is an instrument designed to measure platelet aggregation in a suspension of citrated whole blood or PRP. Specimens are collected and managed in compliance with standard laboratory protocol as described in the section entitled Preparation of Hemostasis Specimens for Assay, and maintained at ambient temperature (18° C to 24° C) until testing begins. Specimens for PRP-based light-transmittance aggregometry must stand undisturbed for 30 minutes after centrifugation while the platelets regain their responsiveness. Specimens for impedance whole blood aggregometry are diluted 1:1 with normal saline and tested immediately. Specimens must be tested within 4 hours of collection to avoid spontaneous in vitro platelet activation and loss of normal activity. Platelet aggregometry is a high-complexity laboratory test requiring a skilled, experienced operator.

Platelet aggregometry using platelet-rich plasma

PRP aggregometry is performed using a specialized photometer called a light-transmittance aggregometer (PAP-8E Platelet Aggregation Profiler; Bio/Data Corp., Horsham, PA).31 After calibrating the instrument in accordance with manufacturer instructions, the operator pipettes the PRP to instrument-compatible cuvettes, usually 500 μL; drops in one clean plasticized stir bar per sample; places the cuvettes in incubation wells; and allows the samples to warm to 37° C for 5 minutes. The operator then transfers the first cuvette, containing specimen and stir bar, to the instrument’s reaction well and starts the stirring device and the recording computer. The stirring device turns the stir bar at 800 to 1200 rpm, a gentle speed that keeps the platelets in suspension. The instrument directs focused light through the sample cuvette to a photodetector (Figure 42-3). As the PRP is stirred, the recorder tracing first stabilizes to generate a baseline, near 0% light transmittance. After a few seconds, the operator pipettes an agonist (aggregating agent) directly into the sample to trigger aggregation. In a normal specimen, after the agonist is added, the shape of the suspended platelets changes from discoid to spherical, and the intensity of light transmittance initially (and briefly) decreases, then increases in proportion to the degree of shape change. Percent light transmittance is monitored continuously and recorded (Figure 42-4). As platelet aggregates form, more light passes through the PRP, and the tracing begins to move toward 100% light transmittance. Platelet function deficiencies are reflected in diminished or absent aggregation; many laboratory directors choose 40% aggregation as the lower limit of normal.

Image 

FIGURE 42-3 Analysis of platelet-rich plasma in an optical aggregometer. Desired platelet count is approximately 200,000/μL. Platelets are maintained in suspension by a magnetic stir bar turning at 800 to 1200 rpm. Source: (Courtesy Kathy Jacobs, Chrono-log Corp., Havertown, PA.)

Image 

FIGURE 42-4 Optical aggregometry tracing showing five phases of platelet aggregation: baseline at 0% aggregation, shape change after the addition of the agonist, primary aggregation, release of adenosine diphosphate and adenosine triphosphate, and second-wave aggregation that forms large clumps. The % aggregation is measured by amount of light transmittance through the test sample. Source: (Courtesy Kathy Jacobs, Chrono-log Corp., Havertown, PA.)

Whole-blood platelet aggregometry

In whole-blood platelet aggregometry, platelet aggregation is measured by electrical impedance using a 1:1 saline–whole blood suspension (Model 700 Whole Blood/Optical Lumi-Aggregometer; Chrono-log Corp., Havertown, PA).32 The operator pipettes aliquots of properly mixed whole blood to cuvettes and adds equal volumes of physiologic saline. Suspension volume may be 300 to 500 μL. The operator drops in one stir bar per cuvette and places the cuvettes in 37° C incubation wells for 5 minutes. The operator transfers the first cuvette to a reaction well, pipettes an agonist directly into the specimen, and suspends a pair of low-voltage cartridge-mounted disposable direct current (DC) electrodes in the mixture. As aggregation occurs, platelets adhere to the electrodes and one another, impeding the DC current (Figure 42-5). The rise in impedance, which is directly proportional to platelet aggregation, is amplified and recorded by instrument circuitry. A whole-blood aggregometry tracing closely resembles a PRP-based light-transmittance aggregometry tracing, as shown in Figure 42-4.

Image 

FIGURE 42-5 In whole-blood platelet aggregometry, aggregating platelets form a layer on the electrodes and the platelet layer impedes current. Resistance (in ohms) is proportional to aggregation, and a tracing is provided that resembles the tracing obtained using optical aggregometry. Source: (Courtesy Kathy Jacobs, Chrono-log Corp., Havertown, PA.)

Platelet lumiaggregometry

The Chrono-log Whole Blood/Optical Lumi-Aggregometer may also be used for simultaneous measurement of platelet aggregation and the secretion of adenosine triphosphate (ATP) from activated platelet dense granules.33 The procedure for lumiaggregometry differs little from that for conventional aggregometry and simplifies the diagnosis of platelet dysfunction.34 As ATP is released, it oxidizes a firefly-derived luciferin-luciferase reagent (Chrono-lume; Chrono-log Corp.) to generate cold chemiluminescence proportional to the ATP concentration. A photodetector amplifies the luminescence, which is recorded as a second tracing on the aggregation report.35

Lumiaggregometry may be performed using whole blood or PRP.36 To perform lumiaggregometry, the operator adds an ATP standard to the first sample, then adds luciferin-luciferase and tests for full luminescence. The operator then adds luciferin-luciferase and an agonist to the second sample; the instrument monitors for aggregation and secretion simultaneously. Thrombin is typically the first agonist used because thrombin induces full secretion. The luminescence induced by thrombin is measured, recorded, and used for comparison with the luminescence produced by the additional agonists. Normal secretion induced by agonists other than thrombin produces luminescence at a level of about 50% of that resulting from thrombin (Table 42-4). Figure 42-6 depicts simultaneous aggregation and secretion responses to thrombin; Figure 42-7 is a scanning electron micrograph of resting and activated platelets.

Image 

FIGURE 42-6 Normal lumiaggregometry tracing illustrating monophasic aggregation curve with superimposed release (secretion) reaction curve. Aggregation is measured in ohms (Ω) using the left y-axis scale; release is measured in μM of adenosine triphosphate (ATP) based on luminescence using the right y-axis scale. Curve illustrates full aggregation and secretion response to 1 unit/mL of thrombin. Source: (Courtesy Margaret Fritsma, University of Alabama at Birmingham.)

Image 

FIGURE 42-7 Scanning electron micrograph of resting (A) and activated (B) platelets.

TABLE 42-4

Typical Normal Ranges in Platelet Lumiaggregometry

Agonist

Final Concentration

Aggregation Recorded as Impedance

ATP Secretion

Thrombin

1 unit/mL

Not recorded, as thrombin often causes clotting

1.0–2.0 nM

TRAP

10, 50, 100 μM

15–27 Ω

1.0–2.0 nM

Collagen

1 μg/mL

15–27 Ω

0.5–1.7 nM

5 μg/mL

15–31 Ω

0.9–1.7 nM

ADP

5 μM

1–17 Ω

0.0–0.7 nM

10 μM

6–24 Ω

0.4–1.7 nM

Arachidonic acid

500 μM

5–17 Ω

0.6–1.4 nM

Ristocetin

1 mg/mL

> 10 Ω

Not recorded

TRAP, Thrombin receptor-activating peptide; ATP, adenosine triphosphate; ADP, adenosine diphosphate.

Platelet agonists (activating agents) used in aggregometry

The optical PRP-based aggregation method is employed most frequently in clinical practice, and the agonists used are thrombin or synthetic thrombin receptor-activating peptide (TRAP), adenosine diphosphate (ADP), epinephrine, collagen, arachidonic acid, and ristocetin. Table 42-5 lists representative concentrations and platelet activation pathways tested by each agonist. Small volumes (2 to 5 μL) of concentrated agonist are used so that they have little dilutional effect in the reaction system.37

TABLE 42-5

Platelet Aggregometry Agonists, Reaction Concentrations, and Platelet Receptors

Agonist

Typical Final Concentration

Platelet Membrane Receptors

Thrombin

1 unit/mL

PAR1 and PAR4; GP Ibα and GP V

ADP

1–10 μM

P2Y1, P2Y12

Epinephrine

2–10 μg/mL

α2-adrenergic receptor

Collagen

5 μg/mL

GP Ia/IIa, GP VI

Arachidonic acid

500 μM

TPα, TPβ

Ristocetin

1 mg/mL

GP Ib/IX/V in association with von Willebrand factor

GP, Glycoprotein; PAR, protease-activatable receptor; P2Y, platelet membrane ADP-receptor; TP, thromboxane receptor.

Thrombin (or TRAP) cleaves two platelet membrane protease-activatable receptors (PARs), PAR-1 and PAR-2, both members of the seven-transmembrane repeat receptor family (Chapter 13). Thrombin or TRAP also cleaves glycoprotein (GP) 1bα and GP V. Internal platelet activation is effected by membrane-associated G proteins and both the eicosanoid and the diacylglycerol pathways. Thrombin-induced activation results in full secretion and aggregation. In lumiaggregometry, the operator ordinarily begins with 1 unit/mL of thrombin or TRAP (agonist concentrations are expressed as final reaction mixture concentrations) to induce the release of 1 to 2 nM of ATP, detected by the firefly luciferin-luciferase luminescence assay. Other agonists—for instance, 5 μg/mL of collagen—induce the release of at most 0.5 to 1.0 nM of ATP. Thrombin-induced secretion may be diminished to less than 1 nM in storage pool deficiencies (Chapter 41), but it is relatively unaffected by membrane disorders or pathway enzyme deficiencies.

Reagent thrombin is stored dry at –20° C and is reconstituted with physiologic saline immediately before use. Leftover reconstituted thrombin may be divided into aliquots, frozen, and thawed for later use. Thrombin has the disadvantage that it often triggers coagulation (fibrin formation) simultaneously with aggregation. The use of TRAP avoids this pitfall.

ADP binds platelet membrane receptors P2Y1 and P2Y12, also members of the seven-transmembrane repeat receptor family. ADP-induced platelet activation relies on the physiologic response of membrane-associated G protein and the eicosanoid synthesis pathway. The end product of eicosanoid synthesis, thromboxane A2, raises cytosolic free calcium, which mediates platelet activation and induces secretion of ADP stored in dense granules. The secreted ADP activates neighboring platelets.

ADP is the most commonly used agonist, particularly in aggregometry systems that measure only aggregation and not luminescence. The operator adjusts the ADP concentration to between 1 and 10 μM to induce “biphasic” aggregation (Figure 42-4). At ADP concentrations near 1 μM, platelets achieve only primary aggregation, followed by disaggregation. The graph line deflects from the baseline for 1 to 2 minutes and then returns to baseline. Primary aggregation involves shape change with formation of microaggregates, both reversible. Secondary aggregation is the formation of full platelet aggregates after release of platelet dense-granule ADP. At agonist ADP concentrations near 10 μM, there is simultaneous irreversible shape change, secretion, and formation of aggregates, resulting in a monophasic curve and full deflection of the tracing. ADP concentrations between 1 and 10 μM induce a biphasic curve: primary aggregation followed by a brief flattening of the curve called the lag phase and then secondary aggregation.

