Giese s ‘LIntraduction Sections | About Introduction Welcome to the Inhibitor-Inactivator module! This module consists of 6 sections. Prothrombin Time (PT) In this module we cover: Activated Partial Thromboplastin + Acquired inhibitors of blood coagulation Time (APTT) + Principles behind the APTT inhibitor screening assay and the complete inhibitor-nactivator test Thrombin Time (TT) + Interpretation of inhibitor-inactivator test results Factor V Assay ome “This module is pat of a series. Click the right arrow to continue.eee nee 4. Introduction Sections | About Learning Objectives After completing this module, you will be able to: + Describe the APTT inhibitor screening assay. Interpret the possible outcomes of an inhibitor-inactivator screening assay. Describe when an inhibitor-inactivator assay should be ordered. Describe the Bethesda Titer Technique. Define an antiphospholipid antibody (lupus anticoagulant) and explain how it may affect the APT. Explain the difference between an antiphospholipid antibody, a nonspecific inhibitor and an antibody to a factor. When given a set of numbers, analyze them to determine if the patient has a factor deficiency, lupus anticoagulant, nonspecific inhibitor or an antibody to a factor. Determine when confirmatory testing is required and how to interpret i. | Page 20110 D> UNIVERSITY oF MINNESOTAaan 4. Introduction ‘The Purpose of the Int itor/inactivator Test ‘The purpose of the inhibitor/inactivator testis to screen for ‘a possible inhibitor or inactivator in a patient. Sometimes it also detects a factor deficiency. + An inhibitor slows down the ability of blood to coagulate. + An inactivator, since itis an antigen-antibody reaction, destroys a factor and prevents complete coagulation from taking place. ‘We will discuss inhibitors and inactivators in more detail throughout the module. Click the right arrow to continue. (lees ‘Sections | About Inhibitor Slows down the ability of blood to coagulate. Inactivator Destroys a factor and prevents complete coagulation from taking place. cach nianAPTT Inhibitor-Inactivator Cee elo ‘Introduction Sections | About Screening Tests In this module we describe principles of the inhibitor screening test and the inhibitor-inactivator test. APTT inhibitor screening assay GUIneArT bnaeceeans canine be Dierentiates between inhibitors and Siferontctes between inhbiors and factor deficiencies in factor deficiencies in patients patients. If this screening test is positive for an inhibitor (or Fan inactivator s suspected ina hemophilia), then the | Caplets wiibhiorwectvatr teats pecemred Complete inhibitor/inactivator test inhibitors, (2) The complete inhibitorfinactivator test. This test ponpstesetakingeeansnosinen Essesses factor deficiencies, inhibitors, inacvator, and antbocies Click the right arrow to continue. J Pagesotto D>Gan ee ees An Example Click the right arrow to continue. 4. Introduetion Cee es ‘Sections | About Consider the following example: Bill Smith, a hemophiliac with a factor VIll deficiency underwent total knee replacement last week. He is being treated with factor Vill concentrate and being followed with factor Vill assays. With concentrate, his factor Vill level remained about 150% since his surgery. But today, his factor Vill went down to 10%, ‘The physician wants to know if the concentrate was bad or if Bil has developed a factor VIII antibody to the VIII concentrate with which he is being treated. The doctor orders an inhibitor-inactivator study. |. Page sot 10. > Paceitor-Inactivator CoE ‘Introduction Sections | About APTT Inhibitornactivator tests are done using a procedure called the APTT, which stands for “Activated Partial Thromboplastin Time Activated Since many laboratories don’t do the inhibitor-inactivator Partial procedure anymore, we don't cover the specifics of the lab Procedure n fle module. rowover, You hee ' thuersand Ie prnepleg behind tne APT AMI Thromboplastin Inaevator tot end be ele fo nerret i resus oe Click the right arrow to continue. «|. rage 60110 D> iranaan ‘Introduction Sections | About Acquired Inhibitors Can Develop in People with No Underlying Condi ‘Accoagulation inhibitor can act at any place in the hemostatic system. ‘Acquired inhibitors develop in both + Patients with certain disease states + Some people who have no apparent underlying conditions Click the right arrow to continue. (lees cach nianGinieeneees ‘Introduction Sections | About Acquired inhibitors can develop in people who have no apparent underlying conditions. Select the best answer and click Check Answer. Check Answer Correct! Acquired inhibitors can develop in patients with certain disease states and in some people who have no apparent underlying conditions. ansima eeneee en Ce escisesiad ‘LIntroduetion Sections | About The suspicion of a coagulation inhibitor is often prompted by the unexplained prolongation of the PT or APTT in a patient. OTe © False Select the best answer and click Check Answer. Check Answer Incorrect. The suspicion of a coagulation inhibitor is often prompted by the unexplained prolongation of the PT or APTT in a patient. ‘These inhibitors can act at any place in the hemostatic system. ‘Some inhibitors react immediately and some are time