HEMA 2 LEC: Laboratory Evaluation of Hemostasis

Venous whole blood

specimen of choice for hemostasis testing

3.2% Buffered Sodium Citrate

standard anticoagulant used for coagulation studies

9:1

blood to anticoagulant ratio for coag studies

Whole blood

specimen used for Platelet aggregometry and Platelet count

Platelet rich plasma (PRP)

specimen used for Platelet aggregometry and Platelet function tests

Platelet poor plasma (PPP)

specimen used for Coagulation testing/assays

30 minutes

Patients need not fast, but they should avoid vigorous activities and should rest quietly for ______________ prior to collection for hemostasis testing.

Falsely prolonged PT, APTT

Underfilling of tubes may result in excess citrate, which may cause a __________________________

Aspirin

drug intake may affect most platelet function (esp platelet aggregation)

Coumadin (warfarin)

drug intake may affect factor IX, X, VII, II → results to prolonged PT.

Heparin

therapy for thrombotic disorder; patient undergoing said therapy may cause prolonged APTT

Penicillin

alters blood vessel walls; intake may induce hemolysis

Plastic blue-stopper

collection tube used in most hemostasis specimens; sterile evacuated blood collection tubes containing a measured volume of 0.105-0.109 M (3.2%) buffered sodium citrate anticoagulant.

Uncoated soda-lime glass tubes

collection tube that are unsuitable because their negative surface charge activates platelets and plasma procoagulants.

Siliconized (plastic-coated) glass tubes

collection tube available, but their use is waning because of concern for potential breakage, with consequent risk of exposure to blood borne pathogens.

Non-additive

f the hemostasis specimen is part of a series of tubes to be filled from a single venipuncture site, it must be collected first or immediately after __________________ tube.

Prolonged

A short draw generates erroneously ____________________ clot-based coagulation test results.

False prolonged

short draw may result to excess anticoagulant, which causes ________________________ coagulation tests

Procoagulant substances

Excess needle manipulation (Fishing) may promote the release of ____________________________ from the skin and connective tissue (damaged blood vessels).

False shortened

Test results from specimens collected during a traumatic venipuncture may be ____________________ and unreliable.

Blood stasis

Must be remove within 1 minute of its application to avoid ______________________.

► Condition in which venous flow is slowed.

False shortened

Blood stasis results in the local accumulation of factor VIII and von Willebrand factor (VWF), which may result in _________________ clot-based coagulation test results

20-/21-gauge

needle gauge used if overall specimen is 25 mL or less

19-gauge

needle gauge used if overall specimen is >25 mL

23-gauge

acceptable needle gauge used for pediatric patients or patients whose veins are small

3.2% Buffered Sodium Citrate

- Anticoagulant used for hemostasis testing.

- Binds calcium ions to prevent coagulation; buffer stabilizes specimen pH as long as the tube stopper remains in place.

- BLOOD TO AC RATIO: 9:1 (0.3 mL of anticoagulant is mixed with 2.7 mL of whole blood)

EDTA

- Not used for coagulation testing because calcium ion chelation is irreversible (interferes w/ coagulation assays)

- INHIBITS: Thrombin-fibrinogen reaction

- May be required for specimens used for molecular diagnostic testing, such as testing for factor V Leiden mutation or the prothrombin G20210A mutation.

Heparin

- Have never been validated for use in plasma coagulation testing but may be necessary in cases of platelet satellitosis (satellitism) as a substitute for specimens collected in EDTA or sodium citrate

- Also used for platelet retention test/glass bead retention test (test for platelet adhesion).

Citrate Theophylline Adenosine Dipyridamole (CTAD)

- Used for quantitative tests of PF4, TAT, and p-selectin.

- Used to halt in vitro platelet or coagulation activation for specialty assays such as those for the platelet activation markers platelet factor 4 (PF4) and platelet surface membrane P-selectin (measured by flow cytometry) or the coagulation activation markers prothrombin fragment 1+2 and thrombin-antithrombin complex.

Shortened

use of oxalate anticoagulant results to _________________ clotting time because it forms insoluble complexes/precipitates.

Increased

pH remains constant as long as the specimen is sealed: ________________ pH deteriorates clotting factors (prolonged coagulation tests).

18-24°C

Specimens are maintained at ________________ (ambient temperature), NEVER at refrigerator temperatures

Factor VII, XI

activated if specimen is stored at 1-6°C → platelet activity is destroyed through uncontrolled activation (premature activation), and causes the cryoprecipitation of large VWF multimers.

