Presentation for haematology

1. PREPARATION OF A GOOD
QUALITY BLOOD FILM
Presenters
Edwin Aliko
Hilda B Mwaipopo
Superviser; Irene Mshana

2. OBJECTIVES
• Introduction to blood film
• Purpose and importance of preparing blood film
• Types of blood film
• Preparation of blood film
• Precautions to observe when preparing blood
film
• Precautions to be observed for proper staining of
a blood film

3. INTRODUCTION
• A blood film is a thin layer of blood smeared on a
microscope slide and stained in such a way to
allow various blood cells to be examined
microscopically.
• There are three main blood cells that the test focuses
on:
I. Red cells, which carry oxygen throughout the body
II. White cells, which help your body fight against
infections and other inflammatory diseases
III. Platelets, which are important for blood clotting

4. Cont.…
• Irregularities in the number or shape of your
red blood cells can affect how oxygen travels
in your blood.
• These abnormalities are often caused by a
mineral or vitamin deficiency, but they can
also be caused by inherited medical
conditions, such as sickle cell anaemia.

5. Cont.…
• White blood cells are an integral part of your
body’s immune system, which is a network of
tissues and cells that help your body fight
infection.
• Having too many or too few white blood cells can
indicate a blood disorder.
• Disorders affecting these cells often result in the
body’s inability to eliminate or control infections
or other inflammatory problems.

6. Cont.…
• Platelet disorders affect your blood’s ability to
clot, which can lead to excessive or prolonged
bleeding or blood clotting. They often occur
when the body produces too many or too few
platelets.

7. AIM OF PREPARATION
• Blood films are usually examined to investigate
haematological problems(disorders of blood) and,
occasionally, to look for parasites within the blood such
as Malaria.
• Examination of thin blood film is important in the
investigation and management of anaemia,infections
and other haematological conditions which produce
changes in the appearance of blood cells.
• A blood film report can provide rapidly and at low cost,
useful information about patient’s conditions which
can help doctors diagnose certain blood disorders or
other medical conditions.

8. TYPES OF BLOOD FILM
• The peripheral blood film is of two types:
Thin blood film
Thick blood film
• Qn; What are the differences between thin
blood film and thick blood film?

9. THIN BLOOD FILM

10. PARTS OF A THIN BLOOD FILM
• A peripheral blood film consists of three main
parts:
i. Head ie; the portion of blood film near the
drop of blood
ii. Body ie; the main part of the blood film
iii. Tail ie; the tapering end of the blood film

11. FEATURES OF A GOOD BLOOD FILM
• It should be free from lines and holes.
• It should not have irregular tail.
• It should not cover the entire surface of the
slide ie; it should be 2/3 of the slide.

12. A well made PBS

13. Cont.…

14. Unacceptable PBS

15. Cont.…
• Thin peripheral blood film can be prepared
from anticoagulated blood obtained by
venipuncture blood by any of the following
three techniques:
i. Slide method(Wedge)
ii. Cover glass method
iii. Spin method(Automated)

16. STEPS FOR THE PREPARATION
• There are three basic steps on making thin
blood film:
i. Preparing thin blood film
ii. Fixing thin blood film
iii. Staining thin blood film

17. 1.PRINCIPLE OF PREPARING THIN
BLOOD FILM
• A small drop of blood is smeared on a clean
glass slide to create a single layer of cells
which when stained they can be easily
differentiated and counted.

18. PROCEDURES OF PREPARING THIN
BLOOD FILM
1. Place a drop of blood onto a clean glass slide, it
should be placed at one end (frosted end) and a
drop of blood should not be too thick.At the
frosted part, the patient name and number
should be written by using a pencil.
2. Place another slide(clean smooth edge
spreader) infront of the drop of blood at an
angle of 30.
3. Draw the spreader back to touch a drop of blood
and allow the blood to extend along the edge of
the spreader.

19. Cont.…
4. Spread the drop of blood to make a film, A
film should be about 40-50mm in length which
is equivalent to two-third of the slide.
5. Immediately allow the film to air dry, protect
the dried film from dust.

20. Illustration

21. 2.FIXING THIN BLOOD FILM

22. STAINING OF A GOOD QUALITY
BLOOD FILM
PRESENTERS;
Edwin Aliko
Hilda B Mwaipopo
SUPERVISER: MSHANA IRENE E.

23. INTRODUCTION
• Staining is an auxiliary technique used in
microscopy to enhance contrast in the
microscopic image.
• Stain is a colour (a material or object) by
applying a penetrative dye or chemical.

