Practical Hematology for BDS Komal Marwaha
INDEX
ABCDEFGHILMNOPRSTUVWZ
A
ABO blood grouping 84
Acetone 59
Acid citrate dextrose (ACD) 22
Acidified glycerol lysis test (AGLT) 122
Advantages of
Sahli's method 75
Westergren method 112
Wintrobe method 106, 114
ZSR method 115
Anemia 42
classification based on blood indices 108
Assessment of stained blood smear 60
Autologous transfusion 87
Automated method 103
B
Bart's Hb 76
Bleeding
disorders 94
time 90, 124
Blood
determination 87
indices 124
transfusion 87
BT tests 94
C
Calculation of
dilution factor 41, 49
RBC count 41
volume of fluid examined 41, 49
Capillary blood sample 11
Causes of
decrease in hemoglobin concentration 78
increase in hemoglobin concentration 78
Cell volume 103
Centrifugal method 58
Charging chamber 32
Citrate phosphate dextrose-adenine 22
Clinical significance of ESR 117
Clinically important blood group systems 86
Clotting time 90, 124
Coagulation defects 94
Collection of venous blood 14, 15
Composition of Hayem's fluid 39
Copper sulphate falling drop method 77
Counting
chamber 29
cells 41, 48
Cover slip method 57
CT tests formation of clot 96
Cytoplasm 64
to nucleus ratio 64
D
Decrease in HCT 108
Decreased
osmotic fragility 123
RBC count 43
Derivatives of hemoglobin 75
Determination of
BT 90
CT 92
erythrocyte sedimentation rate (ESR) 111
hemoglobin concentration in blood 72
osmotic fragility of RBCs 120
Diagnostic use of ESR 117
Dicumarol 25
Different types of microscopes 8
Differential leukocyte count 67, 124
Diluting pipettes 28
Disadvantages of
Sahli's method 75
ZSR method 115
Disposable syringe 14
Distribution of cells in smear 64
Double oxalate 23
Duke's method 90
E
Ear lobe puncture 13
Errors of hemocytometry 33
Erythrocyte sedimentation rate 124
Estimate of iron content 77
Ethylenediaminetetraacetic acid sequestrene/versene 21
Examination of
peripheral blood smear 55
stained smear under oil immersion 60
F
False
negative results 85
positive results 85
Fate of WBC in RBC count 43
Favouring factors 116
Features of
ideal blood film 57
well-stained blood smear 60
Fixing smear 59
Focusing counting grid 31
Functions of
constituents of Turk's fluid 47
hemoglobin 75
G
Gasometric method 77
Giving false
high value 75
low value 75
Glass slide method 55
Granules 60
H
Heel puncture 13
Hematocrit 103
Hemoglobin concentration 124
Hemoglobinometery 77
Hemoglobinopathies 77
Hemophilia 96
Heparin 20, 24
I
Identification of
cells 60
leukocytes 61
Improved Neubauer chamber 30
In vitro anticoagulants 20
In vivo anticoagulants 24
Increase in HCT 108
Increased RBC count 43
Indications of determining BT and CT 94
Inherent errors 34, 50
Ivy's method 91
L
Landsteiner's law 86
Lee and White test tube method 93
Leukemia 51
Leukemoid reaction 51
Leukocyte pool in body 50
Leukocytosis 50
Leukopenia 51
Leukostasis 51
Lobes 64
M
Major cross matching 87
Manual method 103
Method of
determination
of blood group 82, 103
using microscope 7
Micro-ESR 115
Microhematocrit method 106
Minor cross matching 87
MNS and P system 87
Morphological classification of anemias 107
N
Naked eye
appearance 60
single tube red cell osmotic fragility test 122
Normal
count 50
differential leukocyte count 68
hematological values 124
range of RBCs 42
Normal value 113, 121
by Wintrobe method 114
Nucleus 64
O
Opposing factors 115
Osmotic fragility 124
test 120
Other
blood groups 87
fetal hemoglobins 76
Oval field of view 8
P
Packed cell volume 124
Par focal system 6
Parts of microscope 4
Pathological
causes of leukocytosis 51
conditions 117
Photoelectric colorimetry 77
Physiological causes of leukocytosis 51
Pink test 122
Pipetting 31, 48
Plasma factors 116
Polycythemia 42
Poor illumination 8
Preparation of peripheral blood smear 55
Prerequisites of blood transfusion 87
Principle of staining procedure 59
Purpura 95
R
RBC
diluting fluid 38
pipette 28
Reasons of false counts 34
Red cell factors 116
Rhesus system 86
Role in ESR 116
S
Sahli's method for determination of Hb 72
Significance of osmotic fragility tests 122
Simplate method 92
Size of cell 60
Slide method of blood group determination 82
Sodium
citrate 21
fluoride 24
Spectrophotometric method 77
Stages of ESR 115
Staining of peripheral smear 58
Study of
anticoagulants 20
hemocytometry 27
T
Tallquist's method 77
Tests for determination of
bleeding time (BT) 90
clotting time (CT) 92
Thalassemia 76
Total
erythrocyte count 38
leukocyte count 47
RBC count 43
red blood cell count 124
white blood cell count 124
True hematocrit 107
U
Under
high power 60
low power 60
Universal
donor 87
recipient 87
Uses of
HCT determination 107
RBC pipette 28, 43
WBC pipette 29
V
Vacuum blood collection system 15
Variation in WBC count 50
Varieties of Hb 76
Venous blood sample 14
Vessel wall defects 94
von Willebrand disease 95
W
Warfarin 25
Water free methyl alcohol 59
WBC pipette 29
Wedge method 55
Westergren method 111, 124
Whole body HCT 107
Wintrobe method 103, 113, 124
Wright's capillary glass tube method 92
Z
Zeta sedimentation ratio (ZSR) 115
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Chapter Notes