Factor V, VIII

storage of specimen at temperatures greater than 24°C causes deterioration of what factors (2)

PT

Specimens collected for ______ testing may be held at 18-24°C and tested within 24 hours of the time of collection.

APTT

Specimens collected for ______ testing may also be held at 18-24°C, but tested within 4 hours of the time of collection (provided that the specimen does not contain unfractionated heparin anticoagulant).

Unfractionated heparin therapy (UFH)

If a patient is getting ________________________________, specimens for APTT and PT testing must be centrifuged within 1 hour of the time of collection, and the plasma, which should be PPP, must be tested within 4 hours of the time of collection.

Whole Blood

sample must be collected with 3.2% sodium citrate and held at 18-24°C until testing.

*chilling destroys platelet activity → aggregometry should be started immediately and must be completed within 4 hours of specimen collection

200,000-300,000/uL

platelet count for Platelet Rich Plasma specimens

Platelet Rich Plasma

- sample should be Sodium citrate-anticoagulated blood.

- first checked visually for clots and then centrifuged at 50xg for 30 minutes with the stopper in place to maintain the pH.

- supernatant is transferred by a plastic pipette to a clean plastic tube, and the tube is sealed and stored at 18-24°C (ambient temperature) until the test is begun

<10,000/uL

platelet count for Platelet Poor Plasma specimens

Platelet Poor Plasma

- specimen required for clot-based plasma coagulation tests.

- sodium citrate-anticoagulated whole blood is centrifuged at 1500xg for 15 minutes in a swinging bucket centrifuge to produce supernatant.

StatSpin Express 2

machine/centrifuge that generates 4400 xg and can produce PPP within 3 minutes.

<100,000/uL

Platelet count that is the most common cause of clinically important bleeding.

Phase Contrast Microscope method

- Reference method: described by Brecher and Cronkit.

- Gold standard for quantifying platelets: EDTA whole blood is diluted 1:100 with 1% ammonium oxalate to lyse the nonnucleated RBCs.

- Platelets are counted in the 25 small squares of the central large square (1 mm2) of the hemocytometer.

Wright stained PBS

The accuracy of the manual thrombocyte count from phase contrast microscopy method must be verified by performing a platelet count estimate on a ____________________ made from the same specimen.

1:100

dilution factor for EDTA whole blood sample for phase contrast microscopy method of platelet count

1% ammonium oxalate

reagent used with diluted whole blood for phase contrast microscopy method of platelet count; makes platelets swell, hence more visibility

Light Microscope method

- AKA Tocantin's/Reese and Ecker method.

- Visualizing the platelets may be more difficult.

- Uses brightfield microscope.

- Dilution factor of 1:200 with the use of Toncantin reagent

1:200

dilution factor for EDTA whole blood sample for light microscopy method of platelet count

Toncantin reagent

reagent used with diluted whole blood for light microscopy method of platelet count; contains formalin to lyse RBCs and brilliant cresyl blue, which stains platelets

Peripheral Blood Smear (Platelet count estimation)

- Relies on number of RBCs (unreliable).

- To determine the approximate number of platelets per field, examine the thin area of the slide (where only a few red blood cells slightly overlap) using the oil immersion objective.

7-21

In PBS, a normal (wedge) blood smear should demonstrate approximately _______ cells per OIO field.

Automated Platelet count

- Most commonly used in laboratories.

- Required 2-20 fL size so machine will count platelets.

False increased

platelet count is _______________________ whenever there is presence of WBC fragments, RBC fragmentation or microcytic RBC.

False decreased

platelet count is _______________________ in presence of giant platelets, platelet satellitism (platelet clumping), and sample clot.

Light-transmittance Platelet aggregometry

- Designed to test platelet-rich plasma (plasma with a platelet count of 200,000-300, 000/uL)

- To produce adequate PRP, the original specimen must measure .

- Test is UNRELIABLE when the patient's whole-blood platelet count is <100, 000/uL.

Platelet Lumiaggregometry

- Production of luminescence

- Special method that measures platelet aggregation and ATP release (from dense granules) or granule secretion.

- SAMPLE: Whole Blood Diluted with Saline or PRP

Thrombin

first agonist added to specimen in platelet lumiaggregometry test to measure platelet aggregation, granule secretion.

