LABORATORY ACTIVITY 5 AND LABORATORY ACTIVITY 6 Flashcards
Within [?] after whole blood is allowed to clot in a clean glass tube at [?], the clot will begin to shrink and retract from the walls of the tube.
1 hour
37OC
Serum is expressed and the clot becomes [?].
denser
This retraction process is maximal at [?], by which time it occupies almost half of the original blood volume
24 hours
PROCEDURE:
Modified MacFarlane Serum method
- Collect [?] of venous blood samples. Note the time of collection.
5 ml
- Dispense into a [?] noting exact amount of blood used.
graduated centrifuge tube
- Place an [?] at the center of tube.
applicator stick
- Incubate test tubes at [?] in a water bath for [?].
370C
2 hours
- Examine and record degree of retraction (?)
partial, complete, or no retraction
- Pull [?] with clotted blood and describe the clot.
applicator stick
- Serum expressed is centrifuged for [?] at [?].
3-5 minutes
3,500 rpm
- Calculate % of serum expressed as follows:
Volume of Serum =
% serum expressed =
total volume of serum in tube – volume of packed red cells
Volume of serum in ml/Volume of blood used in ml x (100)
- Report results in
%
This measures the time required for blood to clot after it has been removed from the body.
WHOLE BLOOD COAGULATION TIME
This is a measure of the overall intrinsic and common pathways of coagulation.
WHOLE BLOOD COAGULATION TIME
Micro Method –
capillary blood
slide method
Macro Method –
Whole blood
Lee and White Method
- Do a finger puncture. Start stopwatch at the time of appearance of [?] from the puncture
blood
- Place on a clean glass slide [?]
3 separate drops of blood.
- Allow to stand for [?] at room temperature. Check clot formation by drawing the blood with a needle or lancet and observe for thread formation.
2 minutes
- Record [?] from the start to fibrin thread formation.
clotting time
- Label three uniformly sized tubes [?].
1, 2 and 3
- Make a clean venipuncture using [?] and note the time at which blood enters the syringe. Draw [?] of blood.
20-gauge needle
four (4) ml
- Carefully dispense [?] to tube 3, then [?] to tube 2 and [?] to tube 1. [?] the remaining blood.
1 ml
Discard
- Incubate all tubes in a water bath at
37 0C (+ 0.5 0C )
- At exactly [?], tilt tube number 1 to a [?] angle and observe for clotting. Repeat every [?] until tube can be completely inverted without spilling contents. Note time of clotting.
5 minutes
450
30 seconds
- After[?], tube 1 is clotted, proceed to tube 2 and repeat the preceding procedures. Repeat procedures with tube 3.
30 seconds
- Record coagulation time as the time elapsed between the [?] and the [?] in tube 3.
withdrawal of blood
completion of coagulation
CLOT RETRACTION - otherwise known as
“contraction of clot”
CLOT RETRACTION - otherwise known as
“contraction of clot”
- requires great amount of ATP and Calcium
CLOT RETRACTION
- requires great amount of ATP and Calcium
CLOT RETRACTION
: compress to become denser
Contraction
: moves out of surface
Retraction
Mainly involves platelet
CLOT RETRACTION
In vivo or in vitro indication of plt function test (normal platelet function and number)
CLOT RETRACTION
Affected by both qualitative (inability to adhere) and quantitative (thrombocytopenia)
CLOT RETRACTION
based on platelet normal function
CLOT RETRACTION
: released by plt when activated; dense granules
ATP and Calcium
plt trapped in the clot releases [?] found in the cytoplasm of plt
factor XIII
= denser and more compact clot to be able to retract from the vessel wall
continuous cross-linking of XIII
: plays a role in contraction (retraction from the wall)
Calcium
allows platelet to go back to its original shape after contraction
Calcium
: produces energy
ATP
nonspeific screening test for in vivo plt function
CLOT RETRACTION
CLOT RETRACTION Principle:
When [?] is complete, clot normally undergoes [?].
blood coagulation
retraction
[?] is expressed and clot becomes [?]
Serum
denser
Normal clot retraction:
30 mins (initial)/24 hrs (completely/maximal)
contracts the clot
Platelet
Indication that clot has been retracted
Serum is expressed
Clot settles at the [?] or attaches to the applicator stick
bottom
happens before fibrinolysis
Clot retraction
before it dissolves
clot retracts
retraction does not mean the clot has been removed from the bv, but it simply [?] and proceeds to the bottom of the bv initiating plasmin to dissolve the clot = healing of bv
detaches
to preven thrombosis and establish blood flow
Fibrinolysis (dissolution of clot)
to give space for blood to start flowing again
Retraction
initially restores blood flow
Retraction
restored after fibrinolysis
Retraction
Factors affecting clot retraction
Adequate amount of plt
Calcium
ATP
Fibrinogen
normal interaction of plt and fibrinogen
(form platelet and bones)
Calcium
(from food, other cells, muscle breakdown)
ATP
adhesion of platelet to bv:
Ib-IX-V
adhesion of platelet to platelet:
fibronectin and fibrinogen
: plt will not aggregate = weak release of XIII
↓ Fibrinogen
plt needs to attach to the fibrin clot (no problem in the fibrin-plt interactiom)
normal interaction of plt and fibrinogen
normal interaction of plt and fibrinogen
qualitative defect
Other factors
thrombostenin
plasma volume: red cell mass
nature of the surface where CRT is being measured
contains actomyosin for muscle
thrombostenin
provides contractile force for plt
thrombostenin
stretches the plt
thrombostenin
equal in proportion
plasma volume: red cell mass
affects clot retraction if unequal
plasma volume: red cell mass
preferably glass = slower retraction in plastic; normal in glass(in vitro)
nature of the surface where CRT is being measured
prosthetic heart valve and fistula are dangerous since attachment of clot will not retract leading to obstruction of bv
nature of the surface where CRT is being measured
Process of Clot Retraction
Platelets → Thrombostenin → Actin/Myosin (Myosin II) → Clot retraction → BV edge retracts
[?] are entrapped in fibrin threads while continuously releases ?
