laboratory approach to bleeding disorders

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Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: [email protected] Website: pathologybasics.wix.com/notes

LABORATORY APPROACH TO

BLEEDING DISORDERS

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OVERVIEW

1. Normal coagulation homeostasis 2. Classification of bleeding disorders 3. Clinical evaluation of bleeding disorders 4. Laboratory evaluation of bleeding disorders

a. Screening tests SCREENING TESTS FOR PRIMARY HEMOSTASIS

1. Peripheral smear 2. Platelet count 3. Bleeding time 4. Platelet function analysis

SCREENING TESTS FOR SECONDARY HEMOSTASIS 1. Clotting time 2. Prothrombin time 3. Activated partial thromboplastin time 4. Thrombin time

Summary of screening tests

b. Specific tests TESTS FOR PLATELET FUNCTIONS

1. tests for platelet adhesion 2. Test for platelet aggregation 3. Test for platelet release reaction 4. Test for clot retraction 5. Test for platelet procoagulant activity 6. Test for glycoproteins on platelet surface 7. Test for abnormalities in arachidonic acid metabolism

TESTS FOR COAGULATION FACTORS 1. coagulation factor assays 2. Quantitative estimation of fibrinogen 3. Thromboplastin generation test 4. Mixing test based on PT or aPTT 5. FXIII qualitative assay 6. Paracoagulation test

TESTS FOR FIBRINOLYSIS 1. Detection of fibrinogen and fibrin degradation products 2. Detection of cross linked fibrin D dimmers

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* NORMAL COAGULATION HOMEOSTASIS

1. Normal coagulation involves interaction of vessel walls, platelets and coagulation factors 2. Problems at any one of these steps may lead to abnormal homeostasis

Vessel injury Collagen exposure Platelet adhesion FXII activatn Tissue thrombo plastin

Vasoconst Platelet release reaction BLOOD COAGULATION riction 5-ht

TXA2

Platelet aggregation Fibrinogen + thrombin Primary hemostatic plug Fibrin Stabilized hemostatic plug

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* CLASSIFICATION OF BLEEDING DISORDERS

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* CLINICAL EVALUATION OF BLEEDING DISORDERS History from the patient can tell whether the disorder is –

1. Localized or generalized 2. hereditary or acquired 3. Platelet disorder or coagulation disorder

Localized Generalized

1. Localized bleeding 1. recurrent episodes of bleeding in response to trivial trauma 2. bleeding from more than one site 3. spontaneous bleeding 4. excess bleeding following minor surgeries like tooth extraction, tonsillectomy, circumcision

Hereditary Acquired

1. onset early in life 2. similar disorder in close relatives 3. past history of similar episodes 4. history of disorder only in males on

the maternal side for many generations – X linked Hemophilia A or B

5. history of consanguineous marriage, both males and females only from the current generation affected – Autosomal recessive disorders like afibrinogenemia, von willebrand disease, Factor V or Factor X deficiency

6. Bleeding in both males and females, bleedin in one parent, bleeding in all generations – Autosomal dominant disorders like von willebrand disease, hereditary hemorrhagic telengectasia

usually secondary to disorders of 1. liver 2. uremia 3. hematologic malignancies 4. carcinoma 5. sepsis

Platelet disorders/

Vascular disorders Coagulation

disorders Sex affected Female Male Family history Negative Positive Petechiae, bleeding gums, epistaxis, menorrhagia

Common Rare

Deep hematoma in muscle and joints

Rare Common

Delayed bleeding from same site (12-24 hrs after)

Rare Common

Previous history of bleeding Not present Present since childhood

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* SCREENING TESTS FOR PRIMARY HEMOSTASIS

(i) Platelet count 1. MANUAL METHOD: Blood + 1% Ammonium oxalate counted on neubauer’s chamber (small roughly spherical refractile particles) Normals:

Platelet count 1,50,000 to 4,00,000 lakhs/mm3

2. AUTOMATED METHOD:

1. A cell counter analyses platelets based on its size. 2. Other platelet associated parameters like MPV, PDW and reticulated platelets are also

obtained MPV (mean platelet volume) In some platelet disorders, abnormally large platelets are released in circulation

MPV increased MPV normal 1. myeloproliferative disorders 2. peripheral destruction of platelets

1. Thrombocytopenia due to impaired platelet production like thrombocytopenia

PDW (platelet distribution width) Measure of degree of variation in platelet size

PDW increased PDW normal Myeloproliferative disorder Secondary or reactive thrombocytosis

Reticulated platelets: Concept analogous to reticulocytes.

