1. Tissue thromboplastin contamination 2. Inappropriate container 3. Improper temperature: labile factors 4. Hemolysis

Laboratory Evaluation of Coagulation and Fibrinolysis Flashcards by Arriane Cyrel (MTI)

Things to consider:

Tissue thromboplastin contamination
Inappropriate container
Improper temperature: labile factors
Hemolysis

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Inappropriate container

a) Glass surface
b) Polystyrene tubes/ glass tube with silicone- preferred

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preferred

Polystyrene tubes/ glass tube with silicone

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@room temp: _______

V & VIII

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@ref temp: _________

VII & XI

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PT plasma capped stability for 24 hours @: _____

RT (18-24oC)

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APTT samples stable for 4 hours @: ___________

RT (18-24oC)

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Factor assays stable for 4 hours @: ___________

RT (18-24oC)

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Hemolysis

-prolonged tourniquet application
-moisture/contamination
-inappropriate needle bore
-Frothing of sample
-improper transfer from barrel to tube
-excessive mixing mixing

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▪ Adult (≤25 mL):

G20 -21; 1 - 1.25 in.

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▪ Adult (≥25 mL):

G19; 1 or 1.25 in.

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▪ Child or adult with small, friable, or hardened veins:

G23

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▪ Syringe w/ winged-needle set:

G20, 21, or 23 (mostly)

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: used in blood donations

▪ G16-18

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Type of Syringe:

Plastic/Silicone coated- single draw
2-way needle and holder-multiple draw
Winged-needle sets

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Anticoagulants:

A) Sodium oxalate
B) Trisodium citrate
4) Heparin
3) EDTA

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: Interfere with Spectrophotometric reading (OD)

A) Sodium oxalate

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: -Preserves FV and FVIII

B) Trisodium citrate

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Ratio of 9:1 (blood: anticoagulant)

B) Trisodium citrate

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B) Trisodium citrate Prolonged

Incomplete filling/high hematocrit

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B) Trisodium citrate
- Two concentrations:

3.2% and 3.8%

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-acts with antithrombin III to inhibit all stages of coagulation

4) Heparin:

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-used for plt retention test

4) Heparin:

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-unsuitable for coagulation

3) EDTA

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-inhibits thrombin-fibrinogen reaction

3) EDTA

-Factor V is unstable in its presence

3) EDTA

Evacuated tube arrangement: 2-way needle collection

a. Red stopper b. Blue stopper (3.2%) c. Lavander stopper d. plastic syringe (for additional tests)

1) Effects of pH: _______

PROLONG THE CT

2) Temperature: (as mentioned during the first slide)

PPP preparation: __________ for _________

2000 x g for 10 mins

Storage: 4 deg within 2 hours/ rapid freezing @:_______________

-20oC

PRP preparation: __________ for________@___

60-100 x g for 10 mins @ RT

Principle: when venous blood is put into a glass tube, it will form a solid clot.

Lee and White Method

-the time response is a measure of overall intrinsic and common pathway

Lee and White Method

-Insensitive to factor deficiencies.

Lee and White Method

Lee and White Method NV:

7-15 mins

Principle: Presence of an activator (DIATOME) and by keeping the blood constant at 37 deg celsius, the test is more reliable and rapid.

Activated Clotting Time

Activated Clotting Time NV:

75-120 sec.

Principle: Time required for blood to clot after the addition of calcium

Plasma Recalcification Time (PRT)

-Modified Lee and White

Plasma Recalcification Time (PRT)

Plasma Recalcification Time (PRT) NV:

PRP: 100-150 sec PPP: 130-240 sec

Routine screening of coagulation disorders in intrinsic system and common pathway

Activated partial thromboplastic time (APTT/PTT)

- Detects the presence of circulating anti-coagulant

Activated partial thromboplastic time (APTT/PTT)

- Monitors heparin therapy

Activated partial thromboplastic time (APTT/PTT)

Principle: Measures all coagulation factors needed for the generation of Intrinsic prothrombinase except for Calcium and PPL

Activated partial thromboplastic time (APTT/PTT)

When Calcium is added with incomplete thromboplastin (plt substitute- CEPHALIN), intrinsic prothrombinase is generated.

