Common Laboratory Tests in the Diagnosis of

A i h h li id S dAntiphospholipid Syndrome:some Technical Aspectssome Technical Aspects

ل ف ف ض نشانگان ضد فسفوليپيدنشانگانجنبه هاي فني آزمايشهاي تشخيصي آنو

Dr. M. Mahdi Mohammadi, LMD, PhD, MPHKerman University of Medical SciencesKerman University of Medical Sciences

Dr.Mohammadi@kmu.ac.ir

TerminologyTerminology• APS Anti-Phospholipid SyndromeAPS• APLS• PL

Anti Phospholipid Syndrome

Phospholipid, Lipo.P, Phosphatidyls• P• PLDCT

p pPhosphorusPL-Dep. Coagulation Tests

, p , p y

• APA• APLA

p gAnti-Phospholipid Antibody

• aCL• ACLA

β2G

Anti- Cardiolipin Antibody

A i b 2 Gl i 1 (Ab)• a−β2GPI• LA

Anti-beta2 Glycoprotein 1 (Ab)Lupus Anticoagulant

Diagnosis of APLS, Detection of APLAg ,

The diagnosis of APLS should be seriously considered in fcases of:

• thrombosis, cerebral vascular accidents in individuals younger than 55 years of age oryounger than 55 years of age,or:

• pregnancy morbidity in the presence of livedo reticularisor thrombocytopeniaor thrombocytopenia.

In these cases APLA antibodies should be measured.

At least one clinical and one laboratory criterion ensures the diagnosis even in the presence of other causes ofthe diagnosis even in the presence of other causes of thrombophilia.

HPIM, ed18, 2012

Historical description of A ti h h li id A tib di (APLA )Antiphospholipid Antibodies (APLAs)

Simple Algorithm for the Diagnosis of APLSAPLS

Different APLA specificitiesDifferent APLA specificities

Some Risk Factors For Thrombosis

fl i

Hyperlipidemia,Atherosclerosis

GeneticHereditary 

Thrombophilia

Inflammation& Trauma(eg surgery)

Thrombosis

Thrombophilia (eg surgery)

Hormones(eg Esteroids),

OTC &

Some

Malignancies OTC & Pregnancy

Hospitalization, Immobility

Malignancies

Immobility

Lab Criteria for the Diagnosis of APLS

Laboratory criteria include:

g

• (1) LA,• (2) anticardiolipin (aCL) and/or• (3) anti‐2GPI antibodies,at intermediate or high titers,g ,on two occasions, 12 weeks apart.

Coombs-positive hemolytic anemia and thrombocytopenia are laboratory findingsthrombocytopenia are laboratory findings associated with APS.

HPIM, ed18, 2012 (Pt15, Sec2, Ch323)

Some Lab Facts about APLA• APLA is against Phospholipid binding plasma proteins• APLA is against Phospholipid-binding plasma proteins.• APLA occur in 1–5% of general population.• APLA prevalence increases with age.• One-third of APLA positive individuals experience thrombotic events or pregnancy

morbidity.• One-third of patients with SLE possess APLA.• APLA in other autoimmune connective tissue disorders such as SS, Scl (syst.

sclerosis), DerMyo and RA ranges from 6% to 15%.• ACLA or Ab against cardiolipin (a neg. charged PL) is detected by ELISA.g p ( g g ) y• Antibodies against phospholipids/cholesterol complexes are also detectable as

biologic false-positive serologic test for syphilis (VDRL+, FTA-Abs_)

