1

LABORATORY
APPROACH TO
BLEEDING DISORDERS
Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar
...
2

OVERVIEW
1.
2.
3.
4.

Normal coagulation homeostasis
Classification of bleeding disorders
Clinical evaluation of bleedi...
3

* NORMAL COAGULATION HOMEOSTASIS
1. Normal coagulation involves interaction of vessel walls, platelets and coagulation ...
4

* CLASSIFICATION OF BLEEDING DISORDERS

Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar
Co...
5

* CLINICAL EVALUATION OF BLEEDING DISORDERS
History from the patient can tell whether the disorder is –
1. Localized or...
6

* SCREENING TESTS FOR PRIMARY HEMOSTASIS

(i) Platelet count
1. MANUAL METHOD:
Blood + 1% Ammonium oxalate

counted on ...
7

(ii) Peripheral Smear
For details on peripheral smear examination, refer to separate notes on the same. Only
salient po...
8

(iii) Bleeding time
Definition:
Time required for bleeding to cease following skin puncture/incision.
Methods:
1. Duke
...
9

(iv) Platelet function analysis
Principle:
Blood aspirated through
Capillary into machine
collagen
epinephrine/
ADP

Bl...
10

* SCREENING TESTS FOR SECONDARY HEMOSTASIS

(i) Clotting time
Definition:
Time required for whole blood to clot in a g...
11

$ see next page for coagulation pathways details
Pathways of coagulation

Notes on Laboratory approach to bleeding dis...
12

Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Websit...
13

(ii) Prothrombin time (PT) and Activated partial thromboplastin time
(aPTT)
Precautions and prerequisites:
1. Venepunc...
14

Principle:
1. Tissue thromboplatin activates factor VII and initiates the extrinsic pathway of
coagulation
2. Thrombop...
15

ACTIVATED PARTIAL THROMBOPLASTIN TIME:
Method:
1. Prepare platelet poor plasma as above
2. Take 25 µl sample in cuvett...
16

(iii) Thrombin Time (TT)
It is a test for plasma fibrinogen levels.
Method:
1. Platelet poor plasma + thrombin reagent...
17

*SUMMARY OF SCREENING TESTS
DEFECT IN

BT

Thrombocytopenia
Extrinsic pathway
Intrinsic pathway

N
N

N
N

Common path...
18

*TESTS FOR PLATELET FUNCTION

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

Blood

Platelets counted before...
19

(ii) Test for platelet aggregation
Method:
Prepare platelet rich plasma
Add aggregating agent and put in aggregometer
...
20

Aggregating agents used
ADP / Epinephrine

Curve type
Biphasic

Collagen / ristocetin / Arachidonic acid

Monophasic c...
21

Abnormals:
1. GLANZMANN’S THROMBASTHENIA

1.
2.
3.
4.

In Glanzmann’s thrombasthenia, Platelet GPIIb/IIIa is defective...
22

2. BERNARD SOULIER SYNDROME AND VON WILLEBRAND DISEASE

1. In Bernard soulier syndrome there is deficiency of GpIb/IXa...
23

3. STORAGE POOL DEFECTS
1. In this disorder, there is defective granule release from platelets
2. Due to defective rel...
24

(iii) Tests for platelet release reaction

Indirect Tests

Direct Tests

The secondary wave of aggregation in
Response...
25

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

(iv) Tests for clot retraction
1. Clot ...
26

(vii) Tests for defects in Arachidonic acid metabolism (AA)
1. Arachidonic acid is necessary for TXA2 generation which...
27

*TESTS FOR COAGULATION FACTORS

(i) Coagulation factor assays
Principle:

Patient’s plasma

+

standard plasma deficie...
28

(ii) Quantitative estimation of fibrinogen
Normals:
Fibrinogen levels

200-400 mg/dl

Abnormals:
Decreased fibrinogen
...
29

(iii) Thromboplastin generation test (TGT)
Method:
STAGE I
Sample
+
Phospholipid
+
Calcium

FXa-V complex
(aka prothro...
30

Ad Plasma substitution defective
Serum substitution defective
Both substitution defective