Luciferin-luciferase

second agonist added to specimen in platelet lumiaggregometry which measures ATP release → results in cold luminescence

Bleeding time

- Time required for skin to stop bleeding.

- A screening test for detecting disorders of platelet function and VWD.

- NOT affected by the coagulation mechanism

Duke's method

- Bleeding time test that uses either the earlobe or the finger as the puncture site.

- Blot drop of blood with filter paper every 30 secs (filter paper MUST NOT touch the wound).

NV = 2-4 mins

Ivy method

- Bleeding time test that uses a standard pressured of 40 mmHg and the volar surface of the forearm as puncture site.

- Blot the drop of blood from each puncture site on 2 separate filter paper every 30 secs. (filter paper MUST NOT touch the wound).

NV = 1-7 mins

Template Bleeding time

- An Ivy bleeding time test modification by C.H. Mielke, Jr.

- uses a template containing a standardized slit in place of the disposable lancets; replaced by several commercially made devices [e.g., Simplate, Surgicutt]

- Standard skin wound size must be 5mm x 1mm by the use of spring-loaded lancet.

GENERAL REFERENCE RANGE: 2-9 mins

Rumple-Leede test

- An example of a capillary fragility test (particularly a tourniquet test).

- 15 minutes after release of pressure cuff, choose an area of 1-inch diameter anywhere on the areas mentioned above; number of petechiae formed is counted within the 1-inch diameter area and graded.

NV: (grade of 2+ above = capillary weakness)

Scurvy

Rumple Leede test is most commonly used in patients with _____________ (fragile capillaries = petechiae)

Clot retraction tests

- test to measuresThrombosthenin (a contractile protein important for CR tests).

- PRINCIPLE: Normal blood clot contracts (in so doing, it expresses serum); Degree of clot retraction is measured on the amount of expressed serum.

Hirschboeck/Castor oil test

- Qualitative method of clot retraction test.

- Place few drops of castor oil into a clean test tube, place 1 drop of fresh blood (without anticoagulant) into the castor oil. - Observe for extrusion of droplet-like serum on top portion of the drop of blood = normal aggregation of plt, normal thrombosthenin.

- Stop the timer as soon as "dimpling" of serum is detected.

NV: 14-15 mins

Dimpling phenomena

extrusion of droplet-like serum on top portion of the drop of blood; a result of normal thrombosthenin and aggregation of platelet

McFarlane method

- Quantitative method of clot retraction test.

- Uses wire loop to check for clot, then water bath.

- Checked after 1-hr: Blood volume should decrease up to 50%

NV = 44-67%

Clotting time tests

test that evaluates clotting factors for secondary hemostasis.

Slide/Drop method

- Micro method for clotting time (micro amounts of blood used).

- For children; uses small amount of drop.

- Do capillary puncture, wipe first drop of blood; start timing after second drop of blood wait for 30 secs and check for formation of fibrin strand (hair-like projections).

- INTERVAL: 30 secs

Capillary Tube method (Dale & Laidlaw's method)

- Uses capillary tube (with blue mark; contains no anticoagulant).

- Do skin puncture and place blood in tube; break every 30 secs and check for formation of fibrin strand.

- Prone to skin puncture.

Lee & White Whole blood Coagulation time

- Macro method for clotting time (venous blood is used).

- Insensitive to factor deficiencies.

- First laboratory procedure used to assess coagulation.

- Based on the fact that when whole blood is placed in a test tube, it will form a solid clot (NO longer used).

Activated Clotting time

- Used for patients undergoing Heparin therapy.

- Uses an activator: Diatomaceous Earth

- Samples are incubated at 37°C.

NORMAL: 75-120 secs

PX UNDERGOING HEPARIN THERAPY: 140-185 secs

Diatomaceous earth

activator used for activated clotting time test to stimulate clotting factors. ; contains 12 mg diatomite.

Activated Partial Thromboplastin Time (APTT)

- Used to screen deficiencies in intrinsic & common pathways.

- Used for monitoring heparin therapy.

- Measures all factors EXCEPT: Factors VII and XIII

SPECIMEN: citrated PPP (Incubated @ 37C)

REF RANGE: 20-45 secs

Rabbit phospholipid

Activators: Kaolin, Cellite, Silica, Ellagic acid

APTT reagents

0.025 M CaCl2

added after APTT reagent; test is started after addition

Prothrombin Time (PT)

- AKA Quick's Test (invented by Dr. Armand Quick).

