t
males and 4-5 Liters in adult females.
-Its colour is bright scarlet in arterial blood and to a dull brick red in
venous blood.
Composition of blood:
a) Cellular elements: 45% and include RBCs, WBCs and platelets
b) Plasma : 55 %
- It is formed of water (90%) in which more than 100 different
substances are dissolved.
- Dissolved substances include nutrients, gases, hormones, various
wastes and metabolites, many types of proteins, and electrolytes.
Chapter I: Blood Practical
3
a- ABO system
Blood experiments:
1- Determination of blood groups.
2- Determination of haemoglobin content.
3-Determination of haematocrite value.
4-Analysis of complete blood picture.
5-Determination of erythrocyte sedimentation rate.
6-Haemostatic function tests.
-The blood of human is classified into many groups according to certain
antigens present on the surface of RBCs.
-There are hundreds of different antigens have been found on the surfaces
of human RBCs.
-Most of these antigens are weak and are mainly of importance in genetic
studies.
-They include ABO system, Rh system, MNS system, Lewis system and
Kell system
-This system includes 2 related antigens (or agglutinogens) A and B.
-According to the presence or absence of these 2 antigens, the human
blood is normally classified into 4 major groups:
Group A Group B Group AB Group O
% 41 % 9% 3% 47%
Agglutinogen
(antigen)
A B A&B ----
Agglutinin
(antibody)
B A ----- A&B
Genotype AA or AO BB or BO AB OO
Blood groups according to ABO system
1-Determination of blood groups
Chapter I: Blood Practical
4
Determination of ABO system:
1. Mix one drop of blood with 1ml of isotonic saline in test tube.
- Put two separate drops of the diluted blood on a glass slide.
- Add one drop of anti-A serum (blue coloured) to one blood drop and
one drop of anti B serum (yellow coloured) to the other blood drop.
- Mix the blood with anti-A and anti-B sera gently using the blunt end
of two separate pins.
- After 2 min, examine for agglutination:
a. if agglutination occurs with anti-A serum only, the blood
group is type A.
b. If agglutination occurs with anti–B serum only, the
blood group is type B.
c.If agglutination occurs with both anti-A and anti –B sera
the blood group is type AB.
d. If no agglutination with either anti – A or anti- B sera,
the blood group is type O.
Chapter I: Blood Practical
5
b-Rh system (Rh factor)
Anti-A Anti-B Anti-D
A +ve
B +ve
AB +ve
O -ve
Typing of ABO blood grouping in humans
Importance of ABO system
- Blood transfusion:
- blood grouping and cross matching test should be done to choose
the compatible groups.
- Disputed paternity:
- It is a good -ve test that excludes paternity rather than prove it.
- Medicolegal use: to prove or disprove the claim of the victim.
- Susceptibilty to various diseases:
a- O group people are susceptible to peptic ulcer.
b- AB group people are susceptible to diabetes mellitus.
-There are six common types of Rh antigens named C, D, E, c, d, and e.
-The type D-antigen is considerably the most antigenic than the others,
so;
a. Presence of D-antigen→ Rh +ve.
b. Absence of D-antigen→ Rh -ve.
Chapter I: Blood Practical
6
Rh +ve Rh –ve
White races 85% 15%
Egyptians 90% - 95% 5%- 10%
Importance of Rh factor:
In blood transfusion:
-When Rh -ve person , receives Rh +ve blood , he will develop
agglutinins against Rh-factor.
-If this person receives Rh+ ve blood again agglutination &
haemolysis occur.
Determination of Rh group:
-Put 2 drops of diluted blood on a glass slide.
-Add one drop of anti-D serum to the blood and mix gently with
the blunt end of a pin.
-Examine for agglutination after 3min.
-If agglutination occurs, the blood group is Rh +ve.
-If not, the blood group is Rh -ve.
Cross matching (compatibility التوافق ( test:
- this is a direct test of compatibility of donor cell and recipient serum.