• Antibodies against b2GPI is detected by ELISA.Antibodies against b2GPI is detected by ELISA.• LA (Lupus Anticoagulant) constitutes a heterogeneous group of antibodies directed

also against PL binding proteins, mainly 2GPI and prothrombin.• LA antibodies induce elongation in vitro of some PL based clotting times:• LA antibodies induce elongation in vitro of some PL-based clotting times:

aPTT, kaolin clotting time, dilute Russel viper venom test (dRVVT).• LA is detected by functional clotting assays. HPIM, ed18, 2012

dRVVT

TextarinEcarin

Reptilasepaaaa

The Complemnt System

Main Tests & Adjunct TestsMain Tests & Adjunct Tests• VDRL, RPR• PLT, Hb, Coombs, Autoimmune assessment• aPTT, mixed aPTT, dilute aPTT,• Lupus Sensitive aPTT• Dilute PT (TF inhibition if 50 500 times diluted)• Dilute PT (TF inhibition, if 50‐500 times diluted)• KCT (only by kaolin, no PL)• dRVVT• Neutralization/ Correction Tests (using PLT PRP or Hexagonal PL)Neutralization/ Correction Tests (using PLT, PRP or Hexagonal PL)• Textarin & Ecarin clotting Time (Heparin Independency)• ELISA tests for APLA (Standard or non standards!)

Sens & Spec, Qualitative vs Quantitative, Class & subclass, Titer, Serial or p , Q Q , , ,Parallel panel, Not transient, False Positive/ Negative states,  drugs, pregnancy, age, etc

• BT, FDP, D‐dimer

LAC in the Lab and Clinic:2 dissimilar shadows from a solid object!j

!نی از اين خور و نی از آن

A Puzzle of Dilemmatic Words!• Anticoagulant & Procoagulant ( !پيش انعقادی ) • Lupus AC, Prothrombotic ( !پيش لخته ای )

h b l• Thromboplastin• Tissue Thromboplastin, Tissue Factor, F III• Histoplastin!!Histoplastin!!• Cephaloplastin• Placentoplastin & Pneumoplastin!• Plasma Thromboplastin, F IX• Extrinsic & Intrinsic (Coagulation Pathways)E t l S f C t t F t• External Surfaces, Contact Factors

• TT: Thrombin Time (F II & F I evaluation)• PT: Prothrombin Time, Quick Test (Temp De Quick)PT: Prothrombin Time, Quick Test (Temp De Quick)• PTT & aPTT (which word does the adjective PARTIAL refer to? To TIME or to Throm?!)

زمان، نسبی است يا معرف ترومبوپالستين؟؟

زمان نسبی است يا فعال شدن نسبی است يا معرف ترومبوپالستين؟؟

!!!

Lupus Anticoagulants and upus t coagu a ts a dthe Antiphospholipid Syndrome

• The lupus anticoagulant may be discovered incidentally when routine coagulation assaysincidentally when routine coagulation assays reveal prolongations in the PT, the aPTT, or bothboth

Cecil, ed24, 2012

Lupus Anticoagulant( )

•The term LA is a double misnomer!

(LAC or LA)

•The term LA is a double misnomer!•The LA test is founded on a wide range of clot‐based assays.y•According to the Guidelines, a three‐step procedure is required (Prolongation, Mixed test, Correction)h l b h l b b•The laboratory diagnosis should be based on:1) Prolongation of a phospholipid dependent clotting tests,2) Lack of correction of the prolonged clotting time by2) Lack of correction of the prolonged clotting time by 

addition of a small amount of normal plasma,3) Confirmation through correction by the presence of the 

higher concentration of phospholipidshigher concentration of phospholipids.

Lupus Anticoagulant Tests (functional)Lupus  Anticoagulant Tests (functional)

• In the past, many different tests were used in the process of d iLA detection:

• Kaolin clotting time (KCT) (Galli et al., 1995)• Silica clotting time (SCT) (Dragoni et al., 2001)• diluted prothrombin time (dPT) (Liestøl et al., 2002) & dPTT• Activated partial thromboplastin time sensitive/insensitive ratio 

(aPTT ratio) (Ames et al., 2001)/ ( )• Textarin/Ecarin clotting time (Triplett et al., 1993)

• Recently, only two following tests have been recommended f d i b d i li d l b l ifor LA detection based on practicality and global experience:diluted Russell viper venom time (dRVVT) and aPTT.

