Prezentace aplikace PowerPoint · isotonic hypotonic more hypotonic A/ Isotonic solutions –the same osmotic pressure as plasma, optimum for function of Ery B/ Hypertonic solutions

Blood Physiology

Practical 3

© Katarína Babinská, MD, PhD. MSc., 2018

[email protected]

ContentsBlood plasma, osmotic pressure and oncotic

pressure. Erythrocyte osmotic resistance.

Blood density and its determinants.

Haemostasis

Practical tasks1. Determination of erythrocyte osmotic resistance

2. Determination of blood density

3. Blood coagulation time determination by the

Lee – White method

4. Determination of bleeding time by the Duke

method

5. Determination of the thromboplastin time by the

Quick test

Determination of the erythrocyte osmotic resistance

-plasma (but also all body fluids) contains dissolved substances that are osmotically

active and give rise to osmotic pressure

Osmosis - diffusion of solvent through semipermeable membrane from space with

lower concentration of solute into the space with higher concentration

- semipermeable membrane - permeable only for solvent, not for dissolved

substances

Osmotic pressure – water (solvent) passes the semipermeable membrane under

pressure called osmotic pressure

- the bigger the difference in concentration, the higher is the osmotic pressure

selectively permeable membrane

Osmotic pressure of blood plasma

lower concentration higher concentration

vodap

water

- normal value 690 kPa

- depends on concentration od osmotically active substances

- osmolarity of plasma (concentration of osmotically active substances):

290 - 300 mmol.l-1

- osmotic forces are generated mainly by Na+, Cl-, glucose, albumin

Osmotic pressure of blood plasma

Some functions in human body are based on osmotic pressure, e.g.:

• regulation of water balance - hypothalamus monitors osmolarity of plasma

• absorption in gut

• water reabsorption in kidney

• osmotic pressure needs to be considered when patient is given an infusion, or in

laboratory experiments with blood

water plasma

690 kPa

- blood plasma and blood elements – are isoosmotic (isotonic)

= osmotic equilibrium – no water gain/loss

isotonic

hypertonic

hypotonic

Osmotic resistance of erythrocytes – the ability to

resist small changes in osmotic pressure

lysisisotonic hypotonic more hypotonic

A/ Isotonic solutions

– the same osmotic pressure as plasma, optimum for function

of Ery

B/ Hypertonic solutions

– higher osmotic pressure

– if a cell („isotonic solution“) is put into a hypertonic solution,

it loses water, shrinks and may malfunction or die due to

rupture of its membrane (haemolysis)

C/ Hypotonic solutions

– lower osmotic pressure

– water flow is directed into the erythrocyte

– cause expansion of cells, their malfunction and eventually

destruction and death (haemolysis)

In intravenous administration of solutions

(fluids, nutrients, drugs)

• their concentration of osmotically active substances is adjusted to isotonicity

• isotonic solutions:

– 0,9 % NaCl (physiological solution)

– 5 % glucose

– they can be infused without danger of disturbing osmotic equilibrium

• non – isotonic solutions may be used in

special circumstances

– e.g. hypertonic solution in cerebral

oedema – water is attracted from

brain tissue into the circulation

osmotic equilibrium can be broken in dehydration, after infusion of non-isotonic solution

- a component of osmotic pressure

- exerted by plasma albumins

- normal value: 3,7 kPa

(out of 690 kPa of the total osmotic pressure)

proteinwater

tissue blood capillary

Function:

- plays significant role in water reabsorption in capillaries

- it prevents water loss from circulation

Oncotic pressure (colloid-osmotic

pressure of plasma proteins)

1. are permeable for low molecular weight

substances (e.g. ions)

- ions freely cross the capillary membrane in

both directions (tissue - capillary)

- the osmotic pressure of low-molecular weight

substances in capillaries = 0

- no net changes in water volume

Na+

Cl-

HCO3-

proteins

Na+

Cl-

HCO3-

proteins

tissue blood capillary

2. are impermeable for plasma proteins (macromolecules)

- plasma proteins exert oncotic (colloid-osmotic) pressure on capillary wall

- concentration of proteins in plasma >> concentration of proteins in tissue fluid