dependent. | Page9.of10 > RecsGan ee ees Cees ‘.Introduetion Sections | About ‘Summary + Inhibitors (sometimes called circulating anticoagulants) are substances produced by the body that inhibit coagulation. + Acquired inhibitors develop in both patients with certain disease states and some people who have no apparent underlying conditions. + The suspicion of a coagulation inhibitor is often prompted by the unexplained prolongation of the PT or APTT in a patient. ‘You have reached the end of this section. Click the right arrow to continue to the next section. | Pace 100t10APTT Inhibitor-Inactivator ‘Two Main Types of Inhibitors/Inactivators In this section, we will discuss two main types of inhibitors/inactivators: (1) Factor antibodies (2) Antiphospholipid inhibitors (lupus anticoagulant) 2. Principles Sections | ‘Two main types of inhi inactivators: (1) Factor antibodies (2) Antiphospholipid inhibitors (lupus anticoagulant) minesGiese s (Meese 2.Principles Sections | About Factor Antibodies ‘The most common antibodies against factors are found in hemophiliacs who are treated. These antibodies are usually directed against factor Vill or factor IX. Ifthe antibody is directed against factor Vill itis called alloanti-Vill antibody. Factor antibodies are seen in about 10% - 20% of factor Vill deficient hemophiliacs and 1%-3% of factor IX deficient hemophiliacs. Antibodies most often develop in severely affected hemophiliacs after replacement therapy for their factor deficiency. J ave 20116 D> ansGites 2.Principles "Sections | About Factor Antibodies (continued) Factor VIll antibodies can also be seen in: + Non-hemophiliacs during or following pregnancy (especially with a retained placenta) + In.some immunologic disorders + Spontaneously in elderly individuals + Following penicillin reaction ‘The clinical course of nonhemophillac patients who develop an antibody to a factor can resemble that of patients with severe hemophilia, which is severe bleeding, ‘These antibodies against factors are time and temperature dependent. ‘These antibodies are called autoanti-Vill antibodi | Pageaot16 >aes 2.Principles | Sections | About Factor Antibodies With alloanti-Vil antibodies (those seen in treated hemophiliacs) there is progressive invitro neutralization of Factor Vill activity with complete inactivation in antibody excess. This is calledType I Kinetics. Type | Kinetics Type Il Kinetics Alloantibodies Autoantibodies 2 2 3 < 2 s f Factor Activity With autoanti-Vill antibodies there is early rapid loss of Factor Vill activity, however residual remains. This is called Type ll Kinetics. |, Pegesorts D> ti cmcon ansGini eeneees 2. Principles | Antiphospholipid Inhibitors Besides factor antibodies, the most common inhibitor is the antiphospholipid inhibitor. ‘The most common types of antiphospholipi (1) Antcardioipin antibody (ACA) Inhibitors are: (2) Lupus anticoagulants (LA) ‘These react against Be - glycoprotein | (an apolipoprotein), prothrombin, and kininogen. ‘Sections | About Common types of antiphospholipid inhibitors: (1) Anticardiolipin antibody (ACA) (2) Lupus anticoagulants (LA) | Pave sot16 D> en nanane e 2. Principles Lupus Anticoagulant ‘One type of antiphospholipid inhibitor is the lupus anticoagulant (LA). It received its name when it was discovered in patients with systemic lupus erythematosus (SLE). Approximately 6% to 16% of patients with SLE develop LA. ‘Though its called the lupus inhibitor, it can also be seen in other disease processes like autoimmune diseases, infections, neoplasias and after certain drugs. ‘Sections | About The lupus anticoagulant (LA) Received its name when it was discovered in patients with systemic lupus erythematosus (SLE) ect ceaGin eee 2.Principles Sections | About Lupus Anticoagulants are Immunoglobulins ‘The lupus anticoagulants are part of a family of autoantibodies that were thought to be directed against phospholipid (antiphospholipid antibodies). ‘They are immunoglobulins (IgG, \gM, or both) which interfere with ane or more of the inviro phospholipid dependent reactions in coagulation, resulting in a prolonged APTT. | raa0 70116 D>Gini eeneees (elie ese 2.Principles | Sections | About How the Lupus Anticagulant is Formed: A Recent Theory ‘The antigen-antibody complexes react with anionic phospholipid or phospholipid-plasma protein complexes in the test system. Recent theory suggests that the lupus anticoagulant is formed in vivo after damage to endothelial cells exposes anionic phospholipids that bind prothrombin and trigger antibody formation. | Paves ot16 D> atedaes 2.Principles | Sections | About Clinical Manifestation ‘The lupus inhibitor by itself rarely or never causes abnormal bleeding. However, ifthe lupus patient has other hemostatic abnormalities such as a low factor Il or a low platelet count, bleeding may occur. While bleeding is rare, thrombosis is associated with the lupus inhibitor in 30% to 40% of affected patients. For this reason, lupus anticoagulant assays are often part ‘ofa thrombosis workup in a patient. Clotting episodes ‘connected to lupus anticoagulants are more often associated with venous thrombosis than arterial thrombosis, | Pageoot1s >> uatedeen 2. Principles | Heparin Its important