• The use of other tests has been discouraged mainly due toThe use of other tests has been discouraged mainly due to the limited experience rather than their poor performance.

Algorithm for the detection ofl l ( )lupus anticoagulant (LAC)

Lupus  Anticoagulant Tests

• The detection of LA is delicate and sometimes impossible when the ti t i l d t t d ith l ti l t

some Technical Aspects

patient is already treated with oral anticoagulants.• Pre‐analytical variables (e.g. quality of sample collection, 

centrifugation, temperature of storage) strongly influence final resultsresults.

• The specimen is sodium citrate anticoagulated blood which requires immediate, quick, double centrifugation.

• If the test is not immediately performed, the sample needs to beIf the test is not immediately performed, the sample needs to be frozen in a deep freezer.

• One of the extremely important aspects ensuring the good test results is the process of the blood collection. The smooth blood fl i i f h l iflow prevents activation of the coagulation processes.

• At least 4 times inversion of blood tube without shaking.

Dil ti ! A i l i i th d t kDilution! A simple inexpensive method to make a more sensitive reagent (of PTT or PT) for testing APLA.

Effects of Heparin anticoagulation on L b T tLab Tests

• The aPTT (and thrombin times) are prolonged even with minimal amounts of heparin in the circulation or with contaminatingamounts of heparin in the circulation or with contaminating indwelling catheters from which blood specimens are obtained.

• The reptilase time can be used to distinguish heparin from other causes of thrombin time prolongation (e g fibrin degradationcauses of thrombin time prolongation (e.g., fibrin degradation products, abnormal fibrinogens).

• The PT may be prolonged in the presence of large concentrations of heparinof heparin.

• Heparin functions as a circulating inhibitor, so that mixing studies of patient plasma with normal plasma do not result in correction of the aPTTof the aPTT.

• LMWH preparations do not affect the aPTT but may affect the thrombin time, depending on the thrombin concentration used in the assayin the assay.

• The anticoagulant properties of LMWHs can be monitored by the anti‐factor Xa assay. Cecil, ed24, 2012

Collection, Transport, and Processing ofBlood Specimens for

Testing Plasma Based Coagulation AssaysTesting Plasma-Based Coagulation Assays• Specimen Collection

Obtaining Blood Specimens (2 tube gentle mix)Obtaining Blood Specimens (2‐tube, gentle mix)Anticoagulant (Na Citrate, dihydrate, 105 to 109 mmol/L, 3.13% to 3.2%)Blood/Anticoagulant Ratio (1:9, tube in tubeCitrate Concentration Adjustments (HCT dependent)Citrate Concentration Adjustments (HCT dependent)Needle Gauge, hemolysis, winged blood collection sets, etc

• Specimen Transport, Processing, and Sample StorageSuitable Plasma Specimen (no clot, PLT‐poor , not icteric, lipemic or hemolysed)Storage (& Transportation Time)Temperature? (RT/Frig/Freezer, low/deep/ultra deep, no frost?)centrifuged? Plasma on top of the cells?

• Performance of Coagulation AssaysSingle vs. Duplicate Determinations, Incubation (Prewarming time, activation time)

• Reporting of Resultslt f th PT t t t th t h lf f d l d th t f APTT t t tresults of the PT test to the nearest half of a second or less and that of APTT test to 

the nearest second or less

CLSI, H21‐A4 (Approved Guideline, ed 4)

CLSI, H21‐A4 (Approved Guideline, ed 4)

!غHenry’s CD&MLM (2011, ed 22)

!غ

Citric Acid / Sodium Citrate/(and the water content or آب تبلور)

• Hydrated or Dessicated?• Made in Iran or somewhere else?Made in Iran or somewhere else?• Merck?• Sigma?!!g• Fluka, Panreac, Aldrich, Rankem, LobaChemie?

با افتخار؛ ايرانی بخرناصرخسرو( از )نه )نه از ناصرخسرو(

!سری به سالنھای بيرون بزنيم

Let’s have a look outside here,Let s have a look outside here,over the counter in the booths! 

