DR.G BALAJI.PROF.DR.G. SUNDARAMURTHY’S UNIT

Disorders of blood coagulation:

Inherited or acquired coagulation disorders. Disorder of vessels

Disorders of platelets- quantity or function

HemostasisBV Injury

PlateletPlateletAggregation

PlateletActivation

Blood VesselBlood Vessel Constriction

CoagulationCoagulation Cascade

Stable Hemostatic Plug

Fibrin formation

Reduced

Blood flow

Damage/contact.

Primary hemostatic plug

Neural

•CBC-Plt•BT,(CT)•PT•PTT

Platelet studyAntibody testsFactor Assay

Contact

Type of Bleedingecchymosespetechiaeepistaxisdeep soft tissue bleedhemarthrosesGI bleeding

Tests of Hemostasis:Screening tests:

Bleeding.T - 10m. Platelet & BV function Prothrombin.T – Extrinsic, aPTT – IntrinsicThrombin.T – common path.

Specific tests:Factor assays – hemophilia.Tests of thrombosis – TT, FDP, Platelet function studies:

Adhesion, Aggregation, Release tests.Bone Marrow study

Pre-analytic errors Problems with blue-top tube

Partial fill tubes Vacuum leak and citrate

evaporation

Problems with phlebotomy Heparin contamination Wrong label Slow fill Underfill Vigorous shaking

Biological effects• Hct ≥55 or ≤15

• Lipemia, hyperbilirubinemia, hemolysis

Laboratory errors• Delay in testing

• Prolonged incubation at 37°C

• Freeze/thaw deterioration

Bleeding timeMeasure of efficiency of vascular or

platelet phases. Do not discriminate between vascular

defects , thrombocytopenia or platelets dysfunction.

Not reproducible.Not a screening test . normal bleeding time do not exclude a

bleeding disorder- american society of clinical pathologists.

Platelets enumerationDirect or automated methods.Normal: 1.5 lakhs- 4.5 lakhsFalsely low platelets counts-

PSEUDOTHROMBOCYTOPENIA platelets agglutinins, paraproteinemias, giant platelets, lipemia, EDTA induced

platelets clumping.Flasely high platelets counts: Microspherocytes, WBC/RBC

fragments, pappenheimer bodies.

Platelets function assaysPlatelets aggregation using platelets rich

plasma.- standard method.Nephelometric or photometric measurements.ADP- produce platelets aggregation directly

irrespective of release of ADP from platelets.RISTOCETIN- antibiotic that induce platelets

aggregation in presence of von Willebrand factor.

Collagen, epinephrin, thrombin- cause platelet aggregation by release reaction.

Platelet function analyser- PFA 100hemoSTATUS SYSTEM- modified activated

clotting time test. To monitor cardio pulmonary bye-pass system.

Verify now systemHemostasis analysis system.DiaMed- Impact-R System

Coagulation testsAll tests on citrated plasma except D- Dimer.Nine parts of blood to one part of citrate.3.2% sodium citrate.Blue topped vacuum blood collection tubes.

Blood Coagulation & TestsBlood Coagulation & Tests

Activated partial thromboplastin timeIntrinsic and common pathway.When mixture of plasma and phospholipid

platelets substitute is re calcified, fibrin is formed in the presence of factors in intrinsic pathway and common pathway.

Platelet substitute-chloroform extract of brain, Inosithin- soyabean. Partial thromboplastin. Do not activate the

extrinsic pathway which require complete tissue thrombo plastin.

APTTPTT is used to detect factor deficiency ,

screen for lupus anti coagulantMonitor heparin anti coagulation.More sensitive to deficiency of VIII and

IX.Yields abnormal results if any factor level is

< than 15-30% of normal.PTT may be shortened by a high level of any

single factor. m/c is factorVIII.

APTTContact activation was provided by the glass

tube.Activators- ellagic acid, particulate silicates

such as celite or kaolin.No significant differences between adult and

children between 7-17 years.Shortened PTT Values is an independent risk

factors for death, thrombosis ,bleeding and morbidity.

PROTHROMBIN TIME:Extrinsic and common path way.Tissue factor, factor VII,X,V, prothrombin,

fibrinogen.Plasma is recalcifed in the presence of tissue

factor.Independent of platelet count.PT is used for controlling anti coagulant

therapy.More sensitive to deficiency of VII and X.

Prothrombin timeChildren between 7-17 years have PT value 1

second longer than adults.Use INTERNATIONAL NORMAL RATIO.STYPVEN time- Russel viper venom directly

initiate coagulation with out factor VII. Used to differentiate factorVII deficiency from factor- X deficiency.