Steps:
- obtain 1 ml blood from the recipient 's vein and allow it to
clot, when clot retracts the serum can be pipetted off.
- prick the finger of the donor , one drop of blood should be placed
in 1 ml saline and mixed.
3- place one drop of the donor 's red cells on a glass slide and over
it place one drop of recipients serum.
4- wait 10 minutes and observe agglutination.
Chapter I: Blood Practical
7
Indications of blood transfusion:- To restore the whole
blood as in haemorrhage.
2- To restore one element of the blood when dificent e .g
- RBCs in severe anaemia,
- WBCs in severe Leukopenia,
- Platelets in severe thrombocytopenia,
- Clotting factors in severe haemophilia.
Precautions before blood transfusion:
1- The transfused blood should be compatible with recipients blood
as regards ABO system & Rh system.
2- HB content of transfused shouldn't be less than 90 %.
3- The transfused blood should be free of infections e.g AIDS,
viral hepatitis , & malaria.
4- The transfused blood should be fresh not frozen (stored at 4
oC
& less than 21 days).
5- Cross matching test should be done before transfusion.
Relation of blood group to blood transfusion:
i-AB blood is considered universal recipient as it has no antibodies in
its plasma.
ii- O blood is considered universal donor as it has no antigens on its
RBCs.
The possible transfusions between various groups
are:
Group Gives Takes from
AB AB All groups
O All groups O
A A & AB A & O
B B & AB B & O
Blood transfusion
Chapter I: Blood Practical
8
Effects of incompatible blood transfusion:
- If the blood is transfused from a person with certain blood group to
a person with unsuitable blood group, transfusion reactions occur.
-The cause is that the donor „s RBCs are agglutinated by recipient „s
antibodies → haemolysis of the transfused RBCs.
Effects:
1- Blockage of blood capillaries by the clumped RBCs → several
pains e.g back pain & joint pain.
2- Hazards of intravascular haemolysis.
a- Circulatory shock:
-The released histamine → arterial blood pressure.
b- Hyperkalaemia:due to K+
released from haemolysed RBCs.
c- Jaundice.
e- Acute renal failure.
Dangers of blood transfusion:
1- Incompatible blood transfusion ( see before).
2- Allergic reactions.
3- Transmission of diseases e.g AIDS, malaria & viral hepatitis.
Chapter I: Blood Practical
9
-Haemoglobin (Hb) is a protein having a M.W of 66.000. It represents
34% of RBC volume. It consists of four heme groups combined with one
molecule of globin.
Normal value:
Adult male 13.5 - 16 gm % (average 15gm %)
Adult female 11.5 - 16 gm % (average 14gm %)
Newly born infant 18-20 gm %
Children 11 gm %
Sahli’s Method for Hb determination:
Principle:
-This method depend upon converting Hb into acid hematin (has a dark
brown color) by adding dilute HCL.
-The intensity of this color depends on the concentration of acid hematin
which in turn, depends on the concentration of Hb.
-The color of the solution after dilution with water is matched with the
colour of standard tubes.
-The readings are obtained in gm%.
Reagents and equipments:
-N/10 solution of hydrochloric acid.
-Distilled water.
Sahli apparatus (haemoglobinometer): consists of:
a- Central graduated tube which has 2 graduations, one indicating the
amount of Hb in gm/100 cc, while the other indicating the Hb%.
b- 2 standard coloured tubes on its side.
2-Determination of Haemoglobin Content
Chapter I: Blood Practical
10
- Special pipette (= 0.02ml or 20 mm3
) capacity
- A glass rod and a droper.
Special pipette
Sahli Apparatus
Procedure:
- Place N/10 HCl in the central tube up to mark 20 on the
percentage graduation.
- Draw a blood by the pipette up to 0.02 mark.
- Wipe the tip of the pipette.
