HEMATLOGY 2 UIC-MLS

Clinical Hematology
Prelim Topic 5- Laboratory Evaluation of Secondary Hemostasis

[TRANS] TOPIC 5: PRELIM TOPIC 5- LABORATORY EVALUATION OF SECONDARY HEMOSTASIS

 Principle:
o Addition of activators in the test system
OUTLINE maximizes activation of contact factors. Added
1) Coagulation Tests
A. Clotting Time phospholipids substitute for platelet factor 3.
B. Prothrombin Time APTT measures activity of all coagulation
C. Activated Partial Thromboplastin Time factors except VII and XIII.
D. Stypven Time STYPVEN TIME
E. Thrombin Time
F. Reptilase Time  A.k.a Russell’s Viper Venom Time
G. Ducker’s TIme  Monitors coagulation factors of common pathway
2) Fibrinolysis Tests o Prolonged results indicate an abnormality of
A. Whole Blood Clot Lysis Time one or more common pathway coagulation
B. Euglobulin Clot Lysis Time
C. Protamine Sulfate Test factors
D. Ethanol Gelation Test  Used to detect deficiencies in fibrinogen,
E. Latex D-dimer Test prothrombin, factor V, and factor X
 Used to differentiate CF VII or X deficiencies
COAGULATION TESTS o CF X: ↑ PT, ↑ ST
CLOTTING TIME o CF VII: ↑ PT, ⓃST
 Measures the period required for free formation of  Differs from PT in which factor VII deficiency is NOT
blood to clot after it has been removed from the body detected
 Used to screen for problems in the blood clotting or  Reagent: East Indian Viper venom ( Vipera russelli )
coagulation mechanism o Capable of bypassing action of factor VII and
METHODS: directly activating factor X to Xa
 Normal value: 6-10 second
 Capillary Blood Method
THROMBIN TIME
o Slide Drop Method
 Detects fibrinogen deficiency
 Normal value: 2-4 minutes
 Sensitive test in detecting heparin therapy
o Capillary Method (Dale and Laidlaw’s)
 Prolonged in:
 Whole Blood (Lee and White Method)
o i. Fibrinogen deficiency and abnormalities
o Normal value: 7-15 minutes
o ii. Presence of thrombolytic/antithrombotic
PROTHROMBIN TIME
agents
 Monitors coagulation factors of the extrinsic and
o iii. Presence of FDP and FSP
common pathways
o iv. Presence of heparin
o Prolonged PT results indicate an abnormality of
 Sample: PPP (2000 g x 10 minutes)
one or more extrinsic or common pathway
 Reagents:
coagulation factors
o i. Thrombin
 Used to monitor oral anticoagulant therapy
o ii. Calcium Chloride
(coumadin/warfarin – vitamin K antagonists)
 Principle:
 Sample: PPP (2000 g x 10 minutes)
o Addition of thrombin to plasma bypasses all
 Reagents:
coagulation factors, except polymerization of
o i. Thromboplastin
fibrinogen. Thus, it measures conversion of
o ii. Calcium Chloride
fibrinogen to fibrin and is not influenced by
ACTIVATED PARTIAL THROMBOPLASTIN TIME
other coagulation factor deficiencies
 Monitors coagulation factors of the intrinsic and
REPTILASE TIME
common pathways
 Reptilase – thrombin-like enzyme from the reptile
o Prolonged APTT results indicate an
Bothrops atrox
abnormality of one or more intrinsic or common
o Converts fibrinogen to fibrin
pathway coagulation factors
 Detects fibrinogen deficiency
 Used to monitor heparin therapy (heparin –
 Unaffected by heparin therapy
antithrombin)
 Prolonged in:
 Sample: PPP (2000 g x 10 minutes)
o i. Fibrinogen deficiency and abnormalities
 Reagents:
o ii. Presence of thrombolytic/antithrombotic
o Activators (MECK)
agents
 Micronized silica, Elagic acid, Celite, Kaolin
o iii. Presence of FDP and FSP
o Phospholipids
 Sample: PPP (2000 g x 10 minutes)
o Calcium Chloride
 Reagents:

