Blood PhysiologyPractical 2

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

katarina.babinska@fmed.uniba.sk

Contents

• Erythrocytes – sedimentation rate, hemolysis

• Blood plasma, osmotic, oncotic pressure

• Blood groups

Practical tasks

• Hemolysis

• Determination of erythrocyte osmotic resistance

• Measurement of erythrocyte sedimentation rate

• Determination of blood groups of the ABO system

• Determination of the Rhesus system (Rh factor)

• The cross matching test

Haemolysis

- destruction of erythrocyte membrane, haemoglobin is released from erythrocyte

- can be observed in a tube: opaque pink blood suspension transparent pink

solution

osmotic

- hypertonic solution

- hypotonic solution

chemical

- acids, bases, tensides

physical

- mechanic or thermic energy, irradiation

immunologic

- transfusion of incompatible blood

toxic

- cell lysis caused by enzymes in poison of snakes, wasps, spiders, plants

- daily approx 1% of Ery do haemolyze – old elements

- hemolytic anaemia – decreased Hb concentration due to excessive haemolysis

HaemolysisIntroduction

http://www.heftpathology.com/cache/com_zoo/images/learn_more1_879bccc4a5cf3c9e06cc1d987b32281f.jpg

https://upload.wikimedia.org/wikipedia/commons/thumb/7/7f/Hemolysis.jpg/220px-Hemolysis.jpg

- ether and saponine are substances that cause chemical hemolysis

• put physiological solution into 2 tubes (approx ½ of a tube)

• add a few drops (5) of venous blood (with citrate) into both tubes

• add 1 ml ether or 1 small spoon of saponine to 1 tube

• mix well (cca 1 min)

• describe the changes in appearance of the tube content

HaemolysisProcedure

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 Ery

B/ Hypertonic solutions

– higher osmotic pressure than plasma or intracellular fluid

– cells („isotonic solution“) in hypertonic solution lose water,

shrink and may malfunction or die due to rupture of the cell

membrane (erythrocytes haemolyse)

C/ Hypotonic solutions

– lower osmotic pressure

– water flow is directed into the cell (erythrocyte)

– expansion of cell volume, 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

- 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 blood cells hemolyse and less

survive

• use the prepared 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 in a test-tube rack

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)

− start to read the results from the tube with highest NaCl concentration !!!

Result 1. Read the minimum osmotic resistance

- 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

Measurement of the sedimentation rate

of erythrocytes

(FW - Fahraeus – Westergren method)

Blood examination: Erythrocyte sedimentation rate

Blood is a suspension (type of solution containing solid particles)

- plasma (water, dissolved substances)

- blood elements - solid particles that are heavier than plasma

If blood sample is put into a tube

(with anticoagulant to prevent clotting)

- erythrocytes sink to the bottom (because they are heavier)

- leave behind transparent upper layer of plasma

= Er sedimentation

Determination of sedimentation rate

- in tubes (e.g. Westergren tubes, Sedivettes)

- size of the plasma layer in the sample is measured

- in 1 hour

- in 2 hours

1st hour

males 2 – 5 mm (up to 15 mm)

females 3 – 8 mm (up to 20 mm)

2nd hour

two times the value in 1st hour or

less (but not more !)

start 1st hour 2nd

hourNormal values (normal FW)

https://www.sarstedt.com/fileadmin/produkte/bilder/_processed_/csm_90.1090_2402_a3f8824e35.png

Example of a normal value: FW = 7/14 mm

Abnormalities of sedimentation rate

(higher sedimentation rate, lower sedimentation rate)

Causes

• if the blood composition is abnormally changed (less red blood cells, too much

proteins, ....) the sedimentation rate may become abnormal

• the most common cause of high sedimentation rate

is inflammation – due to increased concentration of

inflammatory proteins

• ESR can be slightly higher during periods or pregnancy

Why do the females have higher sedimantation rate ?

- lower erythrocyte count

- higher concentration of fibrinogen (plasma protein)

erythrocytes - charged

plasma proteins + charged

• the sedimantation rate – a non-specific marker of inflammation

start 1 h 2 h

- a 2.8 ml volume of blood is drawn into the S-Sedivette® pre-

dosed with 0.7 ml citrate (pull the piston to the very end, the

respective volume of blood will be sucked to the tube)

- remove the needle from the tube, discard

- mix genly (the tube contains an anticoagulant)

- break the puller of the piston

- place the S-Sedivette® into the sedimentation rack, use the

thumb screw to adjust the sample level to zero

- leave standing

Determination of the sedimentation rate

Procedure

Result

- read the size of clear plasma column (in mm)

a/ in 1 h, b/ in 2 h

FW = 1st hour value/2nd hour value (e.g. FW=5/10)

Conclusion

- evaluate the result: is the value normal, or not,

- If not normal what may be the cause?