Operators expend considerable effort to discover the ADP concentration that generates a biphasic curve with a visible lag phase because the appropriate concentration varies among patients. This enables operators to use aggregometry alone to distinguish between membrane-associated platelet defects and storage pool or release defects.

Lumiaggregometry provides a clearer and more reproducible measure of platelet secretion, rendering the quest for the biphasic curve unnecessary. Secretion in response to ADP at 5 μM is diminished in platelet membrane disorders; eicosanoid synthesis pathway enzyme deficiencies; or aspirin, NSAID, or clopidogrel therapy. Secretion is absent in storage pool deficiency when thrombin or TRAP is used as the agonist.

Reagent ADP is stored at –20° C, reconstituted with physiologic saline, and used immediately after reconstitution. Leftover reconstituted ADP may be aliquotted and frozen for later use.

Epinephrine binds platelet α-adrenergic receptors, identical to muscle receptors, and activates the platelets through the same metabolic pathways as reagent ADP. The results of epinephrine-induced aggregation match those of ADP, except that epinephrine cannot induce aggregation in storage pool disorder or eicosanoid synthesis pathway defects no matter how high its concentration. Epinephrine does not work in whole-blood aggregometry.

Epinephrine is stored at 1° C to 6° C and reconstituted with distilled water immediately before it is used. Leftover reconstituted epinephrine may be aliquotted and frozen for later use.

Collagen binds GP Ia/IIa and GP VI, but it induces no primary aggregation. After a lag of 30 to 60 seconds, aggregation begins, and a monophasic curve develops. Aggregation induced by collagen at 5 μg/mL requires intact membrane receptors, functional membrane G proteins, and normal eicosanoid pathway function. Loss of collagen-induced aggregation may indicate a membrane abnormality, storage pool disorder, release defect, or the presence of aspirin.

Most laboratory managers purchase lyophilized fibrillar collagen preparations such as Chrono-Par Collagen (Chrono-log Corp.). Collagen is stored at 1° C to 6° C and used without further dilution. Collagen may not be frozen.

Arachidonic acid assesses the viability of the eicosanoid synthesis pathway. Free arachidonic acid agonist at 500 μM is added to induce a monophasic aggregometry curve with virtually no lag phase. Aggregation is independent of membrane integrity. Deficiencies in eicosanoid pathway enzymes, including deficient or aspirin-suppressed cyclooxygenase, result in reduced aggregation and secretion.

Arachidonic acid is readily oxidized and must be stored at –20° C in the dark. The operator dilutes arachidonic acid with a solution of bovine albumin for immediate use. Aliquots of bovine albumin–dissolved arachidonic acid may be frozen for later use.

Platelet aggregometry tests in von willebrand disease

Ristocetin-induced platelet aggregation. 

Although this test is usually called the ristocetin-induced platelet aggregation (RIPA) test, ristocetin actually induces an agglutination reaction that involves little platelet shape change and little secretion. A normal RIPA result may imply that normal concentrations of functional VWF are present and that the platelets possess a functional VWF receptor, GP Ib/IX/V (Chapter 13).38

Using light transmittance aggregometry, ristocetin at 1 mg/mL final concentration induces a monophasic aggregation tracing from a normal specimen. Specimens from patients with VWD, except for subtype 2B VWD, produce a reduced or absent reaction, although all other agonists generate normal tracings (Table 38-4). Exogenous VWF from normal plasma restores a normal RIPA reaction, confirming the diagnosis (Chapter 38). In patients with Bernard-Soulier syndrome, a congenital abnormality of the GP Ib or IX portion of the GP Ib/IX/V receptor results in a diminished RIPA reaction that is not corrected by the addition of VWF (Chapter 41).

In VWD subtype 2B, a VWF gain-of-function mutation, aggregation occurs even when reduced ristocetin concentrations (down to 0.1 mg/mL final concentration) are added. This response illustrates the increased affinity of large VWF multimers for platelet receptors. The low-dose or low-concentration RIPA, sometimes called the ristocetin response curve, is used to diagnose type 2B VWD.

The RIPA test is qualitative and is diagnostic in only about 70% of cases. Most laboratory managers have dropped RIPA from their test menus because of its poor predictive value. There is considerable variation in laboratory results from one patient to another in the same kindred and from time-to-time in a single patient. Consequently, the laboratory director must include the ristocetin cofactor test, the VWF antigen immunoassay, and the coagulation factor VIII activity assay in the VWD profile. Many laboratories also offer the VWF activity immunoassay and the VWF collagen-binding assay. Ultimate confirmation and characterization of VWD is based on gel immunoelectrophoresis to characterize VWF monomers (Chapter 38).39

Ristocetin cofactor assay for von willebrand factor activity. 

One essential refinement of ristocetin aggregometry is the substitution of formalin-fixed or lyophilized normal “reagent” platelets for the patient’s platelets.40 When reagent platelets are used, the test is called theristocetin cofactor or VWF activity assay. The medical laboratory practitioner prepares the patient’s PPP; mixes it with reagent platelets; adds ristocetin; and performs optical, not impedance, aggregometry. The ristocetin cofactor assay yields a proportional relationship between VWF activity and the aggregometry response of the reagent platelets. Comparison of the aggregation results for patients’ PPP with those for standard dilutions of normal “reagent” PPP permits a quantitative expression of the VWF activity level. The ristocetin cofactor test also is available as an automated assay on the BCT coagulometer and the BCS coagulometer (Siemens Healthcare Diagnostics, Deerfield, IL), which use latex particles in place of preserved platelets.

VWF activity immunoassay and vwf activity collagen binding assay. 

Although the ristocetin cofactor assay has been used for many years to measure VWF activity, it offers consistently poor precision, as illustrated by external quality assurance survey results.41 Two additional assays, the VWF Activity Immunoassay (for instance, the REAADS von Willebrand Factor Activity enzyme immunoassay, DiaPharma, West Chester, OH) and the VWF Collagen Binding Assay (Technozym VWF:CBA ELISA Collagen Type I, DiaPharma, West Chester, OH) are available. The former employs a monoclonal antibody specific for an active VWF epitope, and the latter mimics VWF’s in vivo collagen adhesion property. Both reflect VWF activity rather than concentration and offer improved precision when compared to the ristocetin cofactor assay.

Summary of lumiaggregometry agonist responses in various circumstances

Thrombin produces maximum ATP release through at least two membrane-binding sites. Laboratory practitioners use collagen, ADP, and epinephrine to test for abnormalities of their respective membrane binding sites and the eicosanoid synthesis pathway. Arachidonic acid is the agonist that practitioners use to check for eicosanoid synthesis deficiencies. Ristocetin is used to check for abnormalities of plasma VWF in VWD. The following conditions may be detected through platelet lumiaggregometry.

Therapy with aspirin, other nonsteroidal anti-inflammatory drugs, and clopidogrel. 

NSAIDs such as aspirin, ibuprofen, indomethacin, and sulfinpyrazone permanently inactivate or temporarily inhibit cyclooxygenase. The thienopyridine antiplatelet drugs clopidogrel and prasugrel irreversibly occupy the ADP receptor P2Y12 whereas the nucleoside ticagrelor is reversibly bound (Chapter 41). The NSAIDs limit or eliminate the aggregation and secretion responses to arachidonic acid and collagen. The P2Y12inhibitors suppress aggregation and secretion responses to ADP.42 Platelet aggregometry is employed to monitor response to these antiplatelet drugs (Chapter 41).43 The VerifyNow system (Accumetrics, San Diego, CA) with specific assays for monitoring the effect of aspirin, the P2Y12 inhibitors, and the GP IIb/IIIa inhibitor antiplatelet drugs is gaining favor in point-of-care settings (Chapter 44). The physician or medical laboratory practitioner must instruct the patient to discontinue all antiplatelet drugs at least 1 week before blood is collected for aggregometry unless aggregometry is ordered to monitor the effects of these drugs.

Platelet release (secretion) defects: Eicosanoid pathway enzyme deficiencies. 

Congenital or acquired deficiencies of cyclooxygenase, thromboxane synthase, protein kinase C, or any enzyme in the eicosanoid activation pathway limit or prevent secretion. Thrombin may induce normal responses, but secretion and aggregation are diminished in response to ADP, collagen, and arachidonic acid. Because the aggregation responses resemble the responses seen during the use of NSAIDs, release defects are often called aspirin-like disorders.

Storage pool deficiency. 

In a congenital or acquired storage pool defect, dense granules are empty or missing. ATP release in response to thrombin is reduced, as it is in response to ADP, arachidonic acid, and collagen (). Table 42-6

TABLE 42-6

Expected Platelet Lumiaggregometry Results in Storage Pool Disorder for a Variety of Agonists

Agonist

Final Concentration

Aggregation Recorded as Impedance

ATP Secretion

Thrombin

1 unit/mL

Not recorded*

< 0.1 nM

TRAP

10, 50, 100 μM

20 Ω

< 0.1 nM

Collagen

5 μg/mL

20 Ω

< 0.1 nM

Arachidonic acid

500 μM

12 Ω

< 0.1 nM

* Thrombin aggregation is not recorded as thrombin typically causes clotting, which interferes with the aggregation response. TRAP avoids the clotting response.

ATP, Adenosine triphosphate.

Platelet membrane defects: Thrombasthenia. 

Glanzmann thrombasthenia, a membrane defect characterized by dysfunction or loss of the GP IIb/IIIa receptor site, may be diagnosed by its characteristically diminished secretion and aggregation responses to all agonists except thrombin or its modest response to arachidonic acid.

Acquired platelet disorders. 

Platelets may become either dysfunctional or hyperactive in acquired hematologic and systemic disorders such as acute leukemia, aplastic anemia, myeloproliferative neoplasms, myelodysplastic syndromes, myeloma, uremia, liver disease, and chronic alcohol abuse. The physician looks for these disorders in any case where aggregation is abnormal and no other explanation is available. Platelet aggregometry results may predict the risk of bleeding or thrombosis in the patient with acquired platelet function disorders.44

Testing for heparin-induced thrombocytopenia

A description of the clinical manifestations and mechanism of heparin-induced thrombocytopenia (HIT) is provided in Chapter 39, and a summary of laboratory tests for HIT is provided in Chapter 40. Aggregation tests for HIT include light-transmittance aggregometry, washed platelet light-transmittance aggregometry, washed platelet lumiaggregometry, and whole-blood lumiaggregometry. The washed platelet carbon-14 (14C) serotonin release assay (SRA) is based on platelet activation and secretion. All these tests employ unfractionated heparin as their agonist. The 14C-SRA is available from specialized reference laboratories and is regarded as the reference confirmatory method. Few local institutions provide the 14C-SRA, because a radionuclide license is required. Except for the 14C-SRA, aggregometry and lumiaggregometry tests for HIT have proven to be insensitive and have been largely discontinued.