- Push its contents of blood quickly into the central tube, then mix
the blood with HCl several times.
- Now Hb of the blood is converted to acid haematin (dark brown
colour).
- After 15 minutes add distilled water, drop by drop and mix well
with the glass rod.
- Continue this process until the colour in the central tube will be the
same as in the standard tubes.
- Record your results.
Graduated tube
Standard tubes
Chapter I: Blood Practical
11
Advantages of Sahli Method:
-The method is simple & quick.
- It does not require any costly apparatus.
Significance of Hb determination:
- Diagnosis of anaemia (if Hb is less than its normal value for the same
sex and age) , and polycythaemia (if Hb is more than its normal value)
- Calculations of some blood used for diagnosis of the type of
anaemia.
Questions
- How to diagnose anemia?
- Mention the importance of Hb determination
………………………………………………………………………
………………………………………………………………………
………………………………………………………………………
………………………………………………………………………
………………………………………………………………………
……………………………………………………………………….
………………………………………………………………………
………………………………………………………………………
………………………………………………………………………
Chapter I: Blood Practical
12
- it’s the percent ratio of RBC’s volume to the total blood volume.
Normal values:
Haematocrit e = RBC’s volume
Total blood volume
× 100
In adult male 46 %
In adult female 42 %
In newly born 60 %
Methods of determination of haematocrite:
-A blood sample from the patient is placed in a special tube
(wintrope tube) which contain anticoagulant.
-The sample is then centrifuged.
- It’s noticed that
the RBCs are packed at the bottom of the
tube, while clear plasma remains above.
WBCs platelets form a small layer just above RBCs column.
- the haematocrite is then calculated by dividing RBCs
column by the total blood column. Then multiply x l00
Factors affecting haematocrite value:
-It's affected by changes in plasma & RBCs in that:
Increased in Decreased in
- Polycythemia.
-Dehydration.
- Anaemia.
- Overhydration.
Uses of haematocrite value:
1- Calculation of blood indices.
2- Diagnosis of anemia & polycythemia
3- Haematocrite value = Packed cell
volume, PCV
Chapter I: Blood Practical
13
-Complete blood count (CBC) is one of the most commonly ordered
blood tests.
-The complete blood count is the calculation of the cellular elements of
blood.
-These calculations are generally determined by special machines that
analyze the different components of blood in less than a minute.
-A major portion of the complete blood count is the measure of the
concentration of white blood cells, red blood cells, and platelets in the
blood.
-CBC, lists a number of many important values. Typically, it includes the
following:
1-Red blood cell count (RBC or erythrocyte count)
2-Hemoglobin (Hbg)
3-Hematocrit (Hct)
4-Blood indices:
a-Mean corpuscular volume (MCV).
b-Mean corpuscular hemoglobin (MCH)
c-Mean corpuscular hemoglobin concentration (MCHC).
5-White blood cell count (WBC or leukocyte count)
6-WBC differential count
7-Platelet count
Analysis of complete blood count (CBC)
Chapter I: Blood Practical
14
1- RBCs count:
-The normal RBCs count differs according to age and sex:
In adult males 5 - 5.5 million / mm3
In adult females 4.5 - 5 million / mm3
In new born 6 - 8 million / mm3
In children 3.5 – 4.5 million / mm3
Average 5 millions
-Lower values than normal occur in anaemia and higher values than
normal occur in polycythemia.
2- Hemoglobin content:
-The normal HB content differs according to age and sex:
In adult male 13.5 - 18 average 16 gm %.
In adult female 11.5 - 16 average 14 gm %.
In new born may reach 18 gm %.
Average 15 gram %
-Lower values than normal occur in anaemia and higher values than
normal occur in polycythemia.
3- Hematocrit (Hct) value:
- it‟sthe percent ratio of RBC’s volume to the total blood volume.
In adult male 46 %
In adult female 42 %
In newly born 60 %
Average 45 %
-Lower values than normal occur in anaemia and higher values than
normal occur in polycythemia.
