Laboratory Evaluation of Coagulation Inhibitors (Endang)

7/30/2019 Laboratory Evaluation of Coagulation Inhibitors (Endang)

1/5laboratorymedicine> august 2003> number 8> volume 34

84

case study[coagulation and hematology]

Laboratory Evaluation of Coagulation InhibitorsAmy L. Adams, MD, Yara M. Audeh, BS, Regina de Luna, MD, Monette S. Baker, MD, Marisa B. Marques, MDDepartment of Pathology, Division of Laboratory Medicine, University of Alabama School of Medicine, Birmingham, AL

DOI: 10.1309/YLB7RF56KRNHTL94

Patient #: 1 2 3 4

Age/Sex: 11-year-old female 43-year-old male 11-year-old male 69-year-old femaleChief Complaint: Epistaxis, fever, Ischemic stroke Epistaxis that occurred 1 week Easy bruising within the p

malaise, and anorexia prior to current evaluation; treated few weekswith fresh frozen plasma at anotherinstitution where a diagnosis ofprothrombin deficiency was made

Physical Exam Afebrile, well-nourished Apparently healthy child in no acute Large hematoma at the siteFindings: child with cervical distress with no active bleeding IM injection; multiple

lymphadenopathy ecchymoses in leftarm with diffuse swelling

Past Medical Unremarkable Hypertension Fever and lymphadenopathy approxi- Hypertension, diabetes meHistory: mately 6 months prior to presentation rheumatoid arthritis, and

asthmaSurgical History: Tonsillectomy at age None Circumcision and tonsil lectomy with- Cholecystectomy and

9 years without out excessive bleeding hysterectomy withoutincreased bleeding increased bleeding

Family History: No relatives with a Noncontributory Negative for bleeding disorder No relatives with a bleedinbleeding disorder disorder

Drug History: Aspirin for fever Multivitamins None Acetaminophen, atenolol,azithromycin, estradiol,glyburide, pravastatinsodium, prednisone, ram

and theophylline

Principal Laboratory Findings:

[T1]

Questions:

1. What are the most striking laboratory results for each of

these patients?

2. What are the common causes of an abnormal PTT with

normal PT?

3. What clinical factors should be considered when evalu

patients with abnormal PTT and normal PT results?

4. What is your assessment of the most striking laborator

results for each of the four patients presented in these



studies? Include in your assessment the most likely diag-

nosis for each of the 4 patients presented, how each condi-

tion should be treated, and the role of the laboratory in

managing these patients.

Possible Answers:

1. One or more abnormal screening tests: PT (Patient #3),

PTT (Patients #1-4), PTT-LA (Patients #1-4); abnormal PTT

mixing study results (Patients #1-4); abnormal confirmatorytest for the presence of an inhibitor, Staclot LA delta (Pa-

tients #1,3, and 4) and/or dRVVT ratio (Patients #2 and 3);

low factor VIII activity with an increased factor VIII inhibitor

level (Patient #4).

2. The most common reasons for prolongation of the PTT

with a normal PT are heparin therapy, intrinsic factor deficien-

cies, presence of a lupus anticoagulant (LA), and/or an intrin-

sic factor inhibitor (eg, an antibody against factor VIII).

3. When assessing a patient with abnormal coagulation

screening tests such as PT and/or PTT, several clinical factors

are important to consider in guiding further evaluation in a

cost-effective manner: gender, age, drug history, signs/symp-

toms, and concurrent medical conditions.

4. Patient #1: Since this patient is female, the probability of a

factor deficiency is low, because the most common factor defi-

ciencies, hemophilia A (deficiency of factor VIII) and hemophilia

B (deficiency of factor IX), are X-linked disorders, and therefore

only males are affected. Hemophilia carriers have decreased lev-

els of factors VIII or IX, but these patients still have enough fac-

tor activity to provide a normal PTT result. Heparin is also an

unlikely explanation for this patients prolonged PTT result

because she was not hospitalized and did not have an indwe

catheter. Finally, an antibody to factor VIII is very unlikely b

cause the patient is a child, and specific factor inhibitors occ

mainly in older individuals or in association with pregnancy.

Thus, the most likely explanation for the prolonged PTT res

this patient is the presence of a LA.