Determination of blood groups of the ABO system

Blood groups

Blood type must be considered in:

- transfusions - transplantations- gynecology and obstetrics

Major clinical importance (out of all existing blood systems):

1. ABO system2. Rh system

- in case of ABO / Rh mismatching transfusion – high risk of - serious health consequences - death

GENERAL RULE: USE MATCHING BLOOD (POSSIBLY THE SAME BLOOD TYPE)

– are determined by

• antigens (agglutinogens) A and/or B in the membrane of erythrocytes

• antibodies (agglutinins) anti -A or anti - B in the plasma

Antigen

• a chemical substance in the cell membrane

• determines individual identity (different people – different antigens)

• if a foreign antigen enters a body (e.g. mismatching blood)

– it is able to trigger production of antibodies

– it is able to react with antibodies (e.g. anti A + A; anti B + B)

– reaction with antibodies starts the immune response -the

foreign cell „marked“ by an antibody is destroyed

• (weak antigens – show only weak or no immune response)

Blood groups in ABO system

X

Er

Er

ABO – blood groups

Erythrocytes

Agglutinogen

Antigen

PlasmaAgglutininsantibodies

Blood group

A (48%)

B (9%)

AB (4%)

0 (39%)

A

B

A,B

H

anti B

anti A

anti A, B

not present

substance H is not an antigen

Principle

blood groups are assessed on the basis of reaction between blood and a known diagnostic serum containing antibodies

- anti A serum – contains anti-A antibodies against agglutinogen A

- anti B serum – contains anti B antibodies against agglutinogen B

Determination of a blood group

• if the antibodies in diagnostic serum

„find“ antigen, they react with it and cause

blood agglutination

• agglutination = proof that the respective

agglutinogen is present in membrane of

Ery

• no agglutination – the respective

agglutinogen is not present in

membrane of Ery

anti A + - + -

anti B - + + -

blood

groupA B AB 0

Procedure

• take a testcard

• place a drop of Anti-A serum into the preprinted area

on the testcard

• place a drop of Anti-B serum into the preprinted area

on the testcard

• puncture a fingertip, wipe the first drop of blood

• place 2 drops of blood into a pre printed places on the testcards

• take a stick

– use one end to stir a one blood drop with anti A serum

– use the other end to stir the second blood drop and anti B serum

• Result: observe agglutination (b)

• Conclusion: determine the blood group

+

recipient (patient) donor

A / anti B B / anti A

recipient (patient) donor

A / antiB A / anti B

=

=

• incompatible blood (mismatched)

• compatible (matching) blood

Blood groups and transfusion

X X

+

Transfusion

of full blood

A

B

AB

0

A B AB 0

+

+

+

+

-

-

-

-

- -

--

Donor

Recipient

--

-

-

0 – universal donor?

0 +- - -

A B AB 0

+ +

Full blood

Erythrocytes

0 + +

Donor

Recipient

Transfusion of

erythrocytes

A

B

AB

0

A B AB 0

+

+

+

+

-

-

-

--

Donor

Recipient

-

-

+

+

+++

Reaction after transfusion of mismatching blood

• the agglutinins are attached to agglutinogens in Er membranes

• this reaction is called agglutination

• In agglutination aggregates of Er are formed and are visible in the blood sample

What is really dangerous about mismatching transfusion?

• possible consequences - more or less serious:

– immune reaction and circulatory shock (breathlessness, pain in chest, nausea,

sweating...), death

– haemolysis, icterus, kidney failure, death

• symptoms usually occur soon after the transfusion has started – in this case

immediately STOP the transfusion

donor recipient

A / anti B B / anti A

=+ X X

agglutination

Determination of Rhesus system (Rh factor )

Rh system

Determined by 3 antigens in the membrane of Er:

(genetically determined) C or c

D or d

E or e

• Rh positivity (Rh+) – 85% of population

– determined by the presence of antigen D

in the erythrocyte membrane

– CDE, CDe, cDe, cDE

• Rh negativity (Rh-) – 15% of population

– d antigen present: CdE, Cde, cde, cdERh-

Rh+

C

c

D

d

e

e

Antibodies in Rh system - normally not present

However!!!