Increased Normal

Peripheral destruction Aplastic anemia

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(ii) Peripheral Smear For details on peripheral smear examination, refer to separate notes on the same. Only salient points related to platelet examination will be described here. Examination of platelets on peripheral smear helps to rule out falsely low counts on automated methods, like due to clumping.

Total count Associated abnormalities in other cell lines RBC Fragmented RBCs indicate DIC with thrombocytopenia WBC Abnormal cells with thrombocytosis/thrombocytopenia (leukemias) Platelet Normal count 1 platelet per 500-1000 red cells Giant platelets Myeloproliferative neoplasms

Bernard soulier syndrome ITP

Discrete isolated platelets without clumping in finger prick smear

Glanzmann’s thrombasthenia Uremia

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(iii) Bleeding time Definition: Time required for bleeding to cease following skin puncture/incision. Methods:

1. Duke a. lobe is punctured b. method is not standardized 2. Ivy

a. On volar surface of forearm with lancet b. Depth of 2 – 2.5 mm under standard venous pressure of 40 mm Hg c. Blood oozing is blotted with filter paper d. Time taken to stop bleeding is noted

3. Template a. uses same site as Ivy b. larger surgical blade is used for incision, depth 6-9 mm

Normals:

Ivy’s method 2 to 7 minutes Causes of prolonged bleeding time: See chart on page 4

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(iv) Platelet function analysis Principle: Blood aspirated through Capillary into machine collagen epinephrine/ ADP Blood passes through Aperture in Machine which is coated with Platelet agonists such as Collagen/epinephrine/ADP The PFA 100 ANALYSER

Platelet adhesion, activation And aggregation closes the Aperture overtime This time known as closure time is indicator of Platelet function Abnormals: Closure time is prolonged in

1. Platelet disorders not prolonged in vascular disorders 2. Von willebrand disease

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* SCREENING TESTS FOR SECONDARY HEMOSTASIS

(i) Clotting time Definition: Time required for whole blood to clot in a glass test tube at 37˚ C Normals:

Clotting time 5-15 mins Abnormals:

1. Prolonged in defects of common/intrinsic pathway$ 2. But is less sensitive, prolonged only when factor levels drop to <1% of normal, time is

not prolonged in mild / moderate deficiency

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$ see next page for coagulation pathways details Pathways of coagulation

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(ii) Prothrombin time (PT) and Activated partial thromboplastin time (aPTT) Precautions and prerequisites:

1. Venepuncture the anticubital vein, with minimal trauma to avoid tissue thromboplastin release

2. Donot collect from indwelling catheters to avoid mixing with tissue fluids 3. Use plastic syringes with 20-21 G needles. Donot use glass syringes/bottles as glass

activates contact factors and initiates clotting through intrinsic pathway. 4. Prolonged tourniquet should be avoided as it causes increased fibrinolysis 5. Anticoagulant used in sodium citrate (3.2%) as it causes rapid chelation of calcium & FV

and FVIII remain relatively more stable in it, blood:anticoagulant ratio = 1:9 6. Allow blood to flow gently down the sides of tube/container 7. thoroughly mix blood and anticoagulant 8. transport the tightly sealed tube to lab without delay 9. if labile coagulation factors are to be measured, maintain tube at 4˚C 10. Coagulation studies should be carried out within 2 hours of collection

PROTHROMBIN TIME: Method:

1. Prepare platelet poor plasma (PPP) Allow blood in citrate tube to stand centrifuge at 3000-4000 rpm for 15/20 min

Platelets, RBC and WBC will settle down

Use supernatant (for platelet function studies, we need to use platelet rich plasma) 2. Take 25 µl PPP in a cuvette A. Take 25 µl tissue thromboplastin + 25 µl calcium chloride

in another cuvette B. 3. Place PPP cuvette in reader 4. Start optic reader 5. Put contents of cuvette B (total 50 µl) into cuvette A 6. Obtain readings of PT and INR 7. A control sample must be run along with patient’s

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Principle: 1. Tissue thromboplatin activates factor VII and initiates the extrinsic pathway of

coagulation 2. Thromboplastin also provides phospholipids for certain coagulation reactions 3. PT measures activity of Extrinsic and common pathways FVII F X, V, II, I

CONCEPT OF INR

1. The international normalized ratio (INR) was introduced in an attempt to standardize the PT.

2. In its original manifestation, the PT was very variable because different thromboplastins were non-standardized and derived from many varied sources. PTs performed on the identical specimen by different laboratories were inconsistent.