The term lupus anticoagulant refers to a heterogeneous

group of autoantibodies that react with protein-binding pho

lipids and thereby affect clotting tests that use a limited suppof phospholipid to form the clot (eg, PTT, PTT-LA, and dR

tests).1 Thus, LAs and anticardiolipin antibodies are called a

tiphospholipid antibodies. Anticardiolipin antibodies, howev

do not prolong clot-based tests and are assayed by ELISA. T

International Society on Thrombosis and Haemostasis (IST

has defined the following 4 criteria for the diagnosis of LA

at least one abnormal screening test (PTT, PTT-LA, or

dRVVT) result; (2) lack of correction of the prolonged scr

ing test result after a mixing study has been performed; (3

shortening of an abnormal clotting time with the addition

excess phospholipid (eg, Staclot LA); and (4) exclusion

specific factor inhibitor.2 Evaluation of the coagulation resu

for Patient #1 against these criteria indicated that she was lik

to have an LA in her plasma [T2]. Screening tests such as P

PTT-LA, and dRVVT are very sensitive to interference by a

phospholipid inhibitor and are typically prolonged in the pr

ence of an LA. Because an abnormal screening test by itself

not specific for LA, additional testing must be done to conf

the diagnosis. A mixing study is helpful in this scenario and

performed by repeating the initial screening test with the

patients plasma diluted 1:1 with normal plasma containin

adequate levels of all clotting factors. This mixture would b

expected to correct an abnormal screening test result that wa

due to a specific coagulation factor deficiency. In the case o

Principal Laboratory Findings

Test Results for Patient # Reference Range

1 2 3 4

Platelet count 287 324 130-400 x 103/LPT, sec 13.8 12.5 25.0 11.2PT reference interval, sec 11.3-14.6 11.3-14.6 10.7-13.0 10.7-13.0INR 2.3 1.00Prothrombin activity 82 6 50-150%PTT 55.0 78.0 62.0 99.5 25.0-34.0 secPTT mixing study 43.0 42.0 60.4 Correction to 25.0-34.0PTT-LA 108.0 90.0 113.0 131.4 36.1-50.1 sec

Staclot LA delta 20.4 61.5 19.9



86

abnormal screening test due to the presence of an inhibitor such

as an LA, the test result obtained on the mixture will still be ab-

normal because the inhibitor will interfere with the clotting fac-

tors from both plasma sources. The Staclot LA (Diagnostica

Stago M, Parsippany, NJ) test is a commonly used confirmatory

test in the workup of a possible LA. Using the Staclot LA pro-

cedure, 2 PTT-based clotting times are performed, 1 with a lim-

ited amount of phospholipid (tube 1) and the other with an excess

of hexagonal phospholipids (tube 2). If the difference (or delta)

between the clotting time values from tubes 1 (low phospholipid)

and 2 (excess phospholipid) is greater than 8 seconds, a phospho-

lipid-binding antibody such as an LA may be present. However, a

decrease in the clotting time after the addition of excess phospho-

lipid is not specific for an LA because this can happen in other

conditions with a prolonged PTT, such as the presence of factor

VIII antibodies. Although an LA is more common than any spe-

cific coagulation factor inhibitor, factor VIII antibodies are the

second most common inhibitors, and their presence must be ruled

out in order to establish the presence of an LA. Factor VIII in-

hibitors are measured with the Bethesda assay, which quantifies

the amount of inhibitor based on the ability of the patientsplasma to inhibit the activity of factor VIII present in normal

plasma. One Bethesda unit is the amount of antibody capable of

decreasing the activity of normal factor VIII by 50%. A positive

Bethesda assay in conjunction with a low factor VIII activity con-

firms the presence of a factor VIII inhibitor. The factor VIII activ-

ity for this patient was normal, however, thus ruling out the

presence of a factor VIII inhibitor. In this case the patients pro-

longed PTT, PTT-LA, and diluted Russell viper venom time

(dRVVT) [T1], along with a mixing study that did not correct the

PTT and a high Staclot LA delta, confirm the presence of a LA.

Lupus anticoagulants are only rarely associated with

bleeding. More often they are asymptomatic or are associated

with thrombosis. This patients PT and prothrombin activitywere normal. Therefore, this patients epistaxis was a red

herring and may have been due to thrombocytopenia from a

viral illness or from the effect of aspirin therapy on her

platelets. Most LAs in children are associated with viral infec-

tions, are transient, and are not expected to cause thrombosis

under these clinical conditions.3

Patient #2: Similar to Patient #1, the coagulation results

for Patient #2 are consistent with the presence of an LA. The

PTT, PTT-LA, and dRVVT results were abnormal, and the

dRVVT mixing study did not correct the dRVVT check re

[T1]. In the case ofPatient #1, the presence of a LA was c

firmed using the Staclot LA test. For Patient #2, the conf

matory test was the dRVVT ratio. The dRVVT is a clotting

that utilizes the ability of Russell viper venom to activate f

tor X. As a screening test for the presence of a LA, the tes

performed with a low amount of phospholipid (dRVVT

check). If abnormal, the dRVVT may be repeated using a

mixture of the patients plasma with normal plasma (ie, pl

known to contain all clotting factors in adequate amounts

no substances that will interfere with any of the componen

the coagulation cascade). If an inhibitor is identified by lac

correction in the mixing study, another dRVVT with conce

trated phospholipid is performed (dRVVT sure). If an L

present, it will essentially be neutralized by the excess pho

pholipid, allowing the dRVVT to correct. A dRVVT chec

dRVVT sure ratio greater than 1.3 is consistent with the

presence of a phospholipid inhibitor such as an LA. Moreo

the presence of a factor VIII inhibitor in this patients plas

was ruled out by his high factor VIII activity level, probabdue to an acute phase reaction. Because this patient exhibi

