General Approach of

Haemostasis

Lupus Anticoagulant

Islamic University of Gaza

.ANTIBODIES
 Anti-phospholipid antibodies are a heterogeneous family of
autoantibodies that react with epitopes on proteins that are
themselves complexed with negatively charged phospholipids.
 The blood vessel problems can then lead to complications such
as stroke, heart attack, and miscarriage.
 The two most commonly measured kinds of Antibodies are:
 Lupus anticoagulant
 Anticardiolipin antibody
LUPUS ANTICOAGULANTS
(LUPUS ANTIBODY, LA, OR LUPUS INHIBITORS)

 Lupus anticoagulants (LA) are heterogeneous IgG or IgM autoantibodies

which inhibit phospholipid-dependent assays of blood coagulation by
binding to plasma phospholipid-binding proteins such as beta 2-
glycoprotein I (β2-GPI) or prothrombin
 Lupus Anticoagulant was found in patient with SLE.
 Most patients with a lupus anticoagulant do not actually have lupus
Erythematosus, and only a small proportion will proceed to develop this
disease.
 It is a prothrombotic agent, that is, presence of Lupus anticoagulant
antibodies precipitates the formation of thrombi in vivo.
 In vivo, it is thought to interact with platelet membrane phospholipids,
increasing adhesion and aggregation of platelets; thus its in vivo
prothrombotic characteristics.
 The term "anticoagulant" accurately describes its function in vitro, but in
vivo, it is now known that it functions as a coagulant
 Lupus anticoagulants impair the in vitro phospholipid dependent
activation of factor X, factor IX, and Prothrombin.
 Presence of these antibodies causes an increase in aPTT.
 The initial workup of a prolonged PTT is a mixing test.
 If the mixing test indicates an inhibitor, diagnosis of a lupus
anticoagulant is then confirmed with phospholipid-sensitive functional
clotting testing, such as the dilute Russell's viper venom time, or the
Kaolin clotting time
 The presence of an LA is usually not associated with a bleeding problem
unless accompanied by
oThrombocytopenia,
oFactor II deficiency,
oPlatelet dysfunction
oOr drug administration (e.g., aspirin).
 The ISTH(International Society on Thrombosis and Haemostasis )
recommends that the laboratory diagnosis of lupus anticoagulants (LA)
should be carried out on double-centrifuged plasma following a four-step
procedure adhering to these principles:
 Prolongation of a phospholipid-dependent coagulation test.
,Extrinsic (dPT)
Intrinsic (aPTT, dilute aPTT, KCT, colloidal silica clotting time), Final common
pathway (dRVVT, Taipan venom time, Textarin and Ecarin time)
 Evidence of inhibitory activity on mixing tests.
 Evidence of phospholipid dependence.
Correction of the prolonged coagulation time after addition of excess
phospholipid or platelets
 Lack of specificity for any one coagulation factor
Lack of specifi c inhibition of one coagulation factor (such as FVIII:C,FIX:C, or
.FXI)
 .ANTI-CARDIOLIPIN ANTIBODIES
 There is many classes of anticardiolipin antibody, abbreviated as
IgG, IgM, and IgA.
 The anticardiolipin antibody is measured in an ELISA test.
 The IgG type of anticardiolipin antibody is the type most often
associated with complications.
 Some lupus patients with very high IgM anticardiolipin antibody
have a problem called hemolytic anemia, in which their immune
system attacks their red blood cells.
ANTI-PHOSPHOLIPID ANTIBODY SYNDROME

 APS is present if at least one of the clinical criteria and one of

the laboratory criteria that follow are met:
1. Clinical criteria: vascular thrombosis or pregnancy morbidity
2. Laboratory criteria:
A. Lupus anticoagulant present in plasma on two or more occasions at least 12
weeks apart.
B. Anticardiolipin antibody, IgG or IgM positive in medium or high titer on
two or more occasions at least 12 weeks apart
C. Anti-β2GPl antibody, IgG or IgM present on two or more occasions at least
12 weeks apart.
 The positive laboratory criteria and the clinical criteria should
occur within 5 years of each other.
LABORATORY INVESTIGATIONS
 Immunological Assays
 Individuals with Lupus anticoagulants may also show the presence
of other anti phospholipid antibodies. The most frequent finding is
the presence of Anti- cardiolipin antibodies.
 The commonly employed method is the ELISA technique where
the solid phase is coated with cardiolipin and β-2 GPI (as a
cofactor). The ELISA method detects IgM, IgG and IgA class of
anti-cardiolipin antibodies.
 An important point to note is that Lupus anticoagulants and anti-
cardiolipin antibodies differ in their phospholipid-binding
characteristic hence detection of anti cardiolipin antibodies is not
specific for the presence of Lupus anticoagulants though they may
be present in the same patient.
Clot based assays
1. APTT ( Activated Partial Thromboplastin Time)

