Download Blood clot

Physiology of Blood
Practical 4
Contents
The types of leukocytes
Haemostasis
Practical tasks
1. The blood smear and leukogram
2. Blood coagulation time determination by
the Lee – White method
3. Determination of bleeding time by the
Duke method
4. Determination of the thromboplastin time
by the Quick test
© Katarína Babinská, MD, PhD. MSc., 2010
katarina.babinska@fmed.uniba.sk
Types of leukocytes
• granulocytes
-specific granules (vesicles)
-lobulated nucleus - polymorphonuclears
1. neutrophilic 56 -64%
2. eosinophilic
1-3%
3. basophilic
0,5-1%
• agranulocytes
-do not contain specific granules
-mononuclear – simple shape nucleus
4. monocytes
3 - 8%
5. lymphocytes 24 - 40%
2
1
4
5
3
differential white blood cell count (leukogram)
- examination of the % of individual types of leukocytes in %
- helps to make diagnosis - individual types of Le are involved in different functions
!!! in children – the most prevalent type of Le are lymphocytes
Neutrophilic granulocytes (56 – 64 % Le)
Properties
• nucleus 1 – 5 segments (lobes)
- number of segments indicates age of the cell
- young cells – one segment („stick“)
- by maturation the number of segments increases
• cytoplasmic granules - purple colour (lysosomes with hydrolytic enzymes)
Function
- professional phagocytes (microphages) – ingest and destroy foreign material
- participate in non-specific immune reactions
- high motility, first line defence – first arrive to the place of invasion (of all WBC)
Kinetics (fate)
formation in the bone marrow
mature elements enter the blood - they spend 7-8 h here
pass from blood into tissues through capillary wall
-survive here for 4-5 days, then die
-if involved into phagocytosis, they die soon afterwards (i.e. earlier than in 4-5 d)
Eosinophilic granulocytes (1-3% Le)
- dense purple cytoplasmic granules
- nucleus - 2 segments
- weak ability of phagocytosis
- involved in:
- the defence against parasites
- allergic reactions
Basophilic granulocytes (0,5 – 1% Le)
-
dark blue granules in cytoplasm
-
two segment nucleus
-
release active substances:
- histamin – causes vascular dilatation – increases blood flow into areas of
tissue damage, facilitates the movement of leucocytes into tissues
- heparin - anticoagulant
- eosinophilic and basophilic granulocytes have similar kinetics as neutrophilic granulocytes
Monocytes (3 – 8% Le)
-
largest blood elements
large kidney-shaped nucleus
Function:
- professional phagocytes (non-specific immunity)
- take part also in specific immune reactions
Kinetics
in blood 10-20 h
from blood - migrate into tissues → here maturate and transform to
macrophages
free macrophages – actively move in tissues
fixed macrophages – in the sites of potential invasion of the pathogens
skin (histiocytes)
lung
liver
lymph nodes
Lymphocytes (24 – 40% Le)
-
large round nucleus
narrow cytoplasm
Types
- T-Ly
- B-Ly
- NK cells (natural killers)
- K (killer) cells, LAK cells
involved mainly in the specific type of immunity
recirculate:
blood
lymph
Life span – years
tissues
Defensive properties of leukocytes
diapedesis – ability to squeeze and pass through the capillary wall
ameboid motion – active movement in tissues, the cell projects protoplasmic
extensions and follows them
chemotaxis – direction and speed of movement influenced by chemical
substances (i.e. produced in the focus of infection)
tigmotaxis – tendency to stick to solid surfaces
phagocytosis (especially neutrophils and macrophages)
White blood cells and immunity
Immunity
- capacity to resist foreign substances that tend to damage tissues and organs
- microorganisms
- molecules
- own abnormal cells (cancer cells, old cells)
The immune system
- organs positioned throughout the body
(thymus, lymph nodes, lymphoid tissue in gut, spleen, etc.)