24. Cont.…
• Differential Stain is a stain which uses more
than one chemical stain(dye).E.g. Leishman
stain which has both basic and acidic dye
• Simple stain can be used to determine cell
shape, size and arrangement. True to its
name, simple stain is a very simple
staining procedure because it involves only
one stain E.g. basic stains such as methylene
blue

25. Cont.…
• Romanowsky stains are used universally for routine
staining of blood films.
• Romanowsky stain is a prototypical stain that was
the forerunner of several distinct but similar
methods, including Giemsa, Jenner, Wright, Field,
May Grunwald stain and Leishman stains, which are
used to differentiate cells in Haematological
specimens.
• The remarkable property of the Romanowsky dyes
depend on two components—azure B
(trimethylthionin) and eosin Y (tetrabromo-
fluorescein).

26. Colour responses of blood cells to
Romanowsky staining
• Nuclei
• Chromatin -Purple
• Nucleoli -Light blue

27. Cont.…
Cytoplasm
• Erythrocyte- Dark pink
• Lymphocyte- Blue
• Monocyte Grey–blue
• Neutrophil -Pink/orange
• Basophil- Blue
• Erythroblast- Dark blue
• Reticulocyte Grey–blue
• Metamyelocyte- Pink
• Myelocyte- Pink
• Promyelocyte- Blue

28. Factors giving rise to faulty staining
Too blue, nuclei Eosin concentration too low
Blue to black Incorrect preparation of stock
Stock stain exposed to bright daylight
Batch of stain solution overused
Impure dyes
Staining time too short
Staining solution too acid
Smear too thick
Inadequate time in buffer

29. Cont.…
Too pink Incorrect proportion of azure
B-eosin Y
Impure dyes
Buffer pH too low
Excessive washing in buffer solution
Pale staining Old staining solution
Overused staining solution
Incorrect preparation of stock
Impure dyes, especially azure A and/or C
High ambient temperature

30. Cont.…
Neutrophil granules not stained Insufficient azure B
Neutrophil granules
Dark blue/black (pseudo-toxic) Excess azure B
Other stain anomalies Various contaminating dyes and metal salts
Stain deposit on film Stain solution left in uncovered jar
Stain solution not filtered
Blue background Inadequate fixation or prolonged storage before fixation
Blood collected into heparin as anticoagulant

31. 3.Staining Thin Blood Film.
• During staining we use Leishman stain as one
of the Romanowsky stains.
• Romanowsky stains are neutral stains
composed of a mixture of oxidized methylene
blue(Azure) dyes and Eosin.

32. Preparation of Leishman stain
• Weigh out 0.2 g of leishman powder and transfer
it into a dry conical flask.
• Using a complete dry cylinder, measure 100mls of
methanol and add to the conical flask which
contains leishman powder.
• Mix well at an interval until the dye has
dissolved. Warming the solution in a 50 ̊c will
help the dye to dissolve for 15minutes.
• Label well the bottle.
• Store it at room temperature in a dark and keep
the bottle tightly stoppered.

33. Principle of staining
• Leishman stain consist of both acidic dye and
basic dye, the acidic dye(Eosin) stains the
cytoplasm which is basic into pink whereas
the basic dye(Methylene blue) stains the
nucleus which is acidic into blue.

34. Materials and Equipments
a)Whole uncloted EDTA blood b
b)Microscope glass slides
c)Spreading glass slide
d)Fixatives(10% methanol)
e)Coplin jar ,stainind rack ,
d)Leishman stain

35. Procedure of staining
• Flood the slide with leishman stain for 3minutes.
• Add double the volume of buffered water and
leave for 10minutes.
• Wash the slide with cleaning running tape water.
• Clean back of the slide.
• Allow the slide to air dry.
• Observe the slide microscopically.

36. X’stics of a well stained blood film
An optimally stained film has the following
characteristics:
1. The red blood cells (RBCs) should be pink to salmon.
2. Nuclei are dark blue to purple.
3. Cytoplasmic granules of neutrophils are lavender to
lilac.
4. Cytoplasmic granules of basophils are dark blue to
black.
5. Cytoplasmic granules of eosinophils are red to orange.
6. The area between the cells should be colorless, clean,
and free of precipitated stain.

37. A well stained blood film

38. SICKLING TEST
FRANCO SHADRACK
SABRINA MBINGALALA
SUPERVISOR; MISS IRENE MSHANA

39. INTRODUCTION
Sickling test is one among the test that are
useful in looking for abnormal hemoglobin HbS
in the blood that causes the disorder called
sickle cell disease.