Quantitative measurement of platelet markers

Immunoassay for the anti–platelet factor 4 (heparin-induced thrombocytopenia) antibody

Amiral and colleagues developed a HIT screening immunoassay based on their discovery that PF4 is the target for the heparin-dependent antiplatelet antibody that causes HIT (Chapter 40).48 One adaptation of this principle is the PF4 Enhanced Solid Phase ELISA (Hologic Gen-Probe Inc., San Diego, CA). Patient plasma is incubated in microtiter plate wells that are coated with a solid-phase complex of purified PF4 and polysulfonate, a plastic molecule integral to plate construction that resembles heparin. Heparin-dependent anti-PF4 antibodies bind the PF4-polysulfonate complex. Bound antibodies are detected using enzyme-conjugated anti–human immunoglobulin G (IgG), IgA, and IgM antibodies and a substrate chromophore. This test is more sensitive than the 14C-SRA and detects antibodies early in the development of HIT, but it may detect antibodies that are unaccompanied by clinical symptoms, known as biologic false positives.49 A second kit that uses only enzyme-conjugated anti-human IgG is more specific. Other immunoassay kits employ PF4-heparin in place of PF4-polysulfonate and have similar sensitivity and specificity characteristics.

Assays for platelet activation markers

Elevated plasma levels of the platelet-specific proteins β-thromboglobulin and PF4 may accompany thrombotic stroke or coronary thrombosis.50 The implication that in vivo platelet activation contributes to the condition or that the measurement of these proteins is of diagnostic or prognostic significance is under investigation.51 Diagnostica Stago, Inc. (Parsippany, NJ) produces enzyme immunoassay kits for PF4 and β-thromboglobulin (β-TG) under the brand names Asserachrom PF4 and Asserachrom β-TG. Special collection techniques are necessary because PF4 and β-TG test results may be invalidated by platelet activation during and even subsequent to specimen collection.52 CTAD tubes (refer to the section on Hemostasis Specimen Collection above) are required for specimen collection for PF4 and β-TG assays. Plasma must undergo extraction before PF4 and β-TG assays are performed because several eicosanoids cross-react with kit antibodies and falsely raise the results.

Thromboxane A2, the active product of the eicosanoid pathway, has a half-life of 30 seconds, diffuses from the platelet, and spontaneously reduces to thromboxane B2, a stable, measurable plasma metabolite (Chapter 13). Efforts to produce a clinical assay for plasma thromboxane B2 have been unsuccessful because specimens must be collected in CTAD tubes to prevent in vitro platelet activation and must undergoan extraction step before the assay is performed. Thromboxane B2 is acted on by liver enzymes to produce an array of soluble urine metabolites, including 11-dehydrothromboxane B2, which is stable and measurable.53 Immunoassays of urine 11-dehydrothromboxane B2 are employed to characterize in vivo platelet activation.54 These assays require no special specimen management and can be performed on random urine specimens. The urinary 11-dehydrothromboxane B2 assay also may be used to monitor aspirin therapy and to identify cases of therapy failure or aspirin resistance.55

Clot-based plasma procoagulant screens

The Lee-White whole-blood coagulation time test, described in 1913, was the first laboratory procedure designed to assess coagulation.56 The Lee-White test is no longer used, but it was the first in vitro clot procedure that employed the principle that the time interval from the initiation of clotting to visible clot formation reflects the condition of the coagulation mechanism. A prolonged clotting time indicates a coagulopathy (coagulation deficiency). A 1953 modification, the activated clotting time (ACT) test, utilizes a particulate clot activator in the test tube, which speeds the clotting process. The ACT is still widely used as a point-of-care assay to monitor heparin therapy in high-dosage applications such as percutaneous intervention (cardiac catheterization) and coronary artery bypass graft surgery (Chapter 43).

The standard clot-based coagulation screening tests—PT, PTT, fibrinogen assay, and thrombin clotting time (TCT)—use the clotting time principle of the Lee-White test. Many specialized tests, such as coagulation factor assays, tests of fibrinolysis, inhibitor assays, reptilase time, Russell viper venom time, and dilute Russell viper venom time, are also based on the relationship between time to clot formation and coagulation function.

Prothrombin time

Prothrombin time principle

PT reagents, often called thromboplastin or tissue thromboplastin, are prepared from recombinant or affinity-purified tissue factor suspended in phospholipids mixed with a buffered 0.025 M solution of calcium chloride.57 A few less responsive thromboplastins are organic extracts of emulsified rabbit brain or lung suspended in calcium chloride. When mixed with citrated PPP, the PT reagent triggers fibrin polymerization by activating plasma factor VII (Figure 42-8). Calcium and phospholipids participate in the formation of the tissue factor–factor VIIa complex, the factor VIIIa–factor IXa complex, and the factor Va–factor Xa complex. The clot is detectable visually or by optical or electromechanical sensors. Although the coagulation pathway implies that the PT would be prolonged in deficiencies of fibrinogen, prothrombin, and factors V, VII, VIII, IX, and X, the procedure is most sensitive to factor VII deficiencies, moderately sensitive to factor V and X deficiencies, sensitive to severe fibrinogen and prothrombin deficiencies, and insensitive to deficiencies of factors VIII and IX.5859 The PT is prolonged in multiple factor deficiencies that include deficiencies of factors VII and X and is used most often to monitor the effects of therapy with the oral anticoagulant Coumadin (Chapter 43).

Image 

FIGURE 42-8 Prothrombin time (PT) reagent (thromboplastin) consists of tissue factor (TF), phospholipid (PL), and ionized calcium (Ca++). The reagent activates the extrinsic and common pathways of the coagulation mechanism beginning with factor VII (see colored area in figure). The PT is prolonged by deficiencies of factors VII, X, and V; prothrombin; and fibrinogen when the fibrinogen level is less than 100 mg/dL. The PT is prolonged in Coumadin therapy because Coumadin suppresses production of factor VII, factor X, and prothrombin. Factor VII has a 6-hour half-life and has the earliest effect on the PT. The PT does not detect factor XIII deficiency. HMWK, High-molecular-weight kininogen (Fitzgerald factor); Pre-K, prekallikrein (Fletcher factor); Pro, prothrombin (II, zymogen); Thr, thrombin (activated factor II, or IIa; serine protease); Va, VIIIa, activated factors V and VIII (serine protease cofactors); VIIa, IXa, Xa, XIa,activated factors VII, IX, X, XI (serine proteases); XIIa, activated factor XII (serine protease, but not part of in vivo coagulation); XIIIa, activated factor XIII (transglutaminase).

Prothrombin time procedure

The tissue factor-phospholipid-calcium chloride reagent is warmed to 37° C. An aliquot of test PPP, 50 or 100 μL, is transferred to the reaction vessel, which also is maintained at 37° C. The PPP aliquot is incubated at 37° C for at least 3 and for no more than 10 minutes. Aliquots that are incubated longer than 10 minutes become prolonged as coagulation factors begin to deteriorate or are affected by evaporation and pH change. A premeasured volume of reagent, 100 or 200 μL, is directly and quickly added to the PPP aliquot, and a timer is started. As the clot forms, the timer stops, and the elapsed time is recorded. If the procedure is performed in duplicate, the duplicate values must be within 10% of their mean or the test is repeated for a third time. Most laboratory practitioners perform PTs using automated instruments that strictly control temperature, pipetting, and interval timing (Chapter 44). With automated instruments, duplicate testing is unnecessary.

Prothrombin time quality control

The medical laboratory practitioner tests normal and prolonged control PPP specimens at the beginning of each 8-hour shift or with each change of reagent. Although lyophilized control PPPs are commercially available, the laboratory manager may choose to collect and pool PPP specimens from designated subjects to make “laboratory-developed” controls. In this case, the specimens must be collected and managed using the same tubes, anticoagulant, and protocol that are used for patient plasma specimen collection. The samples are pooled, tested, and aliquotted. Regardless of whether commercial or locally prepared controls are used, the control is tested alongside patient specimens using the same protocol as for patient PPP testing.

The normal control result should be within the reference interval, and the prolonged control result should be within the therapeutic range for Coumadin. If the control results fall within the stated limits provided in the laboratory protocol, the test results are considered valid. If the results fall outside the control limits, the reagents, control, and equipment are checked; the problem is corrected; and the control and patient specimens are retested. The operator records all the actions taken. Control results are recorded and analyzed at regular intervals to determine the long-term validity of results.

Reporting of prothrombin time results and the international normalized ratio

The medical laboratory practitioner reports PT results to the nearest tenth of a second, along with the PT reference interval. If the PT assay is performed in duplicate, the results are averaged, and the average is reported.

For Coumadin monitoring, to compensate for the inherent variations among thromboplastin reagents, most laboratories report the international normalized ratio (INR) for patients with a stable anticoagulation response using the following formula:60

Image

where PTpatient is the PT of the patient in seconds, PTgeometric mean of normal is the PT of the geometric mean of the reference interval, and ISI is the international sensitivity index. Reagent producers generate the ISI for their thromboplastin by performing an orthogonal regression analysis comparing its PT results on a set of plasmas, with the results obtained using the international reference thromboplastin preparation (World Health Organization human brain thromboplastin). Most responsive thromboplastin reagents have ISIs near 1, the assigned ISI of the WHO reagent. Automated coagulation instruments “request” the reagent ISI from the operator or incorporate it from the reagent label bar code and compute the INR for each specimen. INRs are meant to be computed only for samples from patients who have achieved a stable anticoagulation response with Coumadin. During the first week of Coumadin therapy, the physician should interpret PT results in seconds, comparing them with the reference interval. Chapter 43 provides a full discussion of Coumadin therapy monitoring.

Localized ISI calibration is replacing reagent manufacturer-generated ISIs as it produces a laboratory-specific ISI value that is likely to be more accurate than a distributor-provided ISI.61 The laboratory practitioner performs PTs on a set of four to five calibrator plasmas—for instance, ISI Calibrate (Instrumentation Laboratory, Bedford, MA). The calibrators arrive with predetermined PT values. If calibrators are not available, the practitioner may use a series of 100 patient specimens. The practitioner prepares a linear graph with the preestablished calibrator PTs or the PT values of the 100 patient specimens using the lab’s current PT reagent on the Y scale and local PTs using the new reagent on the X scale and computes the slope. The reference ISI provided by the manufacturer for the new PT reagent is multiplied by the slope value to produce the local ISI of the new PT reagent.

The same approach may be applied to lot-to-lot calibrations of PT reagents; however, in most lot-to-lot validations the operator need only assay a three-level validation plasma set—for instance, ISI Validate (Instrumentation Laboratory, Bedford, MA). If the lot values determined using the new reagent are within predetermined limits, the lot may be placed in everyday operation without a change; if not, it is necessary to recalibrate the ISI value of the new lot of the PT reagent.

Prothrombin time reference interval

The PT reference interval, computed from PT values of healthy individuals, varies from site to site, depending on the patient population, type of thromboplastin used, type of instrument used, and pH and purity of the reagent diluent. Each center must establish its own range for each new lot of reagents, or at least once a year. This may be done by testing a sample of at least 30 specimens from healthy donors of both sexes spanning the adult age range over several days and computing the 95% confidence interval of the results. A typical PT reference interval is 12.6 to 14.6 seconds.

The prothrombin time as a diagnostic assay

The PT is performed diagnostically when any coagulopathy is suspected. Acquired multiple deficiencies such as disseminated intravascular coagulation (DIC), liver disease, and vitamin K deficiency all affect factor VII activity and are detected through prolonged PT results. The PT is particularly sensitive to liver disease, which causes factor VII levels to become rapidly diminished (Chapter 38).

