Laboratory Methods Flashcards
Causes of Shortened Clotting time
Premature Activation
* Contamination of tissue factor
* Use of Glass container
Hemolysis
Overfilling
Causes of Prolonged Clotting time
Polycythemia vera (Hct: >50%)
Underfilling
Overanticoagulation
Change in pH
Sodium Citrate
* Ratio:
* Concentration:
* Centrifugation:
Sodium Citrate
* Ratio (blood:anticoagulant): 9:1
* Concentration: 3.2% (preffered)
* Centrifugation: 2000 g for 10 minutes
Changes in pH can ________ clotting time
prolong
Loss of CO2 changes pH
Note: Should be unopened if testing not done
Prolonged tourniquet application - Statis can elevate ____ and ________ ; falsely decreases finrinolytic parameters and falsely ________ clotting time
Prolonged tourniquet application - Statis can elevate vWF and Factor VIII ; falsely decreases finrinolytic parameters and falsely shortened clotting time
Common gauge for hemostatis collection
20G
Can be used for platelet retension test
Heparin
Factor VII and XI
Prematurely activated at 4C
Modification of Lee and White Clotting time
Plasma Recalcification Test
Time required for blood to clot after Ca2+ is added
Reference Range - Plasma Recalcification Time
* PRP
* PPP
Reference Range - Plasma Recalcification Time
* PRP - 100-150 seconds
* PPP - 130-240 seconds
PRP should be 20 seconds faster than PPP
aPTT
* Sample:
* Reagent:
* NV:
aPTT
* Sample: Platelet poor plasma (PPP)
* Reagent: Phospholipid (platelin) + Activator + CaCl2
* NV: 20-45 seconds
Partial thromboplastin
Contains phospholipid (platelin) only and is a substitute for platelet factor 3
Activator in aPTT
Kaolin
Celite
Ellagic acid
Silica
Negatively charged particles that activate PF XII
Monitor Heparin therapy
aPTT
modification of partial thromboplastin time
Tests for Intrinsic Pathway
Lee and White
Plasma Recalcification time
aPTT
Activator - Activated Clotting Time
Diatomite
Test for Extrinsic Pathway
Prothrombin Time
PT
* Specimen:
* Reagent:
* NV:
PT
* Specimen: PPP
* Reagent: Thrombolastin (Simplastin) + CaCl2
* NV: 10-12 seconds
Therapy monitored in PT
Vitamin K antiagonist - Warfarin & Coumarin
INR
International Normalized Ratio
provide a standardized PT result and monitor warfarin/coumarin therapy
INR - Warfarin therapy
2.0 - 3.0
Manchester Reagent
Rabbit brain tissue thromboplastin
Most sensitive PT reagent
Steypven Test
a.k.a Russel Viper venom test
Detect deficiency in common pathway
Particularly deficiency in PF X
Stypven Test
* Reagent:
* Snake:
* NV:
Stypven Test
* Reagent: Russel’s viper venom
* Snake: Vipera russeli
* NV: 6-10 seconds
Bypass plasma factor VII and directly activate plasma factor X
Stypven Test
Stypven Test
* Abnormal:
* Normal:
Stypven Test
* Abnormal: Factor X
* Normal: Factor VII
Test also help distinguish between factor X and factor VII deficiencies
5M Urea Solubility test
a.k.a Dickhert’s Test
Detect deficiency in Factor XIII
5M Urea Solubility Test
* Reagent:
* Substitute:
5M Urea Solubility Test
* Reagent: 5M Urea
* Substitute: 1% monochloroacettic acid or 2% aceticacid
5M Urea Solubility Test
* Normal:
* Abnormal:
5M Urea Solubility Test
* Normal: Insoluble - F XIII is present
* Abnormal: Soluble - Deficiency in F XIII
A stable fibrin clot with FXIII is resistant to 5M urea
Thrombin Time
addition of exogenous thrombin to activate clot formation
Detect deficiency in **Fibribogen **in patient not undergoing heparin therapy
Not indication for Heparin therapy patient
Thrombin time
Thrombin Time
- Sample:
- Reagent:
- Heparin
- NV:
Thrombin Time
- Sample: PPP
- Reagent: Thrombin
- Heparin: Affected
- NV: 10-14 seconds
Greatly Prolonged Thrombin Time
Hypofibrinogenemia
<100 mg/dL
Prolonged in Thrombin Time
Hypofibrinogenemia
Dysfibrinogenemia
FDP
Heparin therapy
Reptilase Test
Detect deficiency in **Fibribogen **in patient undergoing heparin therapy
Reptilase Test
- Reagent:
- Heparin:
- Snake:
- NV:
Reptilase Test
- Reagent: Reptilase in snake venom
- Snake: Bothrops atrox
- NV: 10-15 secopnds
Enzyme found in Bothrops atrox venom that has similar activity to thrombin
Reptilase
Greatly Prolonged in Reptilase Test
Dysfibrinogenemia
Claus Factor I assay
Estimates functional fibrinogen level
Claus factor I assay
* Specimen:
* Reagent:
* Dilution:
* Buffer:
Claus factor I assay
* Specimen: PPP
* Reagent: Bovine thrombin (50 NIH units/mL)
* Dilution:** 1:10**
* Buffer: Owren Buffer
Tests for Primary Hemostasis
Bleeding Time
Capillary Resistance Test
Clot Retraction Test
Platelet Count
Platelet Adhesiveness
Platelet Aggregation
Actual time it takes for a wound to stop bleeding
Bleeding Time
Methods - Bleeding Time
Mnemonics: BleeDing IVY
- Duke Method (Earlobe)
- Ivy Method (Pressure cuff at 40 mmHg)
Duke’s Method
* Site:
* NV:
Duke’s Method
* Site: Earlobe
* NV: <8 minutes
Ivy Method
* Site:
* NV:
Ivy Method
* Site: Volar surface of the forearm
* NV: 3-6 minutes
uses filter paper strips after 2 minutes and again every after 30 sec.