40. What is sickle cell disease?
• Sickle cell disease is an inherited disorder that
cause red blood cells to become sickle shaped.
• With sickle cell disease the cell die early, resulting
to shortage of healthy red blood cells(sickle cell
anemia) and can block the blood flow causing pain.
• Most this disease affects people from Africa, India,
Middle East and Mediterranean region, causing
high mortality rate especially in childhood.

41. Cont.
• The main forms of sickle cell disease found in
tropical countries are;
I. Homozygous sickle cell Anaemia(HbSS), this
occurs when a person inherits a haemoglobin S
(HbS) gene from both parents.
II. Sickle cell haemoglobin C(HbSC), this occurs when
a person inherits HbS gene from one parent and
HbC gene from the other parent.
Note: heterozygous sickle cell HbAS is termed as
sickle cell carrier.

42. Cont.
• The following are the tests used to investigate
suspected sickle cell disease and to identify sickle
cell trait carriers;
I. Measurement of Haemoglobin, example by using
HPLC
II. Sickle cell slide test to detect HbS
III. HbS solubility filtration test
IV. Examination of a Romanowsky stain thin blood
film for features associated with sickle cell
disease.

43. SICKLE CELL SLIDE TEST
Why sickle cell slide Test:
• This test is simple to perform and requires a
single reagent.
• It is useful when it is not possible to perform
HbS solubility filtration test.
• To identify the abnormal haemoglobin S
which will undergo sickling when exposed to
an oxygen deficient atmosphere.

44. Principle of the Test
• Blood is mixed with on a slide with a chemical
reducing agent (sodium metabisulpite)
covered with a cover glass and incubated at
room temperature. The reducing agent
deoxygenates the haemoglobin in the red cells
providing the conditions for cells containing
HbS to sickle.

45. Equipment, Reagent and supplies
• Sodium metabisulphite, Glass slide, Pasteur
pipette, Cover slip, Weigh balance, spirit lamp,
distilled water, match box, petroleum jelly,
microscope.

46. Preparation of the Reagent (2%)
• 0.2g of sodium metabisulphite powder and transfer it
into a clean tube.
• Using a micropipette, measure 10mls of distilled water
and add to a tube which contain sodium metabisulphite
powder.
• Mix them well until they dissolve completely.
NOTE: The chemical is unstable. It can be used only
on the day it is prepared(up to 8 hours)

47. Procedures of the Test
• Place one drop of patient blood on a slide.
• Add an equal volume of fresh reducing agent
mix and cover with a cover glass, exclude any
air bubble.
• Using a Pasteur pipette, cover the smear with
petroleum jelly around the edges of the cover
slip such that no any oxygen penetrate in.

48. Cont.
• Place the slides in container(plastic box or Petri
dish) with a damp piece of blotting paper or
tissue in the bottom to prevent drying of the
preparation. Close the container and leave it at
room temperature.
• After 10-20 minutes examine the patient's
preparation microscopically for sickle cell. Focus
the cells first with the 10x objective and
examine for sickling using the 40x objective

49. Sources of Error
• Not using the reducing agent at the correct
concentration or using too much reagent to
the volume of blood.
• Using reagent that is more than 8 hours old.
• Not using a clean glass slide or clean cover
glass

50. Limitation of sickle cell slide test
• The test can only be used to detect the
presence of HbS, it cannot tell which
other HB is present.
• It cannot differentiate between sickle cell
disease HbSS and sickle cell traits HbAS.

51. Interpretation of the Results
• A positive sickle cell test appears crescent
shaped with pointed ends.
• Reporting of the test is in two ways;
Sickle cell test positive
Sickle cell test negative

52. Cont.
• A positive sickle cell test indicates that a person
red blood cells contain HbS.
• A negative sickle cell test indicates that a
person red blood cells do not contain HbS.
NOTE: The negative control preparation should
show no sickle cell.

53. Appearance on the Microscope

54. References
• Monica Cheesbrough District Laboratory
Practice in Tropical Countries, Part 2
Cambridge University Press.
• Dacie and Lewis Practical Haematology Tenth
Edition

55. Supervisor
Ms. Irene mshana
Presenters
Robinson Paul Mabongo
Issaya D Macha

56.  ESR, is the rate at which RBC’S sediments in a
period of one hour.
 It’s non specific test
 It’s raised in a wide range of infection,
inflammation and malignant conditions which
associate with changes in plasma protein
particularly increase in fibrinogen,
immunoglobulins and c-reactive protein.

57.  It’s also affected by many other factors
include
anaemia,pregnancy,haemoglobinopathies and
treatment with Anti-inflammatory drugs.
 Moderate raised sedimentation rates can
sometimes found in healthy people,
particularly those living in tropical countries.
 Normal ESR cannot exclude disease.