F V / VIII abnormal
F IX/X ...
31

(iv) Mixing test based on PT/aPTT
Done to differentiate clotting factor deficiency from prolonged PT/aPTT due to inhib...
32

(v) FXIII Qualitative assay (Urea clot lysis test)

Done when all other tests for hemostasis are normal.
FXIII provide...
33

*TESTS FOR FIBRINOLYSIS
These are latex agglutination tests for

Fibrinogen and Fibrinogen degradation
Products (FDPs)...
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Laboratory approach to bleeding disorders

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Laboratory approach to bleeding disorders

  1. 1. 1 LABORATORY APPROACH TO BLEEDING DISORDERS Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  2. 2. 2 OVERVIEW 1. 2. 3. 4. Normal coagulation homeostasis Classification of bleeding disorders Clinical evaluation of bleeding disorders 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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  3. 3. 3 * 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 Vasoconst Platelet release reaction riction 5-ht Tissue thrombo plastin BLOOD COAGULATION TXA2 Platelet aggregation Fibrinogen + thrombin Primary hemostatic plug Fibrin Stabilized hemostatic plug Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  4. 4. 4 * CLASSIFICATION OF BLEEDING DISORDERS Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  5. 5. 5 * 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 1. Localized bleeding Generalized 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 onset early in life similar disorder in close relatives past history of similar episodes 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 1. 2. 3. 4. Sex affected Family history Petechiae, bleeding gums, epistaxis, menorrhagia Deep hematoma in muscle and joints Delayed bleeding from same site (12-24 hrs after) Previous history of bleeding Acquired usually secondary to disorders of 1. liver 2. uremia 3. hematologic malignancies 4. carcinoma 5. sepsis Platelet disorders/ Vascular disorders Female Negative Common Coagulation disorders Male Positive Rare Rare Common Rare Common Not present Present since childhood Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  6. 6. 6 * 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 1. myeloproliferative disorders 2. peripheral destruction of platelets MPV normal 1. Thrombocytopenia due to impaired platelet production like thrombocytopenia PDW (platelet distribution width) Measure of degree of variation in platelet size PDW increased Myeloproliferative disorder PDW normal Secondary or reactive thrombocytosis Reticulated platelets: Concept analogous to reticulocytes. Increased Peripheral destruction Normal Aplastic anemia Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  7. 7. 7 (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 RBC WBC Platelet Associated abnormalities in other cell lines Fragmented RBCs indicate DIC with thrombocytopenia Abnormal cells with thrombocytosis/thrombocytopenia (leukemias) Normal count 1 platelet per 500-1000 red cells Giant platelets Myeloproliferative neoplasms Bernard soulier syndrome ITP Discrete isolated platelets Glanzmann’s thrombasthenia without clumping in finger prick Uremia smear Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  8. 8. 8 (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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  9. 9. 9 (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 2. Von willebrand disease not prolonged in vascular disorders Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  10. 10. 10 * 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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  11. 11. 11 $ see next page for coagulation pathways details Pathways of coagulation Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  12. 12. 12 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  13. 13. 13 (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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  14. 14. 14 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 INR 11-16 Seconds 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 Treatment of pulmonary embolism Prevention of systemic embolism Tissue heart valves Acute myocardial infarction Atrial fibrillation 2.0 - 3.0 Recurrent embolism Mechanical heart valve Antiphospholipid antibodies 2.5 - 3.5 3. DIC – exhaustion of coagulation factors 4. Inherited deficiency of coagulation factors Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  15. 15. 15 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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  16. 16. 16 (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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  17. 17. 17 *SUMMARY OF SCREENING TESTS DEFECT IN BT Thrombocytopenia Extrinsic pathway Intrinsic pathway N N N N Common pathway N PT APTT N N N Platelet fuction/ vascular disorders/ vW defects PLATELET COUNT N N N Multiple pathways (DIC) Clot stabilization N N N/ N N N N COMMON CAUSES Von willebrand disease Storage pool defects Other platelet affecting disorders All causes of thrombocytopenia See causes of prolonged PT See causes of prolonged aPTT Causes of either prolonged PT or prolonged aPTT DIC/Liver disease FXIII deficiency Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  18. 