Vitamin K deficiency is seen in severe malnutrition, during use of broad-spectrum antibiotics that destroy gut flora, with parenteral nutrition, and in malabsorption syndromes. Vitamin K levels are low in newborns, in which bacterial colonization of the gut has not begun. Hemorrhage is likely in vitamin K deficiency, and the PT is the best indicator. To distinguish between vitamin K deficiency and liver disease, the laboratory practitioner determines factor V and factor VII levels. Both factor V and factor VII are reduced in liver disease; only factor VII is reduced in vitamin K deficiency. Chapter 38 provides details regarding liver disease and vitamin K deficiency.

The PT is prolonged in congenital single-factor deficiencies of factor X, VII, or V; profound prothrombin deficiency; and fibrinogen deficiency when the fibrinogen level is 100 mg/dL or less. When the PT is prolonged but the PTT and thrombin clotting time (TCT) test results are normal, factor VII activity may be deficient. Any suspected single-factor deficiency is confirmed with a factor assay. The PT is not affected by factor VIII or IX deficiency, because the concentration of tissue factor in the reagent is high, and those factors are bypassed in thrombin generation.

Minimal effectiveness of prothrombin time as a screening tool

Preoperative PT screening of asymptomatic surgical patients to predict intraoperative hemorrhage is not supported by prevalence studies, unless the patient is a member of a high-risk population.6263 No clinical data support the use of the PT as a general screening test for individuals at low risk of bleeding, and the PT is not useful for establishing baseline values in Coumadin therapy.64 The therapeutic target range for Coumadin therapy is based on the INR, not the baseline PT result or PT control value.

Limitations of the prothrombin time

Specimen variations profoundly affect PT results (). The ratio of whole blood to anticoagulant is crucial, so collection tubes must be filled to within tube manufacturers’ specifications and not underfilled or overfilled. Anticoagulant volume must be adjusted when the hematocrit is greater than 55% to avoid false prolongation of the results. Specimens must be inverted five times immediately after collection to ensure good anticoagulation, but the mixing must be gentle. Practitioners must reject clotted and visibly hemolyzed specimens because they give unreliable results. Plasma lipemia or icterus may affect the results obtained with optical instrumentation. Table 42-7

TABLE 42-7

Factors That Interfere with the Validity of Clot-based Test Results

Problem

Solution

Blood collection volume less than specified minimum

PT falsely prolonged; recollect specimen.

Hematocrit ≥55%

Adjust anticoagulant volume using formula and recollect specimen using new anticoagulant volume.

Clot in specimen

All results are affected unpredictably; recollect specimen.

Visible hemolysis

PT falsely shortened; recollect specimen.

Icterus or lipemia

Measure PT using a mechanical coagulometer.

Heparin therapy

Use reagent known to be insensitive to heparin or one that includes a heparin neutralizer such as polybrene.

Lupus anticoagulant

PT result is invalid; use chromogenic factor X assay instead of PT.

Incorrect calibration, incorrect dilution of reagents

Correct analytical error and repeat test.

Heparin may prolong the PT. If the patient is receiving therapeutic heparin, it should be noted on the order and commented on when the results are reported. The laboratory manager selects thromboplastin reagents that are maximally sensitive to oral anticoagulant therapy and insensitive to heparin. Many reagent manufacturers incorporate polybrene (5-dimethyl-1,5-diazaundecamethylene polymethobromide, hexadimethrine bromide, Sigma-Aldrich, St. Louis, MO) in their thromboplastin reagent to neutralize heparin. The medical laboratory practitioner may detect unexpected heparin by using the TCT test, which is described subsequently.

Lupus anticoagulants (LAs) prolong some thromboplastins. LAs are members of the antiphospholipid antibody family and may partially neutralize PT reagent phospholipids. Coumadin often is prescribed to prevent thrombosis in patients with LAs, but the PT may be an unreliable monitor of therapy in such cases. Patients who have an LA and are taking Coumadin should be monitored using an alternative system, such as the chromogenic factor X assay.6566

Reagents must be reconstituted with the correct diluents and volumes following manufacturer instructions. Reagents must be stored and shipped according to manufacturer instructions and never used after the expiration date.

Partial thromboplastin time

Partial thromboplastin time principle

The PTT (also called the activated partial thromboplastin time, or APTT) is performed to monitor the effects of unfractionated heparin therapy and to detect LA and specific anticoagulation factor antibodies such as anti-factor VIII antibody. The PTT is also prolonged in all congenital and acquired procoagulant deficiencies, except for deficiencies of factor VII or XIII.67

The PTT reagent contains phospholipid (previously called partial thromboplastin) and a negatively charged particulate activator such as silica, kaolin, ellagic acid, or celite in suspension. The phospholipid mixture, which was historically extracted from rabbit brain, is now produced synthetically. The activator provides a surface that mediates a conformational change in plasma factor XII that results in its activation (Figure 42-9). Factor XIIa forms a complex with two other plasma components: high-molecular-weight kininogen (Fitzgerald factor) and prekallikrein (Fletcher factor). These three plasma glycoproteins, termed the contact activation factors, initiate in vitro clot formation through the intrinsic pathway but are not part of in vivo coagulation. Factor XIIa, a serine protease, activates factor XI (XIa), which activates factor IX (IXa) (Chapter 37).

Image 

FIGURE 42-9 Partial thromboplastin time (PTT) reagent (partial thromboplastin) consists of phospholipid (PL), a negatively charged particulate activator (NCS), and ionized calcium. It activates the intrinsic and common pathways of the coagulation mechanism through the contact factors XII, prekallikrein (Pre-K; also called Fletcher factor), and high-molecular-weight kininogen (HMWK; also called Fitzgerald factor), none of which is significant in the in vivo coagulation mechanism (see colored area in figure). The PTT is prolonged by deficiencies in Pre-K; HMWK; factors XII, XI, IX, VIII, X, and prothrombin; and fibrinogen when the fibrinogen level is less than 100 mg/dL. The deficiencies for which the PTT reagent is specifically calibrated are factors VIII, IX, and XI. The PTT is prolonged in heparin therapy because heparin activates plasma antithrombin, which neutralizes all the plasma serine proteases, particularly thrombin (IIa) and activated factor X (Xa). The PTT is prolonged in the presence of lupus anticoagulant because the anticoagulant neutralizes essential reagent phospholipids. The PTT does not detect factor XIII deficiency. TF, Tissue factor; Pro, prothrombin (II, zymogen); Thr, thrombin (activated factor II, or IIa; serine protease); Va, VIIIa,activated factors V and VIII (serine protease cofactors); VIIa, IXa, Xa, XIa, activated factors VII, IX, X, XI (serine proteases); XIIa, activated factor XII (serine protease, but not part of in vivo coagulation); XIIIa, activated factor XIII (transglutaminase).

Factor IXa binds calcium, phospholipid, and factor VIIIa to form a complex. In the PTT reaction system, ionic calcium and phospholipid are supplied in the reagent. The factor IXa–calcium–factor VIIIa–phospholipid complex catalyzes factor X (Xa). Factor Xa forms another complex with calcium, phospholipid, and factor Va, catalyzing the conversion of prothrombin to thrombin. Thrombin catalyzes the polymerization of fibrinogen and the formation of the fibrin clot, which is the endpoint of the PTT.

The factors whose deficiencies are associated with hemorrhage and are reflected in prolonged PTT results, taken in the order of reaction, are XI, IX, VIII, X, and V; prothrombin; and fibrinogen, when fibrinogen is 100 mg/dL or less. Most PTT reagents are designed so that the PTT is prolonged when the test PPP has less than approximately 0.3 units/mL (30% of normal) of VIII, IX, or XI.68 The PTT also is prolonged in the presence of LA, an immunoglobulin with affinity for phospholipid-bound proteins, and is prolonged by anti-factor VIII antibody, antibodies to factor IX and other coagulation factors, and therapeutic heparin. Factor VII and factor XIII deficiencies have no effect on the PTT. Deficiencies of factor XII, prekallikrein, or high-molecular-weight kininogen prolong the PTT but do not cause bleeding.

Partial thromboplastin time procedure

To initiate contact activation, 50 or 100 μL of warmed (37° C) reagent consisting of phospholipid and particulate activator is mixed with an equal volume of warmed PPP. The mixture is allowed to incubate for the exact manufacturer-specified time, usually 3 minutes. Next, 50 or 100 μL of warmed 0.025 M calcium chloride is forcibly added to the mixture, and a timer is started. When a fibrin clot forms, the timer stops, and the interval is recorded. Timing may be done with a stopwatch or by an automatic electromechanical or photo-optical device. If the PTT is performed manually, the test should be done in duplicate, and the two results must match within 10%.

Partial thromboplastin time quality control

The medical laboratory practitioner tests normal and prolonged control plasma specimens at the beginning of each 8-hour shift or with each new batch of reagent. The laboratory director may require more frequent use of controls. Controls are tested using the protocol for patient plasma testing.

The normal control result should be within the reference interval, and the abnormal control result should be within the therapeutic range for unfractionated heparin (Chapter 43). If the control results fall within the stated limits in the laboratory protocol, the test results are considered valid. If the results fall outside the control limits, the reagents, control, and equipment are checked; the problem is corrected; and the control and patient specimens are retested. The operator records each control run and all the actions taken. Control results are recorded and analyzed at regular intervals to determine the long-term validity of results.

Reagents must be reconstituted with the correct diluents and volumes following manufacturer instructions. Reagents must be stored and shipped according to manufacturer instructions and never used after the expiration date.

Specimen errors that affect the PT similarly affect the PTT (Table 42-7).

Partial thromboplastin time reference interval

The PTT reference interval varies from site to site, depending on the patient population, type of reagent, type of instrument, and pH and purity of the diluent. One medical center laboratory has established 26 to 38 seconds as its reference interval. This range is typical, but each center must establish its own interval for each new lot of reagent, or at least once a year. This may be done by testing a sample of 30 or more specimens from healthy donors of both sexes spanning the adult age range over several days and computing the 95% confidence interval of the results.

Monitoring of heparin therapy with partial thromboplastin time

Since the early 1970s, the PTT has been the standard method for monitoring unfractionated heparin therapy, which is used to treat patients with venous thrombosis, pulmonary embolism, myocardial infarction, and several other medical conditions.69 The laboratory practitioner establishes a PTT therapeutic range and publishes it to all inpatient units. A typical therapeutic range is 60 to 100 seconds; however, the range varies widely and must be established locally.70 The range must be reestablished with each change of PTT reagent, including each lot change, and upon instrument recalibration. Details on monitoring of heparin therapy and establishment of the PTT therapeutic range are provided in Chapter 43.