58.  ESR measurements have been discontinued
because they add little to diagnose disease,
assessing it’s progress and monitoring
response to treatment.
 Therefore, when performed test results must
be interpreted in conjuction with other results
of other laboratory tests.

59.  ESR is determined by the interaction between
factors that promote (fibrinogen) and resist
(negatively charge of RBCs-that repel each other)
sedimentation
 Normal RBCs settle slowly as they do not form
rouleaux or aggregate together. Instead they
gently repel each other due to the negative
charge on their surface
 Plasma proteins especially fibrinogen adhere to
the red cell membrane and neutralize the surface
negative charges, promoting cell adherence and
rouleaux formation.
 Rouleaux are stacks of many RBCs that become
heavier and sediment faster.
 Increased rouleaux formation contribute to high
ESR

60.  There are two main methods to determine
ESR:
 Westergren’s Method
 Wintrobe’s Method
 Westergren’s method is preferably as an
indication for the patient’s conditions.

61. The westergren’s tube is open at both ends
and it’s 30cm length and 2.5mm in diameter.

62.  When an anticoagulated blood is allowed to
stand in a tube undisturbed for a period of
time, the RBC’S under the influence of gravity
settle out from plasma. The rate at which it
settles is measured as the number of
millimeters of clear plasma present at the top
of the column after one hour(mm/hr).

63.  It involves three stages;
i. Stage of Aggregation, it’s the initial stage in
which pilling up of the RBC’S occurs.
 It’s also known as rouleaux formation.
 It occurs in the first 10-15minutes.
ii. Stage of sedimentation, this is the stage of
actual falling of the RBC’S in which
sedimentation occurs at constant rate.
 It occurs in 30-40minutes out of 1hr.

64. iii. Stage of packing, this being the final stage
or stationary phase.
 It’s known as the slower rate falling due to
overcrowding of the cells.
 It occurs in the final 10minutes in 1hour.

65.  Westergren tube
 Westergren tube rack
 Test tube(anticoagulated)
 Rubber tit(rubber bulb)(sucker)
 Stop watch
 Tri sodium citrate 3.8%

66. i. Weigh 3.8g of Tri-sodium citrate.
ii. Dissolves in 100mls of distilled water.
iii. Mix them well until the mixture is
completely dissolved.
NB: Keep the solution in a refrigerator of 4-8oC
and if the solution is cloudy or if it contains
particles then discard it.

67. i. Arrange the tubes according to the number
of tests to be performed.
ii. Add 0.25mls of sodium citrate solution into
the test tube.
iii. Pipette 1ml of blood into the test tube
using the calibrated rubber teat/syringe
and mix them together with sodium citrate
solution.
iv. Pipette the mixture using the graduated ESR
pipette.

68. v. Stand the mixture upright 90oC away from
direct sunlight.
vi. Start a stop watch and leave undisturbed
for 1hour.
vii. After exactly 1hour,read the level at which
the plasma meets the red cells in
millimeters.

69.  The wintrobe method is performed similarly
except that the wintrobe tube is smaller in
diameter than Westergren tube and only by
100mm long.
 EDTA anticoagulated blood without extra
diluent is drawn into the tube and rate of
fall of red blood cells is measured in
millimeters after 1hr.

71.  Mix the anticoagulated blood thoroughly
 Fill the wintrobe tube by using paster pipette
up to “0” mark
 Place the tube vertically in the wintrobe
sedimentation rack
 Note the ESR at the end of 1hr

72.  The reference ranges varies between men and
women therefore,
 Men 0-15mm/hr. (over 50years 1-20mm/hr)
 Women 0-20mm/hr. (over 50 years 1-
30mm/hr)
 Children 1-10mm/hrs

73.  Autoimmune disease e.g. Rheumatoid
arthritis, systemic lupus erythematosus
 Infection e.g. HIV
 Pregnancy
 Tissue injury (acute or chronic)

74.  Polycythaemia vera
 Sickle cell disease
 Dehydration and other conditions associated
with haemoconcentration

75.  Temperature-the test should be done between
18-25˚C. higher temperatures raise the ESR
 Not observing the standard time of 1hr when
taking the reading
 Not standing the tube in an absolute vertical
position. Inclination of the tube rises ESR
 Using wrong anticoagulant
 Delay in performing the test. The test should be
done within 2 hrs of blood collection
 Wrong tube dimensions and material
 Clotted blood sample

76. RETICULOCYTE
COUNTING
Presenters
Mr Shadrack M.
Mc Margareta M.
Supervisor
Mr Sephord N.

77. Outline
 Introduction
 Reticulocyte Index
 Supravital staining
 Counting of reticulocytes
 Clinical implication
 QC in reticulocyte count

78. 13.1.Introduction
 Reticulocytes (Retics) are juvenile red cells which
contain remnants of the ribosomal RNA
 The most immature reticulocytes are those with the
largest amount of precipitable material
 In the least immature Retics, only a few dots or strands
are seen.