Thrombin time:When thrombin is added to plasma the time required

for clot formation is a measure at which fibrin forms.Abnormal value when fibrinogen level is <70 to 100

mg/dl.Prolonged by heparin.Prolonged in qulitatively abnormal fibrinogen,

elevated FDP, paraproteinemias, hyper fibrinogenemias.

REPTILASE CLOTTING TIME- un affected by heparin.

D-DIMERS and FDP:FDP results from proteolytic cleavage of fibrin

by plasmin. Increased in DIC and fibrinolysis.RA factor or residual fibringen may yield false

positive results.D-DIMER- specific fibrin degradation

product.DDE- TRIMER.UN POLYMERISED FIBRIN MONOMER.

UREA CLOT LYSIS ASSAY:FACTOR XIII assay.XIII Stabilises fibrin.Deficiency leads to premature clot lysis.Screening test. Abnormal results should be

confirmed by quantitative measurements of XIII.

TESTS FOR FIBRINOLYSISEUGLOBULIN CLOT LYSIS TIME:Plasma euglobulin contains plasminogen

activators and fibrinogen.Rate of lysis of a fibrin clot prepared from

euglobin fraction is a measure of plasminogen activators.

EACA can inhibit plasminogen activators but not free plasmin. Shortened euglobulin lysis time in presence of EACA indicated presence of free plasmin.

Bio assays for coagulation factorsExtent to which an unknown sample corrects

the abnormality in plasma with a known deficiency is proportional to the content of the deficient factor in the sample.

Measured in units or percentage of normal.Normal- 50-150 units/dl or 50-150% of

normal.Factor V, VIII, X , prothrombin.

Tests for inhibitors of coagulationMixing studies or inhibitor screens.Small amount of patient’s plasma if contains

inhibitors of coagulation will impair coagulation in normal samples.

Based on PT or APTT.HEPARIN presence can be confirmed by

thrombin time or reptilase time, or correction with protamine sulphate.

Automated coagulation methodsThrombo elasto graph- demonstrates

changes during blood coagulation and fibrinolysis. Used in surgical settings.

Activated clotting time- whole blood clotting assay.

Used to monitor intrinsic pathway.In cardio pulmonary bye pass, dialysis,

cardiac catheterisation.

Chromogenic and flurogenic methodsArtificial substances when cleaved by

coagulation factors release chromogenic or fluroscopic peptides.

Prothrombin, VII, IX, X, anithrombin, heparin, plasmin.

More precise and less time consuming.Used mainly in research and large reference

labs.

Evaluation of the patientHistoryPhysical ExaminationLaboratory Evaluation

HistoryAre you a bleeder?

surgical challengesaccidents & injuriesdental extractionsmenstrual history

HistoryGeneralized haemostatic defect or local

defectBleeding from multiple sites, spontaneous

bleeding or excessive bleeding after injury

Inherited defect or acquired Family H/O bleeding disorder, age of onset of

disease or any other event ppting. the bleeding

Is Bleeding suggestive of – Vascular, Platelet or coagulation disorderVascular /platelet disorders – Easy bruising ,

spontaneous bleeding from small vessels esp into skin – Purpura and echymosis .

Mucous membrane bleeding – Epistaxis, mouth –gums and Menorrhagia.

Coagulation disorders – Hemarthrosis, muscle bleeds, bleeding after injury or surgery

Does it sound genetic?duration of bleeding historycongenital v. acquiredfamily history

examine pedigreedetermine inheritance

Medical Historyliver diseaserenal diseasemalignanciespoor nutrition (Vit. K or C deficiency)

FIVE DRUGS THAT INTERFERE WITH HEMOSTASIS

ASPIRIN ANTICOAGULANTS ANTIBIOTICS ALCOHOL ANTICANCER

Laboratory AssessmentGuided by historyScreening tests

PTaPTTplatelet countfibrinogenthrombin time

Partial Thromboplastin Time(PTT)

Prothrombin time(PT)

Intrinsic pathwayExtrinsic pathway

Common pathwayThrombin timeThrombin

Surface activating agent (Ellagic acid, kaolin)PhospholipidCalcium

Thromboplastin Tissue factor Phospholipid

Calcium

Fibrin clotFibrin clotFibrin Clot

Initial Evaluation of a Bleeding Patient - 1Normal PT

Normal PTT

Consider evaluating for: Mild factor deficiency Monoclonal gammopathy Abnormal fibrinolysis Platelet disorder (2 anti-plasmin def) Vascular disorder Elevated FDPs