Chapter I: Blood Practical
15
4-Blood indices:
-they are a group of values designed to assess the type of anaemai:
a-Mean corpuscular volume (MCV):
Def: is the average volume of a single RBC in micron (µ
3
)
Equation MCV = haematocrite value x 10
RBCs count in millions/mm3
Normal values 45 x 10 / 5 = 90 µ
3 + 7 i.e 83 to 97 µ
3
Abnormalities -lower values than 83 µ
3 occur in microcytic anaemia as iron
deficiency anaemia.
-higher values than 97 µ
3 occur in macrocytic anaemia as
megaloblastic anaemia.
b- Mean corpuscular HB (MCH):
Def: is the average amount of HB a single RBC in pg.
Equation MCV = Hb content x 10
RBCs count in millions/mm3
Normal values 15 x 10 / 5 = 30 pg + 3 i.e 27 to 33 pg.
Abnormalities -lower values than 27 pg occur in microcytic anaemia as iron
deficiency anaemia.
-higher values than 33 pg occur in macrocytic anaemia as
megaloblastic anaemia.
N.B: anemia may be:
1-Microctytic anemia e.g iron deficiency anaemia.
2-Macrocytic anemia e.g megakoblastic anemia.
3-Normocytic anemia e,g aplastic anemia.
3- Mean corpuscualr haemoglobin concentration (MCHC):
Def: is the average HB concentration in a single RBC.
Equation MCHC= Hb content x 100
Haematocrite value
Normal values 15 / 45 x 100 = 33 % + 2 i.e 31 to 35 pg.
Abnormalities higher values than 35 % occur in hereditary spherocytosis
(a type of hereditary haemolytic anaemia).
Chapter I: Blood Practical
16
-normally from 150,000 to 400,000 /mm3
.
-higher values > 400,000 are called thrombocytosis.
-lower values < 150,000 are called thrombocytopenia.
5-White blood cell count (WBC or leukocyte count):
-Number: 4000 - 11,000 /mm3
6-WBC differential count:
Granular leukocytes
Neutrophil 60 - 70% of leukocytes.
-Higher values are called neutrophilia and occurs in bacterial
infection.
-Lower values are called neutropenia
Eosinophils 1 - 5% of leukocytes
-Higher values > 5% are called esinophilia and occur in allergy and
parasitic infection.
-Lower values are called esinopenia
Basophils 0 - 1 % of leukocytes
-Higher values are called basophilia
-Lower values are called basopenia
A Granular leukocytes
Monocytes 3-8% of leukocytes
-Higher values are called monocytosis
-Lower values are called monocytopenia.
Lymphocytes 20-30 % of leukocytes
-Higher values are called lymphocytosis
-Lower values are called lymphopenia.