to remember that heparin is a type of circulating anticoagulant or inhibitor. For this reason, any. plasma to be tested for an inhibitor should first have a TT (thrombin time) done on it to ensure that no heparin is Present, ‘Sections | About Heparin Is a type of circulating anticoagulant or inhibitor | Page 100118 > cena!APTT Inhibitor-Inactivator Corre el 2.Principles Sections | About Indications for Ordering an Inhibitor/Inactivator Screening ‘There are several reasons why inhibitor/inactivator screenings are ordered: + To detect an antibody in 2 hemophiliac before elective surgery or the appearance of an antibody in a hemophiliac during replacement treatment. The detection of the antibody is important as it affects treatment options. + Lupus inhibitors are often ordered as part of a thrombosis workup. In women who have had several spontaneous a lupus anticoagulant may be ordered as part ofthe infertility workup, since LA can cause miscarriages. + Inhibitor/inactivator studies are also ordered to explain an unexplained long APTT in a patient. Page 110118 p> ener eanaa enee es 2. Principles Bethesda Titer Technique ‘Once an antibody has been detected by the inhibitor- inactivator test, quantitation of the antibody that develops in ‘a hemophiliac who has been treated is accomplished by Bethesda Titer Tech the Bethesda Titer technique. In this technique, normal plasma with a Factor Vil activity Pelt ep pet opel det near 100% is mixed at incrementally increasing dilutions 100% is mixed at incrementally increasing with the patient plasma and incubated for 2 hours at $7 lutions with the patient plasma and eqrece Galas, incubated for 2 hours at 37 degrees Celsius. ‘The Factor Vill antibody in the patient wll destroy part of the Factor Vill in the normal plasma. The residual Factor Vill is measured. One Bethesda unit (BU) is defined as the ‘quantity of inhibitor that neutralizes 60% of the Factor Vil in normal plasma in 2 hours at 37 degrees Celcius. The titer result, or Bethesda units, helps the physician to ‘choose the correct therapy for treatment. {| Page 120f 16 >Genres 2.Principles "Sections | About The most common inhibitors against factors are found in hemophiliacs who are treated and are directed against factor VIll or factor IX. etme O False Ss Knowledge Selact the best answer and click Check Answer. Check es Correct! Antibodies most offen develop in severely affected hemophiliacs after replacement therapy for their factor deficiency. The most common inhibitors ‘against factors are found in hemophiliacs who are treated and are directed against factor Vill or factor IX. Factor inhibitors are seen in about 10% of factor Vill deficient hemophiliacs and 1%-3% of factor IX deficient hemophiliacs, J Page 13018.Gin eee 2.Principles Sections | About Although the lupus anticoagulant (LA) is called the lupus inhibitor, it can also be seen: In autoimmune diseases ~ In infections % In neoplasias After certain drugs Select all that apply and click Check Answer. Check Answer Incorrect. All of these are correct. Though it s called the lupus inhibitor, it can also be seen in other disease processes like autoimmune diseases, infections, neoplasias and after certain drugs. 4 Pose Hots6 >eure 2.Principles | Sections | About Any plasma to be tested for an inhibitor should first have a TT done on it to ensure that no heparin is present. OTe © False Select the best answer and click Check Answer. Incorrect. itis important to remember that heparin is a type of circulating anticoagulant or inhibitor. For this reason, any plasma to be tested for an Inhibitor should first have a TT done on it to ensure that no heparin is present. | Page 15 0f16 > cate icaneee nee 2.Principles. "Sections | About Summary * The most common antibodies agains! factors are found in hemophiliacs who are treated and ae directed againt factor Vil or factor | + A.common inhibitor is the lupus anticoagulant (LA). It got its name when it was discovered in patients with systemic lupus erythematosus (SLE). + Itis important to remember that heparin is a type of circulating anticoagulant or inhibitor. For this reason, any plasma to be tested for an inhibitor should first have a TT done on it to ensure that no heparin is present, n- Click the right arrow to continue tothe next section.) age ot 6 D> eerGuanes 53. Sereening Test Introduction In this section we describe the APTT screening assay. This isa screening test that differentiates between inhibitors and factor deficiencies in patients. Click the right arrow to continue. Sections | About APTT Screening Assay Differentiates between inhibitors and factor deficiencies Page 10f 11. > ReaGuanes Cee 3: Sereening Test | Sections | About The APTT Screening Assay ‘The APTT screening assay consists of diluting patient plasma with an equal amount of normalicontrol plasma (which contains about 100% of al factors) and then doing an APTT on the mixture, Because the APTT assay is only sensitive to 40-50%, a + = mixture should correct to the normal range ifa factor deficiency was found in the patient plasma (100% in normal plasma + 0% in the patient stil equals 50%). patient plasma normal plasma (contains 100% ofall factors) “The ost is performed by mixing equal parts of patient plasma and normal plasma, Click the right arrow to continue. of Page 20111. D>Giese s (Meese 3.Sereening Test Sections | About APTT Ifthe APTT corrects into the normal range, a factor deficiency and not an inhibitor is suspected. Ifthe APTT remains prolonged, an inhibitor should be suspected and the complete inhibitor-inactivator test performed. (This test is described in more detail in the next section.) Patient plasma normal plasma (contains 100% of all factors) Click the right arrow to continue. 