- white blood cells - main cells of the immune system
Immunity
1. innate
2. acquired (adaptive)
1. Innate immunity (defense mechanisms present from birth)
Characteristics
• the immune response is non-specific
(it is not targeted at a specific antigen, but is rather equal to different antigens)
•
the first line defense - provides an immediate response
Types of leukocytes involved and mechanisms*
a) phagocytosis by the neutrophils and macrophages (professional phagocytes)
b) eosinophils - phagocytize the antigen-antibody complexes
- kill parasites (attach to the parasite and attack its cell membrane)
c) action of some types of lymphocytes:
• NK (natural killer) and LAK (lymphokine activated cells) cells - recognize absence
of normal “self” antigens in the body´s infected and tumour cells and destroy them
• K (killer) cells - recognize, bind and kill cells coated with antibodies
*(not all mechanisms, but only those performed by the white blood cells are mentioned)
2. Acquired immunity
A/ Active immunity
develops after exposure to a foreign antigen
(by an infection, vaccination) - active response of a host
– first exposure - a weak immune response
– in next exposure the response is strengthened
permanent immunity
specific = targeted at foreign material that induced the response
therefore highly effective
exhibits immunological memory (permanent immunity)
mediated by B and T lymphocytes
http://www.virology.ws/wpcontent/uploads/2009/07/im
mune-memory.jpg
Active immunization - vaccination
- vaccines contain weakened or dead microbes, that induce an immune response
- acitve response/ first response weak, further responses faster, stronger
Passive immunization
• transfer of antibodies from exogenous source (e.g. from an immunized donor to a patient, from mother to a newborn via the breastmilk)
• temporary protection (weeks) - no active response of the immune system occurs
Effector cells of acquired immunity
B-Lymphocytes
- formation and maturation in bone marrow
- mediate humoral type of immunity
B-Ly after recognizing the foreign agent - transform into plasma cells
produce specific molecules of antibodies (immunoglobulins): M, A, G, D, E
antibodies are bind to the invading agent (antigen)
the complex of antibody+antigen is destroyed and removed by immune reactions
• antibody binds with the foreign agent (e.g. the bacteria)
and marks it for destruction (by phagocytosis or by other means)
• activation of B lymphocytes requires assistance of the
TH lymphocytes
X
T-lymphocytes (25%)
- exhibit cell mediated immunity
- formation in bone marrow, maturation in thymus
- differentiate into different types
• TH (helper)
1. required for activation of B-Ly (without their cooperation the B-Ly cannot
recognize majority of antigens – failure of the immune system)
2. capable of direct killing of some types of microorganisms and infected cells
3. produce cytokines – regulate immune functions
• TC (cytotoxic) – directly destroy the target cells by releasing substances that
attack their cell membranes, e.g.
- cells containing viruses
- cancer cells
• TS (suppressor) - close down the immune response after invading organisms are
destroyed and the immune response has achieved its goal
Task: Blood smear (Leukogram)
•• proportion of individual types of white blood cells in % is assessed
© Katarína Babinská, MD, PhD. MSc., 2010
Procedure
•
make a capillary blood collection – place a drop of blood on a glass slide
•
make a thin blood smear using another glass slide (see picture)
•
leave to dry
Staining according to Pappenheim
•
place the slide horizontally on a holder
•
drop the May-Grünwald stain so that it covers the smear – wait 3 min
•
drop distilled water (do not let the stain to float away), wait 1 min
•
remove the stain with distilled water
•
dilute 15 drops of Giemsa-Romanovski stain in 10 ml of distilled water
•
cover the smear with stain, wait 15 – 20 min
•
remove the stain, wash the smear with water
•
put the smear into a holder in vertical position, leave to dry
© Katarína Babinská, MD, PhD. MSc., 2010
• observe in microscope at 1000x
enlargement (use oil)
• count individual Le types in one
visual field
• move the sample, observe next
visual fields – move the glass
slide in a way shown in picture
• evaluate 100 Le
Ne
Ly
Mo
Ba
Eo
//// //// ////
//// //
//
/
Result
- % of individual Le types
Conclusion
- compare your result with normal
values
© Katarína Babinská, MD, PhD. MSc., 2010
Haemostasis – bleeding arrest
- a complex action preventing from blood loss
• maintenance of normal blood volume – vital
• massive bleeding eventually leads to cardiovascular collapse and death
- constituted by simultaneous interrelated events:
1/ reaction of the vessel - vascular constriction
2/ formation of a platelet plug
3/ blood clotting (coagulation)
1. Reaction of the vessel
- contraction of the smooth muscle in the vessel wall – vascular constriction
- as direct response of the injured vessel
- maintained by chemical substances released from platelets
(serotonin, TXA2)
Effect: decreases the blood flow through the ruptured vessel
and thus the blood loss
2. Formation of the platelet plug
- includes several steps:
A/ ADHESION OF PLATELETS
- normal smooth endothelial lining of
vessels – repels platelets
- vessel trauma – exposure of
subendothelial collagen tissue – it
activates the platelets
- thrombocytes stick to collagen (it
has receptors for thrombocytes)
B/ CHANGE OF THE SHAPE AND THE RELEASING REACTION
thrombocytes swell and become spheric
- caused by contractile fibres
in cytoplasm (actin and myosin)
formation of pseudopods protruding from the surface
- easier contact with - other thrombocytes
- collagen
- fibrin threads
releasing reaction – degranulation
- platelets release substances active in hemostasis into the blood
- e.g. serotonin, ADP, thromboxane A2 (TXA 2), platelet factors, etc.