2. TTI (Tissue Thromboplastin Inhibition test)

3. KCT (Kaolin Clotting Time)

4. PNP (Platelet Neutralization procedure)

5. Hexagonal Phase Phospholipids (Staclot-LA)

APTT ( ACTIVATED PARTIAL THROMBOPLASTIN TIME)
 The specificity of the APTT to LA is reduced:
 Different reagents have varying sensitivity for the
presence of Lupus anticoagulants on the basis of their
phospholipid composition ( variability of the test)
 The APTT test is affected by the presence of inhibitors to
Factor VIII, IX and XI.
 The APTT test is also the test of choice for monitoring
heparin therapy.
TTI (TISSUE THROMBOPLASTIN INHIBITION TEST)
 The Tissue Thromboplastin Inhibition test utilizes a diluted PT reagent.
The results are expressed as ratio of patient values to normal control
values.

 The TTI test is affected by numerous variables:

1. Species and tissue origin of thromboplastin can affect the test

results as different sources of thromboplastin have varying
sensitivity and responsiveness.
2. Choice of “Normal” reference plasma is the most critical
variable, because depending on the laboratory the choice of
reference plasma could be lyophilized plasma, a frozen plasma pool
or fresh plasma. The ratio of patient to normal can therefore change
according to the choice of “normal” plasma.
3. Some IgM Lupus anticoagulants do not prolong the TTI test
KCT (KAOLIN CLOTTING TIME)
KCT is similar to APTT, the difference being that KCT reagent is devoid of
phospholipids and incorporates Kaolin as contact activator. The test is
performed on a range of mixture of normal and patient’s plasma. Different
patterns of response are obtained indicating the presence of Lupus
anticoagulants or the deficiency of one or more coagulation factors.
The KCT test though sensitive is not specific for LA, additionally:
• It cannot be automized

• The test shows prolonged results with factor VIII, IX, XI & XII
deficiency or corresponding inhibitors
• The test is also highly sensitive for the presence of heparin.
PNP (PLATELET NEUTRALIZATION
PROCEDURE)

 The platelet neutralization procedure (PNP) is based on the ability

of platelets to significantly correct in vitro coagulation
abnormalities.
 The disrupted platelet membranes present in the freeze-thawed
platelet suspension neutralize phospholipid antibodies present in
the plasma of patients with LA.
 After the patient plasma is mixed with the freeze-thawed platelet
suspension, the APTT will be shortened when compared with the
original baseline APTT. But if an inhibitor is directed against
specific coagulation factor, the clotting time is not shortened.
 A correction of the baseline APTT of a defined amount of time
(i.e., 3 to 5 seconds or more) by the platelet suspension as
compared with the control is indicative of the presence of an LA.
• Due to limited stability the platelet preparations loose their activities on
storage hence do not show reproducible results.
HEXAGONAL PHOSPHOLIPID
NEUTRALIZATION ASSAY
• The hexagonal phospholipid neutralization assay uses the same
principle as the PNP assay, normalization of the aPTT in the
presence of added phospholipid, but this assay specifically uses a
phospholipid in a hexagonal conformation, Neutralization by this
hexagonal form in an assay with a very lupus-anticoagulant
sensitive aPTT reagent, is a more sensitive confirmation test than
the PNP.
Comment Specimen collection, centrifugation, and processing are
critical when testing for the presence of an LA.
CONFIRMATORY TESTS
FOR LUPUS ANTICOAGULANTS

 Confirmatory tests to identify an LA include those that utilize a low

concentration of phospholipid in the test system, thereby increasing the
LA effect such as
 The tissue thromboplastin inhibition test (TTIT),
 Dilute Russell's viper venom time (dRVVT),
 And the kaolin clotting time (KCT), or
 Those that increase the phospholipid, thereby neutralizing the LA
effect, such as the platelet neutralization
DILUTE RUSSELL’S VIPER VENOM TIME
(DRVVT)

 dRVVT : The test of choice for screening and confirmation of LA

– Indicating the phospholipid dependence of LA
– Achieving maximum sensitivty for the precence of LA’s.

