Bleeding disorders – How can it be evaluated?

Abstract

AIM: To review the control of bleeding disorders using clinical laboratory methods.

METHODOLOGY: Various scientific procedures were consulted to demonstrate the tests applicable to bleeding disorders and its clinical implications.

CONCLUSION: All bleeding disorders investigation should start with the screening tests and when necessary, specific coagulation factor assays should be undertaken.

KEYWORDS: Bleeding, Disorders, Evaluation

Introduction

Most hemostasis laboratory procedures require plasma from a whole blood specimen collected by venepuncture and mixed 9:1 with a 3.2 % solution of sodium citrate anticoagulant. Those incharge of blood collection must adhere closely to published protocols for specimen collection ,transport and management. The supervisor is responsible for the current validity of specimen collection and handling protocols and insures that personnel employ approved techniques. Patients need not fast for hemostasis testing and little special preparation is required before collection of hemostasis samples. Most samples are collected in plastic blue stopper sterile blood collection tubes containing a measured volume of 0.105 to 0.109 mol/l (3.2%) of buffered sodium citrate anticoagulant. Tubes of uncoated soda- lime glass are unsuitable because their negative surface charge activates platelets and plasma procoagulants.

Hemostasis sample collection tubes

The person incharge of blood collection must observe certain rules in hemostasis specimen collection, the rules are as follows:

1.If the hemostasis samples is part of a series of tubes from a single venepuncture site, it must be collected first or immediately after a non additive tube Gottfried & Adachi (1997). The hemostasis tube may not immediately follow tubes that contain heparin, ethylenediamine tetra acetic acid (EDTA), sodium fluoride or clot promoting materials as are found in plastic red stopper or in serum separator (gel, red and gray stopper) tubes. These additives may transfer to the hemostasis specimen on the stopper needle and invalidate all hemostasis test results.

2.The ratio of whole blood to anticoagulant should be 9:1. Evacuated tubes are designed so that the vacuum draws the correct volume of blood. Collection tube manufacturers indicate the allowable range of collection volume error. In most cases, the volume of blood collected must be within 90% of the calibrated volume. A short draw generates erroneously prolonged clot based coagulation test results because the relative excess anticoagulant neutralizes test reagent Calcium, Adcock D M et al (1998). Smaller tubes provide less tolerance for short draws.

3.When blood samples are collected using winged needle butterfly sets, the phlebotomist must compensate for the internal volume of the tubing, usually 0.5ml. If a single tube is collected using a needle set, the phlebotomist should collect a non additive discard tube first. This ensures that the needle set tubing is filled before the hemostasis specimen is collected.

4.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 six times to mix the blood and anticoagulant and prevent clot formation. The clinical laboratory technologist must visually examine for clots again just before centrifugation and testing.

5.Excessive specimen agitation causes hemolysis, procoagulant activation and platelet activation, the phlebotomist must never shake the tube.

6.The test results from visibly hemolyzed specimens are unreliable and the specimen must be recollected.

7.Specimen that are lipemic or icteric may give erroneous results when the laboratory employs optical coagulometers for clot-based testing. When specimens are cloudy or highly colored, the technologist employs an alternative mechanical detection system. Lipemia and icterus may cause a recollect Ma Z et al (1997).

8.Slowed or stopped venous circulation is stasis. Stasis causes local concentration of factor V??? / Von Willebrand factor (VWF), which may result in false shortening of clot based coagulation test results. When collecting blood the phlebotomist must remove the tourniquet within 1 minute of its application to avoid stasis Palkuti H S (1988).

Hemostasis specimen management

Specimens collected for platelet aggregometry are maintained at 18oc to 24oc never at refrigerator temperatures (2oc to 4oc). Specimens collected for the PT may be maintained at 18oc to 24oc and the PTT at 2oc to 4oc or maintain room temperature during transport but should never be stored at temperatures greater than 24oc. Heat destroys the activity of coagulation factor V???. Specimens collected for PT testing are held at 18oc to 24oc and tested within 24 hours of the time of collection Ens & Newlin (1995).

Specimens collected for PT testing may be held at either 18oc to 24oc or 2oc to 4oc and tested within 24 hours of the time of collection. Specimens collected for PTT testing also may be held at the same temperature as that of the PT but must be tested within 4 hours of the time of collection, provided that specimen does not contain unfractionated heparin anticoagulant. If a patient is receiving unfractionated heparin therapy, specimens for PTT testing must be centrifuged within 1 hour of the time of collection and the plasma, which should be platelet poor plasma(PPP) ,must be tested within 4 hours of the time of collection, Adcock & Kressin(1998).