- water moves from tissues (interstitial fluid) into capillaries

Blood capillaries

venous end of capillary (blood pressure here lower than at the arterial end)

blood pressure + oncotic pressure of interstital fluid < oncotic pressure of plasma(2 + 0,7 < 3,7 kPa)- reabsorption – water passes from interstitium into capillaries

• arterial end of capillary

blood pressure + oncotic pressure of interstitial fluid > oncotic pressure of plasma

(4 + 0,7 > 3,7 kPa)

-filtration - from capillary into interstitial space

- hypoproteinemia (e.g. due to starvation, liver diseases, kidney disease)

- decrease in plasma oncotic pressure – oedema formation

tissue

capillary

Oedema- more water

filtrated, less

water reabsorbed

due to decreased

oncotic pressure

filtration reabsorption

hypoproteinaemia

tissue

capillary

Capillary filtration and reabsorption effect of hydrostatic (blood pressure) and oncotic pressure

- 0,9 % solution (9 g /L) of NaCl is isotonic - optimum for Ery,

they survive in this solution and do not hemolyze

- hypertonic/hypotonic – may cause hemolysis

- HOWEVER!!! To some extent erythrocytes are able to survive even

in slightly hypotonic/hypertonic environment

- osmotic resistance – ability of Ery to resist slightly

hyper/hypotonic environment

- the more hyper/hypotonic solution, the more red blodd cells

hemolyze = the less survive

• prepare set of tubes with decreasing Na Cl concentration

(0,72 - 0,68..... 0,24 – 0,2 g NaCl/l)

• collect venous blood into syringe

• put 3 drops of blood into each tube

• allow to stand for 2 – 3 hours

Determination of the erythrocyte osmotic resistance

Procedure

Read the results

solution in the tube is clear = no hemolysis

sediment of Er at the bottom of the tube (settle down because they are

heavier than water)

Result 1. Read the minimum osmotic resistance

Start to read the results from the tube with highest NaCl concentration

= Find the first tube with pinkish content and sediment of erythrocytes

Minimum osmotic resistance

= NaCl concentration in which red blood cells begin to hemolyze

the colour of the solution turns pink (indication of hemolysis)

usually in concentration: 0,44-0,4 g .l-1 NaCl

some Er remain unhemolyzed – settle down at the bottom of the tube

Result 2. Read the maximum osmotic resistnace

= find the 1st tube with dark pink content without sediment

Maximum osmotic resistance

NaCl concentration in which all red blood cells hemolyze

the first tube with no sediment at the bottom

(i.e. all erythrocytes are hemolyzed)

normal value 0,34-0,30 g.l-1 NaCl

SPECIFIC GRAVITY (DENSITY)

- in a healthy human – a constant value

blood 1,052 – 1,063 g.cm-3 (ml)

blood elements 1,090 g.cm-3 (ml)

plasma 1,026 – 1,031 g.cm-3 (ml)

depends mainly on:

erythrocyte count

plasma protein concentration

Determination of the blood densityIntroduction

1 ml

- comparative method (indirect Philips – Van Slyke method)

- if a drop of blood is put into a tube with solution (copper sulphate CuSO4)

- of the same specific gravity – it remains in the middle of the fluid

- with higher specific gravity – it remains on the top

- with lower specific gravity – the drop falls down to the bottom of the tube

Procedure

- add a drop of blood into each tube in a set of tubes containing solutions

(CuSO4) with increasing density (1,042 – 1,064 g.cm-3)

- determine the specific gravity of blood (=result)

- conclusions: compare the result with normal values – is the obtained value

normal? If not – try to explain.