The partial thromboplastin time as a diagnostic assay

The physician orders a PTT assay when a hemorrhagic disorder is suspected or when recurrent thrombosis or the presence of an autoimmune disorder points to the possibility of an LA.71 The PTT result is prolonged when there is a deficiency of one or more of the following coagulation factors: prothrombin; factor V, VIII, IX, X, XI, or XII; or fibrinogen when the fibrinogen level is 100 mg/dL or less. The PTT also is prolonged in the presence of a specific inhibitor, such as anti-factor VIII or anti-factor IX; a non-specific inhibitor, such as LA; and interfering substances, such as fibrin degradation products (FDPs) or paraproteins, which are present in myeloma.

DIC prolongs PTT results because of consumption of procoagulants, but the PTT results alone are not definitive for the diagnosis of DIC. Vitamin K deficiency results in diminished levels of procoagulant factors II (prothrombin), VII, IX, and X, and the PTT is eventually prolonged. Because factor VII deficiency does not affect the PTT, however, and because it is the first coagulation factor to become deficient, the PTT is not as sensitive to vitamin K deficiency or Coumadin therapy as the PT. The PTT is not prolonged in deficiencies of factor VII or XIII. No clinical data support the use of the PTT as a general screening test for individuals at low risk of bleeding.72

Partial thromboplastin time mixing studies

Lupus anticoagulants

LAs are IgG immunoglobulins directed against a number of phospholipid-protein complexes.72 LAs prolong the phospholipid-dependent PTT reaction. Most laboratories employ a moderate-phospholipid or high-phospholipid PTT reagent in their primary PTT assay to monitor heparin therapy and detect coagulopathies. Laboratories use a second low-phospholipid PTT reagent such as PTT-LA (Diagnostica Stago, Parsippany, NJ), which is more sensitive to LA, as their LA screen (Chapter 39). Because they have a variety of target antigens, LAs are called nonspecific inhibitors. Chronic presence of LAs confers a 30% risk of arterial or venous thrombosis; every acute care laboratory must provide a means for their detection. Together, chronic and transient LAs are found in 1% to 2% of randomly selected individuals.

Specific factor inhibitors

Specific factor inhibitors are IgG immunoglobulins directed against coagulation factors. Specific inhibitors arise in severe congenital factor deficiencies during factor concentrate treatment. Anti-factor VIII, the most common of the specific inhibitors, is detected in 10% to 20% of patients with severe hemophilia, and anti-factor IX is detected in 1% to 3% of factor IX-deficient patients. Autoantibodies to factor VIII occasionally may arise in individuals without hemophilia, usually in young women, where they are associated with a postpartum bleeding syndrome or in patients over 60 with autoimmune disorders. The presence of these types of antibodies is called acquired hemophilia (Chapter 38). Alloantibodies and autoantibodies to factor VIII are associated with severe anatomic hemorrhage.

Detection and identification of lupus anticoagulants and specific inhibitors

LA testing is part of every thrombophilia profile (Chapter 39). An unexpectedly prolonged screening PTT may also trigger an LA investigation. PTT mixing studies are necessary for the initial detection of LAs.73Mixing studies also distinguish LAs from specific inhibitors and factor deficiencies and should be available in all coagulation laboratories.74

When the initial PTT is prolonged beyond the upper limit of the reference interval, the laboratory practitioner first determines if heparin is present by performing the TCT. A TCT result that exceeds the upper limit of the TCT reference interval is evidence for the presence of heparin. In fact, heparin often prolongs the TCT to 30 to 40 seconds. Heparin may be neutralized using polybrene or heparinase (Hepzyme; Siemens Healthcare Diagnostics, Tarrytown, NY), and the treated sample may be used for PTT mixing studies.

The heparin-free or heparin-neutralized patient plasma is then mixed 1:1 with reagent platelet-poor normal plasma (PNP; Figure 39-1). Several manufacturers make PNP—for example, frozen Cryocheck Normal Reference Plasma (Precision BioLogic, Inc, Dartmouth, Nova Scotia). A new PTT is performed immediately on the 1:1 mixture. If the mixture PTT corrects to within 10% of the PNP PTT (or to within the reference interval) and the patient is experiencing bleeding, a coagulation factor deficiency (coagulopathy) is presumed.75

Some LAs are time dependent and temperature dependent. Most anti-factor VIII inhibitors are temperature-dependent IgG4-class antibodies. If the immediate PTT corrects, a new mixture is prepared and incubated 1 to 2 hours at 37° C. If the incubated mixture’s PTT fails to correct to within 10% of the incubated PNP PTT, an inhibitor may be present. If the patient is bleeding, a specific inhibitor such as anti-factor VIII is suspected, and a factor VIII activity assay is performed. Although anti-factor IX and other inhibitors have been documented, anti-factor VIII is the most common. The Bethesda titer procedure, discussed later in this chapter, is used to confirm the presence of specific anti-coagulation factor antibodies.

If the PTT of the initial or incubated mixture fails to correct and the patient is not bleeding, the laboratory practitioner suspects LA and automatically orders an LA profile, as described in Chapter 39. LA profiles are available from tertiary care facilities and specialty reference laboratories.

Thrombin clotting time

Thrombin clotting time reagent and principle

Commercially prepared bovine thrombin reagent at 5 National Institutes of Health (NIH) units/mL cleaves fibrinopeptides A and B from plasma fibrinogen to form a detectable fibrin polymer (). Figure 42-10

Image 

FIGURE 42-10 Thrombin clotting time (TCT, also reptilase time) coagulation pathway. The reagent activates the coagulation pathway at the level of thrombin and tests for the polymerization of fibrinogen (see colored area in figure). The TCT is prolonged by unfractionated heparin; direct thrombin inhibitors; fibrin degradation products; M-proteins; and dysfibrinogenemia, hypofibrinogenemia, and afibrinogenemia. The reptilase time is unaffected by heparin but is prolonged by dysfibrinogenemia, hypofibrinogenemia, and afibrinogenemia. Neither the TCT nor reptilase time detects factor XIII deficiency. HMWK, High-molecular-weight kininogen (Fitzgerald factor); NCS, negatively charged surface; Pre-K,prekallikrein (Fletcher factor); PL,phospholipid; TF, tissue factor; Thr, thrombin (activated factor II, or IIa; serine protease); Va, VIIIa, activated factors V and VIII (serine protease cofactors); VIIa, IXa, Xa, XIa, activated factors VII, IX, X, XI (serine proteases); XIIa, activated factor XII (serine protease, but not part of in vivo coagulation); XIIIa, activated factor XIII (transglutaminase).

Thrombin clotting time procedure

Reagent thrombin is warmed to 37° C for a minimum of 3 and a maximum of 10 minutes. Thrombin deteriorates during incubation and must be used within 10 minutes of the time incubation is begun. An aliquot of PPP, usually 100 μL, is also incubated at 37° C for a minimum of 3 and a maximum of 10 minutes. The operator pipettes 200 μL of thrombin into the PPP aliquot, starts a timer, and records the interval to clot formation. TCT tests may be performed in duplicate and the results averaged.

Thrombin clotting time quality control

The medical laboratory practitioner tests a normal control sample and an abnormal control sample with each batch of TCT assays and records the results. The normal control results should fall within the laboratory’s reference interval. The abnormal control results should be prolonged to the range reached by the TCT in moderate hypofibrinogenemia. If the results fall outside the laboratory protocol’s control limits, the reagents, control, and equipment are checked; the problem is corrected; and the control is retested. The actions taken to correct out-of-limit tests are recorded. Control results are analyzed at regular intervals (weekly is typical) to determine the longitudinal validity of the procedure.

Specimen errors that affect the PT likewise affect the TCT (Table 42-7).

Reporting of thrombin clotting time results and clinical utility

A typical TCT reference interval is 15 to 20 seconds, although the reference interval should be established locally. The TCT is prolonged when the fibrinogen level is less than 100 mg/dL (hypofibrinogenemia) or in the presence of antithrombotic materials such as FDPs, paraproteins, or heparin. Afibrinogenemia (absence of fibrinogen) and dysfibrinogenemia (presence of fibrinogen that is biochemically abnormal and nonfunctional) also cause a prolonged TCT. Before a prolonged TCT may be considered as evidence of diminished or abnormal fibrinogen, the presence of antithrombotic substances, such as heparin, FDPs, or paraproteins, must be ruled out. The TCT is part of the PTT mixing study protocol and is used to determine whether heparin is present whenever the PTT is prolonged.76

The TCT may also assess the presence of the oral direct thrombin inhibitor dabigatran. The TCT provides binary (qualitative) evidence for dabigatran; if drug is present, the TCT is markedly prolonged. A normal TCT rules out dabigatran. A TCT modification, the plasma-diluted TCT, provides a quantitative measure of dabigatran when used with calibrators of specific drug concentrations.77

The fibrinogen assay described in a subsequent section is a simple modification of the TCT. In the fibrinogen assay, the concentration of reagent thrombin is 50 NIH units/mL, or about 10 times that used in the TCT, and the patient specimen is diluted 1:10. This dilution minimizes the effects of heparin or antithrombotic proteins. The reptilase time procedure described below is identical to the TCT procedure, except that the reptilase reagent is insensitive to the effects of heparin.

Reptilase time

Reptilase time reagent and principle

Reptilase is a thrombin-like enzyme isolated from the venom of Bothrops atrox that catalyzes the conversion of fibrinogen to fibrin (Pefakit Reptilase Time; Pentapharm, Inc., Basel, Switzerland). In contrast to thrombin, this enzyme cleaves only fibrinopeptide A from the fibrinogen molecule, whereas thrombin cleaves both fibrinopeptides A and B.78 The specimen requirements, procedure, and quality assurance protocol for the reptilase time test are the same as those for the TCT. The reagent is reconstituted with distilled water and is stable for 1 month when stored at 1° C to 6° C. Reptilase time reagent is a poison that may be fatal if it directly enters the bloodstream.

Reptilase time clinical utility

Reptilase is insensitive to heparin but is sensitive to dysfibrinogenemia, which profoundly prolongs the assay time. The reptilase time test is also useful for detecting hypofibrinogenemia or dysfibrinogenemia in patients receiving heparin therapy. The reptilase time is prolonged in the presence of FDPs and paraproteins.

Russell viper venom

Russell viper venom (RVV) from the Daboia russelii viper, which triggers coagulation at the level of factor X, was once used as an alternative to the prothrombin time. The assay was named the Stypven time, but is now obsolete. Russell viper venom is used in a dilute form to detect and confirm lupus anticoagulant, an assay called the dilute Russell viper venom time described in Chapter 39.

Coagulation factor assays

Fibrinogen assay

Fibrinogen assay principle

The clot-based method of Clauss, a modification of the TCT, is the recommended procedure for estimating the functional fibrinogen level.7980 The operator adds reagent bovine thrombin to dilute PPP, catalyzing the conversion of fibrinogen to fibrin polymer. In the fibrinogen assay, the thrombin reagent concentration is 50 NIH units/mL. The PPP to be tested is diluted 1:10 with Owren buffer. There is an inverse relationship between the interval to clot formation and the concentration of functional fibrinogen. Because the thrombin reagent is concentrated and the PPP is diluted, the relationship is linear when the fibrinogen concentration is 100 to 400 mg/dL. Diluting the PPP also minimizes the antithrombotic effects of heparin, FDPs, and paraproteins; heparin levels less than 0.6 units/mL and FDP levels less than 100 μg/dL do not affect the results of the fibrinogen assay provided the fibrinogen concentration is 150 mg/dL or greater.