79. Introduction cont’d
Reticulocytes after counter staining with
Romanowsky dyes (left panel) and supravital
dyes (right)

80. Introduction Cont’d
 Supravital staining is staining of cells while they are
viable
 The number of reticulocytes in the peripheral blood is a
fairly accurate reflection of erythropoietic activity
 Complete loss of basophilic material probably occurs as
a rule in the blood stream after the cells have left the
bone marrow.
 The ripening process is thought to take 2-3 days, of
which about 24 hours are spent in the circulation.
 When there is an increased erythropoietic stimulus as in
hemolytic anemia there will be premature release of
reticulocytes into the circulation as their transit time in
the bone marrow is reduced, the so-called 'stress' or
'shift' reticulocytosis.

81. Clinical significance
 To assist physicians in the diagnosis, treatment or
monitoring of patients with various anemias
 The reticulocyte count is an index of bone marrow red
cell production
 It measures erythropoietic activity

82. Principle
 The count is based on the property of ribosomal RNA to
react with basic dyes such as new methylene blue or
brilliant cresyl blue to form a blue precipitate of granules
or filaments.
Note: Polychromasia on Romanowsky stains are indicative
of Reticulocytosis in PBS
 Only supravital staining techniques enable their number
to be determined with sufficient accuracy.

83. Cont’d
 Better and more reliable results are obtained with new
methylene blue than brilliant cresyl blue, the former
stains the reticulo-filamentous material in the
reticulocytes more deeply and more uniformly than
does the latter
Staining Solution:
 1% New methylene blue (NMB)
 1% Brilliant cresyl blue.

84. Specimen type
 EDTA whole blood:
 Specimen can be stored at room temperature for 8
hours or refrigerated at 2-8oC for 24 hours
Equipments
 Microscope slide
 microscope
 Tes tubes
 dropper

85. Method
1. Deliver 2-3 drops of the dye solution into 75 X 10mm
glass or plastic tube using a Pasteur pipette.
2. Add 2-3 drops of the patient’s EDTA anticoagulated blood
to the dye solution and mix.
3. Plug the tube and incubate at 370C for 15-20 minutes.
4. After incubation, resuspend the cells by gentle
mixing and make films on glass slides in the
usual way
5. Allow to air dry
6. Examine the films in a microscope

86. CONT.......
7.Look into the slide area using 10x objective
8. Add a drop of oil
9. Move to 100x
10. Count all red cells (reticulocytes + RBC) using a man
ual counter
11. Make sure not to count WBC (large nucleated cell)
12. The number of reticulocytes is counted also on outs
ide paper to be able to calculate.

87. Method cont’d
.
 In a successful preparation, the reticulofilamentous material
should be stained deep blue and the non-reticulated cells
stained diffuse shades of pale greenish blue.
1% NMB stained
smear

88. Method cont’d
Counting
 An area of the film should be chosen for the count where
the cells are undistorted and where the staining is good.
 To count the cells:
 eye pieces provided with an adjustable diaphragm
could be used

89. Method cont’d
 Reticulocyte is obtained in a population of 1000 red
blood cells in a specific field.
 Reticulocyte count (%) = Reticulocyte number X 100%
RBC number
 Absolute reticulocyte count = Reticulocyte count (%) X RBC count

90. RETICULOCYTE INDEX
 When there is severe anaemia Reticulocyte count is
expressed as RI
 RI =Observed reticulocyte count(%) X Measured
Hb/Appropriate normal Hb

91. Sources of Error in the Reticulocyte Count
1. Insufficient number of cells counted.
2. Confusion of reticulocytes with red cell inclusions like
Basophilic stippling, Howell-Jolly bodies, Pappenheimer
bodies and
3. Recent blood transfusion
4. Mishandling, contamination, or inadequate refrigeration
of the sample
Pappenheimer bodies Basophilic stippling

92. Interpretation
Reference value
 0.5 - 2.5% of total erythrocytes in adult(or 25 - 85 X 109/l)
 2.0-5% of total erythrocyte in neonates
 Increased numbers (Reticulocytosis):
 Indicates hyperactive erythropoiesis in the bone
marrow
A. An increase in reticulocyte number is seen in the
following conditions:
I. hemolytic anemias
 Immune hemolytic anemias
 Primary RBC membrane defects
 Sickle cell disease
 RBC enzyme defect
 Exposure to toxins

93. Cont’d
II. Following hemorrhage
III. Following treatment of anemias where an increase in
the reticulocyte number may be used as an index of
the effectiveness of treatment.
e.g.
 After doses of iron in iron deficiency anemia, the
reticulocyte count may exceed 20%
 Proportional increase of Retic count when pernicious
anemia is treated by transfusion or vitamin B12
therapy.