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 18oc to 24oc until testing. Chilling destroys platelet activity. Aggregometry may be started immediately and must be completed within 3 hours of specimen collection. The technologist mixes the specimen by gentle inversion. Check the sample for clots just before testing and rejects specimens with clots.

Platelet rich plasma specimens used for platelet aggregometry.

Optical platelet aggregometers are designed to test platelet rich plasma( PRP), Plasma with a platelet count of 200 to 300 × 109/L. Sodium citrate – anticoagulant blood first is checked visually for clots, then centrifuge at 50g for 30 minutes with the stopper in place to maintain the PH.

Platelet poor plasma required for most clot based testing

Most clot based plasma coagulation tests require PPP. PPP is plasma with a platelet count of less than 10 × 109/L Pierangeli & Harris (2005). The technologist routinely counts coagulation plasmas to ensure they are platelet poor and inspects the PPP for hemolysis, lipemia and icterus. Visible hemolysis implies platelet or coagulation pathway activation, so the specimen is rejected, and a new specimen is collected. If the hemostasis test cannot be completed within the prescribed interval, the technologist must centrifuge the sample at a force sufficient to yield PPP. The supernatant PPP must be transferred by plastic pipette to a plastic tube, stoppered, quick- frozen and stored at -20oc for 2 weeks or less or -70oc for 6months or less. At the time of testing, the sample must be thawed rapidly at 37oc , mixed well and tested within 1 hour of the time it is removed from the freezer. If it cannot be tested immediately, the sample may be stored at 2oc to 4oc for 2 hours after thawing.

Platelet function tests

A platelet count is performed and the blood film is reviewed before beginning platelet function tests because thrombocytopenia is a common cause of mucocutaneous hemorrhage, Bick(1992). Qualitative platelet abnormalities are suspected only when systemic hemorrhagic symptoms such as easy bruising, petechiae, Purpura or epistaxis are present and the platelet count exceeds 50 × 109/L. Although, hereditary platelet function disorders are rare, acquired disorders associated with hemorrhage and thrombosis are common. Acquired defects often are associated with liver disease, renal disease, myeloproliferative disorders, myelodysplastic syndromes, myeloma, uremia, autoimmune disorders, anemias and drug therapy.

Bleeding time for platelet function-template method

The bleeding time test is occasionally helpful for diagnosis of an unexplored bleeding disorder, although it fails as a screen, Lind (1991).

Principle: A standard incision is made on the volar surface of the forearm and the time the incision bleeds is measured. Cessation of bleeding indicates the formation of hemostatic plugs, which are in turn dependent on an adequate number of platelets and on the ability of the platelets to adhere to the subendothelium and to form aggregates Rodgers & Levin (1990).

Normal range is 2 – 7 minutes. An upper limit of 4 minute has been reported in one study on men and women who had not used aspirin or other relevant drugs in the ten days before the test, Bain & Forster (1980). Ideally, every laboratory should determine its own normal reference range and if possible ensure that the test is performed by the same operator.

Ivy´s Method

Ivy´s method is similar to the template method, but instead of a standardized incision, two separate punctures 5 – 10 cm apart are made in quick succession using a disposable lancet. Any microlance with a cutting depth of 2.5mm and width of just more than 1mm is suitable; it can be inserted to its maximum depth without fear of penetrating too deeply. A source of inaccuracy with Ivy´s method is the tendency for the puncture wound to close before bleeding has ceased. Normal range is 2- 7 minutes, ideally, every laboratory should determine its own normal reference range and if possible ensure that the test is performed by the same operator.

Interpretation of results

A prolonged bleeding time may result from the following:

• 1. Thrombocytopenia

• 2. Disorders of platelet function

• 3. Von Willebrand disease

• 4. Vascular abnormalities

Limitation of the bleeding time test is that it suffers from inherent variability and does not correlate well with the incidence of clinically significant bleeding.