3. The concept behind the INR is that differences between the thromboplastins are accounted for by a calculation:-

INR = [PT (patient) ÷ PT (Control)]ISI The INR has no units (it is a ratio) and is determined to one decimal place. ISI, or international sensitivity index is a function of the thromboplastin reagent. Normals:

PT 11-16 Seconds INR 0.9 – 1.1

Abnormals: PT is prolonged in

1. Vitamin K deficiency – (F II, VII, IX, and X are vit K dependent factors out of which F VII and X are important in extrinsic and common pathways) Extrinsic and common pathways are affected

2. Oral anticoagulant therapy – PT is standard for monitoring anticoagulant therapy Recommended Therapeutic Range for Oral Anticoagulant Therapy. INDICATION INTERNATIONAL NORMALIZED RATIO (INR) Treatment of venous thrombosis 2.0 - 3.0 Treatment of pulmonary embolism Prevention of systemic embolism Tissue heart valves Acute myocardial infarction Atrial fibrillation Recurrent embolism 2.5 - 3.5 Mechanical heart valve Antiphospholipid antibodies

3. DIC – exhaustion of coagulation factors 4. Inherited deficiency of coagulation factors

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ACTIVATED PARTIAL THROMBOPLASTIN TIME: Method:

1. Prepare platelet poor plasma as above 2. Take 25 µl sample in cuvette, add 25 µl aPTT reagent (which contains activator#) to it 3. Incubate for 180 secs 4. Place cuvette in optic area 5. Add 25 µl calcium chloride when ready 6. A control should always be run along with the patient’s

#Activator is not present in PT reagent, activators used can be kaolin, elite, ellagic acid, silica etc. Normals:

aPTT 30-40 seconds Principle:

1. The activator activates intrinsic pathway of coagulation. 2. Thus PT is altered in intrinsic and common pathway defects.

Abnormals: aPTT is prolonged in:

1. Inherited deficiencies of factor VIII (Hemophilia A) and Factor IX (Hemophilia B) 2. Non specific inhibitor antibodies against F VIII e.g. Lupus inhibitor

a. Donot act directly but block interaction of FVIII with other clotting factors 3. DIC 4. Heparin

a. Inhibits factor XII, XI and X through antithrombin III b. Heparin therapy is monitored through aPTT

5. Vit K deficiency Affects Factor IX

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(iii) Thrombin Time (TT) It is a test for plasma fibrinogen levels. Method:

1. Platelet poor plasma + thrombin reagent 2. Time required for clotting is noted

Principle:

1. Plasma fibrinogen is cleaved by thrombin (activated factor II) to form fibrin clot Normals:

TT 11.6 – 20.7 seconds Abnormals: TT is prolonged in

1. Afibrinogenemia (<100 mg/dl) or dysfibrinogenemia 2. Fibrinogen or fibrin degradation products (interfering substances) 3. presence of heparin in plasma 4. chronic liver disease

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*SUMMARY OF SCREENING TESTS

DEFECT IN BT PLATELET COUNT

PT APTT COMMON CAUSES

Platelet fuction/ vascular disorders/ vW defects

N N N Von willebrand disease Storage pool defects Other platelet affecting disorders

Thrombocytopenia N N All causes of thrombocytopenia

Extrinsic pathway N N N See causes of prolonged PT Intrinsic pathway N N N See causes of prolonged

aPTT Common pathway

N N Causes of either prolonged PT or prolonged aPTT

Multiple pathways (DIC)

N/ DIC/Liver disease

Clot stabilization N N N N FXIII deficiency

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*TESTS FOR PLATELET FUNCTION

(i) Test for platelet adhesion (Glass bead column test)

Blood

Platelets counted before entering glass column Glass column with beads, adhesion of platelets Occurs here Aggregation also occurs, hence is non specific Platelets are counted after passing through Glass column Normals:

>25 % retention of platelets Normal Abnormals:

1. <25% retention is observed with von willebrand’s disease

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(ii) Test for platelet aggregation Method: Prepare platelet rich plasma Add aggregating agent and put in aggregometer