- Useful for monitoring oral anticoagulant therapy.

- Used to detect deficiencies of vitamin K dependent group EXCEPT factor IX.

SPECIMEN: citrated PPP

REF RANGE: 10-12/12-14 secs

Simplastin

PT reagent (contains thromboplastin and CaCl2)

International sensitivity index (ISI)

- A value assigned to each lot of prothrombin thromboplastin reagent to compensate for variations in sensitivities of thromboplastin from different sources

- According to WHO the standard is 1.0 when buying PT reagent.

International Normalized Ratio (INR)

- Standardized report of PT (minimize difference in PT results).

- Used to monitoring patients taking oral anticoagulants therapy.

Formula: 𝐼𝑁𝑅=(𝑃𝑎𝑡𝑖𝑒𝑛𝑡′𝑠 𝑃𝑇/𝐶𝑜𝑛𝑡𝑟𝑜l 𝑃𝑇 )^𝐼𝑆𝐼

Stypven's time/Russel's Viper Venom test (DRRVT)

- Use of venom obtained from Russel's viper (Vipera russeli).

- Venom bypasses/imitates action of Factor VII (directly activates Factor X to Xa.)

- Used to check problems for factor VII and X.

Factor X deficiency

LAB TEST:

• Prolonged PT

• Prolonged ST/DRRVT

Factor VII deficiency

LAB TEST:

• Prolonged PT

• Normal ST/DRRVT

Thrombin time/ Thrombin clotting time

- Sensitive test for Heparin Inhibition

- Use of commercially prepared thrombin.

- Affected by circulating anticoagulants: Heparin

- Prolonged if fibrinogen is below 75-100 mg/dL and impaired fibrinogen function.

SPECIMEN: PPP

Reptilase test

- Has same principle with Stypven's time; only concern of Reptilase test is factor I.

- Use of an enzyme called Reptilase, found in the venom of Bothrops atrox.

- Unaffected by heparin or circulating anticoagulants.

REFERENCE RANGE: 10-15 secs

Fibrinogen/Factor I deficiency

LAB TEST:

• Prolonged TT

• Prolonged RT

Factor XIII assay/Duckert's assay/5M urea solubility test

- Used to check for stability of clot.

- Plasma is mixed with thrombin (activates factor XIII) then 5M urea is added and left to stand for 24 hours.

- OTHER REAGENTS: 1% monochloroacetic acid or 2% hydrochloric acid

Clot is dissolved, soluble to urea = Factor XIII deficiency

Behrichrom assay

confirmatory test for Factor XIII assay

Whole blood clot lysis time (WBCLT)

- Not widely used.

- 2 Tubes Are Used:

• 1st tube: placed in incubator at 37°C (lysed)

• 2nd tube: placed in ref (will serve as control)

- Lysis is measured after 48 hours

Fibrinogen deficiency

WBCLT LAB RESULT:

• 1st and 2nd tube = Lysis

Plasma clot lysis time

- Not widely used.

- Plasma is collected in with an anti-coagulant, centrifuge, and plasma is recalcified.

- Sample incubated at 37°C for 48 hours and checked for lysis of clot

Euglobulin clot lysis time

- Removes inhibitors of fibrinolysis (anti-plasmin and anti plasminogen activators).

SPECIMEN: Citrated platelet poor plasma (PPP), thrombin, 1% acetic acid

DIC

EUGLOBULIN CLOT LYSIS TIME LAB RESULT:

• Clot lysis less than 2 hours = Increased fibrinolytic activity

may indicate _____________.

Euglobulins

are precipitated (bottom of tube) proteins when plasma is diluted with water [e.g., plasminogen, plasmin, fibrinogen, and plasminogen activators].

Protamine Sulfate dilution test

- Detects fibrin monomers (should not be detected if patient is normal).

- Paracoagulation: Detection of Protamine Sulfate of fibrin monomers and early FDP (X and Y) by .

- Protamine sulfate reacts to FDPs = production of paracoagulation phenomena

Ethanol gelation test

- Less sensitive test but more specific than protamine sulfate in detecting fibrin monomers.

- 50% ethanol is added to citrated PPP and observed for gel like clot formation.

Fibrin monomers

presence may indicate venous/arterial thromboembolism or DIC

*Normal patients should have no clot formation in both PSDT and EGT