The interval to clot formation is compared with the results for fibrinogen calibrators. A calibration curve is prepared in each laboratory and updated regularly.

Fibrinogen assay procedure

Fibrinogen assay thrombin reagent. 

Most laboratory managers prefer commercially manufactured diagnostic lyophilized bovine thrombin reagent for fibrinogen assays. Pharmaceutical topical thrombin also may be used. The reagent is reconstituted according to manufacturer instructions and used immediately or aliquotted and frozen. If thrombin is to be frozen, it should be prepared in a stock solution of 1000 NIH units/mL and frozen at –70° C until it is ready for use. When thawed, the thrombin is diluted 1:2 with buffer, is stable for only a few hours, and cannot be refrozen.

Fibrinogen assay calibration curve. 

The laboratory practitioner prepares a calibration curve every 6 months at a minimum and with each change of reagent lot numbers, with a shift in QC, and after major maintenance. The curve is prepared by reconstituting commercially available lyophilized fibrinogen calibration plasma. Using Owren buffer, five dilutions of the calibration plasma are prepared: 1:5, 1:10, 1:15, 1:20, and 1:40. An aliquot of each dilution, usually 200 μL, is transferred to each of three reaction tubes or cups, warmed to 37° C, and tested by adding 100 μL of working thrombin reagent at 50 NIH units/mL. Time from addition of thrombin to clot formation is recorded, results of duplicate tests are averaged, and the values in seconds are graphed against fibrinogen concentration (). Because patient PPPs are diluted 1:10 before testing, the 1:10 calibration plasma dilution is assigned the same fibrinogen concentration value as that of the undiluted reconstituted calibration plasma.Figure 42-11

Image 

FIGURE 42-11 Fibrinogen calibrator curve plotted on log-log axes.

Fibrinogen assay test protocol. 

The laboratory practitioner prepares a 1:10 dilution of each patient PPP and control with Owren buffer. Then 200 μL of each of the diluted PPPs is warmed to 37° C in each of two reaction tubes or cups for 3 minutes. After incubation, 100 μL of thrombin reagent is added, a timer is started, and the mixture is observed until a clot forms. The timer is stopped, values for duplicate runs are averaged, and the interval in seconds is compared with the graph. Results are reported in mg/dL of fibrinogen.

If the clotting time of the patient PPP dilution is short, indicating a fibrinogen level greater than 480 mg/dL, a 1:20 dilution is prepared and tested. The resulting fibrinogen concentration from the graph must be multiplied by 2 to compensate for the dilution. If the clotting time of the original 1:10 patient PPP dilution is prolonged, indicating less than 200 mg/dL of fibrinogen, a 1:5 dilution is prepared. The operator divides the resulting concentration reading from the graph by 2 to compensate for the greater concentration of the specimen.

Fibrinogen assay quality control

All results for duplicate tests must agree within a coefficient of variation of less than 7%. The medical laboratory practitioner tests a normal control sample and an abnormal control sample with each batch of specimens for which fibrinogen levels are measured and records the results. The normal control results should be within the laboratory’s reference interval. The abnormal control results should be less than 100 mg/dL. If either control result falls outside the control limits, the reagents, control, and equipment are checked; the problem is corrected; and the control is retested. The actions taken to correct out-of-limit tests are recorded. Control results are analyzed at regular intervals (weekly is typical) to determine the longitudinal validity of the procedure.

Specimen errors that affect the PTT likewise affect the fibrinogen assay and all factor assays (Table 42-7).

Fibrinogen assay results and clinical utility

One institution’s reference interval for fibrinogen concentration is 220 to 498 mg/dL, although each local institution prepares its own interval. Hypofibrinogenemia, a fibrinogen level of less than 220 mg/dL, is associated with DIC and severe liver disease. Moderately severe liver disease, pregnancy, and a chronic inflammatory condition may cause an elevated fibrinogen level, greater than 498 mg/dL. Congenital afibrinogenemia leads to prolonged clotting times and is associated with a variable hemorrhagic disorder. Dysfibrinogenemia may give the same results as hypofibrinogenemia by this test method, because some abnormal fibrinogen species are hydrolyzed more slowly by thrombin than is normal fibrinogen. Some forms of dysfibrinogenemia may be associated with thrombosis.81

Fibrinogen values measured using immunologic assays and turbidimetric methods (Ellis-Stransky technique; PT-Fibrinogen HS Plus, Instrumentation Laboratory, Bedford, MA) are normal in dysfibrinogenemia. The fibrinogen concentration is estimated from reaction mixture turbidity and reported with each PT.

Fibrinogen assay limitations

Although antithrombotic effects are minimized by the dilution of PPP specimens, heparin levels greater than 0.6 units/mL and FDP levels greater than 100 μg/mL prolong the results and give falsely lowered fibrinogen results. The operator ensures that the thrombin reagent is pure and has not degenerated. Exposure to sunlight or oxidation results in rapid breakdown. The working dilution lasts only 1 hour at 1° C to 6° C and should remain cold until just before testing.

Single-factor assays using the partial thromboplastin time test

Principle of single-factor assays based on partial thromboplastin time

If the PTT is prolonged and the PT and TCT are normal, and there is no ready explanation for the prolonged PTT such as heparin therapy, LA, or a factor-specific inhibitor, the medical laboratory practitioner may suspect a congenital single-factor deficiency. Three factor deficiencies that give this reaction pattern and cause hemorrhage are factor VIII deficiency (hemophilia A), factor IX deficiency (hemophilia B), and factor XI deficiency, which causes a mild intermittent bleeding disorder called Rosenthal syndrome found primarily in Ashkenazi Jews.8283 These deficiencies are most often detected in childhood. The next step in diagnosis of a congenital single-factor deficiency is the performance of a one-stage single-factor assay based on the PTT system.

Although necessary for diagnosis, PTT-based single-factor assays are most often performed on specimens from patients with previously identified single-factor deficiencies. Their purpose is to monitor supportive therapy during bleeding episodes or invasive procedures. Because hemophilia A is the most common single-factor deficiency disorder, this discussion is confined to the factor VIII assay; however, the protocol may be generalized to the assays for factors IX and XI.

The medical laboratory practitioner uses the PTT system to estimate the concentration of functional factor VIII by incorporating commercially prepared factor VIII-depleted PPP in the test system (CryocheckFactor VIII Deficient Plasma; Precision BioLogic Inc, Dartmouth, Nova Scotia). Distributors collect plasma from normal donors and employ immunodepletion, relying on a monoclonal anti-factor VIII antibody bound to a separatory column, to prepare factor VIII-depleted plasma.84

In the PTT-based factor assay system, factor VIII-depleted PPP provides normal activity of all procoagulants except factor VIII. Tested alone, factor VIII-depleted PPP has a prolonged PTT, but when normal PPP is added, the PTT reverts to normal. In contrast, a prolonged result for a mixture of patient PPP and factor VIII-depleted PPP implies that the patient PPP is factor VIII deficient. The clotting time interval for the mixture of patient PPP and factor VIII-depleted PPP may be compared with a previously prepared reference curve to estimate the level of factor VIII activity in the patient PPP. The quantitative factor assay is typically performed on three or four dilutions of patient PPP—for instance, 1:10, 1:20, 1:40, and 1:80—and the results compared with mathematical manipulation. Multiple dilutions contribute to the accuracy of the results.

Factor VIII assay reference curve

To prepare a reference curve for the factor VIII assay, the laboratory practitioner obtains a reference plasma such as CAP FVIIIc RM (College of American Pathologists, Northfield, IL) and prepares a series of dilutions with buffered saline.85 Although laboratory protocols vary, most laboratory practitioners prepare a series of five dilutions, from 1:5 to 1:500. Each dilution is mixed with reagent factor VIII-depleted plasma and tested in duplicate using the PTT system. The duplicate results are averaged and plotted on log-log or log-linear graph paper (Figure 42-12). The 1:10 dilution is assigned the factor VIII assay activity value found on the package insert. When patient PPP is tested, the time interval obtained is entered on the vertical coordinate and converted to a percentage.86

Image 

FIGURE 42-12 Factor VIII assay calibrator curve plotted on linear-log axes.

Factor VIII assay procedure

The medical laboratory practitioner (or the automated coagulometer) prepares 1:10, 1:20, 1:40, and 1:80 dilutions of each patient PPP and control specimen and then mixes each dilution with equal volumes of factor VIII-depleted plasma and PTT reagent. In most cases, 100 μL of PTT reagent is mixed with 100 μL each of patient PPP dilution and factor VIII-depleted plasma mixture. All dilutions of each specimen or control are tested in duplicate. After incubation at 37° C for the manufacturer-specified time, typically 3 minutes, 100 μL of 0.025 M calcium chloride is added, and a timer is started. The interval is recorded in seconds, duplicates are averaged, the mean result is compared with the reference curve, and the percentage of factor VIII activity is reported. Factor activity results for the 1:20, 1:40, and 1:80 dilutions are multiplied by 2, 4, and 8, respectively, to compensate for the dilutions and should match the results of the 1:10 dilutions within 10%. If the results of the dilutions do not match within 10%, they are considered to be nonparallel. An LA may be present, and the assay cannot provide a reliable estimate of factor VIII activity.

Tests for factors IX and XI are performed using the same approach, except that the appropriate factor-depleted plasma is substituted for factor VIII-depleted plasma. Tests for the contact factors XII, prekallikrein, and high-molecular-weight kininogen are seldom requested because deficiencies are not associated with bleeding disorders. Acquired and congenital contact factor deficiencies are relatively common, however, and cause PTT prolongation. Factor XII, prekallikrein, and high-molecular-weight kininogen assays are available from hemostasis reference laboratories, and their use may be necessary to account for an unexplained prolonged PTT.

Expected results and clinical utility of single-factor assays

The reference interval for factor VIII activity is 50% to 186%. Spontaneous symptoms of hemophilia are evident at activity levels of 10% or less. The test is used most often to estimate the plasma level of factor VIII activity during therapy (Chapter 38). Chronically elevated factor VIII predicts an elevated risk of venous thrombotic disease (Chapter 39).

Single-factor assay quality control

All duplicate results must agree within 10%. The medical laboratory practitioner tests a normal and a deficient control specimen with each assay and records the results. The normal control results should fall within the reference interval. The deficient control results should be in the range of 10% factor VIII activity or below. If either control result falls outside the control limits, the reagents, control, and equipment are checked; the problem is corrected; and the control is retested. The practitioner records all actions taken to correct out-of-limit tests. Control results are analyzed at regular intervals (weekly is typical) to determine the longitudinal validity of the procedure.