4 ave sc1t_ D> feces!eee eee Patient plasma normal plasma (contains 100% of all factors) 3. Screening Test mixture ‘Sections | About Question: What should be suspected about this patient? ‘A. The patient is factor deficient ©B. The patient has an inhibitor OC. The inhibitor status of the patient cannot be assessed. Select the best answer and click Check Answer. Check Answer Incorrect. The correct answer is: A ~ the patient is factor, deficient. ‘You can tell because the APTT on the mixture corrected into the normal range (24.2 seconds). Because the patient has a factor deficiency, even if his factor was 0%, when mixed with the 100% in the normal plasma, the result would be '50% and the APTT is not sensitive to this level of deficiency and should correct into the normal range. Remember that the ‘APTT will only remain long if an inhibitor is present. | Pasesot1 > eatenGiese s 3.Sereoning Test Sections | Patient plasma normal plasma (contains 100% of all factors) ‘About What further testing should be done? OA. Factor assays ©. Inhibitorfinactivator study © C. No further testing is needed. Select the best answer and click Check Answer. Wi check answer Incorrect. The correct answer is: A. This patient has a factor deficiency and factor assays should be done to determine which factor is missing. Because the patient has a factor deficiency, inhibitorfinactivator studies will be negative and will not help to further his diagnosis. «| Pages.oft1 > RasAPTT Inhibitor-Inactivator 3. Sereening Test normal plasma mixture (contains 100% of all factors) ‘Sections | About Example 2: What should be suspected about this patient? © A. The patient is factor deficient ©. The patient has an inhibitor ©. The inhibitor status of the patient cannot be assessed, Select the best answer and click Check Answer. Check Answer Incorrect. The correct answer is: B — the patient has an inhibitor. You can tell because the APTT on the mixture stayed long (53.0 seconds) and did not correct into the normal range. (If the patient had a factor deficiency, the APTT would correct into the normal range.) sj rage ott y teasGini eeneees 3. Sereoning Test Patient plasma normal plasma (contains 100% Of all factors) ‘Sections | About What further testing should be done? OA, Factor assays © B, Inhibitorinactivator study CC. No further testing is needed, Select the best answer and click Check Answer. Check Answer Correct. The correct answer is: B. This patient has an inhibitor, s0 doing factor assays on him will not help further his diagnosis. A complete inhibitoriinactivator study Would help define the type of inhibitor this patient has. J Pasezertt easGages 3. Screening Test | Sections | About Drag each of these to the appropriate test result. a ae factor dficlaney and not an inhibitor is suspected. the Vir APTT remains prolonged, an inhibitor shouldbe suspected {and the compe inibtor inactivator tet performed Sean Check AnswerGan ee ees Cees 3.Sereening Test Sections | About If the results of an APTT screening assay show that a patient has an inhibitor, doing factor assays on the patient should be done to further the patient's diagnosis. OTe © False Select the best answer and click Check Answer. Check Answer Correct! if a patient has an inhibitor, doing factor assays will not hele further the patient's diagnosis. A complete inhibitor-inactivator study ‘would be done to help define the type of inhibitor the patient has. | rege vottt_ y enna!Gages 3.Screening Test | Sections | About If the results of an APTT screening assay show that a patient has a factor deficiency, inhibitor-inactivator assays will not help to further the patient's diagnosis. True OF alse Select the best answer and click Check Answer Wi check answer Correct! If patient has a factor deficiency, factor assays should be done to determine which factor is missing. Inhibitor-nactivator studies will be negative and will not help to further the patient's diagnosis. 