Activated partial thromboplastic time (APTT/PTT)

Activated partial thromboplastic time (APTT/PTT) Specimen:

Citrated PPP

Activated partial thromboplastic time (APTT/PTT) Reagent:

0.025M CaCL2 Phospholipid (plt sub)

Activated partial thromboplastic time (APTT/PTT) Activators:

kaolin, celite or ellagic acid

Activated partial thromboplastic time (APTT/PTT) NV:

25-35 seconds/ 22-34 seconds

Prolonged PTT:

1. Deficiency in intrinsic/common pathway factors 2. Presence of inhibitor 3. <60 mg/dL fibrinogen 4. Increased FSP

-screening for the extrinsic and common pathway coagulation.

PROTHROMBIN TIME (Quick’s Test)

-monitors oral anticoagulant with COUMADIN

PROTHROMBIN TIME (Quick’s Test)

PROTHROMBIN TIME (Quick’s Test) Principle: When tissue extract of (?) is added to (?), along with (?), it reacts with (?) to activate (?).

thromboplastin (complete) PPP Calcium FVII FX

PROTHROMBIN TIME (Quick’s Test) Sample:

Citrated PPP

PROTHROMBIN TIME (Quick’s Test) Reagents:

Thromboplastin 0.025M CaCL2

INR=

International Normalized Ratio

-standardized way of reporting PT in monitoring anticoagulant therapy

International Normalized Ratio

-calculated as a ratio of the px PT to a control PT (standardized for the potency of the thromboplastin rgt)

International Normalized Ratio

ISI=

International Sensitivity Index (calibration index)

Reference Values for INR

2.0-3.0 2.5-3.5

2.0-3.0

▪ in prevention and treatment of viscous thrombosis ▪ treatment of pulmonary embolism ▪ prevention of stroke in myocardial infarction ▪ peripheral arterial disease ▪ prevention of systemic embolism in atrial fibrillation ▪ cardia valve replacement (tissue valves)

2.5-3.5

▪ in prevention of recurrent MI ▪ reduction of mortality in MI ▪ mechanical prosthetic heart valve (high risk)

PROTHROMBIN TIME (Quick’s Test) NV:

10-12 seconds

PROTHROMBIN TIME (Quick’s Test) Prolonged in:

-extrinsic and common pathway factor deficiencies - Fibrinogen of <100 mg/dL - Dysfibrinogenemia - Vit K deficiency and certain liver disease - Increased FDP’s - False prolongation: heparin contamination

PROTHROMBIN TIME (Quick’s Test) Shortened in:

-Traumatic venipuncture

is a thromboplastin-like substance

Venom “Vipera ruselli”

STYPVEN/RUSELL’s VIPER VENOM Principle: Addition of the (?) bypasses the activation of(?) and directly activates (?).

venom VII FX

STYPVEN/RUSELL’s VIPER VENOM NV:

6-10 seconds

What is the function of Dilute Russel Viper Venome time (dRVVT)?

Detects the presence of lupus anticoagulant

INDIRECT TESTS

Otherwise known as “Fibrinogen Deficiency Test”

THROMBIN TIME

THROMBIN TIME Principle: Addition of thrombin bypasses all coagulation pathways except:

polymerization of Fibrinogen

.-measures the availability of functional fibrinogen

THROMBIN TIME

-measures the conversion of fibrinogen to fibrin

THROMBIN TIME

THROMBIN TIME NV:

17-25 seconds

Prolonged TT:

Fibrinogen level: 75-100 mg/dL Impairment of fibrinogen Presence of Heparin and FDP Normally prolonged in newborn and multiple myeloma

REPTILASE TIME NV:

18-20 seconds

REPTILASE TIME Prolonged in:

Fibrinogen deficiency Presence of FSP

Heparin Therapy; NF1 Prolonged

THROMBIN TIME

Heparin Therapy; NF1 Normal

REPTILASE TIME

FSP: NF1 Prolonged

THROMBIN TIME REPTILASE TIME

Hypofibrinogenemia Greatly prolonged

THROMBIN TIME

Hypofibrinogenemia Prolonged

REPTILASE TIME

Dysfibrinogenemia Prolonged

THROMBIN TIME

Dysfibrinogenemia Greatly prolonged

REPTILASE TIME

Test for FXIII deficiency

DUKERT TEST (5M Urea Solubility Test)