Limitations of single-factor assays

Interlaboratory coefficients of variation for the factor VIII assay reach 80%, which implies undesirable variation in the interpretation of therapeutic monitoring results from unrelated institutions. To reduce inherent variation, the medical laboratory practitioner uses assayed commercial plasma to prepare the reference curve and selects reference dilutions that correspond to only the linear portion of the curve. The laboratory must assay three or more dilutions of patient PPP to check for inhibitors. The practitioner also selects a matching reagent-instrument system with a demonstrated coefficient of variation of less than 5% and uses factor-depleted substrates with no trace of the depleted factor.87 As with the PTT test, good specimen management is essential. Clotted, hemolyzed, icteric, or lipemic specimens are rejected because they give unreliable results. Reagents must be reconstituted with the correct diluents and volumes following manufacturer instructions. Reagents must be stored and shipped in accordance with manufacturer instructions and never used after the expiration date.

Bethesda titer for anti–factor VIII inhibitor

The Bethesda titer is used to confirm the presence of and quantify an anti-factor VIII inhibitor, which is typically an IgG4-class immunoglobulin.88 In this method, 200 μL of patient PPP is incubated with 200 μL of reagent normal plasma for 2 hours at 37° C. A control specimen consisting of 200 μL of imidazole buffer at pH 7.4 mixed with 200 μL of reagent normal plasma is incubated simultaneously. During the incubation period, anti-factor VIII from the patient PPP neutralizes a percentage of the reagent normal plasma factor VIII activity. The degree of factor VIII activity neutralized is proportional to the level of inhibitor activity. After incubation, residual factor VIII activity in the patient PPP–reagent normal plasma mixture is measured using the specific factor activity assay as described in the section on factor assays using the PTT system.

The titer of inhibitor is expressed as a percentage of the control. If the patient PPP-reagent normal plasma mixture retains 75% of the residual factor VIII activity of the control, no factor VIII inhibitor is present. If the residual factor VIII level is 25% that of the control, the patient PPP factor VIII inhibitor level is titered using several dilutions of the patient specimen in reagent normal PPP. One Bethesda unit of activity is the amount of antibody that leaves 50% residual factor VIII activity in the mixture.

Single-factor assays using the prothrombin time test

If the PTT and the PT are both prolonged, the TCT is normal, and there is no ready explanation for the prolonged test results, such as liver disease, vitamin K deficiency, DIC, or Coumadin therapy, the medical laboratory practitioner may suspect a congenital single-factor deficiency of the common pathway (Chapter 37). Three relatively rare factor deficiencies that give this reaction pattern and cause hemorrhage are prothrombin deficiency, factor V deficiency, and factor X deficiency. If the PT is prolonged and all other test results are normal, factor VII deficiency is suspected. The next step is the performance of a one-stage single-factor assay based on the PT test system, which is a relatively rare event. The principles and procedure described in the section on single-factor assay using the PTT system may be applied except that PT reagent replaces the PTT reagent in the test system, and the PT protocol is followed. Factor II (prothrombin)-depleted, factor V-depleted, factor VII-depleted, and factor X-depleted plasmas are available (Table 42-8).

TABLE 42-8

Factor Assays Using the TCT, PT, and PTT Test Systems

Factor

System

Fibrinogen (I)

Clauss method: modified thrombin clotting time

Prothrombin (II)

Prothrombin time

Factor V

Prothrombin time

Factor VII

Prothrombin time

Factor VIII

Partial thromboplastin time

Factor IX

Partial thromboplastin time

Factor X

Prothrombin time

Factor XI

Partial thromboplastin time

Factor XIII

Chromogenic assay

PT, prothrombin time; PTT, partial thromboplastin time; TCT, thrombin clotting time.

Factor XIII assay

Coagulation factor XIII is a transglutaminase that catalyzes covalent cross-links between the α and γ chains of fibrin polymer.89 Cross-linking strengthens the fibrin clot and renders it resistant to proteases. This is the final event in coagulation, and it is essential for normal hemostasis and normal wound healing. Factor XIII from plasma, platelets, and tissue function identically. Neither the PT nor the PTT is prolonged by factor XIII deficiency.

Inherited factor XIII deficiency, an autosomal recessive disorder, affects both sexes in all races. The first report of the deficiency appeared in 1960, and the frequency is estimated at 1 in 2 million. Factor XIII levels also may be low in chronic DIC secondary to Crohn disease, leukemias, ulcerative colitis, sepsis, inflammatory bowel disease, surgery, and Henoch-Schönlein purpura. In these cases, the factor XIII level decreases to 50% of normal, not low enough to create symptoms, although occasionally acquired factor XIII deficiencies produce low enough levels to cause mild bleeding. Acquired factor XIII inhibitors have been described in patients treated with isoniazid, penicillin, valproate, and phenytoin.90 These drugs may cause complete absence of factor XIII.

Factor XIII activity levels lower than 5% result in hemorrhage. In congenital factor XIII deficiency, bleeding is evident in infants, with seepage at the umbilical stump.91 In adults, bleeding is slow but progressive, accompanied by poor wound healing and slowly resolving hematomas. Recurrent spontaneous abortion and posttraumatic hemorrhage are common. Acquired factor XIII inhibitors cause severe bleeding that does not respond to therapy.

When a patient comes for treatment of bleeding and poor wound healing and the PTT, PT, platelet count, and fibrinogen level are normal, the laboratory practitioner may recommend a factor XIII assay such as the Technochrom Factor XIII (DiaPharma Group, Inc., West Chester, OH).92 In this representative assay, quantitation of factor XIII activity is based on the measurement of ammonia released during an in vitro transglutaminase reaction. Plasma factor XIII is first activated by reagent thrombin. The resultant factor XIIIa then cross-links the fibrin amine substrate glycine ethyl ester to the glutamine residue of a peptide substrate, releasing ammonia. The concentration of ammonia is monitored in a glutamate dehydrogenase–catalyzed reaction that depends on NADPH, the reduced form of nicotinamide adenine dinucleotide phosphate. NADPH consumption is measured by the decrease of absorbance at 340 nm. The absorbance is inversely proportional to factor XIII activity. Several manufacturers market immunoassays for factor XIII, which provide factor XIII concentration but do not identify functional factor XIII abnormalities.

Tests of fibrinolysis

Quantitative D-dimer immunoassay

Physiology of fibrin degradation products and D-dimers

During coagulation, fibrin polymers become cross-linked by factor XIIIa and simultaneously bind plasma plasminogen and tissue plasminogen activator (TPA) (Chapter 37). Over several hours, bound TPA activates nearby plasminogen to form plasmin. The bound plasmin cleaves fibrin and yields the FDPs D, E, X, and Y and D-dimer. The FDPs represent original fibrinogen domains, and D-dimers are covalently linked D domains reflecting the cross-linking effects of factor XIIIa. Assays for FDPs, including D-dimer, are convenient for detecting active fibrinolysis, which indirectly implies the occurrence of thrombosis. Normally, FDPs, including D-dimer, circulate at concentrations of less than 2 ng/mL. Fibrinolysis yields FDPs and D-dimer at concentrations greater than 200 ng/mL. Increased FDP and D-dimer concentrations are characteristic of acute and chronic DIC, systemic fibrinolysis, deep vein thrombosis, and pulmonary embolism.93 FDPs, including D-dimer, also are detected in plasma after thrombolytic therapy.94

Principle of the quantitative d-dimer assay

Plasma D-dimer immunoassays abound, and several diagnostics distributors offer automated quantitative immunoassays for plasma D-dimers that generate results within 30 minutes.95 Microlatex particles in buffered saline are coated with monoclonal anti–D-dimer antibodies. The coated particles are agglutinated by patient plasma D-dimer; the resultant turbidity is measured using turbidometric or nephelometric technology. Sensitivity varies, depending on the avidity of the monoclonal anti–D-dimer and the detection method; however, most methods detect concentrations as low as 10 ng/mL.

Clinical value of the quantitative D-dimer assay

The quantitative D-dimer assay is essential for ruling out venous thromboembolic disease in patients with low pretest probability and is required for detecting and monitoring DIC (Chapter 39).96 The D-dimer assay helps rule out acute myocardial infarction and ischemic stroke and may be used to monitor the efficacy of Coumadin therapy.97 The various quantitative D-dimer assays have negative predictive values of 90% to 95% and may be used to rule out deep vein thrombosis and pulmonary thrombotic emboli in patients at low risk without resorting to compression ultrasonography, tomography, or venous imaging.98,99Because of the high sensitivity but low specificity (60% to 70%) of the quantitative D-dimer assay, laboratory practitioners do not use this assay to positively diagnose venous thromboembolic disease but only to rule it out. Because any chronic or acute inflammation is accompanied by elevated D-dimer concentrations, the assay cannot be used to “rule in” thromboembolic disease. The upper limit of the reference interval for the quantitative D-dimer assay varies with the methodology, ranging from 250 ng/mL to 500 ng/mL. In DIC, D-dimer levels may reach 10,000 to 20,000 ng/mL.

Qualitative D-dimer assay

The automated quantitative D-dimer assay has largely replaced manual D-dimer or FDP assays. The SimpliRED D-dimer assay (BBInternational, Inc., Dundee, United Kingdom) is a manual method that uses visible latex particles coated with monoclonal antibody. The SimpliRED D-dimer assay is suited to low-volume or near-patient (point-of-care) applications. The manufacturer reports the clinical sensitivity for pulmonary embolus to be 94% and the specificity to be 67%.100

Fibrin degradation product immunoassay

Although the FDP assay has largely been replaced by the automated quantitative D-dimer assay or the manual semiquantitative D-dimer assay, FDPs may be detected using a semiquantitative visible agglutination immunoassay.101 One such method is the 1972 Thrombo-Wellcotest (Remel, Inc., Lenexa, KS).102 Polystyrene latex particles in buffered saline are coated with polyclonal antibodies specific for D and E fragments calibrated to detect FDPs at a concentration of 2 μg/mL or greater. The assay usually is performed on serum collected in special tubes that promote clotting and prevent in vitro fibrinolysis, although plasma-based assays are also available.

Plasminogen chromogenic substrate assay

Excessive fibrinolytic activity occurs in a variety of conditions. Inflammation and trauma may be reflected in a radical increase in circulating plasmin that has the potential to cause hemorrhage. Bone trauma, fractures, and surgical dissection of bone, as in cardiac surgery, may raise fibrinolysis activity.103 Fibrinolysis deficiencies occur when TPA or plasminogen levels become depleted or when excess secretion of PAI-1 depresses TPA activity. Plasminogen, the precursor of the trypsin-like proteolytic enzyme plasmin, is produced in the liver and circulates as a single-chain glycoprotein (Chapter 37). When bound to fibrin, plasminogen is converted to plasmin by the action of nearby TPA. Bound plasmin degrades fibrin, whereas a circulating inhibitor, α2-antiplasmin, rapidly inactivates free plasmin.

Congenital plasminogen deficiencies are associated with thrombosis in some families.104 Acquired plasminogen deficiencies are seen in DIC and acute promyelocytic leukemia.105 Thrombolytic therapy is ineffective when plasminogen activity is low. Plasminogen is readily measured in PPP using a chromogenic substrate assay, available from several manufacturers.