٩/٣٢/٧ ٩X٢/٧= ٠/٣

CLSI, H21‐A4 (Approved Guideline, ed 4)

جداره ٢لوله ھای •The inner tube is usually made out ofpolypropylene (PP) and prevents the i l i f icitrate solution from evaporating.

•Polypropylene (PP) is ideal for sensitivecoagulation parameters, due to its inert characteristics.

سابقه•g p ,

•The outer tube is made of polyethyleneterephthalate (PET) and ensures a longshelf life for the vacuum

سابقه•اھداف•

shelf‐life for the vacuum.

•The minimum and maximum fill levelensures a reliable, correct blood to additive

اھداف•ا ا مزايا•

ا

ratio. مزايا•معايب• معايب•معايب•

CLSI, H21‐A4 (Approved Guideline, ed 4)

Just for Warming‐up!

كشيدن دندان دارد چند بار سابقه او . ساله اي كانديد جراحي پالستيك بيني شده است 25آقاي :اوليه او به صورت زير مي باشدآزمايشهاي.كه در اين مورد خونريزي نداشته است ريزي و ور ين ير ه ي بز ي زير ور ب و ي و

PLT: 400,000PT: 13 secPT: 13 sec

PTT: 110 secBT: 4 min

است؟ تر محتمل فاكتو كدام زادي ماد كمبود مادرزادي كدام فاكتور محتمل تر است؟كمبود

كمبود فاكتور سيزده) الفهشت)ب فاكت د ك كمبود فاكتور هشت)بكمبود فاكتور يازده) جدوازدهكمبود فاكتور ) د

The most common, simple Blood Coagulation Tests

aPTT Normal aPTT Prolonged

Deficiencies of prekallikreinHK

T Normal

HKfactor XIIfactor XI

factor s IX & VIII

Von Willebrand diseaseDeficiencies of factor XIIIOther factors (cases in IRAN)Mild Carrier StatesReagent Insensitivity!PT Inhibitor / Ab to factor VIII

Dysfibrinogenemias

Lupusanticoagulant

Heparintherapy

Reagent Insensitivity!

Deficiencies of factor XFactor Von

ged

Deficiency of factor VII

y g

Factor VProthrombinFibrinogen

Massive TransfusionC

PT Prolo Deficiency of factor VII

Warfarin therapyLiver disease

DICVitamin k deficiency

Red Indicates Bleeding or even Hemorrhage! Yellow indicates Inhibitor rather than Deficiency

More Warming‐up!

كشيدن دندان دارد كه در اين چند بار سابقه او . ساله اي كانديد جراحي پالستيك بيني شده است 25خانم :اوليه او به صورت زير مي باشدآزمايشهاي.مورد خونريزي نداشته است ز ر و هور يز ر ز ور و و

PLT: 400,000PT: 13 sec

PTT: 110 secBT: 4 min

كدام مورد محتمل تر است؟

كمبود فاكتور سيزده) الفكمبود فاكتور هشت)بكمبود فاكتور يازده)جدوازدهكمبود فاكتور ) دAPLA) ره

Hexagonal PLHexagonal PL

• Adjunctive tests involve tests directed at inhibition of jin vitro clotting (e.g., activated partial thromboplastintime, dilute Russell's viper venom time), recognizing the discordancy between in vivo thrombosis and inthe discordancy between in vivo thrombosis and in vitro anticoagulation.

• The mechanism by which antibodies to phospholipids y p p pand other clotting factors induce thrombosis in vivo is unknown, although they may interact with the surface of cells (e g endothelium) and with solublesurface of cells (e.g., endothelium) and with soluble clotting factors to promote a prothrombotic state.

