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Blood PhysiologyPractical 2
© Katarína Babinská, MD, PhD. MSc., 2018
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