DUKERT TEST (5M Urea Solubility Test) Principle: clot formed in (?) is insoluble in (?) during a (?).

normal plasma 5M urea 24 hr incubation

Indicator: If Factor XIII is deficient, the clot is dissolved in less than 24 hours by urea

DUKERT TEST (5M Urea Solubility Test)

Clot + 5M Urea NORMAL FXIII Deficiency Excessive fibrinolysis

Clot +NaCl NORMAL FXIII Deficiency Excessive fibrinolysis

-screening procedure for the assessment of fibrinolysis

EUGLOBULIN TEST

-Euglobulin fraction:

Plasminogen, activators of plasminogen, fibrinogen

-EUGLOBULIN TEST Principle: Plasma euglobulins are precipitated with (?). Precipitates are redissolved and clotted with (?). The clot is incubated and the time for complete lysis @ (?) is measured.

1 %Hac thrombin 37 deg celcius

EUGLOBULIN TEST NV:

2-4 hours

: abnormal fibrinolytic activity

<2 hours

EUGLOBULIN TEST Increase fibrinolytic activity is seen on:

-circulatory collapse -adrenalin injection -sudden death -pulmonary surgery -pyrogen reaction -obstetric complications

: Rapid clot breakdown

Primary

-increase in the circulating tPA binding to fibrin

Primary

-Excess tPA

Primary

-Excess tPA 2:

decreased hepatic clearance decreased fibrinolytic inhibitors

Secondary: Secondary to

systemic hypercoagulability

-systemic or microvascular

Secondary

-not localized

Secondary

-commonly assoc with DIC

Secondary

-detects fibrin monomers

PROTAMINE SULFATE TEST and ETHANOL GELATION TEST

-distinguishes primary vs secondary fibrinolysis

PROTAMINE SULFATE TEST and ETHANOL GELATION TEST

-screening procedure in diagnosing DIC

PROTAMINE SULFATE TEST and ETHANOL GELATION TEST

PROTAMINE SULFATE TEST and ETHANOL GELATION TEST Principle: (?) displaces the (?) from (?) and spontaneously form (?) (paracoagulation)

Protamine sulfate/ 50% ethanol secondary FDPs fibrin monomer gel-like clots

PROTAMINE SULFATE TEST and ETHANOL GELATION TEST Normal:

no gel formation

arises from the degradation of cross linked fibrin

D-d

-measures fibrinolysis but not fibrinogenolysis

D-Dimer Test

-uses latex bead with monoclonal ab

D-Dimer Test

-NV: <200 ng/mL

D-Dimer Test

D-Dimer Test Increased in:

DIC Thrombosis Phlebitis

Test for FSP

THROMBO-WELCOTEST

-THROMBO-WELCOTEST Whole blood is added to (?’ to ensure complete clotting and (?) after incubation, the px serum is diluted and mixed with (?) that have been coated with (?).

thrombin soya bean enzyme inhibitor latex particles anti-fibrin split products

caused by traumatic phlebotomy

TISSUE THROMBOPLASTIN CONTAMINATION

: contains tissue factor (TF) released from injured cells that causes premature activation of extrinsic pathway (starts coagulation prior to testing)

tissue thromboplastin

e.g., release of [?] during probing, through and through puncture to the vein

: probing is detrimental to the results

sodium citrate in coagulation tests (PT, APTT)

: probing can be considered

EDTA

TISSUE THROMBOPLASTIN CONTAMINATION effect:

falsely shortened test result

Effect: use of plastic centrifuge tubes

alsely shortened coagulation time

room V and VIII easily deteriorates

Room Temperature (18-24 C)

stable for 24 hrs:

• PT plasma (remained capped until use)

stable for 4 hrs:

• APTT samples • factor assay samples

VII and XI deteriorates

Ref/Cold Temperature

prevention: by pulling the plunger in sync with the speed of blood flow to the tube

frothing of sample

improper transfer from barrel to tube (frothing on top layer of blood) • remedy: [?] (remove cap and let the blood run to the walls of tube)

recap

• transfer of blood from syringe to the rubber ([?]): only done in microbiology (bacteria is still present in hemolyzed samples)