 In general dRVVT based tests comprise of:

– SCREENING REAGENT, containing limited amount of
phospholipids with RVV (Russell’s Viper Venom)
– CONFIRMATION REAGENT, containing additional phospholipids
with same amount of Russell’s Viper Venom, to confirm the presence
of phospholipid dependent Lupus anticoagulants.
PRINCIPLE OF DRVVT FOR LA DETECTION
 Russell’s Viper Venom directly activates Factor V and X in presence of
phospholipid and calcium ions, bypassing Factor VII of the extrinsic
pathway and the contact and antihaemophilic factors of the intrinsic
pathway.
 In normal plasma, in the absence of lupus anticoagulants, Factor V and X
is directly activated by Russell’s Viper Venom, which in presence of
phospholipid and calcium ion leads to clot formation.
 In patients with LA, autoantibodies bind the epitopes of reagent
phospholipids thereby preventing the activation of prothrombinase
complex. This results in a prolongation of clotting time with
SCREENING reagent.
 The CONFIRMATION reagent incorporates additional phospholipids to
neutralize the LA. Once LA are neutralized clot formation proceeds
relatively uninterrupted achieving a lower clotting time, to prove the
phospholipids dependence of the autoantibodies.
 INTERPRETATION OF RESULTS
WITH DRVVT TEST
• If SCREEN TIME is prolonged, to confirm the presence of lupus
anticoagulants the plasma sample is tested with CONFIRMATION
REAGENT.
• If CONFIRM TIME results shows a lower clotting time as compared to
SCREEN TIME, it indicates the presence of phospholipid dependant Lupus
Anticoagulants.
• Also the results can be expressed as ratio,

• The results expressed, as ratio is further useful in classifying the patient as

normal, moderate, high and very high LA.
• If results of the ratio are borderline, mixing studies may be done further
with the sample specimen. The mixing studies should be carried out with a
50:50 mixture of test plasma and normal plasma.
General Approach of
Haemostasis

D-Dimer
D-DIMER
(FRAGMENT D-DIMER; FIBRIN DEGRADATION
(FRAGMENT
 D-dimer is a fibrin degradation product (or FDP), a small protein
fragment present in the blood after a blood clot is degraded by
fibrinolysis. It is so named because it contains two crosslinked D
fragments of the fibrinogen protein.
 D-dimer test used to diagnose
 thrombosis,
 disseminated intravascular coagulation.
 The circulating enzyme plasminncleaves the fibrin gel in a number of
places. The resultant fragments, "high molecular weight polymers", are
digested several times more by plasmin to lead to intermediate and then to
small polymers (fibrin degradation products or FDPs). The cross-link
between two D fragments remains intact, however, and these are exposed
on the surface when the fibrin fragments are sufficiently digested. The
typical D-dimer containing fragment contains two D domains and one E
domain of the original fibrinogen molecule
 the D-dimer assay depends on the binding of a monoclonal
antibody to a particular epitope on the D-dimer fragment.
 Several detection kits are commercially available; all of them
rely on a different monoclonal antibody against D-dimer. Of
some of these it is known to which area on the D-dimer the
antibody binds. The binding of the antibody is then measured
quantitatively by one of various laboratory methods.
 D-dimer testing is of clinical use when:
 There is a suspicion of deep Venous Thrombosis (DVT)
 Pulmonary Embolism (PE)
 Disseminated Intravascular Coagulation (DIC).
 It can also rise Postoperatively.
 It is under investigation in the diagnosis of Aortic Dissection
INTERPRETATIONS OF RESULTS
 A very high score, or pretest probability, a D-dimer will make little
difference and anticoagulant therapy will be initiated regardless of
test results, and additional testing for DVT or pulmonary embolism
may be performed.
 For a moderate or low score, or pretest probability
 A negative D-dimer test will virtually rule out thromboembolism: the degree
to which the D-dimer reduces the probability of thrombotic disease is
dependent on the test properties of the specific test used in the clinical
setting: most available D-dimer tests with a negative result will reduce the
probability of thromboembolic disease to less than 1% if the pretest
probability is less than 15-20%
 If the D-dimer reads high, then further testing (ultrasound of the leg veins or
lung scintigraphy or CT scanning) is required to confirm the presence of
thrombus. Anticoagulant therapy may be started at this point or withheld
until further tests confirm the diagnosis, depending on the clinical situation.
 False positive readings can be due to various causes:
 liverdisease,
 high rheumatoid factor,
 inflammation, malignancy, trauma, pregnancy, recent surgery
as well as advanced age[citation needed]
 False negative readings can occur
 ifthe sample is taken either too early after thrombus
formation
 or if testing is delayed for several days.
 Additionally, the presence of anti-coagulation can render the
test negative because it prevents thrombus extension.
 Likelihood ratios are derived from sensitivity and
specificity to adjust pretest probability.