thrombotic manifestation (stroke) in conjunction with an L

this patient can be diagnosed as having the antiphospholip

syndrome (APS). Phospholipid antibodies are found in 18

ischemic stroke patients younger than 44 years of age. In a

tion, neurological symptoms are more likely to occur in su

patients in the presence of other risk factors, such as hyper

sion and smoking.4 Lastly, since this patients baseline PT

was prolonged due to the presence of a LA, the PTT could

be used to monitor heparin therapy. The anti-Xa (activated

tor X) assay is helpful in this situation because it is not

affected by the presence of an LA and it provides a very re

able measurement of anticoagulation in a patient receivingparin. The laboratory should alert the patients physician t

request the anti-Xa assay in this clinical setting.

Patient #3: This patients prolongation of both the PTT

and PT results suggests either the presence of an inhibitor

the deficiency of a specific coagulation factor within the c

mon pathway of the coagulation cascade. However, the ab

mal screening tests, the lack of correction with a mixing st

and the abnormal confirmatory test (dRVVT ratio and Stac

Evaluation of Coagulation Test Results for Patients 1 Through 4 Against the ISTH Criteria for theDiagnosis of a Lupus Anticoagulant

Criterion Patient #

1 2 3 4

Prolonged coagulation screening tests (PTT, PTT-LA, or dRVVT) + + + +Uncorrectable screening test by mixing study + + + +Shortening of abnormal clotting time with addition of excess PL + + + +Exclusion of a specific coagulation factor inhibitor + + - -

+, criterion met; -, criterion not met;

ISTH, International Society on Thrombosis and Haemostasis; PTT, partial thromboplastin time; PTT-LA, partial thromboplastin time-lupus anticoagulant; dRVVT, dilute Russell vip

venom time; PL, phospholipids.

T



LA delta) results indicate an inhibitor, most likely LA, as the

probable culprit [T1]. In patients with an LA and bleeding, it is

important to check for hypoprothrombinemia. Occasionally, an

LA is directed against prothrombin (factor II), leading to im-

mune clearance of this factor from the plasma. In such cases,

the prothrombin level is low, and the PT is prolonged. This

scenario should be suspected if the INR is greater than 1.4.

Because the INR for Patient #3 was 2.3, while the prothrombin

activity was low [T1], and all of the ISTH criteria for the pres-ence of an LA were met [T2], these findings are highly sugges-

tive of an LA with specificity for prothrombin.

A history of a preceding viral illness is common in pa-

tients with an LA, but an episode of bleeding, as seen in Pa-

tient #3, is not. Lupus anticoagulants are more often

associated with thrombosis. If the LA is directed at prothrom-

bin, however, a hypoprothrombinemia can develop resulting

in epistaxis, easy bruising, hematoma formation, menorrha-

gia, and prolonged bleeding after trauma or surgery. As noted

in T1, the prothrombin activity level was low (6%). This type

of acquired prothrombin deficiency is more likely than a con-

genital form of prothrombin deficiency, as this patient had

previously undergone both a tonsillectomy and circumcision

without excessive bleeding.

Fortunately, even with acquired prothrombin deficiency,

the prognosis of LAs in children is good, as most are tran-

sient and resolve without specific treatment.3 Supportive ther-

apy with management of bleeding episodes as they occur is

the appropriate treatment for this patient. When bleeding

episodes occur, fresh frozen plasma (FFP) is used to control

bleeding. Fresh frozen plasma contains all of the clotting

factors at an activity level of 100%. In vivo, a prothrombin

activity level of 20% to 40% is generally adequate for achiev-

ing hemostasis. This level is often reached with the amount

of prothrombin in FFP, in which case no other treatment isneeded. While there is not a specific prothrombin concentrate

available, a prothrombin complex concentrate exists which

contains, in addition to prothrombin, factors VII, IX, and X.

The only currently available formulation on the market is Be-

bulin, which contains a high level of prothrombin activity.