4] Page 10.0011 >aes 3.Screening Test | Sections | About Summary + The screening test for a possible inhibitor or factor deficiency is to dilute the patient with an equal amount of normal plasma (which ‘contains about 100% of all factors) and do an APT on the mixture. + Because the APTT assay is only sensitive to 40-50%, a mixture should correct to the normal range ifa factor deficiency was found in the patient plasma (100% in normal plasma + 0% in the patient still equals 60%). + Ifthe APTT corrects into the normal range, a factor deficiency and not an inhibitor is suspected. + Ifthe APTT remains prolonged, an inhibitor should be suspected and the complete inhibitor-inactivator test performed. You have reached the end ofthis section, Click the right arrow to continue tothe next section.) pase itofti > tataanaes 4. Interpreting the Results | Sections | Introduction In this section we explain how to perform and interpret the results of the complete inhibitor-inactivator assay in more detall. Recall from the previous section that this testis done if the screening test is positive for an inhibitor or if an inactivator is suspected in a hemophiliac. | Page 10125 > Susa een e 4. Interpreting the Results Inhibitors Inhibitors (sometimes called circulating anticoagulants) are substances produced by the body that inhibit coagulation, The suspicion of an acquired coagulation inhibitor is often prompted by the unexplained prolongation of thePT (Prothrombine Time) or APTT (Activated Partial ‘Thrombopiastin Time) in a patient PT and APTT are measures of the time it takes for the plasma to clot. ‘The majority of these inhibitors are immunoglobulins, either IgG or IgM, either alloantibodies or autoantibodies. ‘Sections | PT Prothrombine Time APTT Activated Partial Thromboplastin Time Page 20125 D> ansGatien 4. Interpreting the Results | Sections | About The Difference Between an Inhibitor and an Inactivator ‘The difference between an inhibitor and an inactivator is, that the inhibitor reacts at 0 time and an activator takes time a to react. An inhibitor Reacts at 0 time An inactivator Takes time to react |, raseset2s D> teen)Gaerne 4. Interpreting the Results Sections | About Inhibitor-Inactivator Assay ‘The first step in an inhibitor-inactivator test is to dilute the - patient serially in normal plasma (control plasma). If i possible, the normal plasma should have an APTT at least 10 seconds shorter than the patient's APT. Page 4025 D> ansaaa ee 4. Interpreting the Results | Sections | About Inhibitor-Inactivator Assay (continued) (Once the dilutions are made, an APTT is performed on: + The normal plasma (control plasma) + Patient plasma + Each dilution The tubes of plasma are then corked, wrapped with parafilm, and set in a 37°C waterbath for 2 hours. After 2 hours, APTTs are done and compared to the APTS at zero time, rast © i rlGian Coie es 4. Interpreting the Results Sections |About The Results ‘The APTT assay worksheet will look something like this: | ave 6ot25 D> necesGites 4. Interpreting the Results Two Important Columns Sections | About ‘The two columns that are important when interpreting an APTT Inhibitor-inactivator are: + The 0 (zero) time column + The Difference column (also known as the “increase or decrease" column) (Note: The 2 hour column is just a working column—no interpretation is done with it.) decrease ‘column | rage 70125 D> (esasaa enee es 4.Intorproting the Results | Sections | About Looking for Factor Deficiency or Inhibitor ‘The 0 column is used to assess factor deficiencies and inhibitors, nace)Giese s (Meese 4. Interpreting the Results Sections | Interpretation of the 0 Column Piensa prolonging the APTT, when the Patient and Control oTime (normal) plasma are mixed together, the APTT should Control 30.2 Even if a patient has 0% of a factor, the Control (normal) 36.4 plasma should have approximately 100% of all factors, so as the equal mix will have 50% of the factor. cena)What does this example show? In this example, you can see that the APTT at 1:2 (56.8) is longer than the initial patient APTT (51.8). The lupus inhibitor destroyed some of the added phospholipid reagent, thus lengthening the time, CoxeGenres Coie es 4. Interpreting the Results Sections | Dilution ‘The inhibitor is continually diluted as more normal plasma is added with each dilution. Eventually the inhibitor is dilute enough that the APTT will return to the normal range. This is seen in the 1:32 dilution of this example. Example of Lupus (inhibitor) | Page 11 of 25 > Resaeee 4. Interpreting the Results “Lupus Like” Inhibitors ‘Sections | ‘About Another result that is sometimes seen in the 0 column is the time not lengthening, but also not returning to the normal range. This is indicative of some inhibitor other than a classic lupus inhibitor (there are “Lupus like” inhibitors) or may indicate a very strong inactivator which is beginning to work at zero time. What does this example show? Click the link for an explanation of this test result. APTT time not lengthening, but also not returning to normal range. 4 age 120125 p> eatenWhat does this example show? In this example, you can see that the APTT at 1:2 Is not longer than the patient, but does not correct into the normal range. Closeaaa eee ce 4. Interpreting the Results. Sections | About Looking for Inactivators ‘The Difference (Increase or Decrease) column is used to detect inactivators or antibodies, which take time to react. Recall: The difference between an inhibitor and an inactivator is that the inhibitor reacts at 0 time and an activator takes time to react. | Page 13 of25 > edaGian 4. Interpreting the Results An Example ‘Sections | ‘About Cee elas In this example, you can see that the increase in the 1:2 rows 156, ‘The increase in the Control row is 3.5. For an inactivator to be present, the increase in row 1:2 would have to be 4 seconds more than the increase indicated in the Control row (35+4= 7.) AA value of 7.6 or greater would indicate an inactivator. Itis okay