94. Cont’d
IV. Physiologic increase in pregnancy and in infants
B. Decreased levels: means that the bone marrow is not
producing enough erythrocytes.
Conditions associated with low Retic count:
 Iron deficiency anemia
 Aplastic anemia
 Radiation therapy
 Untreated pernicious anemia
 Tumour in marrow

95. Quality control
 Use of commercially available controls
 Strict adherence to SOP (preparation, counting, etc)

96. Review Questions
1. What are reticulocytes?
2. How could the number of reticulocytes in the peripheral
blood be a fairly accurate reflection of erythropoietic
activity in the bone marrow?
3. Define supravital staining.
4. What is the difference between a Romanowsky and
supravital stains
5. How do you manage to count the number of
reticulocytes in each field of the microscope after you
stain the cells with supravital dyes?
6. How do you calculate the relative number of
reticulocytes in the patient sample?
7. Briefly discuss RI.

97. Review Questions/Summary
7) What is the clinical interpretation of an increase in the
number of reticulocytes in the peripheral blood in
general terms?
8) list sources of errors in reticulocyte counting and their
remedies

98. REFFERENCE
 https://www.sciencedirect.com/topics/medicine-and
-dentistry/new-methylene-blue
 Dacie and Lewis practical page 36-40
 American society for clinical pathology

99. Preparation of Sensitized
Red Blood Cells
Sephord S. Ntibabara
21/06/2022

100. INTRODUCTION
Sensitization of RBC is the process of removing the inhibiting
blood component such as proteins and activate the binding sites
by addition of the Ant D (IgG) that makes them reactive.
For efficient screening of the antibodies the sensitized cells/
control cells has to be collected from multiple donors
These sensitized red blood cells has to be prepared from
BLOOD GROUP O POSITIVE

101. INTRODUCTION cont..
Sensitized Red Blood Cells
are the pool of red blood cells collected from different individuals
that are used to confirm the validity of Anti globulin Tests
SRBC makes the cell suspension to have the wide range of
antigens which offer maximum screening capacity.
These antigens are the possible cause of agglutination during
transfusion.
These are
IgG
Complements such as C3b or C3d
other free or attached Antigen of the RBCS such as duffy,Kelly
etc

102. FUNCTION OF THE COOMBS
CONTROL CELLS
i. Validate function of AHG reagent
ii. Validate the agglutination tests i.e. DAT, IAT, compatibility etc

103. Principle
• O+ve cells + Anti D IgG = SENSITIZED CELLS
• AHG Reagent + SENSITIZED CELLS =Agglutination

104. Materials and reagents
• Anti D IgG
• Anti Human Globulin
• Normal saline
• Glass test tubes
• 10 mls test tube
• Water bath with thermometer
• Centrifuge

105. Procedures
i. Collect blood from many blood donor (5 individuals
minimum)
ii. Make a pool by taking 0.5 mls from each donor into a 10mls
test tube to make a total of 2.5 mls
iii. Add 7.5 mls of normal saline to the 2.5 mls of poled blood
(3ml of normal saline into 1ml of pooled EDTA blood)
iv. Wash the cells by centrifuging at the speed of 3600RPM for
5minutes
v. Remove the supernatant without mixing with the infranatant
vi. Repeat step iii to v above for other 2 times
vii. Make 5% of the cell suspension
viii. Dilute the incomplete anti-D reagent in 1:5 in normal saline

106. ix. Add 1 volume of cell suspension to 9 volumes of the diluted
anti D
x. Incubate at 37oC for 20 mins
xi. Wash the cells 3 times with normal saline as in iii above
xii. Make 5% cell suspension of the red cells
TO CONFIRM IF THE CELLS ARE SENSITIZED
ix. Take 1 drop of the 5% cell suspension to one drop of AHG
reagent and spin for 1 minute.
x. Agglutination of the cells shows that the cells were
sensitized.