18. 18 *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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  19. 19. 19 (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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  20. 20. 20 Aggregating agents used ADP / Epinephrine Curve type 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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  21. 21. 21 Abnormals: 1. GLANZMANN’S THROMBASTHENIA 1. 2. 3. 4. In Glanzmann’s thrombasthenia, Platelet GPIIb/IIIa is defective. This receptor is important for platelet aggregation. All agents induce aggregation through this receptor except ristocetin. Hence in Glanzmann’s thrombasthenia, aggregation is defective for all agents except ristocetin. Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  22. 22. 22 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. Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  23. 23. 23 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 Disorder Aggregating agents ADP/EPINEPHRINE/COLLAGEN PRIMARY WAVE SECONDARY WAVE BS syndrome N N vWD N N Glanzmann’s D D Storage pool$ N D $ Aspirin like D D Aspirin like in N D response to AA$ (AA used) (AA used) N normal D deficient AA arachidonic acid $ Measure of platelet release reaction Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes RISTOCETIN D D N N D D
  24. 24. 24 (iii) Tests for platelet release reaction Indirect Tests Direct Tests The secondary wave of aggregation in Response to ADP/epinephrine/collagen is a measure of release reaction 1. estimation of secretion of dense core granules 2. estimation of secretion of alpha granules Dense core granules Alpha granules Contents of platelet granules adenosine diphosphate (ADP) adenosine triphosphate (ATP) ionized calcium (which is necessary for several steps of the coagulation cascade) histamine serotonin 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) Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  25. 25. 25 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 In plain tube allowed to clot Citrated plasma (same pt) serum with residual prothrombin Perform PT (PT1) Perform 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 Defective in Bernard soulier syndrome GpIIb/IIIa Defective in Glanzmann’s thrombasthenia Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  26. 26. 26 (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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  27. 27. 27 *TESTS FOR COAGULATION FACTORS (i) Coagulation factor assays Principle: Patient’s plasma + standard plasma deficient in Factor to be tested in various Dilutions perform PT and aPTT Normal Prolonged Patients plasma Contains that Factor patients plasma doesn’t contain that factor Normals: All factors 50-150 % or 50-150 U/dl Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  28. 28. 28 (ii) Quantitative estimation of fibrinogen Normals: Fibrinogen levels 200-400 mg/dl Abnormals: Decreased fibrinogen Increased fibrinogen Afibrinogenemia, dysfibrinogenemia, DIC 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. 3. 4. 5. Immunological method (RIA based) Heat precipitation test Chemical precipitation test Weighing of clot Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  29. 29. 29 (iii) Thromboplastin generation test (TGT) Method: STAGE I Sample + Phospholipid + Calcium FXa-V complex (aka prothrombinase) Generation of Prothrombinase 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 + Phospholipids + calcium Adsorbed plasma$$ + Sample + Phospholipids + Calcium estimation of clotting time estimation of clotting time $ prepared serum contains FV and VIII adsorbed plasma – FIX and X Both contain – FXI and XII $$ Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  30. 30. 30 Ad Plasma substitution defective Serum substitution defective Both substitution defective F V / VIII abnormal F IX/X abnormal F XI/XII abnormal PT tests – F V, X aPTT tests – F VIII,IX, XI, XII Result interpretation: History Bleeding Bleeding Bleeding Bleeding Bleeding No bleeding Coagulation screen PT aPTT N P P N N P P N N P N P TGT Plasma defect Plasma defect Serum defect Serum defect Both defect Both defect Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes Interpretation F VIII deficient F V deficient F IX deficient F X deficient FXI deficient F XII deficient
  31. 31. 31 (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 Inhibitor coagulation factor deficiency perform TGT Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  32. 32. 32 (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 +5M urea dissolves Unstable clot FXIII def Doesnot dissolve FXIII 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/ Ethanol gel formed – fibrin monomer +nt (active coagulation +nt) No gel formed – fibrin Monomer absent (active coagulation –nt) Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
  33. 33. 33 *TESTS FOR FIBRINOLYSIS These are latex agglutination tests for Fibrinogen and Fibrinogen degradation Products (FDPs)# D-dimers# #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 Notes on Laboratory approach to bleeding disorders…by Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes

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