This prothrombin complex concentrate has 2 main advantages

over FFP for the treatment of prothrombin deficiency. First, it

is safer because it undergoes a viral inactivation process to

prevent the transmission of HIV, hepatitis B, and hepatitis C

viruses. Second, the concentrate contains a higher level of

prothrombin activity in a smaller volume, so less volume

needs to be given to the patient to achieve hemostasis. This is

particularly important in children or other patients who can-not tolerate excessive fluid. Also, for patients with more se-

vere bleeding or for those being prepared for a surgical

procedure, a hemostatic level of prothrombin can be achieved

more quickly and with less overall volume. The loading dose

of concentrate may be calculated by first determining the pa-

tients blood volume (BV), which is based upon the patients

weight in kilograms. For children and slender adults, blood

volume is presumed to be 70 mL per kilogram of body

weight. For obese adults, the blood volume (BV) is

estimated using 60 mL per kilogram. Next, the plasma vo

ume (PV) is determined by the following formula:

PV = BV x (1 - Hematocrit)

Finally, the dose of concentrate to be given is calcula

as follows:

Dose, IU = (Desired % Activity Current % Activity) x

where 100% activity equals 1 IU/mL.

The amount of each clotting factor present in 1 vial oprothrombin complex concentrate is based upon the amou

of factor IX present, measured in international units (IU)

example, a vial of Bebulin should have 120 IU for every

IU of factor IX. The package insert for each formulation

prothrombin complex concentrate should specify the tota

number of units of factor IX in each vial. To assess the a

propriateness of the dose, the plasma prothrombin level

should be measured approximately 30 minutes after the i

sion. Since the presence of an LA is expected to shorten

half-life of prothrombin, it is advisable to check factor le

prior to deciding on subsequent doses.

Patient #4: As with the other 3 patients, the results of t

coagulation screening tests (PTT and PTT-LA), the PTT m

study, and/or the Staclot LA delta confirmatory test were

normal [T1]. However, this patients dRVVT test was norm

and the patient had a significant bleeding history, a classic

ture of acquired hemophilia. This combination of findings i

consistent with the presence of a specific inhibitor to factor

VIII, a condition known as acquired hemophilia.5 This sus

cion was confirmed by this patients extremely low (5 BU), hemostasis may

attained with alternative compounds which bypass the need

factor VIII, such as activated prothrombin complex concenor recombinant factor VIIa. If the antibody to factor VIII do

not cross-react with porcine factor VIII, this product may a

be of benefit to control bleeding. The role of the laboratory

this disease is to monitor factor VIII and factor VIII inhibit

levels over time.

A summary of the most likely diagnosis and its ration

for each of the four patients presented in this case study is

cluded in T3.



88

Keywords: lupus anticoagulant, coagulopathies, acquired

hemophilia, antiphospholipid syndrome, acquired prothrom-

bin deficiency

1. Greaves M. Antiphospholipid antibodies and thrombosis.Lancet.1999;353:1348-1353.

2. Brandt JT, Triplett DA, Alving B, et al. Criteria for the diagnosis of lupusanticoagulant: An update. Thromb Haemostas. 1995;74:1185-1190.

3. Male C, Lechner K, Eichinger S, et al. Clinical significance of lupusanticoagulants in children.J Pediatr. 1999;134:199-205.

4. Levine SR, Deegan MJ, Futrell N, et al. Cerebrovascular and neurologic dassociated with antiphospholipid antibodies: 48 cases.Neurology.1990;40:1181-1189.

5. Boggio LN, Green D. Acquired hemophilia.Rev Clin Exp Hematol. 2001404.

Summary of Most Likely Diagnosis and Rationale for the Diagnosis in Patients #1 through #4

Patient # Most Likely Diagnosis Rationale

1 LA with thrombocytopenia* Fever, malaise, and cervical lymphadenopathy suggest infectious mononucleosis. Viral infections acommonly associated with decreased platelet count, while aspirin is an inhibitor of platelet functand, therefore, a promoter of bleeding such as epistaxis

High Staclot LA delta valueNormal PT and prothrombin activity values

2 Antiphospholipid (APL) syndrome Phospholipid antibodies (e.g., LA) are found in 18% of ischemic stroke patients less than 44 yearsdRVVT screening and confirmatory test results consistent with the presence of a phospholipid inhi

3 Acquired prothrombin deficiency Markedly increased Staclot LA delta valuedue to presence of an LA INR > 1.4

Markedly low prothrombin activity level4 Acquired hemophilia Occurs typically in individuals over the age of 50

50% of patients with acquired hemophilia have an associated disorder (eg, RA, SLE, a malignancya drug reaction)

Associated with spontaneous bleeding and/or delayed hemostasisMarkedly low factor VIII activityMarkedly increased factor VIII inhibitor level

*Due to the unavailability of the data from the patients complete blood count (CBC), thrombocytopenia could not be confirmed by a platelet count. RA, rheumatoid arthritis; SLE,

systemic lupus erythematosis.

T

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Laboratory Evaluation of Coagulation Inhibitors (Endang)