ifthe 1:4, 1:8, etc, rows are also > 4 seconds ‘more than the control, but they do not need to be. | Page 150125 D> een andaaa ued 4 Interpreting the Results Sections | About Increase and Decrease in Numbers ‘The Difference (increase) in the 1:2 dilution should be more than or equal to the increase in the 1:4 column. The numbers in the 1:2, 4:4, 1:8, etc. should continue to decrease because, if this is an antibody, in each dilution it will continually be diluted more and more by the Control plasma. Ifthe numbers in the Difference column do not decrease, the test is repeated oTme hour —_ifterence: Control 30.0 314 +14 Patient 55.2 5630 +41 4:2 36.3 445 [+82 > should be > 4:4 35.9 46.3 [+ 10.4 + than 1:8 32.4 34.4 +20 41:16 26.2 28.0 | +18 4:32 25.8 26.0 | +02 Wumbers should continue to decrease. If not, repeat the test. In this example, you can see thatthe difference in the 1:4 row is 10.4, while the diference in the 1:2 row is 8.2. Because 10.4 is. ‘greater than 8.2, this does not make sense and the testis repeated | Pave 16.0125 D>Gian 4: Interpreting the Results Inactivators Inactivators are observed in the Difference column since they are usually antigen-antibody reactions which take time to develop. They do not react fast enough to be seen in the 0 column. Factor Vill or IX antibodies are the most common. antibodies seen, Clie el Sections | About Because inactivators take time to develop, they are observed in the Difference column. | Page 17 of25 > eadaaa ued 4 Interpreting the Results Sections | About Interpretation of the Increase or Decrease (Difference) Column. Note: The patient plasma does not need to increase by four Seconds more than the normal for an inactivator to be present. No increase in the patient column means the antibody has already bound-up all the factors in the patient plasma. ‘The 1:2 dilution, however, adds normal plasma. The antibody now can bind-up the factor in the normal plasma ‘and for this reason an increase will occur in the 1:2 dilution. Click the link for an explanation of this test result. Page 18 f25 age 18.025 >What does this example show? In this example, you can see that the patient plasma shows no increase. The 1:2 diution (+7.0) is > 4 seconds longer than the contro! (normal) plasma (+1.0), This indicates an inactivator. GoneAPTT Inhibitor-Inactivator (oe eee sensed 4. Interpreting the Results Sections | About Interpretation of the Increase or Decrease Column (continued) Ifthe increase in the Control plasma Is greater than, the same 2s, or less than four seconds more than the 1:2 dilution, no inactivator is present. Ifthe patient plasma increase is greater than four seconds ‘more than the control, but the 1:2 dilution is not, the results, are hard to interpret and the test is repeated What does this example show? Click the link for an explanation of this test result.Whi ample show? Inthis example, you can see that the patient increases (+7.0) but the 4:2 dilution only increases +2.3, which is very close to the control plasma (+2.2). Thus, no inactivator Is indicated. Because the patient increased significantly, the test ‘wouid be repeated to make sure the 1:2 value was correct. CiosGenres Clinical Labo 4. interpreting the Results Sections | About Strong Inactivator When a strong inactivator is present, an inhibitory curve (times not correcting into the normai range) will often be seen at 0 time, Gonral aE This is not indicative of both an inhibitor and inactivator Patient 102.1 being present, but just a strong inactivator beginning to 4:2 448 work closer to 0 time. 14 36.8 What does this example show? 18 34.4 1:16 32.4 4:32 29.3 oTime Click the link for an explanation of this test result. | Page 20.0125 > a aceon!What does this example show? Inthis example, you can see that there isa strong inactivator. The 1:2 dilution is 29.3 more than the control lution. At O time, the 1:2 did not correct into the normal range, which ‘would normally indicate an inhibitor. But this is just the strong inactivator working close to 0 time, Goss4. Interpreting the Results | Sections |About the pH of the Incubated Plasma To maintain the pH of the plasma being incubated for 2 hours, itis best to put the tubes in a COz environment. This ‘can be accomplished by setting dry ice on the test tube rack of the waterbath (make sure the dry ice does not touch the tubes). Then drape a plastic wrap (plastic bag) over the waterbath to trap the CO, By maintaining the pH, decay of factors V and Vill is retarded, | Page 210125 > ectedGado enee es 4. Interpreting the Results | Sections | About What is the purpose of the 0 column? © A. To detect inactivators or antibodies, OB. To assess factor deficiencies and inhibitors, Select the best answer and click Check Answer Check Answer Incorrect. The correct answer is: B. The 0 column is used to assess factor deficiencies and inhibitors. The Difference (Increase or Decrease) column is used to detect inactivators or antibodies, Inactivators are observed in the Difference column because they are usually antigen-antibody reactions which take time to develop. They do not react fast enough to be seen in the 0 column, <| Page 22.0f25 > [ton casAPTT Inhibitor-Inactivator 4. Interpreting the Results | Sections | About If the patient plasma increase is greater than 4 seconds more than the control, but the ion is not, what does this indicate? A. Itis characteristic of a classic lupus inhibitor. © B. The results are hard to interpret and the test must be repeated. CC. Astrong inactivator Is present. Select the best answer and click Check Answer. Check Answer Correct. The correct answer is: B. If the patient plasma increase is ‘greater than 4 seconds more than the control, but the 1:2 dilution is not, the results are hard to interpret and the test must be repeated, {| Page 23 0f25 p> SeeCree 4. Interpreting the Results" Sections | About Drag each of these to the appropriate test result. Incorrect. The Matching Descriptions are: Factor deficiency: In the 0 column, the patient time is long, but the 1:2 corrected into the normal range. Classic lupus inhibitor. In the 0 column, the 1:2 dilution is as long as or longer than the patient plasma and further dilutions remain long. An APTT lengthening at zero time is characteristic of a classic lupus inhibitor. Inhibitor: In the 0 column, the pationt time is long and the 1:2 dilution corrected part way to the normal range, but not all the way. The 1:2 dilution is not as long as or longer than the patient. Inactivator. In the 0 column, the long patient time is ‘corrected in the 1:2 dilution, in the difference column, the 1:2 dilution is >4 seconds greater than the increase in the control or normal column, In the 0 column, the 1:2 dilution s as long as or longer than the Classic lupus inhibitor errr tet eer iy In the 0 column, the patient time Is long and the 1:2 dilution Cocc Sonne ad Inthe 0 column, the Iong patient time is corracted in tho 1-2 dilution. i oer co ee nea) Zz WJ check Answer | Page 240125 Potato4. Interpreting the Results Sections | About Inhibitors are substances produced by the body that inhibit coagulation, The suspicion of an acquired coagulation inhibitors often prompted by the unexplained prolongation of th®T (Prothrombine Time) or APTT (Activated Partial Thromboplastin Time) ina patient. PT and APTT are measures of the time it takes forthe plasma to cot. The difference between an inhibitor and an inactivator is that the inhibitor reacts at 0 time and an activator takes time to react. The first step in an inhibitor-inactivator tests to dilute the patient serially in normal plasma (control plasma). Once the dilutions are made, an APTT is performed on the normal plasma (control plasma), patient plasma, each dilution, The two columns that are important when interpreting a PTT Inhibitor-inactivator ar + The 0 (zero) time column, which is used to assess factor deficiencies and inhibitors. + The Difference column, which is used to detect inactivators or antibodies, which take time to react You have reached the end of this section. Click the right arrow to continue to the next section. 00250125 > fornten iceGianieeneees ‘Practice Introduction In this section you will be presented with inhibitor/inactivator test results and asked to identify ‘whether the results indicate a factor deficiency, inhibitor, inactivator, or classic lupus inhibitor. Sections | About - Factor deficiency Inhibitor - Inactivator ~ Classic lupus inhibitor | Pagetot7 pyGian Coe Esra 5.Practice Sections | About Example 1: How would you interpret the 0 time of this inhibitor/inactivator study? ©A, Factor deficiency ©. Inhibitor CC. inactivator ©D. Classic lupus inhibitor Select the best answer and click Check Answer. Correct. The correct answer's: A. This isa factor deficiency since the patient time is long, but the 1:2 corrected into the normal range. J Pase20t7 > ean)APTT Inhibitor-Inactivator ‘Practice | Sections | About Example 2: How would you interpret the 0 time of this inhibitor/inactivator study? OA. Factor deficiency ©B. Inhibitor ©C. Inactvator ©D. Classic lupus inhibitor Select the best answer and click Check Answer. Incorrect. The correct answer is: B. This is an ‘example of an inhibitor, but not a classic lupus inhibitor. In this case the patient time is long and the 1:2 dilution corrected part way to the normal range, but not al the way. It is not a classic lupus inhibitor because the 1:2 dilution is not as long as or longer than the patient. This patient would be described as having an inhibitor, but not lupus. It could also be an example of a heparinized patient, 4) rave sot7_ yp (ceo asGini eens Example 3: How would you interpret the 0 time of this inhibitorfinactivator study? OA. Factor deficiency ©. Inhibitor CC. inactivator © D. Classic lupus inhibitor Select the best answer and click Check Answer. Correct. The correct answers: D. This is an ‘example of a classic lupus inhibitor. The 1:2 dilution is as long as or longer than the patient plasma and further dilutions remain long, 5.Practice Sections | About sj Pave sot? yGa eee 5. Practice Example 4: How would you interpret this inhibitorlinactivator study? Sections | About OA, Factor deficiency ©B. Inhibitor OC. Inactivator ©D. Classic lupus inhibitor Select the best answer and click Check Answer. Check Answer Incorrect. The correct answer is: C. This is an example of a patient with a weak inactivator (antibody). In the zero column, the long patient time is