Platelets
FXIII along w/ Ca and ATP
plt changes in shape once denser via [?]
Thrombostenin
: releases microfilaments
Actin
(small filaments in plt that maintains shape; released by actin force)
microfilaments
: assembles microfilaments only in the surrounding/border of plt for support during change of shape
Myosin II
encodes Myosin II in the platelet
MYH9 gene
can make platelet have self-aggregation w/o fibronectin/fibrinogen
Myosin II
Once assembled, microfilaments will work on the
adhesion complex
(dissector of plt; connects plt to bv)
adhesion complex
to down-regulate it to help plt retract from the bv
adhesion complex
- adhesion complex; lacks in BSS; dissector of plt, down-regulating it; connects plt to bv
Gp Ib-IX-V complex
– plt starts to attach to fibrin links; bv is capable of contraction; plt, fibrin clot, bv
platelet spicules attached to fibrin
– by the help of FXIII
firbin meshwork
bv - conrtacts to help clot from detaching itself; capable of
contraction (bv vasoconstriction)
: action of plt, fibrin clot, and bv
blood retraction
to help eject the clot, the bv needs to constrict
BV edge retracts
CRT is poor when plt count is
<100,000/ul (thrombocytopenia)
Normal:
150-400uL
few plt trapped, few
thrombostenin
Clinical significance
Dysfibrinogenia
Hypofibrinogenemia
Paraproteinemias
: abnormal fibrinogen
Dysfibrinogenia
: low fibrinogen
Hypofibrinogenemia
: abnormal proteins
Paraproteinemias
plt needs to adhere to fibrinogen to allow interaction between them)
Dysfibrinogenia
plt has nowhere to attach to for fibrin-meshwork formation; no retraction will take place)
Hypofibrinogenemia
Abnormal thrombostenin: actin-myosin function will not occur (release and assembly of filaments)
Paraproteinemias
Methods
a. Hirschboeck Method (Castor oil Method)
b. Stefanini Method (Test tube)
c. Mac Farlane Method
Qualitative: Test for the presence or absence of retraction
Hirschboeck Method (Castor oil Method)
Formation of dimpling/droplet like serum on the surface of blood drop
Hirschboeck Method (Castor oil Method)
Normal Values = 15 - 45 minutes
Hirschboeck Method (Castor oil Method)
Normal: clot retraction begins within 1 hour, complete within 18 to 24 hours
Stefanini Method (Test tube)
Provides quantitative estimate of the degree of retraction
Mac Farlane Method
Degree of serum expressed = Degree of retraction
Mac Farlane Method
Normal Values = 44% - 67 %
Mac Farlane Method
COAGLULATION TIME - otherwise known as
“clotting time”
COAGLULATION TIME - Principle: measures the [?] for blood to clot after it has been removed from the body within [?]
time requires
1 hour
COAGLULATION TIME Indication
Factor deficiency
Hemophilia A and B
Does not diagnose mild coagulation disorder
Traumatic
Not reliable
it is a nonspecific screening test for intrinsic and common pathway factor deficiencies = prolongs coagulation time
Factor deficiency:
: severe deficiency of factor VIII (A) and IX (B) = prolongs coagulation time in intrinsic pathway
Hemophilia A and B
screening test: not affected by mild deficiency (nonspeific)
Does not diagnose mild coagulation disorder
normal coagulation time in minimal factor deficiencies
Does not diagnose mild coagulation disorder
puncture of finger
Traumatic
not reproducible; cannot be done on the same person
Not reliable
cannot detect mild factor deficiencies
Not reliable
after the prick, blood will automatically suck in the capillary
Capillary method
break the capillary and note the time
Capillary method
NV: 2-4 minutes
Capillary method
add the blood and start the timer
Tube method
keep on tilting the tube every 30 seconds to see clot formation
Tube method
NV: 7-15 mins
Tube method
Factors affecting Clotting Time
Macromethod
Excessive agitation of the tube:
shortens clotting time
:
(allowing FXII to have contact with glass = activation of negative surface or glass)
Excessive agitation of the tube
no quality control: no comparison to a basis; not reliable
Macromethod
not considered as adequate test: affected by factors such as the glass surface
Macromethod
Causes Of Prolonged Clotting Time Are
Coagulation factors deficiencies which may be: Congenital or Acquired
Severe deficiency of any known plasma clotting factors except XIII (fibrin-stabilizing factor) and VII
Drugs like heparin and thrombin inhibitors
Marked hyperheparinemia
Afibrinogenemia
(extrinsic pathway; in vivo)
VII
(normal clotting time; does not help in clot formation)
XIII
: does not cause bleeding disorder but may cause prolonged clotting time
FXII
Anti-thrombin (heparin, warfarin, Coumarin)
prolongs clotting time
Excessive heparin in the system - attacks thrombin = coagulation cascade failure =
prolongs clotting time
No fibrinogen = No plt plug = No conversion of fibrinogen to fibrin =
Prolongs clotting time
Blood clotting mechanism
Three stages
Vascular spasm
Platelets plug formation
Congulation factors activation
Stages of blood clotting
Formation of protrombinase:
1. Intrinsic pathway
2. Extrinsic pathway
Prothrombin —-> Thrombine (enzyme)
Fibrinogen (soluble) ——> Fibrin (insoluble)