SPS in yellow tube or goldtop

blue top: EDTA:

4-5x 8x

Short draw (< 2.7 mL)

PT/PTT falsely prolonged

Failure to mix specimen after collection

PT/PTT falsely prolonged

Excess vigorous mixing

PT/PTT falsely shortened

Hemolysis

PT/PTT falsely shortened

Improper storage: wrong temperature or held too long

PT/PTT falsely prolonged

Chilling in refrigerator or placing on ice

PT/PTT falsely shortened

Inadequate centrifugation

PTT loses sensitivity for lupus anticoagulants and heparin.

Prolonged tourniquet application

Falsely elevates vWF, factor VIII

Drawing coagulation tube after to other anticoagulant tubes

PT/PTT falsely affected

Probing the vein

PT/PTT falsely shortened

Heparin contamination from line draw

PT/PTT falsely prolonged

Lipemia

Test may not work

Winged-needle sets needle gauge:

G23

for difficult or fragile veins; commonly used on babies, young children, and the elderly to draw blood or to administer medication using an IV

Winged-needle sets

▪ disadvantage: insoluble complex or precipitate of calcium oxalate interfere with spectrophotometric reading (optical density) incorporated in the automations of hematology coagulation machines

SODIUM OXALATE

combine with calcium in the blood to form insoluble complex or precipitate of calcium oxalate which causes depletion of calcium necessary for coagulation of blood

SODIUM OXALATE

such as in the use of PPP that is often recalcified (addition of calcium)

SODIUM OXALATE

less commonly used in hematology

SODIUM OXALATE

self-filling tube (due to vacuum)

TRISODIUM CITRATE

: presence of excess unbound citrate (prolongs the test)

incomplete filling

: may cause underfilling

high hematocrit (viscous blood)

(blood: anticoagulant)

9:1 ratio

coagulation reagents are sensitive to

citrate

concentrations:

1. 3.2% (light blue) 2. 3.8% (black)

3.2% (light blue) - formulation:

0.105 M (glass) or 0.109 M (plastic)

- preferred for coagulation tests (PT and APTT)

3.2% (light blue)

3.8% (black) formulation:

0.129 M

overanticoagulation with high hematocrit blood samples (extra unbound citrate)

3.8% (black)

not used on coagulation studies (thrombin is inhibited)

HEPARIN

action: chelation of calcium

EDTA

FV is unstable in its presence (may cause deterioration)

EDTA

: plain discard tube to contain initial blood volume that may be contaminated with tissue thromboplastin)

a. Red stopper

: all other following tubes affect coagulation tests

b. Blue stopper (3.2%)

: chelates calcium thus must follow after tubes for coagualation tests (citrates)

c. Lavender stopper

: for additional tests (emergency purposes)

d. Plastic Syringe

any changes in the pH can

prolong clotting/coagulation time

uncapping and capping of the tube mediates the loss of carbon dioxide making the sample suffer from

alkalosis

rough test for intrinsic and common pathway (not reliable)

LEE AND WHITE METHOD

addition of an activator (diatome) in the blood must cause enhanced clotting time

ACTIVATED CLOTTING TIME

with the same value in Lee and White method (7-15 mins) indicates problem in coagulation

ACTIVATED CLOTTING TIME

normal PPP and PRP contains all necessary components to generate fibrin clot except

calcium

: x calcium; ✓ PPL

- PRP

: x calcium; x PPL

- PPP

PLASMA RECALCIFICATION TIME recalcification (?) must coagulate and form fibrin clot

addition of calcium to PRP and PPP

one of the most important tests

ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT/PTT)

to determine/differentiate whether the patient has factor deficiency or just under heparin therapy

APTT

- used as a treatment for clotting to ensure normal blood flow

heparin therapy

- inhibits thrombin by acting on antithrombin-III

heparin therapy

targets of heparin:

- FII - FX

may be caused by heparin therapy and not by factor deficiency

• prolonged APTT/PT

• fraction of plasma which contains TF and PPL

thromboplastin

thromboplastin • two types:

complete: provides both TF and PPL incomplete: only provides PPL

: provides both TF and PPL

complete thromboplastin

: only provides PPL

incomplete thromboplastin

• in APTT: incomplete thromboplastin is used thus called

partial thromboplastin time

refrigerated thus, shift it in a body temperature

APTT

reagent does not need incubation, just invert it to maintain the suspension and prevent concentration of reagent at the bottom.