107. Storage of the sensitized red cells
• The sensitized RBCs are stored in 2-8oC for 48hrs

108. MICROSCOPE AND
MICROSCOPY
MARY MIGIRE
BRIAN MPUNGU
Supervisor: Miss Irene Mshana
10/05/2022

109. Outlines
Study objectives:
i. Define and explain microscope and
microscopy.
ii. Know types of microscope
iii. Know all important parts of the
microscope and their functions
iv. Differentiate Magnification from
resolution
v. How to handle microscope.

110. Microscope
• Is an instrument used to enhance
visualization of objects which are too small
to be seen by naked eyes.
• Microscopy-The science of investigating
too small objects(can not be seen by naked
eyes) by using microscope.

111. Types of microscope
Many but can be grouped into:
i. Light/optical microscope
ii. Electronic Microscope

112. 1. Optical/Compound light
microscope
• Employs light to see an object.
• First to be inverted.
• Most commonly used microscope type.
• Magnifies 40-1,000times.
• Compound – has 2 types of lenses to
magnify an object. i.eOcular and objective
lenses.

113. Parts of microscope.

114. base
ocular
objective
Illumination
intensity
stage
Nose piece
Specimen holder
Course adjustment
X-axis knob
Condenser
Iris diaphragm
(aperture
diaphragm ring)
Fine adjustment
y-axis knob

115. Know the following Microscope
parts and their function.
• Eyepiece
• Ocular lens
• Nosepiece
• Objective lens
• Stage
• Stage clip
• Light switch
• Light intensity knob
• Fine adjustment
• Coarse adjustment
• Stage manipulator
knobs
• Condenser
• Light source
• Iris diaphragm knob
• Cord holder
• Microscope body

116. .1.The light source.
 Often is the lamp attached to the stand
2. The substage.
 Is found below and usually attached to the stage
 Can be moved up and down
 In most modern microscopes consists of the condenser
and diaphragm
3. The condenser.
 Light from the lamp is directed into the first major
optical component, the condenser
 The main purpose of the condenser is to focus or
concentrate the available light into the plane of the
object.

117. The condenser—cont--
• Within comfortable limits, the more light at
the specimen, the better is the resolution of
the image

118. The condenser—cont--
RESOLUTION – being the ability to see
separate minute adjacent details.
• Many microscopes have condensers capable
of vertical adjustiment, in order to allow for
varying heights or thickness of slides.
• Once the correct position of the condenser
has been established there is no need to
move it.
•

119. The condenser—cont--
• All condensers have an aperture (Iris)
diaphragm with which the diameter of the
light beam can be controlled (regulated)
• Reducing the aperture reduces the amount
of oblique light reaching the subject and has
an effect of increasing the apparent contrast
of the image

120. 4. Body tube:-
Bodytube connect the eye piece to the
objective lens.
5. Nose piece:-
On many modern microscopes, the
objectives are screwed into the revolving
nosepiece that can accommodate FOUR,
FIVE or SIX objectives.

121. 6. Objective lens:-
The next and most important piece of the
microscope’s equipment is the objective,
The type and quality of the objective have
the greater influence on the performance of
the microscope
Are located at the end of the body tube
Provide both image magnification and
image resolution.

122. Objectives cont--
• The objective’s task is to collect light rays
from the object and within them form a
MAGNIFIED REAL IMAGE, some
distance above

123. Objectives - magnification
• The RESOLVING POWER of the microscope is
very important and is defined as;
• The ability to reveal fine detail,or to
discriminate between adjacent details.
• The revolving power is measured as the least
distance between two objects at which they still
can be discerned as two structures rather than a
single blurred object
• With the light microscope object usually must be
separated by at 0.2μm to be seen as two separate
structures.

124. Objectives cont--
• When the white light enters a lens, it is split
(refracted into the colours of the spectrum
• Because colours are refracted at different
points of focus; therefore, an uncorrected lens
will give an image surrounded by colour
fringes. This is known as CHROMATIC
ABBERATION

125. Objective Lens
• There are four objective lenses.
• Red – 4X
• Blue – 10X
• Yellow – 40X
• Oil immersion (white) – 100X

126. Total Magnification
• When the image
travels through the
objective lens and
the ocular lens it
magnifies the
magnification of
the objective lens
by 10X
Ocular
lens
Objective
lens
Total
magnification
10X Red
4X
40X
10X Blue
10X
100X
10X Yellow
40X
400X
10X White
100X
1000X

127. 7. Ocular (Eye) piece:-
These are the final stages in the optical path
of the microscope
Their function is to magnify the image
formed by the objective within the body tube,
and present the eye with the VIRTUAL
IMAGE.
Commonly have a x10 magnification,
although x5 oculars are frequently used on
students microscopes and x 15 oculars are
preferred by some microscopist

128. II:ELECTRONIC
MICROSCOPE
• Type of microscope that can view very
small organisms even less than 0.2µm. ie
viruses and cellular components.
• More accurate than light microscope.