Ureasolubility

Normal

Abnormal Factor XIII

deficiency

Initial Evaluation of a Bleeding Patient - 2

Normal PT

Abnormal PTT

Test for factor deficiency: Isolated deficiency in intrinsic pathway (factors VIII, IX, XI) Multiple factor deficiencies (rare)

Repeatwith

50:50mix

50:50 mix is normal

50:50 mix is abnormalTest for inhibitor activity:

Specific factors: VIII,IX, XI Non-specific (anti-phospholipid Ab)

Initial Evaluation of a Bleeding Patient - 3

Abnormal PTNormal PTT

Test for factor deficiency: Isolated deficiency of factor VII (rare) Multiple factor deficiencies (common) (Liver disease, vitamin K deficiency, warfarin, DIC)

Repeatwith

50:50mix

50:50 mix is normal

50:50 mix is abnormalTest for inhibitor activity:

Specific: Factor VII (rare) Non-specific: Anti-phospholipid (rare)

Initial Evaluation of a Bleeding Patient - 4Abnormal

PTAbnormal

PTT

Test for factor deficiency: Isolated deficiency in common pathway: Factors V, X, Prothrombin, Fibrinogen Multiple factor deficiencies (common) (Liver disease, vitamin K deficiency, warfarin, DIC)

Repeatwith

50:50mix

50:50 mix is normal

50:50 mix is

abnormalTest for inhibitor activity: Specific : Factors V, X, Prothrombin, fibrinogen (rare) Non-specific: anti-phospholipid (common)

Laboratory Evaluation of BleedingOverview

CBC and smear Platelet count ThrombocytopeniaRBC and platelet morphology TTP, DIC, etc.

Coagulation Prothrombin time Extrinsic/common pathwaysPartial thromboplastin time Intrinsic/common pathwaysCoagulation factor assays Specific factor deficiencies50:50 mix Inhibitors (e.g., antibodies)Fibrinogen assay Decreased fibrinogenThrombin time Qualitative/quantitative

fibrinogen defectsFDPs or D-dimer Fibrinolysis (DIC)

Platelet function von Willebrand factor vWDBleeding time In vivo test (non-specific)Platelet function analyzer (PFA) Qualitative platelet disorders

and vWDPlatelet function tests Qualitative platelet disorders

Liver Disease

Decreased synthesis of II, VII, IX, X, XI, and fibrinogen

Prolongation of PT, aPTT and Thrombin Time

Often complicated byGastritis, esophageal varices, DIC

Lab Results in Hemophilia, VWD and Vit K Def

Haemophilia V W Disease Vit K Deficiency

Bleeding Time Normal Increased Normal

PT Normal Normal Increased

APTT Increased + Increased ± Increased

V111 levels

Decreased ++ Decreased Normal

vWF levels Normal Decreased Normal

Hemophilia A Hemophilia B Von Willebrand Disease

Inheritance X linked X linked Autosomal dominant

Factor deficiency VIII IV VWF

Bleeding site(s) Muscle,joint

Surgical

Muscle ,joint Mucous

Skin

Prothrombin time Normal Normal Normal

Activated PTT Prolonged Prolonged Prolonged

Bleeding time Normal Normal Prolonged or normal

Factor VIII Low Normal Normal

VWF Normal Normal Low

Factor IX Normal Low Normal

Platelet aggregation Normal Normal Normal

Coagulation factor deficienciesSummarySex-linked recessive

Factors VIII and IX deficiencies cause bleedingProlonged PTT; PT normal

Autosomal recessive (rare) Factors II, V, VII, X, XI, fibrinogen deficiencies

cause bleedingProlonged PT and/or PTT

Factor XIII deficiency is associated with bleeding and

impaired wound healingPT/ PTT normal; clot solubility abnormal

Factor XII, prekallikrein, HMWK deficienciesdo not cause bleeding

SummarySymptom Platelet Coagulation Petechiae Yes No

Sites Skin & Mucosa

Deep Tissue

Time Immediate Delayed

Ecchymoses/Hematomas

Yes Yes

Summary Hemostatic Disorders BT Plt PT PTT

Vascular Dis - - - -

PLT Disorder - - - -

Factor 8/9 *Congenital - - -

Vit K / Liver*Acquired - - -

Combined (DIC) -

Tests are normal-Now what?simple purpurasenile purpuraFactor XIII deficiencyalpha-2-antiplasmin deficiencymild factor deficiencyamyloidosisvascular disorders

Still more?Hereditary hemorrhagic telangiectasiascurvyEhlers-Danlos syndromeHenoch-Schonlein purpurathe un-diagnosable fibrinolytic defect

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