APTT

APTT Deficiency in intrinsic/common pathway factors:

XI, IX, VIII, V, X, II, I

circulating inhibitor: autoantibodies such as lupus anticoagulant

APTT

: a powerful inhibitor of thrombin in APTT

E dimer/Fragment E

: inhibit thrombin (no cleaving of fibrinogen into fibrin) in APTT

presence of FSP

use of PRP: presence of numerous platelets will try to react potentially thus falsely prolonging the test

APTT

TEST FOR EXTRINSIC AND COMMON PATHWAY involves factors: except

VII, TF, X, V, II, I FXIII

: vitamin K antagonists

coumadin and warfarin

coumadin and warfarin consequently affects Vitamin K-dependent factors:

FII, FVII, FIX, FX (prothrombin group)

DO NOT INCUBATE SAMPLES AT 37 C FOR LONGER THAN 5 MINUTES TO AVOID THE LOSS OF FV and FVIII.

Tilt Tube Technique

Expected PT (Manual): presence of coagulum in 12-15 seconds (if not within the range, defective reagent)

Tilt Tube Technique

Note: A control plasma should be run together with the test to validate the reagents used

Tilt Tube Technique

the closer to 1, the more sensitive the reagent

International Sensitivity Index (ISI)

optimal ISI:

1.3-1.5

the PT reagent is calibrated against WHO thromboplastin reagent

International Sensitivity Index (ISI)

: first to disappear (very labile in vivo) with intake of oral anticoagulant and during liver disease

Factor VII

• affects both PT (falsely prolonged) and APTT (more prominent; prolonged) in PT

heparin contamination

Shortened due to traumatic venipuncture (probing): contamination of tissue factor

assay similar to the prothrombin time

STYPVEN TIME/RUSSELL’S VIPER VENOM TEST

“Vipera ruselli/ Daboia ruselli” with a thromboplastin-like action (has [?])

TF and FVIII

contains a potent activator of FX which in the presence of PPL, prothrombin, and calcium ions cleaves fibrinogen to fibrin

“Vipera ruselli/ Daboia ruselli”

triggers coagulation at the level of factor X

STYPVEN TIME/RUSSELL’S VIPER VENOM TEST

: prolonged Russell viper venom clotting time (> 10 secs)

FX deficiency

- no clotting occurs even with the addition of venom (containing TF and FVII) to PPP

FX deficiency

- prolonged PT and PTT

FX deficiency

: does not prolong (normal) the Russell viper venom clotting time and results to formation of clot

FVII deficiency

• inhibits most coagulation factors

lupus anticoagulant

lupus anticoagulant • usually targets

FV, FVIII and FX

• its presence causes massive bleeding

lupus anticoagulant

• involved in DIC

lupus anticoagulant

addition of thrombin does not require the activation of other coagulation factors in the cascade to cleave fibrinogen

THOMBIN TIME

THOMBIN TIME sample: reagents:

sample: citrated PPP reagents: standardized thrombin solution and calcium

estimates fibrinogen concentration (thrombin time is proportional to the rate of fibrinogen polymerization

THOMBIN TIME

also used to monitor heparin therapy (more accurate than APTT)

THOMBIN TIME

sensitive to presence of increased levels of FSP

THOMBIN TIME

: considers heparin, intrinsic and common pathway factors

- APTT

: determine thrombin concentration and heparin

- thrombin time

- lacks protein fibrinogen or liver only starts to produce it

newborns

- has abnormal proteins (paraproteins) that impairs fibrinogen

multiple myeloma

used to confirm presence of fibrinogen deficiency in patients undergoing heparin therapy as it only affects thrombin ( [?]is a thrombin-like substance only thus not affected)

reptilase

absence of fibrin polymerization with the addition of reptilase indicates

fibrinogen deficiency

confirm presence of FSP through test for fibrinolysis

REPTILASE TIME

FXIII stabilizes fibrin clot so plasmin cannot dissolve it easily