PADAYHAG, RIGIDOR S. | MLS-3D 1

 HEMATLOGY
Clinical Hematology
UIC-MLS
Chapter 24
2
Prelim Topic 5- Laboratory Evaluation of
Secondary Hemostasis
o i. Reptilase/Atroxin ( Bothrops atrox )  Aged Serum
 Principle: o All factors are present except: 1, 2, 5, 8, 13
o Reptilase mimics the action of thrombin,  Adsorbed Plasma
converting fibrinogen to fibrin. Reptilase differs o All factors are present except: 9, 10, 7, 2
from thrombin in which reptilase cleaves  Aged Plasma
fibrinopeptide A, while thrombin cleaves o All factors are present except 5 and 8
fibrinopeptides A and B. IF APTT: ABNORMAL
DUCKERT’S TEST PT: NORMAL
 A.k.a 5M Urea Solubility Test  Interpretation: Problem w/ the intrinsic pathway
 Detects Factor XIII deficiency  Coagulation factors affected: 12, 11, 9, 8
o Factor XIII deficiency – normal platelet count, IF APTT: NORMAL
PT, APTT, and fibrinogen assays PT: ABNORMAL
 Sample: PPP (2000 g x 10 minutes)  Interpretation: Problem w/ the extrinsic pathway
 Reagents:  Coagulation factors affected: 3, 7
o i. 5M Urea IF APTT: ABNORMAL
o ii. 1% monochloroacetic acid PT: ABNORMAL
o iii. Calcium Chloride
 Principle:  Interpretation: Problem w/ the common pathway
o Patient’s plasma is clotted by the addition of  Coagulation factors affected: 10, 5, 2, 1
calcium chloride. 5M urea and/or 1%
APTT: ABNORMAL PT: NORMAL → INTRINSIC
monochloroacetic acid is added to the clot. PATHWAY→12,11, 9,8
When Factor XIII is present, the fibrin clot
formed is insoluble in 5M urea and/or 1% ADSORBED PLASMA: NOT CORRECTED
monochloroacetic acid when left standing for FRESH SERUM: CORRECTED
24 hours at 37 degrees Celsius. FRESH PLASMA: CORRECTED
 Factor XIII deficiency: clot is soluble to 5M urea ↓
 Presence of Factor XIII: clot is insoluble to 5M urea 9, 10, 7, 2
for 24 hours
= Factor IX
Table 1: Test for Coagulation
Test Rodak Steininger Brown
PT 12.6-14.6 10-12 10-12
seconds seconds seconds
12-14 APTT: ABNORMAL PT: NORMAL → INTRINSIC
seconds PATHWAY→12,11, 9,8
APTT 26-38 20-45 25-35 FRESH SERUM: NOT CORRECTED
seconds seconds seconds FRESH PLASMA: CORRECTED
Stypven 6-10 ↓
Time seconds 1,5,8,13,2
Thrombin 15-20 15-20 10-14
Time seconds seconds seconds = Factor VIII
Reptilase 18-20 10-15
Time seconds seconds

MIXING STUDIES APTT: ABNORMAL PT: ABNORMAL → COMMON

 A.k.a Substitution Studies PATHWAY→10,5, 2, 1
 A procedure done to identify specific factor
deficiencies by mixing correction reagents with a AGED PLASMA: NOT CORRECTED
patient’s plasma, and performing PT and/or APTT AGED SERUM: NOT CORRECTED
 Perform PT and APTT→Mix correction FRESH PLASMA: CORRECTED
reagents→Perform PT and APTT→Observe for ↓
corrected results 5,8 - AGED PLASMA
1, 2, 5, 8,13 -AGED SERUM
 Fresh Plasma
o All factors are present.
= Factor V
 Fresh Serum
o All factors are present except: 1, 5, 8, 13, 2
(20%)