Clot- based plasma procoagulant screen

The Lee- White whole blood coagulation time test, described in 1913, was the first laboratory procedure designed to assess coagulation disorders, Lee-White (1913). The Lee- White is no longer used, but it was the first invitro clot procedure to employ a pervasive principle: The time interval from the initiation of coagulation to visible clot formation reflects the condition of the coagulation mechanism. A prolonged clotting time indicates coagulation inadequacy. A 1953 modification , the activated clotting time supplies a particulate clot activator in the collection tube, speeding the clotting process. The activated clotting time is still used to monitor heparin therapy in high dosage applications such as cardiac surgery. The standard clinical battery of clot based coagulation screening tests which consists of the PT, PTT and thrombin clotting time (TCT), uses the clotting time principle of Lee-White and activated clotting time. Many additional specialized tests, such as coagulation factor assay, tests of fibrinolysis, inhibitor assays, Reptilase time, Russell viper venom time, dilute Russell venom time and tests for circulating anticoagulants, also are based on the relationship between time to clot formation and coagulation function.

Prothrombin Time (PT)

Principle: PT thromboplastin reagents are prepared from recombinant or affinity purified tissue factor suspended in phospholipids mixed with a buffered 0.025m solution of Calcium Chloride. When mixed with citrated PPP, the PT reagent triggers fibrin polymerization by activating plasma factorV??. The clot is detectable visually or by optical or electromechanical sensors. Although the coagulation scheme implies that the PT would be prolonged in deficiencies of fibrinogen, prothrombin and factors V, V?? and factor X, the procedure is most sensitive to factor V?? deficiencies, moderately sensitive to factor V and factor X deficiencies, sensitive to severe fibrinogen and prothrombin deficiencies and insensitive to deficiencies of factors V??? and IX ,Talstad (1993). The PT is prolonged in multiple factor deficiencies that include deficiencies of factor V?? and X and is used most often to monitor the effects of oral anticoagulant warfarin (Coumadin) therapy. The PT is tested in duplicate and always tested along with control plasma. PT up to 15 seconds are considered within the normal reference range .

Reporting prothrombin time results and international Normalized Ratio(INR).

In view of the inherent variations among thromboplastin reagents, most laboratories report the INR for stably anticoagulated patients using the formular of Poller (1986);

INR= (PTpatient /PTgeometric mean of normal)ISI where PT patient is PT of 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.

PT as a diagnostic assay

The PT is performed diagnostically in any suspected coagulopathy. Acquired multiple deficiencies such as disseminated intravascular coagulation (DIC), liver disease and vitamin K deficiency all affect factor V?? activity and detected through prolonged PT results. To distinguish between vitamin K deficiency and liver disease, the laboratory technologist performs a factor V and V?? level. Both are reduced in liver disease; only factor V?? is reduced in vitamin K deficiency.

Partial thromboplastin time (PTT)

Principle: The PTT also called the activated partial thromboplastin time (APTT) is performed to monitor the effects of unfractionated heparin therapy and to detect circulating anticoagulants such as Lupus anticoagulant and anti-factor V??? antibody. The PTT also detects all congenital and acquired procoagulant deficiencies except deficiencies of factor V?? or X???. The factor deficiencies that cause hemorrhage and prolonged PTT results taken in the order of reaction are X?, ?X, V???, X and V, prothrombin and fibrinogen. The PTT also is prolonged in the presence of Lupus anticoagulant, an immunoglobulin with affinity for phospholipid- bound proteins and is prolonged further by anti-factor V??? antibody and heparin. Factor V?? and X??? deficiencies have no effect on the PTT. The normal reference range is up 38 seconds depending on the local reference range of the laboratory.

Thrombin clotting time (TCT)

The TCT is prolonged when the fibrinogen level is less than 100mg/dL ( Hypofibrinogenemia) or in the presence of antithrombic materials such as FDPs, paraproteins or heparin. Afibrinogenemia (absence of fibrinogen) or dysfibrinogenemia ( fibrinogen that is biochemically abnormal and non functional) also cause a prolonged TCT. Before a prolonged TCT may be considered to be evidence of diminished or abnormal fibrinogen, the presence of antithrombic activity such as heparin, FDPs or paraproteins must be ruled out. TCT interval is up to 15 to 20 seconds as regards to the local reference range.

Coagulation Factor Assays

Fibrinogen Assay

Principle; The clot based method of Clauss, a modification of the TCT is the recommended procedure for estimating the functional Fibrinogen level. The interval to clot formation is compared with the results of reference plasma. A reference curve is prepared in each laboratory and updated regularly using reference plasma or control plasma that has been calibrated to reference plasma.