129. Scanning Electron Microscope
• Scanning Electron Microscope
– Image appears in 3-dimensions and in
black and white.
• The magnification is usually 1000X to
10,000X.
• It is particularly useful for studying the
surfaces of cells and viruses.

130. Scanning Electron Microscope

131. Red blood cells, color enhanced

132. Transmission Electron Microscope
• The item to be viewed is sliced into very
thin slices, the image is a slice of the cell.
• These images are used to cross sections of
cells and their intracellular structures.
• Magnification can be as much as 10,000X
to 100,000 X.

134. Sperm cross section TEM,
Mycobacterium tuberculosis TEM

135. HOW TO USE A MICROSCOPE

136. i. Place it on a flat and stable table
ii. Make sure it is clean free from dust and
any contamination.
iii. Connect to the power source.
iv. Place the slide with the specimen on the
stage.
v. Make sure the stage clips holds it properly.
vi. Switch on the power source.

137. viii.Start with 4x to 100x objective depending
with the kind of examination you want to
make.
– Wet preparation
– Sickling test -up to 40x
-Stained slides -100x(oil immersion)

138. Using the microscope
•Always observe using the LOWEST POWER objective first.
•Focus using the COARSE ADJUSTMENT KNOB to bring the
object into focus. Bring the object into sharp focus by using the fine
adjustment knob.
•Focus, and then move to a higher power objective, if needed.
•Use only the FINE ADJUSTMENT KNOB when using the
HIGHEST (longest) POWER OBJECTIVE.
•Keep both eyes open to reduce eyestrain.
•Determine total magnification of the object by multiplying the
power of the ocular (10x) the power by the power of the objective.

139. At 10x objective
Field finding
• Lower the condenser
• Decrease light intensity by the diaphragm
• Use course adjustment
Functions of 10x objective
• Evaluate the quality of the field
• Evaluate staining quality
• Evaluate the working area/examination area

140. At 40x
Examination and reporting is done
• Lower the condenser
• Increase light intensity accordingly
• Use fine adjustment/Course adjustment in
case you disturbed the field

141. At 100x
• Morphological examination and reporting is
done
• Raise the condenser
• Increase the right intensity by opening the
diaphragm
• Use only fine adjustment
• Use oil emulsion

142. Distance between lens and slide
• Use only the fine focus knob with the
40X and 100X lens.

143. The Oil Immersion Lens
• The oil immersion lens or 100X lens is used
with special optical oil. It makes the image
clear at a higher magnification
• Your instructor will tell you if you need to
use this lens.
• It is important to remove all the oil if you
use the oil immersion lens.

144. A B
Which of these images would be viewed at a higher power of
magnification?

145. HANDLING AND CARE OF
MICROSCOPE

146. Care of the microscope
• Routine maintenance of the microscope is very
important and should be part of the routine
assurance program
1.Keep the microscope covered when not in use.
2.Clean the lens frequently with lens paper. Do
not use other hard tissue paper.
3.Remove immersion oil immediately after use

147. Care of the microscope cont--
4. Use sparingly xylene on the objectives only
as a last resort, and then use it and remove
immediately.
5. Do not dismantle the objectives.
6. Be careful when using immersion oil that
the high-powered dry lens is not dragged
through the oil
7. Reduce the light to a minimum or turn off
when the microscope is not in use.

148. The proper way to carry your microscope.
• Always carry the
microscope with two
hands, holding it close
to your body.
• One on the arm and
another on the base.

149. Proper usage
• At the lab table, unwrap only
as much cord as you need, the
rest should remain stored
around the cord wrap.

150. • The microscope
should always be
stored with the 4X
(red) lens in place
and the stage in its
lowest position.

151. Cleaning
• Never use dry lens paper to clean eyepieces.
Use the cleaning solution provided or
breathe on the surface to be cleaned, then
wipe.
• Use lens paper on all glass parts of the
microscope.

152. lens paper

153. Safety
• To avoid
contamination,
DON’T operate the
microscope with
groves.
• (But personal safety
first)

154. References
• District Laboratory Practice in Tropical
Countries Part 1 Second EditionMonica
Cheesbrough
• https://www.britannica.com/technology/mic
roscope
• https://www.slideshare.net/sarathy4/microsc
ope-10905635

155. THANK YOU AND GOODLUCKY TO
EVERYONE.
vanree