PADAYHAG, RIGIDOR S. | MLS-3D 2

 HEMATLOGY
Clinical Hematology
UIC-MLS
Chapter 24
2
Prelim Topic 5- Laboratory Evaluation of
Secondary Hemostasis
Presence of
thrombolytic/antithrom
APTT: ABNORMAL PT: ABNORMAL → COMMON
botic agents
PATHWAY→10,5, 2, 1
Presence of FDP and
ADSORBED PLASMA: NOT CORRECTED FSP
AGED SERUM: NOT CORRECTED
FRESH PLASMA: CORRECTED Presence of heparin
↓ Reptilase  Detects  Reptilase/At Fibrinogen deficiency
9,10,7,2 – ADSORBED PLASMA fibrinogen roxin ( and abnormalities
1, 2, 5, 8,13 -AGED SERUM deficiency Bothrops
 Unaffected atrox ) Presence of
by heparin thrombolytic/antithrom
= Factor II botic agents
therapy
Presence of FDP and
APTT: NORMAL PT: ABNORMAL → EXTRINSIC FSP
PATHWAY→3,7 Duckert’s  A.k.a 5M  5M Urea Factor XIII deficiency
Test Urea  1% – normal platelet
ADSORBED PLASMA: NOT CORRECTED Solubility monochloro count, PT, APTT, and
FRESH SERUM: CORRECTED Test acetic acid fibrinogen assays
FRESH PLASMA: CORRECTED  Detects  Calcium
↓ Factor XIII Chloride
9,10,7,2 deficiency

= Factor VII Coagulation Test Factor/s Involve

PT 3,7,1,2,5,10
APTT 12,11,9,8,10,5,2,1
Stypven 10,5,2,1
Table 2: Summary of Coagulation Tests and their Disease TT 1
Associations Reptilase 1
TEST Principle Reagent Prolonged in Duckert’s 13
PT  Monitors  Thrombopla Abnormality of one or
coagulation stin more extrinsic or
Disorder PT APTT Stypven TT Reptilase Duckert’s
factors of  Calcium common pathway
the Chloride coagulation factors Fibrinogen ↑ ↑ ↑ ↑ ↑ N
extrinsic Deficiency
and Factor VII ↑ N N N N N
common Deficiency
pathways Hemophilia N ↑ N N N N
APTT  Monitors  Activators Abnormality of one or A
coagulation (MECK) more intrinsic or Factor XIII N N N N N ↑
factors of  -Micronized common pathway
the intrinsic silica, Elagic coagulation factors Vitamin K ↑ ↑ ↑ N N N
and deficiency
acid, Celite,
common Kaolin
Heparin ↑ ↑ ↑ ↑ N N
Therapy
pathways  Phospholipi
ds
FIBRINOLYSIS TESTS
 Calcium
Chloride WHOLE BLOOD CLOT LYSIS TIME
 Principle:
STYPVEN  Monitors  East Indian Abnormality of one or o Whole blood clots spontaneously when
coagulation Viper venom more common collected in a glass tube without an
factors of ( Vipera pathway coagulation
factors anticoagulant. The clot should remain intact for
common russelli )
pathway  -Capable of
approximately 48 hours at 37 degrees Celsius.
bypassing Clot lysis or dissolution prior to 48 hours is
action of indicative of excessive systemic fibrinolysis.
factor VII  Results:
and directly
activating
o Normal = Clot lysis in >48 hours
factor X to o Increased fibrinolysis = Clot lysis in <48 hours
Xa EUGLOBULIN CLOT LYSIS TIME
 Test that measures overall fibrinolysis
Thrombin  Detects  Thrombin Fibrinogen deficiency
 Screening procedure for the measurement of
Test fibrinogen  Calcium and abnormalities
fibrinolytic activity
deficiency Chloride
 More sensitive than whole blood clot lysis time