Clinical utility of fibrinogen assay

Hypofibrinogenemia is associated with DIC and severe liver disease. Moderately severe liver disease, pregnancy and a chronic inflammatory condition may cause an elevated fibrinogen level. Congenital afibrinogenemia gives prolonged clotting time and is associated with a variable hemorrhagic disorder.

Single factor assay using the partial thromboplastin time test system

If the PTT is prolonged, the PT and TCT are normal and there is no ready explanation for the prolonged PTT such as heparin therapy, lupus anticoagulant or a factor specific inhibitor, the technologist once again may suspect a congenital single factor deficiency. Three factor deficiencies that give this reaction pattern and cause hemorrhage are;

• 1. Factor V???- Hemophilia A

• 2. Factor ?X- Hemophilia B

• 3. Factor X?- Hemophilia C

Because hemophilia A is the most common single factor deficiency disorder, our testing is limited to factor V??? assay only, but the same protocol applies to others as well. The technologist uses the PTT system to estimate the concentration of functional factor V??? by incorporating commercially prepared factor V???- depleted plasma in the test system. In the PTT system, factor V???- depleted plasma provides normal activity of all procoagulants but not factor V???. Tested alone, factor V???- depleted plasma reagent has a prolonged PTT but when normal PPP is added, the PTT reverts to normal. In contrast a prolonged result on a mixture of patient and factor V???- depleted PPP implies that the patient PPP is deficient in factor V???. The clotting time interval of the patient PPP/factor V???- depleted PPP mixture may be compared with a previously prepared reference curve to estimate the level of factor V??? activity in the patient PPP.

Clinical Utility

The reference range for factor V??? activity is 50% to 150%. Symptoms of hemophilia are clinically evident at activity levels of 10% or less.

Single Factor Assay using the Prothrombin Time Test system

If the PTT and PT are prolonged, the TCT is normal and there is no ready explanation for the prolonged test results, such as liver disease, DIC, oral anticoagulant therapy or vitamin K deficiency, the technologist may suspect a congenital single factor deficiency. Three rare factor deficiencies that gives this reaction pattern and cause hemorrhage are prothrombin, factor V or factor X deficiency. If the PT is prolonged and all other test results are normal, factor V?? deficiency is suspected. The Principles and procedure given in the section on single factor assay of PTT system could be applied except that tissue thromboplastin reagent replaces the PTT reagent in the test system and the PT protocol is followed. Prothrombin depleted and factor V depleted, factor V??- depleted and factor X depleted plasma are commercially available.

Factor X??? Assay : 5 mol/l Urea solubility Test

Coagulation factor X??? is a transglutaminase that catalyzes covalent cross links between the a and ? chains of fibrin polymer, Southern D K (1992). Crosslinking strengthens the fibrin clot and renders it resistant to proteases. This is the final event in plasma coagulation and it is essential for normal hemostasis and normal wound healing processes.

Euglobulin clot lysis test

Excessive fibrinolytic activity occurs in a variety of conditions. Inflammation and trauma may be reflected in a radical increase in circulating plasmin that causes hemorrhage. Bone trauma, fractures and surgical dissection of bone as in cardiac surgery may cause increases in fibrinolysis Vinazzer (1988).

Fibrinolysis deficiencies occur when tissue plasminogen activator(TPA) or plasminogen levels become depleted or when excess secretion of plasminogen activator inhibitor (PAI-1) depresses TPA activity. A time honored approach to measurement of fibrinolytic activity is the euglobulin lysis test.

Limitation of euglobulin clot lysis time

Hypofibrinogenemia and factor X??? deficiency affect the euglobulin lysis time. In hypofibrinogenemia, there is less fibrin to be lysed and a short lysis time may be seen without a genuine increase in fibrinolytic activity. In factor X??? deficiency, the original clot quality is poor and dissolution by normal levels of plasmin is more rapid. The euglobulin clot lysis time also may be falsely prolonged in chronic inflammation if the fibrinogen level exceeds 600mg/dl.

Conclusion: In most cases of bleeding disorders only the screening tests are sufficient for the patient and treatment could be successful without further investigation. However, in some patients, more specific factor assays could be undertaken to actually identify and implicate the factor that is responsible.

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Autor:

Peter Ubah Okeke

Student, School of Science & Engineering

Atlantic International University, Hawaii (www.aiu.edu)

2011