As platelets aggregate, more light passes through Two waves are obtained on graph from the machine, primary wave and secondary wave (biphasic) corresponding to degree of platelet aggregation against time

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Aggregating agents used Curve type ADP / Epinephrine Biphasic

Collagen / ristocetin / Arachidonic acid Monophasic curve

Normals: Bi phasic ADP curves Biphasic epineph Monophasic collagen Monophasic ristocetin monophasic AA See control lines in the graphs

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Abnormals:

1. GLANZMANN’S THROMBASTHENIA 1. In Glanzmann’s thrombasthenia, Platelet GPIIb/IIIa is defective. 2. This receptor is important for platelet aggregation. 3. All agents induce aggregation through this receptor except ristocetin. 4. Hence in Glanzmann’s thrombasthenia, aggregation is defective for all agents except

ristocetin.

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2. BERNARD SOULIER SYNDROME AND VON WILLEBRAND DISEASE

1. In Bernard soulier syndrome there is deficiency of GpIb/IXa, whereas in von willebrand

disease, there is deficiency of von willebrand factor. 2. Aggregation is there in response to all agents except ristocetin

BS and VWD can be differentiated by addition of normal plasma. If the aggregation is seen now, it means the disease is VWF because normal plasma is a source of VWF.

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3. STORAGE POOL DEFECTS 1. In this disorder, there is defective granule release from platelets 2. Due to defective release reaction, secondary aggregation is defective 3. Hence there is no secondary wave in response to ADP/epinephrine/collagen and only

partial aggregation in response to ristocetin.

4. ASPIRIN LIKE DEFECT (DEFECT IN COX PATHWAY) 1. Absent aggregation to arachadonic acid. 2. Primary wave aggregation only with ADP. 3. Decreased or absent aggregation with collagen

Aggregating agents ADP/EPINEPHRINE/COLLAGEN

Disorder

PRIMARY WAVE SECONDARY WAVE RISTOCETIN

BS syndrome N N D vWD N N D Glanzmann’s D D N Storage pool$ N D N Aspirin like$ D D D Aspirin like in response to AA$

N (AA used)

D (AA used)

D

N normal D deficient AA arachidonic acid $ Measure of platelet release reaction

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(iii) Tests for platelet release reaction Indirect Tests Direct Tests The secondary wave of aggregation in 1. estimation of secretion of Response to ADP/epinephrine/collagen dense core granules is a measure of release reaction 2. estimation of secretion of alpha granules

Contents of platelet granules Dense core granules adenosine diphosphate (ADP)

adenosine triphosphate (ATP) ionized calcium (which is necessary for several steps of the coagulation cascade) histamine serotonin

Alpha granules insulin-like growth factor 1 platelet-derived growth factor TGFβ platelet factor 4 (which is a heparin-binding chemokine) other clotting proteins (such as thrombospondin, fibronectin, and von Willebrand factor)

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Abnormals: 1. Storage pool deficiency 2. Aspirin like defect (COX deficiency)

(iv) Tests for clot retraction 1. Clot retraction is the "shrinking" of a blood clot over a number of days. In so doing, the

edges of the blood vessel wall at the point of injury are slowly brought together again to repair the damage.

2. Clot retraction is dependent on release of multiple coagulation factors from platelets trapped in the fibrin mesh of the clot. Thus, failure to retract can be a sign of thrombocytopenia or Glanzmann’s thrombasthenia.

(v) Tests for platelet procoagulant activity This test measures amount of residual prothrombin activity after whole blood is allowed to clot completely. Whole blood allowed to clot serum with residual Perform PT In plain tube prothrombin (PT1) Citrated plasma Perform PT

(same pt) (PT2)

PT1

= PLATELET PROCOAGULANT ACTIVITY PT2

Interpretation: Presence of residual prothrombin (PCA >1) may be due to deficiency of coagulation factors or of platelet phospholipids.