PADAYHAG, RIGIDOR S. | MLS-3D 3

 HEMATLOGY
Clinical Hematology
UIC-MLS
Chapter 24
2
Prelim Topic 5- Laboratory Evaluation of
Secondary Hemostasis
 EUGLOBULIN– protein fraction that precipitates  Forms clot if FDP are Present
when plasma is diluted with water and acidified  Detects the presence of fibrin monomers
o i. Plasminogen  Reagent:
o ii. Plasmin o i. 50% ethanol
o iii. Fibrinogen o ii. Sodium hydroxide
o iv. Plasminogen activators  Principle:
 Principle: o In the presence of 50% ethanol, any soluble
o Addition of 1% acetic acid to diluted plasma fibrin monomer complexes will dissociate,
causes euglobulin fraction to precipitate. After resulting in polymerization of monomers and
precipitation, the supernatant is removed. subsequent gel formation
Precipitated euglobulin is subsequently  Results:
dissolved in a buffer solution. Thrombin is o Normal – no gel formation
added to clot the euglobulin fraction. The clot is o Abnormal – gel formation
incubated at 37 degrees Celsius and the time LATEX D-DIMER TEST
of complete clot lysis is noted.  Mixed with antibody to D and E fragments (late
 Citrated Platelet Poor Plasma with acid in a test tube degradation Products)
 Used to evaluate the presence of specific fragments
arising from degradation of fibrin
 D-dimer– evidence of intravascular fibrin formation
 Principle: Enzyme Immunoassay and Latex Bead
o The test uses latex beads coated with
monoclonal antibody specific for D-Dimer
o The test has no specificity for fibrinogen and,
therefore identifies plasmin action towards
fibrin
 NOTE: Single test to differentiate Primary and
Secondary Fibrinolysis
Table 3: Primary Fibrinolysis vs. Secondary Fibrinolysis
Test Primary Secondary
 If the plasminogen in the euglobulin is converted to Fibrinolysis Fibrinolysis
plasmin, it will lyse the clot. WBCLT <48 hours <48 hours
 Results: Euglobulin Clot <2 hours <2 hours
o Normal = Clot lysis in >2 hours Lysis Time
o Increased fibrinolysis = Clot lysis in <2 hours Protamine - +
PROTAMINE SULFATE TEST Sulfate
 Detects the presence of fibrin monomers by causing Ethanol - +
the formation of fibrin strands or gel-like clots – Gelation
paracoagulation Latex D-Dimer - +
 Detection of early and late degradation Products
 Reagent: Protamine Sulfate “If you don’t sacrifice for what you want, what you
 Principle: want will become the sacrifice”
o When protamine sulfate is added to plasma, it “The chapter you’re learning today, is going to save
displaces secondary degradation products someone’s life tomorrow. LABAN Frmt"
from fibrin monomers and primary degradation
products, which will lead to polymerization. REFERENCES
 Primary fibrinolysis: (Degradation fibrinogen, (–) rxn)
→ no available or Present fibrin but with fibrinolytic University of the Immaculate Conception Powerpoint Presentation
system
and Discussion Prepared By Sir Glenn
 Secondary hemostasis: (Degradation of fibrin, (+) rxn)
→there will be fibrinolytic system only if fibrin is
Present Keohane, E. M., Smith, L. J., & Walenga, J. M. (2020). Rodak's
 Results: hematology: Clinical principles and applications (5th ed.). St.
Louis, MO: Elsevier.
o Normal – no gel formation
o Abnormal – gel formation
ETHANOL GELATION TEST Stiene-Martin, E. A., Lotspeich-Steininger, C. A., &amp; Koepke, J. A.
(1998). Clinical hematology: Principles, procedures,
 Less sensitive but more specific test than protamine
correlations. J.B. Lippincott Co.: Philadelphia, Pa.
sulfate test

PADAYHAG, RIGIDOR S. | MLS-3D 4