corrected in the 1:2 dilution, indicating a factor deficiency. In the difference column, the 4:2 dilution is >4 seconds greater than the Increase in the control or normal column, This indicates an inactivator or antibody being present in the sample. The 1:4 dilution is also >4 seconds longer than the normal plasma, but by the 1:8 dilution, the antibody has been diluted enough that itis not seen J Page sot7 > ae aaGian Coe el Sections | About Example 5: How would you interpret this inhibitor/inactivator study? CA, Factor deficiency ©. Inhibitor CC. Inactivator ©D. Classic lupus inhibitor Select the best answer and click Check Answer. Check Answer Incorrect. The correct answer is: C. This is an example of a strong antibody. In the difference column the 1:2 dilution is much greater than 4 seconds greater than the control plasma. “Note the appearance of what appears to be an inhibitor in the 1:2 dilution (56.7). This is actually a strong inactivator beginning to work at zero time, ekeGagne ‘.Practice | Sections | About Summary Remember: + Factor Deficiency: 0 time + Inhibitors: 0 time—immediate acting + Inactivators: Seen in difference column—takes time for antigen-antibody complex to form You have reached the end of this section. Click the right arrow to continue to the next section. Page 7of7 > peedGenerel 6. Lupus Inhibitor Studies Sections | About Introduction slightly different testis performed—the Lupus Inhibitor test. ace escent: Hore (ese Since inhibitors do not need time to react, only the O time is ———— SS run and only a 1:2 and 1:4 dilution is done. CompanyA 28.3 54.6 45.6 43.8 ‘This testis run on several different companies’ APTT CompanyB 22.6 49.6 43.2 43.6 reagents since different companies use different sources of phospholipid and the lupus inhibitor is directed against phospholipid Company 26.7 53.8 50.7 51.9 | Pagerots > ate)Maer Coie Pel 6. Lupus Inhibitor Studies Sections | About Interpretation: To interpret these studies, you look across at one company at a time, With company A, the patient is 54.6 and the 1:2 corrects to 45.6. What do you think this result indicates? Otis a classic lupus anticoagulant © Itis a factor deficiency tis some type of inhibitor Select the best answer and click Check Answer. Incorrect. The correct answer is: C. This is probably some type of inhibitor since the time stayed long, + Itis not a classic lupus anticoagulant since the 1:2 is not as long or longer than the patient. + Itis not a factor deficiency, since the 1:2 did not correct into the normal range. sj ravez0t8 > eneGin eee 6. Lupus Inhibitor Studios Confirmatory Tests ‘Since this is not a classic lupus inhibitor, a confirmatory test isrun. There are several different types of confirmatory tests. One example is the platelet neutralization test. ‘Sections | About Platelet Neutralization Test J Pacoscte ypGuanes 6, Lupus Inhibitor Studies Platelet Neutralization Test When normal plasma is diluted with an equal amount of butfer or lysed platelets, the APTT usually lengthens 2-3 seconds, When plasma from a patient with lupus inhibitor is diluted with an equal amount of buffer, the APTT of the mixture is ‘usually shorter than that of the undiluted patient, (ole ence sie Sections |About When diluted with an equal amount of buffer: Normal plasma ‘The APTT usually lengthens 2-3 seconds. Plasma from a patient with lupus inhibitor The APTT of the mixture is usually shorter than that of the undiluted patient. J Pagesots > ansGian 6. Lupus Ini Platelet Neutralization Test (continued) ‘When lysed platelets (platelets that have been frozen and thawed to release platelet phospholipid) are substituted for buffer, there may be a further shortening of the APTT. ‘Shortening of the APTT with buffer, with addition of lysed platelets or with both indicates a positive platelet neutralization test and confirmation of a lupus inhibitor. Sections |About Plasma Plasma & Buffer Plasma & Frozen Thawed Platelets. Normal Patient 275 79.2 31.0 63.7 31.0 43.8 A platelet neutralization test worksheet would look tke this. 4 Page 510 D> dansGian Celie Dessay ‘6. Lupus Inhibitor Studies Sections | About Platelet Neutralization Test (continued) Confirmatory tests are done when the inhibitor study is unclear as to whether a lupus anticoagulant is present. For example: APTTReagant Normal Patient 1:2 1:4 CompanyA 28.3 38.0 31.2 29.7 cana!Gases 6, Lupus Inhibitor Studies Sections | About Platelet Neutralization Test (continued) ‘Since the patient's APTT was so short to start with, it is hard to see a correction, so it shard to interpret. The confirmatory test would be done, Normal Patient Plasma 28.5 38.9 Plasma & Buffer 30.7 33.2 Plasma & Frozen 30.2 304 Thawed Platelets Page Tot D> cuminGuanes 6. Lupus Inhibitor Studies "Sections | About Summary + When the laboratory is looking for only a lupus inhibitor, a slightly different test is performed, + This test is run on several different companies’ APTT reagents since different companies use different sources of phosp! and the lupus inhibitor is directed against phospholipid. + Confirmatory tests are done when the inhibitor study is unclear as to whether @ lupus anticoagulant is present. You have reached the end of this module! Page 8 of 8 o oo