Determination of the blood density

Procedure

Haemostasis – bleeding arrest

- a complex process which makes a bleeding to stop

• maintenance of normal blood volume – vital

• massive bleeding may lead to cardiovascular collapse and death

- haemostasis includes 3 simultaneous interrelated processes

1/ reaction of the injured vessel

2/ activity of the platelets (platelet plug formation)

3/ blood clotting (haemocoagulation)

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1. Vascular constriction

Effects

- a decrease of the vessel diameter

- diminished blood flow through the ruptured vessel

- a decrease of the blood loss

- contraction of the smooth

muscle in the vessel wall

(circular muscle)

http://images.radiopaedia.org/images/21153/3c503a80bfd516413a65ac8d3aae8d_gallery.jpg

- includes several steps:

A/ Adhesion of platelets

(normal smooth endothelial lining of vessels – repels platelets)

- vessel trauma – damage to endothelium - subendothelial tissue (collagen) has

receptors for platelets – the platelets stick to the vessel wall (to collagen)

2. Formation of the platelet plug

B/ Change of the shape and releasing reaction

▪ thrombocytes swell (become spheric) and form pseudopods

▪ releasing reaction - platelets release substances active in

hemostasis into the blood

C/ Aggregation of platelets

- several layers of platelets stick together

Result: platelet activity results in formation of the platelet plug

- it is sufficient for temporary blocking the blood loss, especially in small vessels

- it does not contain fibrin threads, therefore loose, fragile

- series of enzyme reactions following in definite and rapid sequence

- the reactions involve activation of clotting factors (12 substances)

Result of haemocoagulation:

- formation of fibrin threads - strengthen and stabilize the platelet plug

- net of fibrin threads + platelet plug + trapped blood elements = blood clot

- blood clot - seals the broken vessel until the tissue is repaired

3. Haemocoagulation (blood clotting)

Blood clotting factors

I. fibrinogen

II. prothrombin

III. tissue thromboplastin

IV. Ca2+ ions

V. proaccelerin

VII. proconvertin

VIII. antihemophilic factor

VIII C - antihemophilic globulin

VIII A – von Willebrand factor

IX. Plasma thrombopastin component - Christmas factor

X. Stuart – Prower factor

XI. PTA – Plasma thromboplastin antecedent

XII. Hageman factor

XIII. fibrin stabilising factor

- present in blood

- inactive forms of proteolytic

enzymes (majority)

- blood clotting = a cascade of

chemical reactions leading to

conversion to active forms

..............

- synthesized in liver

- vitamin K – required for

synthesis of factor II, VII, IX, X

F1 F1A

F2 F2A

•HK - High molecular weight kininogen

•PK - Prekallikrein

1. damage of endothelium and

exposure of collagen in the

vessel wall

activates a stepwside (cascade)

activation of clotting factors

referred to as

intrinsic pathway of blood clotting

2. damage of the perivascular

tissue and release of tissue

thromboplastin

activates a stepwside (cascade)

activation of clotting factors

referred to as

extrinsic pathway of blood clotting

Blood clotting - activation

following reactions of both intrinsic

and extrinsic pathway are the same

and they are referred to as

=common pathway

Result of haemocoagulation:

formation of fibrin thread

INTRINSIC PATHWAY- activated bydamage of endothelial layer

- f. XII

COMMON

PATHWAY

EXTRINSIC PATHWAY

- activated bydamage of vessel wall and extravascular tissue- f. III

III

(III)

INTRINSIC PATHWAY- activated bydamage of endothelial layer

- f. XII

EXTRINSIC PATHWAY

- activated bydamage of vesel wall and extravascular tissue- f. III

III

(III)

EXTRINSIC PATHWAY

activated bydamage of vesel wall and extravascular tissue

III

(III)

1. fibrin monomer

2. fibrin polymer

3. cross-linked fibrin polymer

4. stabilization of the cross

linked fibrin polymer

INTRINSIC PATHWAY- activated bydamage of endothelial layer

- f. XII is activated

EXTRINSIC PATHWAY

- activated bydamage of vesel wall and extravascular tissue- f. III

III

(III)

1. fibrin monomer

2. fibrin polymer

3. cross-linked fibrin polymer

4. stabilization of the cross

linked fibrin polymer

In bleeding mostly both pathways are activated

Extrinsic pathway - fast (seconds), is first activated

= initiation phase the hemocoagulation

- causes formation of minute amounts of thrombin

- amplification phase

- thrombin activates factor V, VIII, IX

- propagation phase reactions of intrinsic pathway take

place

- Intrinsic pathway – delayed activation/proceeds slower

New fundamentals in haemocoagulation

Blood clot

▪ network of fibrin threads running in all directions -

adhere to damaged surfaces of vessels

▪ contains trapped blood elements

Retraction of the clot

- occurs 20 - 60 minutes after formation of the clot

- caused by the contraction of thrombocytes

(contain contractile fibres – actin, myosin)