7-Platelet count:
Chapter I: Blood Practical
17
Question 1:
A male patient 26 years old has the following CBC:
-HB =10 gm%
-Haematocrite value =35%
-RBCs count 3,5 million RBCS/mm3
Comment on :
- HB content:
………………………………………………………………………………………
………………………………………………………………………………………
- RBCs count:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCV:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCH:
………………………………………………………………………………………
………………………………………………………………………………………
- Haematocrite value:
………………………………………………………………………………………
………………………………………………………………………………………
- What is the type of anemia in this condition if present:
………………………………………………………………………………………
………………………………………………………………………………………
- What is the commonest cause of this type of anemia:
………………………………………………………………………………………
………………………………………………………………………………………
Chapter I: Blood Practical
18
Question 2:
A female patient 26 years old has the following CBC:
-HB =8 gm%
-Haematocrite value =30%
-RBCs count 3,7 million RBCS/mm3
Comment on :
- HB content:
………………………………………………………………………………………
………………………………………………………………………………………
- RBCs count:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCV:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCH:
………………………………………………………………………………………
………………………………………………………………………………………
- Haematocrite value:
………………………………………………………………………………………
………………………………………………………………………………………
- What is the type of anemia in this condition if present:
………………………………………………………………………………………
………………………………………………………………………………………
- What is the commonest cause of this type of anemia:
………………………………………………………………………………………
………………………………………………………………………………………
Chapter I: Blood Practical
19
Question 3:
A male patient 26 years old has the following CBC:
-HB =11 gm%
-Haematocrite value =30%
-RBCs count 3,4 million RBCS/mm3
Comment on :
- HB content:
………………………………………………………………………………………
………………………………………………………………………………………
- RBCs count:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCV:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCH:
………………………………………………………………………………………
………………………………………………………………………………………
- Haematocrite value:
………………………………………………………………………………………
………………………………………………………………………………………
- What is the type of anemia in this condition if present:
………………………………………………………………………………………
………………………………………………………………………………………
- What is the commonest cause of this type of anemia:
………………………………………………………………………………………
………………………………………………………………………………………
Chapter I: Blood Practical
20
Question 4:
A male patient 26 years old has the following CBC:
-HB =11 gm%
-Haematocrite value =32%
-RBCs count 3,7 million RBCS/mm3
Comment on :
- HB content:
………………………………………………………………………………………
………………………………………………………………………………………
- RBCs count:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCV:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCH:
………………………………………………………………………………………
………………………………………………………………………………………
Question 5:
A male patient 26 years old has the following CBC:
-HB =20 gm%
-Haematocrite value =60 %
-RBCs count 7 million RBCS/mm3
Comment on :
- HB content:
………………………………………………………………………………………
………………………………………………………………………………………
Chapter I: Blood Practical
21
- RBCs count:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCV:
………………………………………………………………………………………
………………………………………………………………………………………
- Calculate MCH:
………………………………………………………………………………………
………………………………………………………………………………………
Chapter I: Blood Practical
22
3-Erythrocyte sedimentation Rate (ESR)
.
-It is the rate of the downward descent of RBCs in a vertical column of
blood.
Materials:
Westergren Method:
1-Westergren sedimentation tubes: straight glass tubes, 30 cm in
length and 2.5 mm in diameter, and graduated from 0-200 mm.
-Special racks with adjustable leveling screws for holding that
sedimentation tubes firmly in an exactly vertical position.
3-3.8% sodium citrate to be used as anticoagulant.
4-5 ml sterile syringes and test tubes.
Procedure:
1-Withdraw 2.0 ml blood in a syringe containing 0.5 ml sodium
citrate solution.
2-The contents of the syringe are transferred into a test tube, shake
the tube to mix the blood with the anticoagulant.
3-Suck the citrated blood into the Westergren tube up to the O
mark.
4-Place the tube in its special stand in a vertical position an fix its
upper end with the clip.
5-The height of the clear plasma on the top of the tube is measured
after one and two hours.
Chapter I: Blood Practical
23
Westergren tube for erythrocyte sedimentation rate
Normal values:
Increased in First hour Second hour
Male 3-5 mm 6-10 mm
Female 6-10 mm 16-20 mm
Factors affecting ESR:
a) Physiological factors:
ESR is increased in ESR is decreased in
-Old age
-Females
-Pregnancy
-During menstruation
-Muscle exercise
-Newborn
-Males
-High altitudes
b) Pathological factors:
ESR is increased in ESR is decreased in
1-Acute inflammation such
as tonsillitis
2-Chronic infections such as
tuberculosis (TB).
3-Malignancy
4-Tissue trauma
5-Rheumatic fevers
6-Fevers
- Polycythemia
- High cholesterol contents
- Hyperviscosity of plasma
Chapter I: Blood Practical
24
Clinical significance of ESR:
ESR is not a specific and diagnostic test but it is prognostic test.
- It detect the presence and severity of disease.