Cecil, ed24, 2012

Other Immuno/ELISA Tests for APLS Dx:some R not preferred to be used clinicallysome R not preferred to be used clinically

(at least currently)Anti‐Annexin V IgG/IgMAnti Annexin V IgG/IgMAnti‐beta‐2‐Glycoprotein I IgAAnti‐beta‐2‐Glycoprotein I IgG/IgMAnti‐beta‐2‐Glycoprotein I Screeny pAnti‐Cardiolipin IgAAnti‐Cardiolipin IgG/IgMAnti‐Cardiolipin Screen WhichOne? Anti‐Phosphatidic Acid IgG/IgMAnti‐Phosphatidyl Inositol IgG/IgMAnti‐Phosphatidyl Serine IgG/IgM

Which One?Anti‐Phosphatidyl Ethanolamine G/MAnti‐Phospholipid Screen IgG/IgMAnti‐Prothrombin IgAA ti P th bi I G/I MAnti‐Prothrombin IgG/IgMAnti‐Prothrombin Screen ThromboCombo IgG/IgM

Anti β2GPI – The most “real” aPL test?

• β2GPI is a 50‐kDA phospholipid binding protein present in plasma in concentrations of approximately 200 mcg/mL.

• The genetic deficiency of β2GPI appears to be not involved with any diseases (Matsuura et al., 2010).

• A wide range of functions: regulation of coagulation, modulation of l t ti it B ddi f b i d id d l fcomplement activity, Bedding of embryo in decidua, and clearance of 

apoptotic cells from the circulation. The real function of β2GPI is yet not clear.

• It has been found recently that β2GPI can play an important role in theIt has been found recently that β2GPI can play an important role in the immunity (De Groot & Meijers, 2011).

• Beta2‐glycoprotein I is a member of the so‐called "complement control protein" (CCP) superfamily, whose members are identified by the presence of one or more of a motif containing a characteristic disulfide bond pattern. These motifs are called CCP or sushi domains.

• There is no evidence that any of β2GPI polymorphism is connected with the presence aβ2GPI antibodies or APS (Swadzba et al 2006)the presence aβ2GPI antibodies or APS (Swadzba et al., 2006).

• β2GPI was designated as apolipoprotein H initially as it could be isolated from VLDL fractions and had high affinity for triglyceride‐rich particles.

Crystal structure of β2GPIy βwith the five Complement Control Protein domains (CCP‐I to CCP‐V)

Proposed model for binding of b2‐GPIi i h h li idto anionic phospholipids

ELISA Tests(non‐functional)

• Undoubtedly, laboratory criteria that define the antiphospholipid syndrome require modification because the two different possible cut‐off values for discrimination between positive and negative aCL(>99th percentile and >40 GPL).

• Not only the presence of antibodies is important butNot only the presence of antibodies is important but also the coexistence of different  classes of antibodies can increase the risk of thrombosis.

• The updated criteria do not separate IgG from IgM• The updated criteria do not separate IgG from IgMclass of antibodies.

• The term “triple positivity” for the presence of all l i APLA i t d ith hi h i k f th b iclassic APLA is connected with high risk of thrombosis.

ELISA Tests(interpretation)

• APLA positive patients with no APLS clinical symptoms are at present excluded from the diagnosis of APLS syndrome.

• LAC and persistently high titers of all groups of aPLantibodies in IgG class probably are connected with even higher risk of thrombosis than moderate aCL of IgM classhigher risk of thrombosis than moderate aCL of IgM class in women with one pregnancy loss in the history.

• The group of patients with combined: LAC and aCL IgG or d β h l k f h bLAC and aβ2GPI IgG, can have similar risk of thrombosis as 

the patients with triple positivity: LAC and two IgMantibodies (aCL and aβ2GPI).( β )

D i i M kiDecision Making when

Purchasing kits

What’s Ur idea about Purchasing these kits?