C/ AGGREGATION OF THROMBOCYTES
-
platelets stuck to the collagen stimulate sticking of their further layers
Platelet activity results in formation of the platelet plug
-
it does not contain fibrin threads, therefore loose, fragile
-
it is sufficient for temporary blocking the blood loss, especially in small vessels
3. Blood coagulation (haemocoagulation, blood clotting)
-
series of enzyme reactions following in definite and rapid sequence
-
blood contains more than 50 substances related to blood clotting
-
major role - plasma clotting factors (12 substances)
Result of haemocoagulation:
formation of fibrin threads - strengthen and stabilize the platelet plug
net of fibrin threads + platelet plug + trapped erythrocytes = blood clot
- blood clot - seals the broken vessel until the tissue is repaired
Blood clotting factors
I. fibrinogen
II. prothrombin
III. tissue thromboplastin
-
present in blood
-
inactive forms of protheolytic
IV. Ca2+ ions
V. proaccelerin
VII. proconvertin
enzymes (majority)
-
blood clotting = a cascade of
chemical reactions leading to
VIII. antihemophilic factor
VIII C - antihemophilic globulin
conversion to active forms
VIII A – von Willebrand factor
F1
IX. Plasma thrombopastin component
- Christmas factor
F2
X. Stuart – Prower factor
XI. PTA – Plasma thromboplastin
antecedent
F1A
↓
F2A
..............
XII. Hageman factor
-
synthesized in liver
XIII. fibrin stabilising factor
-
vitamin K – required for
•HK - High molecular weight kininogen
•PK - Prekallikrein
synthesis of factor II, VII, IX, X
Blood clotting - activation
1. exposure of collagen in
vessel wall
2. releasing of thromboplastin
from damaged tissue
activates a sequence of chemical
reactions referred to as
activates a sequence of chemical
reactions referred to as
intrinsic pathway of clotting
extrinsic pathway of clotting
final reactions of both intrinsic
and extrinsic pathway are the same
and they are referred to as
=common pathway
- result: formation of fibrin thread
EXTRINSIC PATHWAY
- activated by
damage of vessel wall and
extravascular tissue
- f. III
III
(III)
INTRINSIC PATHWAY
- activated by
damage of endothelial layer
- f. XII
COMMON PATHWAY
EXTRINSIC PATHWAY
1. fibrin monomer
- activated by
damage of vesel wall and
extravascular tissue
- f. III
2. fibrin polymer
III
3. cross-linked fibrin polymer
(III)
INTRINSIC PATHWAY
- activated by
damage of endothelial layer
- f. XII
4. stabilization of the cross
linked fibrin polymer
EXTRINSIC PATHWAY
- activated by
damage of vesel wall and
extravascular tissue
- f. III
III
(III)
INTRINSIC PATHWAY
- activated by
damage of endothelial layer
- f. XII
In bleeding both pathways are activated
Intrinsic pathway - slow (6-10 min)
Extrinsic pathway – faster (seconds)
Blood clot
network of fibrin threads running in all directions
- adhere to damaged surfaces of vessels
contains trapped plasma, blood elements,
coagulation factors
Determination of bleeding time by the Duke method
Principle
• after puncturing the 4. finger tip the time of bleeding is measured
• a general, non – specific test of haemostasis
© Katarína Babinská, MD, PhD. MSc., 2010
Procedure
-
disinfect the ball of 4th finger and puncture it
-
at the moment of puncturing turn on the stop watch
-
wipe off the first drop
-
suck off the blood with a strip of filter paper every 30 seconds
-
stop the watch when you cannot see any more blood on the filter
paper
-
normal value 2-5 minutes
Result
- the time
Conclusion
- is the time within the interval of normal values?
© Katarína Babinská, MD, PhD. MSc., 2010
Blood coagulation time determination
by the Lee – White method
• test of the intrinsic pathway of blood coagulation
• glass tube
– wettable surface = mimics the effect of collagen (starts the intrinsic
pathway of blood clotting)
© Katarína Babinská, MD, PhD. MSc., 2010
Procedure
- collect venous blood – 3 ml
- place the last 1 ml of blood into a tube (to avoid the effect of the
tissue factor – that activates the intrinsic pathway)
- turn on the stop watch
- check every 30 -60 sec if the blood has coaglulated or not
tissue factor
- stop the watch when the blood is clotted
- normal value 6 -10 minutes (in 37°C, in a room tempe rature up to
15 minutes)
Result
• the time
Conclusion
• is the value normal?
• if not, try to explain
• write down, which pathway of blood clotting is tested by this test
© Katarína Babinská, MD, PhD. MSc., 2010
Determination of prothrombin time by Quick
•
test of the external pathway of blood clotting
© Katarína Babinská, MD, PhD. MSc., 2010
Procedure
•
collect a sample of venous blood – 0,3 ml citrate + 2,7 ml blood
•
put the blood into a tube, centrifuge
•
put the tube with plasma into the water bath (37º C)
•
keep other reagents in water bath
•
into another clean tube put
– 0,1 ml of plasma
– 0,2 ml of thromboplastin + CaCl
•
immediately turn on the stop watch
•
with a glass hook try to pick fibrin clots
•
when you find fibrin clot stop the watch
•
normal time 12 – 15 sec
Result: the time
Concusion: evaluate if the time is normal, if not – try to explain
Write into your protocol which pathway of blood clotting is examined by this test.
© Katarína Babinská, MD, PhD. MSc., 2010