- It gives an idea about the activity of disease.
- It is used to follow up of disease and effect of treatment.
Questions:
- What are the factors affecting the rate of sedimentation of RBCs?
- What are the physiological factors affecting ESR?
……………………………………………………………………………
……………………………………………………………………………
Chapter I: Blood Practical
25
I-Bleeding time
-Haemostasis refers to the process of stoppage of bleeding after blood
vessels are punctured, cut, or otherwise damaged.
Haemostasis involves the following 4 steps:
- Vasoconstriction (contraction of injured blood vessels).
- Platelets plug formation.
- Formation of a blood clot.
- Fibrinolysis (dissolution of the clot).
Tests for Hemostasis:
The commonly used tests include:
- Bleeding time (BT).
- Clotting time.
- Prothrombin time (PT).
- Prothrombin concentration.
- Activated partial thromboplastin time (APTT).
- International normalized ratio (INR).
Definition:
-It is the time needed for bleeding to stop from small puncture.
Significance:
-give idea about the degree of vasoconstriction (vascular function) and
functions of platelets.
4- Tests for Haemostasis
Chapter I: Blood Practical
26
II-Coagulation (clotting) time
Technique: Duke 's method:
Materials:
- Disposable sterile lancets
- Filter paper
- Stop watch
- %07 alcohol
- cotton
Procedure
- The skin of fingertip or earlobe is cleaned with 70% alcohol, and
then a puncture is made by using disposable lancet.
- Start the stopwatch and note the time needed to stop bleeding.
- Gently remove completely the blood coming from the wound at 30
seconds interval until bleeding stops.
- Every time use fresh area of the filter paper.
Significance
- Normally: about 2-6 minutes.
- It is prolonged in:
a) purpura (platelet deficiency, or vessel wall defects).
b) Von willbrand disease.
c) Use of anti-platelets as aspirin.
Bleeding time is usually normal in hemophilia.
Definition:
-It is the time needed for clot formation.
Slide method:
• Clean filter papers.
• Clean glass slide
Chapter I: Blood Practical
27
• Lancet
• Stopwatch.
Procedure:
- Clean a fingertip with 70% alcohol and dry it.
- Puncture it with a sterile disposable lancet fairly deep, so
that, blood flows freely.
- Put a drop of blood on the glass slide.
- After 1 min, try to dip the tip of lancet on blood drop every
30 seconds until a thread of fibrin appears between the tip
of lancet and drop of blood.
- The time interval between putting the blood into slide and the
appearance of fibrin threads is the coagulation time.
Significance:
- Gives idea about the efficiency and timing of blood clot formation.
- Normally: 5-8 minutes.
- It is prolonged in:
a) hemophilia.
b) deficiency of clotting factors e.g vit K deficiency & liver diseases
c) use of oral anticoagulants,
- Clotting time is usually normal in purpura.
Chapter I: Blood Practical
28
-Prothrombin time is used as a measure of the extrinsic pathway of
coagulation (factors I, II, V, VII, and X).
Procedure:
- Blood is collected and immediately Na citrate, so that
none of prothrombin is converted into thrombin.
- Take 1.0 ml of citrated plasma in a sterile test tube and
keep it in a water bath at 37c.
- Add 1.0 ml of thrombokinase reagent (thromboplastin)
and then add 1.0 ml of 1% CaCl2.
- By using stopwatch, record the time passes between the
addition of CaCl2 and the appearance of a fine mesh of
fibrin.
- The time required for coagulation is the “prothrombin
time”.
Steps of prothrombin time
Significance:
- Normally; prothrombin time is 12-16 seconds.
- It is prolonged if there is a deficiency of one or more factors of the
extrinsic pathway of coagulation e.g. in liver diseases , oral
anticoagulants and vitamin K deficiency.
III- Prothrombin time
Chapter II: Body Temperature Practical
29
CHAPTER II:
BODY TEMPERATURE