- D is a strong antigen (all the remaining are weak antigens)

- if Rh+

Er enter blood of a Rh-person, D is recognized as a „foreign“ antigen and

production of antibodies is started

Principle:

Rh-Factor is assessed on the basis of reaction

between known diagnostic serum

containing antibodies anti-D and blood

Procedure:

• on a glass slide put

– a drop of anti-D serum

– a drop of capillary blood

• mix together with a glass stick

• Result: observe agglutination

(the agglutination may be slow, sometimes

it is necesary to wait for 5 minutes)

• Conclusion: determine the Rh-factor

Rh-

Rh+

C

c

D

d

e

e

anti-D

Rh factor and transfusion

Rh negat donor Rh negat patient• the same blood group - matching

Rh posit donor Rh posit patient• the same blood group - matching

Rh negat donor Rh posit patient• matching - „d“ does not trigger antibody

production

donor recipient

Rh posit donor Rh negat recipient

- production od antibodies can be triggered

if Rh+ erythrocytes are given to a Rh-

individual (e.g. transfusion of Rh

incompatible blood)

A/ 1 st transfusion – no posttransfusion

reaction - no antibodies present in

blood of recipient

B/ Rh+ erythrocytes act as antigen and

stimulate production of antibodies

against antigen D (within weeks) – the

individual becomes sensitized (i.e.

antibodies are present in his blood)

C/ 2 nd transfusion of incompatible Rh+

blood – antibodies react with antigen

D, posttransfusion reaction occurs

(„d“ does not induce production of

antibodies)x

donor: Rh+ recipient: Rh-

Rh+

father + Rh-mother

A/ Rh-fetus (no problem) or

B/ Rh+

fetus (may be a risk)

1st pregnancy

- blood of the mother and the fetus are separated

by placenta that is a barrier for Er

- usually no problems with Rh incompatibilty

- in case of complicated birth, accident, etc.

the Rh+ erythrocytes of the fetus may enter the

blood of the Rh- mother

- antibody production against baby´s Er is

induced in the mother (even as little as 0,5 ml

of blood may start the Ab production)

- antibodies remain in blood of a Rh-mother

Incompatibility of the blood systems of the mother and the fetus

before pregnancy

after birth

complications at birth

xNext pregnancies – production of antibodies is even

more higher (problems in about 3% of 2nd and 10% of 3rd pregnancies)

Treatment and prevention

-anti-D serum latest until 72 hours after termination of the

pregnancy (birth, abortion) is given to the mother

-antibodies anti-D from the serum are attached to the Er of

baby (in mother´s blood)

-the Er marked by anti-D are destroyed, thus antibody

production by the mother´s body is prevented

anti D serum

mother

fetus

2nd pregnancy – Rh-baby

2nd pregnancy

- problems occur if the 2nd baby is also Rh+

- antibodies from mother´s blood enter blood of the

fetus through the placenta, attach to baby´s Er

- agglutination and hemolysis of Er of the fetus

Consequences

- hemolytic disease of the newborn: anaemia,

hypoxia, icterus-risk of brain damage, death in utero placenta

The crossmatching test (simplified version)

Other blood systems

- About 30 blood systems exist

- Clinically significant:

Kell (K, k) MNSs Kidd

Lewis (Lewisa, Lewisb) Diego Lutheran, etc.

• may cause incompatibility of donor´s and recipient´s blood despite compatibility in

ABO and Rh system

• may cause mother/fetus incompatibility

• may cause posttransfusion reaction in individuals who often receive transfusion

Crossmatching test

- assessment of compatibility between blood of donor and recipient

- blood of both donor and recipient is centrifuged, serum is separated fromerythrocytes

- test is done in 2 steps:

1. major crossmatching test:

serum of recipient is mixed with erythrocytes of donor

2. minor crossmatching test:

serum of donor and erythrocytes of recipient

Result:

- no agglutination = blood compatible

- agglutination = mismatching blood

Er Donor Er Recipient

Serum RecipientSerum Donor

Biological test- performed at the beginning of a transfusion

- give 20 ml of blood, then wait about 2-3 minutes

- repeat 2 more times

- check for symptoms of transfusion reaction

- dyspnea, tachycardia, sweating, low blood pressure, dizziness, stc.

Principle

- blood of a donor and a recipient is mixed and the reaction is observed

Procedure (simplified crossmatching)

- make 2 blood collections from 2 different persons (sample 1, sample 2)

- centrifuge the blood

- separate the plasma from the red blood cells

- take 2 glass slides

- with a pipette put on the glass slides (don´t forget to change the tips)

- Slide 1: Ery from sample 1 and serum from sample 2

- Slide 2: Ery from sample 2 and serum from sample 1

- mix together and read the result after 5 minutes

- if required, investigate microscopically

Result: agglutination – yes/no

Conclusion: blood matching/mismatching

Ery sample 1 Ery sample 2

Serum sample 2Serum sample 1

Topics to study

- Hemolysis – definition, causes

- Blood plasma, osmotic and oncotic pressure in physiological processes

- Isotonic solutions for infusion

- Erythrocyte sedimentation rate– normal values, main abnormalities

- Blood groups, system ABO – agglutinogens, agglutinins

- Blood groups, Rh system - antigens

- Minor blood groups and their clinical implications

- Blood groups and transfusion - matching and mismatching blood

- Cross matching test, major and minor crossmatch

- Mother-foetus incompatibility

- Procedures to the tasks