(vi) Tests for detection of glycoproteins on platelet surface (Flow cytometry)

GpIb/IXa GpIIb/IIIa Defective in Bernard soulier syndrome Defective in Glanzmann’s thrombasthenia

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(vii) Tests for defects in Arachidonic acid metabolism (AA) 1. Arachidonic acid is necessary for TXA2 generation which activates platelets. 2. Normal aggregation in response to Arachidonic acid shows defects in arachidonic acid

metabolism pathway

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*TESTS FOR COAGULATION FACTORS

(i) Coagulation factor assays Principle: Patient’s plasma + standard plasma deficient in perform PT Factor to be tested in various and aPTT Dilutions Normal Prolonged Patients plasma patients plasma Contains that doesn’t contain Factor that factor Normals:

All factors 50-150 % or 50-150 U/dl

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(ii) Quantitative estimation of fibrinogen Normals:

Fibrinogen levels 200-400 mg/dl Abnormals:

Decreased fibrinogen Afibrinogenemia, dysfibrinogenemia, DIC

Increased fibrinogen MI, trauma, neoplasia, inflammatory conditions Methods:

1. Coagulable protein method (based on thrombin time) thrombin Fibrinogen fibrin Thrombin time thrombin time α 1 concentration of fibrinogen It means there is a linear relationship between concentration of fibrinogen and thrombin time. By comparing thrombin time of the test sample with thrombin time of known fibrinogen standard in the same system, the concentration of sample can be obtained by using a standard graph. 2. Immunological method (RIA based) 3. Heat precipitation test 4. Chemical precipitation test 5. Weighing of clot

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(iii) Thromboplastin generation test (TGT) Method: STAGE I Sample FXa-V complex + (aka prothrombinase) Generation of Phospholipid Prothrombinase + Calcium Prothrombin thrombin Fibrinogen Fibrin estimation of clotting time STAGE II if clotting time is abnormal, PT and aPTT are done and substitutions studies are carried out to determine deficient factors in plasma and serum. Prepared Serum$

+ Sample estimation of clotting + time Phospholipids + calcium Adsorbed plasma$$

+ Sample estimation of clotting + time Phospholipids + Calcium $ prepared serum contains FV and VIII $$ adsorbed plasma – FIX and X Both contain – FXI and XII

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Ad Plasma substitution defective F V / VIII abnormal Serum substitution defective F IX/X abnormal Both substitution defective F XI/XII abnormal

PT tests – F V, X aPTT tests – F VIII,IX, XI, XII Result interpretation:

Coagulation screen History PT aPTT

TGT Interpretation

Bleeding Bleeding

N P

P N

Plasma defect Plasma defect

F VIII deficient F V deficient

Bleeding Bleeding

N P

P N

Serum defect Serum defect

F IX deficient F X deficient

Bleeding No bleeding

N N

P P

Both defect Both defect

FXI deficient F XII deficient

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(iv) Mixing test based on PT/aPTT Done to differentiate clotting factor deficiency from prolonged PT/aPTT due to inhibitors Method:

Prolonged PT or aPTT in patient suspected of bleeding disorder

Repeat PT/aPTT after adding normal plasma (patient plasma 50% + normal plasma 50%)

PT/aPTT normal PT/aPTT still prolonged Incubate at 37 deg and repeat Due to immediate acting inhibitor PT/aPTT prolonged PT/aPTT still normal Delayed acting coagulation factor deficiency Inhibitor perform TGT

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(v) FXIII Qualitative assay (Urea clot lysis test) Done when all other tests for hemostasis are normal. FXIII provides stability to clot formed. Method: Fibrin Fibrin clot dissolves - FXIII def FXIII +5M urea Unstable clot Doesnot dissolve Normal FXIII

(vi) Paracoagulation tests (protamine sulphate test & ethanol gelation test) Tests done to detect ongoing intravascular coagulation. Rationale:

In circumstances such as after a major surgical procedure or liver disease

We need to know whether active coagulation is going on in plasma

This would be indicated by presence of soluble (non polymerized) fibrin monomers in plasma

Method: Platelet poor plasma + protamine sulphate/ gel formed – fibrin Ethanol monomer +nt (active coagulation +nt) No gel formed – fibrin Monomer absent (active coagulation –nt)

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*TESTS FOR FIBRINOLYSIS

These are latex agglutination tests for

Fibrinogen and Fibrinogen degradation D-dimers# Products (FDPs)# #Actual clot degradation process

plasmin Fibrinogen fibrinogen degradation

products Thrombin

Fibrin polymer

FXIII

Cross linked fibrin Plasmin

D dimer

Application: 1. for detecting DIC – (d dimer more specific) 2. pulmonary embolism 3. DVT 4. severe pneumonia 5. MI

laboratory approach to bleeding disorders

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