- liquid (serum) is extruded from the clot

- wound surfaces are drawn together, tissue repair is

promoted

clot

clot

serum

IN VIVO – Intravascular anticoagulants

- endothelial factors- that prevent blood coagulation and platelet plug formation

- endothelial layer – non-wettable surface

- glycocalyx - layer of mucopolysaccharides on the surface of endothelium

- repels clotting factors and thrombocytes

- thrombomodulin – protein bound to endothelial cells, binds thrombin

- blood flow – homogenously dispels clotting factors, prevents their local

concentration

- anticoagulant substances

- antithrombin III – binds thrombin, inactivates clotting factors

- heparin – produced in basophils and mast cells

- a weak anti-clotting agent

- its anticoagulant effect increases in complex with antithrombin III

(100 – 1000 x) – inactivates thrombin and f. XII, XI, IX, X

- heparin and its derivatives - used for anticoagulant therapy

- fibrin – adsorbs thrombin, prevents further conversion of fibrinogen to fibrin

Anticoagulants – substances that prevent blood clotting

Anticoagulants for clinical use

▪ tubes from special „non-wettable“ materials (silicone)

▪ non-wettable surface = a surface that does not start the blood clotting

▪ decalcification – binding of Ca2+ ions - oxalate, citrate

▪ defibrination – removal of fibrin (e.g. snake toxins)

▪ coumarin derivates (e.g. warfarin)

- block the effect of vitamin K in liver (long-term effect)

- production of vitamin K dependent factors is affected – lack of Ca2+

receptor in their molecule

- are used also as medicaments in anticoagulation therapy

- effective within 12 hours

▪ heparin

Determination of bleeding time by the Duke method

• Test of platelet function and/or vascular function

Procedure

- disinfect the tip of the 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

Report

A. Write down the time

B. Is the time within the interval of normal values?

0 30´´ 60´´ 90´´ 120´´ 150´´

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)

• in clinical settings Activated Partial Thromboplastin Time (APTT) is

used to test the intrinsic pathway

Procedure

- collect venous blood – 3 ml

- place the last 1 ml of blood into a tube

- by using the last 1 ml the tissue factor III is eliminated (in this way it

remains in the bottom of the tube – i.e. the intrinsic pathway will not

get activated)

- turn on the stop watch

- check every 30 - 60 sec if the blood has coaglulated or not

- stop the watch when the blood is clotted

- normal value 4 -10 minutes (in 37° C), in a room temperature

up to 15 minutes)

Report

A. Write down the time.

B. Is the value normal? Compare measured results with standards

tissue

factor

Determination of prothrombin time by Quick

• test of the extrinsic pathway of blood clotting

• tissue thromboplastin (togehter with Ca ions) is added to blood serum

• tissue thromboplastin activates the extrinsic pathway of

haemocoagulation

• in clinical settings the PT (prothrombin time) test or international

normalized ratio (INR) is used to test the extrinsic pathway

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

• put into a clean tube

– 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 11 – 16 sec

Report:

A. Describe your observation of fibrin clot formation.

B. Write down the result

C. Compare your result to standard values - evaluate if the time is normal

Questions to study

• Haemostasis – 3 main processes and their end-results

• Haemocoagulation – extrinsic and intrinsic pathway, common pathway

• Events that activate the intrinsic/extrinsic pathway, sequence of activation of

the factors

• Haemocoagulation factors and their general description and function, the

role of vitamin K in haemocoagulation

• Tests used for assessment of intrinsic and extrinsic pathways

• Platelets – normal count, activity in haemostasis, event that activates the

platelets

• Haemopoiesis – definition, site, characteristics, stem cells, lineages

• Main nutritional factors of haemopoiesis.

• Stimuli for erythropoiesis (erythropoietin, stimuli for its synthesis and

secretion)

• Plasma components and their functions.

• Plasma osmotic and oncotic pressure and their clinical implications.

• Hypoalbuminemia, decreased oncotic pressure and edemas.