Positive Pressure Test
Capillary resistance test
Rumple Leed Test/Tourniquet Test
Negative Pressure Test
Capillary resistance test
Hess Test/Suction Test
Positive/Tourniquet/Rumple-Leed
* Tool:
* Application:
* Observe for petechiae:
Positive/Tourniquet/Rumple-Leed
* Tool: BP cuff
* Application: 5 minutes
* Observe for petechiae: after 5 minutes
Negative/Suction/Hess Test
* Tool:
* Application:
* Observe for petechiae:
Negative/Suction/Hess Test
* Tool: Suction Cup (2 cm)
* Application: 1 minute
* Observe for petechiae: 5 minutes
Brecher - Cronkite Method
Reference Method for Manual platelet count
No. of RBCs in 1 Platelet
* Rodaks:
* Steininger:
No. of RBCs in 1 Platelet
* Rodaks: 25 RBCs
* Steininger: 10-40 RBCs
Test for Platelet adhesiveness
Glass Bead Retention Test/Salzmann Method
normal platelet that have vWF will adhere and aggregate to the beads
Platelet Aggregation Test
In vitro test to determine the ability of platelets to aggregate using aggregating agents which make sample becomes clearer and transmits more light as platelet aggregates
Aggregating agents: Epinephrine, Collagen, ADP, Restocitin
Test - Determination of Lysis Time
Euglobulin Lysis Time
Principle - Euglobulin Lysis Time
Euglobulin is a protein that is precipitated after plasma is diluted with water and acidified and clot is incubated at 37C
Diluted PPP + Acid = Euglobulin + Thrombin = Clot (incubated at 37C)
Gelation
Clot formation indicating increased fibrinolysis
Test for secondary fibrinolysis (DIC)
Protamine Sulfate Gelation Test
* Reagent:
* Principle:
* Result:
Protamine Sulfate Gelation Test
* Reagent: Protamine sulfate
* Principle: Paracoagulation
* Result: (+) Clot formation ; (-) No clot
Protamine replaces secondary degradation products
Ethanol Gelation Test
* Reagent:
* Principle:
* Result:
Ethanol Gelation Test
* Reagent: 50% Ethanol
* Principle: Paracoagulation
* Result: (+) Clot formation ; (-) No clot
Ethanol causes soluble fibrin monomer to dissociate and then polymerizes
Latex D-dimer assay
Specific test to intravascular fibrin formation and diagnose DIC
measure a specific FDP - D-dimer
In D-dimer assay, it is already positive after ____ hours after DIC onset
In D-dimer assay, it is already positive after 4 hours after DIC onset
Intravenous anticoagulant
Heparin
Oral anticoagulant
Warfarin
Heparin Therapy
* Action:
* Administration:
* Treatment:
* Therapy:
Heparin Therapy
* Action: Inhibit thrombin by enhancing anti-thrombin III
* Administration: Intravenous anticoagulant
* Treatment: Protamine sulfate
* Therapy: APTT and ACT
Warfarin Therapy
* Action:
* Administration:
* Treatment:
* Therapy:
Warfarin Therapy
* Action: Antagonize Vitamin K and induce PIVKAs
* Administration: Oral anticoagulant
* Treatment: Vitamin K, FFP
* Therapy: PT
Used during coronary artery bypass graft surgery and during cardiac catheterization and in several medical conditions.
Heparin Therapy
Differentiate Leukemoid reaction vs. CML
Leukocyte Alkaline phosphatase (LAP) test
Increase: Leukemoid reaction; Decrease: CML
Differentiate** AML and Monocytic leukemia **from ALL
MPO
Differentiate AML & Myelomonocytic leukemia from ALL
Sudan Black B
stains Lipids on granulocyte
Periodic Acid Schiff Test (PAS)
Diagnose DiGuglielmo’s Syndrome (M6)
Differentiate Granulocytic from Monocytic cells
Naphthol AS-D Choroacetate esterase
Stain specific esterase
Tartrate-resistant ACP (TRAP)
Diagnose Hairy cell leukemia
Differentiate ALL from AML
Terminal deoxyribonucleotidyl transferase
Fresh Capillary Blood
Recommended specimen in LAP test
Heparinized whole blood
Recommended specimen in NBT