Principle of the plasminogen chromogenic substrate assay

Chromogenic substrates employ synthetic oligopeptides whose amino acid sequences are designed to be specific for their chosen enzymes. Plasmin hydrolyzes a bond in the oligopeptide sequence valine-leucine-lysine (Val-Leu-Lys). A fluorophore or a chromophore such as para-nitroaniline (pNA) is covalently bound to the carboxyl terminus of the oligopeptide and may be released on digestion. S-2251, composed of H-D-Val-Leu-Lys-pNA, is a chromogenic substrate for plasmin. On plasmin digestion, the pNA is released and transforms from a colorless liquid to yellow (). Figure 42-13

Image 

FIGURE 42-13 Assay of plasma plasminogen using the chromogenic substrate method. Reagent streptokinase activates plasminogen to form plasmin. R-pNA designates a chromogenic substrate, where R indicates one of several choices of peptide sequence and pNA (para-nitroaniline) is the chromophore. In the case of plasminogen, the R represents the peptide sequence valine-leucine-lysine (Val-Leu-Lys). Plasmin recognizes the Val-Leu-Lys amide sequence as its enzymatic cleavage site, releasing the pNA, which generates a yellow color.

Streptokinase is an exogenous plasminogen activator derived from cultures of β-hemolytic streptococci. Streptokinase is added to patient PPP, where it binds and activates plasminogen. The resulting streptokinase-plasmin complex reacts with a chromogenic substrate such as S-2251 to release a color whose intensity is proportional to the plasminogen concentration. Several analogous chromogenic and fluorogenic substrates are suitable for plasminogen measurement. Control plasma is tested with the patient plasma, and the results are recorded.

Results and clinical utility of the plasminogen chromogenic substrate assay

The plasminogen reference interval is 5 to 13.5 mg/dL. Plasminogen levels are decreased in thrombolytic therapy, DIC, hepatitis, and cancer. Hereditary deficiencies have also been recorded.101 Decreased plasminogen is associated with thrombosis. Plasminogen rises in inflammation and during pregnancy, and high levels may be associated with hemorrhage. Plasminogen levels may also be elevated in systemic fibrinolysis. Plasminogen assays are seldom offered in acute care facilities but are readily available at specialty reference laboratories.

Tissue plasminogen activator assay

Physiology of tissue plasminogen activator

The two physiologic human plasminogen activators are TPA and urokinase.106107 TPA is synthesized in vascular endothelial cells and released into the circulation, where its half-life is approximately 3 minutes and its plasma concentration averages 5 ng/mL. Urokinase is produced in the kidney and vascular endothelial cells and has a half-life of approximately 7 minutes and a concentration of 2 to 4 ng/mL. Both activators are serine proteases that form ternary complexes with bound plasminogen at the surface of fibrin, activating the plasminogen and initiating thrombus degradation. The endothelial secretionplasminogen activator inhibitor-1 (PAI-1) covalently inactivates both.

Clinical significance of tissue plasminogen activator

The reference interval upper limit for TPA activity is 1.1 units/mL, and the upper limit for TPA antigen is 14 ng/mL. TPA is the primary mediator of fibrinolysis and is the model for synthetic TPA (Activase; Genentech, Inc., South San Francisco, CA). Decreased TPA levels may indicate increased risk of myocardial infarction, stroke, or deep vein thrombosis, although more data are needed to verify a relationship.108Impaired fibrinolysis in the form of TPA deficiency or PAI-1 excess also is associated with deep vein thrombosis and myocardial infarction.109

Specimen collection for the tissue plasminogen activator assay

TPA activity exhibits diurnal variation and rises upon exercise. Further, TPA is unstable in vitro because it rapidly binds PAI-1 after collection. For specimen collection, patients should be at rest, tourniquet application should be minimal, the phlebotomist should record the collection time, and immediate acidification of the specimen in acetate buffer is necessary.110 Acidification may be accomplished using the Stabilyte acidified citrate tube (Diagnostica Stago, Inc., Parsippany, NJ). Supernatant PPP may be frozen at –70° C until the assay is performed.

Principle of the tissue plasminogen activator assay

Plasma concentration of TPA antigen may be estimated by enzyme immunoassay. To measure TPA activity, a specified concentration of reagent plasminogen is added to the patient plasma (Chromolyse TPA Activity; Diagnostica Stago, Inc, Parsippany, NJ). Plasma TPA activates the plasminogen, and the resultant plasmin activity is measured using a chromogenic substrate. The resulting color intensity is proportional to TPA activity (). The system may incorporate soluble fibrin to increase TPA activity. Figure 42-14

Image 

FIGURE 42-14 To assay tissue plasminogen activator (TPA), plasma that contains TPA is added to plasminogen to produce plasmin. Plasmin activity is measured using the same chromogenic substrate system as in the plasminogen assay illustrated in Figure 42-13. The intensity of color is proportional to TPA activity. pNA, Para-nitroaniline; R, variable peptide sequence.

Plasminogen activator inhibitor-1 assay

PAI-1 is produced by vascular endothelial cells and hepatocytes and circulates in plasma bound to vitronectin at an average concentration of 10 μg/L with diurnal variations.111 An inactive form of PAI-1 circulates in high concentrations in platelets.112 PAI-1 inactivates free TPA by covalent binding. Elevated PAI-1 is associated with venous thrombosis and may be a cardiovascular risk factor (Chapter 39). A few cases of PAI-1 deficiency have been reported; however, hemorrhage apparently occurs only in the complete absence of PAI-1.

Blood is collected from patients at rest into an acidified citrate tube (Stabilyte) and centrifuged immediately to make PPP; this avoids in vitro release of platelet PAI-1. Several immunometric and chromogenic substrate methods are available for estimation of PAI-1 antigen and PAI-1 activity, respectively. One enzyme immunoassay for functional PAI-1 uses urokinase to bind PAI-1. The urokinase–PAI-1 complex is immobilized with solid-phase monoclonal anti–PAI-1 and is measured using monoclonal anti-urokinase immunoglobulin as the detecting antibody.113114

Most chromogenic substrate kits for PAI-1 use an indirect measurement approach (Spectrolyse PAI-1; Diagnostica Stago, Inc, Parsippany, NJ). Patient PPP is mixed with a measured amount of reagent TPA. Residual TPA is assayed in the plasminogen system as shown in Figure 42-15. The resulting color intensity is inversely proportional to plasma PAI-1 activity.

Image 

FIGURE 42-15 To assay plasminogen activator inhibitor 1 (PAI-1) activity, plasma containing PAI-1 is added to reagent tissue plasminogen activator (TPA) of known concentration. The residual TPA is assayed, as shown in Figure 42-14. The intensity of color is inversely proportional to PAI-1 level. pNA, Para-nitroaniline; R, variable peptide sequence.

Confirmation of total PAI-1 deficiency may be accomplished using the serum PAI-1 assay. In serum, platelet PAI-1 is expressed in excess. In true PAI-1 absence, no PAI-1 is detectable in serum.

Global coagulation assays

The TEG Thromboelastograph Hemostasis Analyzer (Haemoscope Corporation, Niles, IL, a division of Haemonetics Corporation) and the ROTEM (Tem, Inc, Durham, NC) are global whole-blood analyzers that measure clotting time and dynamics, clot strength, antithrombotic effects, platelet effects on clot dynamics and strength, and fibrinolysis.115 Both are used as a manual coagulometers, mainly in liver and cardiac surgeries and are described in detail in Chapter 44.

Summary

• Proper hemostasis specimen collection, transport, storage, and centrifugation ensure a valid test result.

• Specimens that are short draws, clotted, or hemolyzed are rejected.

• Platelet aggregometry helps determine the cause of nonthrombocytopenic mucocutaneous bleeding.

• VWD is diagnosed and monitored through the judicious selection and performance of platelet-based laboratory tests.

• The plasma markers PF4 and β-thromboglobulin are research applications used to assess platelet activation.

• Clot-based coagulation screening tests include the ACT, PT, PTT, and TCT.

• Mixing studies are used to detect factor deficiencies, LAs, and specific factor inhibitors.

• Coagulation factor assays are used to detect and measure coagulation factor deficiencies.

• Bethesda titers are used to detect and measure coagulation factor inhibitors.

• Tests of fibrinolysis include assays for FDPs, D-dimer, plasminogen, TPA, and PAI-1.

• Thromboelastography and thromboelastometry are widely used global hemostasis assay methods.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. What happens if a coagulation specimen collection tube is underfilled?

a. The specimen clots and is useless

b. The specimen is hemolyzed and is useless

c. Clot-based test results are falsely prolonged

d. Chromogenic test results are falsely decreased

2. If you collect blood into a series of tubes, when in the sequence should the hemostasis (blue stopper) tube be filled?

a. After a lavender-topped or green-topped tube

b. First, or after a nonadditive tube

c. After a serum separator tube

d. Last

3. What is the effect of hemolysis on a hemostasis specimen?

a. In vitro platelet and coagulation activation occur

b. The specimen is icteric or lipemic

c. Hemolysis has no effect

d. The specimen is clotted

4. Most coagulation testing must be performed on PPP, which is plasma with a platelet count less than:

a. 1000/μL

b. 10,000/μL

c. 100,000/μL

d. 1,000,000/μL

5. You wish to obtain a 5-mL specimen of whole-blood/anticoagulant mixture. The patient’s hematocrit is 65%. What volume of anticoagulant should you use?

a. 0.32 mL

b. 0.5 mL

c. 0.64 mL

d. 0.68 mL

6. You perform whole-blood lumiaggregometry on a specimen from a patient who complains of easy bruising. Aggregation and secretion are diminished when the agonists, thrombin, ADP, arachidonic acid, and collagen are used. What is the most likely platelet abnormality?

a. Storage pool disorder

b. Aspirin-like syndrome

c. ADP receptor anomaly

d. Glanzmann thrombasthenia

7. What is the reference assay for HIT?

a. Enzyme immunoassay

b. Serotonin release assay

c. Platelet lumiaggregometry

d. Washed platelet aggregation

8. What agonist is used in platelet aggregometry to detect VWD?

a. Arachidonic acid

b. Ristocetin

c. Collagen

d. ADP

9. Deficiency of which single factor is likely when the PT result is prolonged and the PTT result is normal?

a. Factor V

b. Factor VII

c. Factor VIII

d. Prothrombin

10. A prolonged PT, a low factor VII level, but a normal factor V level are characteristic of an acquired coagulopathy associated with which of the following?

a. Hemophilia

b. Liver disease

c. Thrombocytopenia

d. Vitamin K deficiency

11. The patient has deep vein thrombosis. The PTT is prolonged and is not corrected in an immediate mix of patient plasma with an equal part of normal plasma. What is the presumed condition?

a. Factor VIII inhibitor

b. Lupus anticoagulant

c. Factor VIII deficiency

d. Factor V Leiden mutation

12. What condition causes the most pronounced elevation in the result of the quantitative D-dimer assay?

a. Deep vein thrombosis

b. Fibrinogen deficiency

c. Paraproteinemia

d. DIC

13. What is the name given to the type of assay that uses a synthetic polypeptide substrate that releases a chromophore on digestion by its serine protease?

a. Clot-based assay

b. Molecular diagnostic assay

c. Fluorescence immunoassay

d. Chromogenic substrate assay

14. What component of the fibrinolytic process binds and neutralizes free plasmin?

a. PAI-1

b. TPA

c. α2-Antiplasmin

d. Urokinase

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