√√

√√

√√

Euroimmune®

For Further Studyy

Downloadable from:http://www.iacld.ir/congresscategory/815--13920326

Lab Criteria and Immunological Criteria

Acquired HemophiliasEpidemiology and Pathobiology• Autoantibody inhibitors can occur spontaneously in individuals with 

previously normal hemostasis (nonhemophiliacs).Alth h i t l 50% f ti t h b i d l i• Although approximately 50% of patients have no obvious underlying cause, the remainder of cases are associated with autoimmune diseases, lymphoproliferative disorders, idiosyncratic drug associations, and pregnancy.p g y

Clinical Manifestations and Diagnosis • Patients typically have massive hemorrhagic events, usually much more 

severe than events produced by alloantibodies in patients with congenital hemophilia.

• The laboratory expression of autoantibodies is similar to that of alloantibodies, except that clotting factor activity is not completely neutralized Residual clotting factor activities between 3% and 20% ofneutralized. Residual clotting factor activities between 3% and 20% of normal frequently are observed in patients with autoantibodies.

Treatment:• Porcine factor VIII concentrate Immunosuppressive therapy withPorcine factor VIII concentrate, Immunosuppressive therapy with 

corticosteroids (prednisone) or cytotoxic agents (cyclophosphamide), anti‐CD20 antibody (rituximab)

Cecil, ed24, 2012

Anti‐Phospholipids antibodies(a short REVIEW)

A ti h h li id tib di h t f ilAnti‐phospholipid antibodies are a heterogeneous familyof autoantibodies that react with epitopes on proteinsthat are themselves complexed with negatively chargedp g y gphospholipids.

The blood vessel problems can then lead to complicationsp psuch as stroke, heart attack, and miscarriage.

The most commonly measured kinds of Antibodies are:– Anticardiolipin antibody

– Lupus anticoagulant

– Anti beta 2 GPI

Lupus  Anticoagulants (lupus antibody, LA, or lupus inhibitors)( p y p )

Lupus anticoagulants (LA) are heterogeneous IgG or IgM autoantibodieswhich inhibit phospholipid‐dependent assays of blood coagulation bybinding to plasma phospholipid binding proteins such as beta 2binding 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 lupusErythematosus, and only a small proportion will proceed to develop thisdisease.

It is a prothromboticprothrombotic agentagent, that is, presence of Lupus anticoagulantantibodies precipitates the formation of thrombi in vivo.

I i it i th ht t i t t ith l t l t b h h li idIn vivo, it is thought to interact with platelet membrane phospholipids,increasing adhesion and aggregation of platelets; thus its in vivoprothrombotic characteristics.

The term "anticoagulant" accurately describes its function in vitro, but in vivo, it is now known that it functions as a coagulantLupus anticoagulants impair the in vitro phospholipid dependentactivation 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 functionalanticoagulant is then confirmed with phospholipid sensitive functional clotting testing, such as the dilute Russell's viper venom timedilute Russell's viper venom time, or the Kaolin clotting timeThe presence of an LA is usually not associated with a bleeding problemThe presence of an LA is usually not associated with a bleeding problemunless accompanied byoThrombocytopenia,oFactor II deficiencyoFactor II deficiency,oPlatelet dysfunctionoOr drug administration (e.g., aspirin).

The ISTH(International Society on Thrombosis and Haemostasis ) recommends that the laboratory diagnosis of lupus anticoagulants (LA) h ld b i d t d bl t if d l f ll i fshould be carried out on double‐centrifuged plasma following a four‐step procedure adhering to these principles:

Prolongation of a phospholipid‐dependent coagulation test.E t i i (dPT)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 testsEvidence of inhibitory activity on mixing tests.Evidence of phospholipid dependence.Correction of the prolonged coagulation time after addition of excess phospholipid or plateletsphospholipid or platelets Lack of specificity for any one coagulation factorLack of specifi c inhibition of one coagulation factor (such as FVIII:C,FIX:C, or FXI).)

preliminary criteria for the classification of definite

Antiphospholipid Syndrome (APLS)Antiphospholipid Syndrome (APLS)

•Definite APS is considered to be present if at least one clinical and one laboratory criteria are met.•Positivity should be present on two or more occasions at least 12 weeks apart for any of the tests.

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