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1
1Practical Section

Study of Compound MicroscopeCHAPTER 1

zoom view
Fig. 1.1: Compound microscope
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OBJECTIVE
After completing the practical student should:
  • Know basics of microscopy
  • Be able to identify different parts of compound microscope and state their function
  • Be able to use different objective lenses with appropriate setting of condenser, iris diaphragm and lamp
  • Explain the use of immersion oil for 100X objective.
 
INTRODUCTION
Microscope is a common instrument employed to see very minute objects which are invisible to the naked eye. Anton van Leeuwenhoek invented compound microscope which has two lens system—the objective and the eye piece (in contrast to one lens in simple microscope) used for forming magnified image of an object. Before learning mircoscopy, one should understand few fundamental terms to microscopy.
  1. Resolution: It is an ability to reveal closely adjacent structural details as separate and distinct. Human eye can resolve two points that are 0.25 mm apart; light microscope can resolve points that are 0.25 μm apart. Resolution sets the limit of useful magnification of a microscope.
  2. Limit of Resolution (LR): Resolving power of a microscope is expressed quantitatively as Limit of Resolution (LR). It is the minimum distance between two visible objects at which they are seen as separate and not in contact with one another.
    zoom view
    • Where, W = Wavelength of light rays
    • NA = Numerical aperture of objective lens in use.
  3. Numerical Aperture (NA): NA of a lens is an index of its resolving power. Greater the numerical aperture, greater is the resolving power. As NA increases, the resolution distance (distance between two objects at which they can be seen as separate and distinct) decreases.
Numerical Aperture can also be described as an index of light gathering power of a lens, i.e. amount of light entering the objective. NA of the lens is the ratio of diameter of the lens to its focal length. NA can be increased or decreased by increasing or decreasing the amount of light passing through a lens. Any particular lens has constant NA, which depends on its radius and its focal length.
NA of Low power objective lens = 0.30
NA of High power objective lens = 0.65
NA of oil immersion objective lens = 1.30
Focal Length: It is the distance from the object being viewed to the objective lens. Change in NA of lens can be achieved by decreasing or increasing the FL as radius of the lens fitted in microscope cannot be changed.
 
PARTS OF MICROSCOPE
Two types of Compound Microscope are commonly used.
  1. Monocular (with one eye piece)
  2. Binocular (with two eye pieces).
Both types have these parts.
  1. Base: Usually it is horseshoe shaped. It gives support and stability to the microscope.
  2. Handle: It supports the magnifying system and the adjusting system of the microscope. It can also be used for carrying the microscope.
  3. Body Tube: It is a cylindrical tube through which light travels. Its length determines the mechanical length (distance between upper part 5of objective lens and eye piece) of microscope. Increase in tube length increases the magnification but this may result in loss of clarity of image. The tube length is therefore fixed which is normally 160–170 mm. Tube consists of 2 parts:
    • Outer tube: It bears nosepiece at its lower end to which three objective lenses are fitted.
    • Inner tube: It carries eyepiece at its upper end having magnification of 5X or 10X. Inner tube can be slid inside the outer tube to adjust the mechanical length.
  4. Stage: It has two parts
    • A fixed stage: It is a horizontal platform on which object being observed is placed. There is an aperture in the centre through which a converging cone of light passes.
    • Mechanical Stage: It is fitted on fixed stage. It has a spring-mounted clip to hold the slide or counting chamber in position. There are screws present for moving it to and fro and side ways.
  5. Sub stage: It lies below the stage; it has a condenser and a diaphragm.
  6. Condenser: It is a system of lenses, which focuses light (condenses parallel rays of light into a beam of light) from a light source on the object. It also helps in resolving the image. It is mounted below the stage of the microscope with a rack and pinion mechanism. Raising or lowering the condenser can vary the intensity of illumination of object. It must be correctly positioned to focus the light properly on the object being viewed. Being a lens, it has a fixed numerical aperture. For better resolution numerical aperture of condenser should be equal to or slightly less than the numerical aperture of the objective lens being used. So, the position of condenser must always be adjusted with each objective lens being used, to get maximum focus of light and accurate resolving power of microscope.
    Table 1.1   Types of objectives and position of condenser
    Types of objective used
    Position of condenser
    Low power (10x)
    Lowest level
    High power (40x)
    Raised optimally to mid level
    Oil immersion objective (100x)
    Raised fully to highest level
  7. Iris diaphragm: It is located at the bottom of condenser. It has an aperture, which can be opened or closed for adjusting amount of light that passes to the field under observation. Regulating the light by adjusting diaphragm (by reducing field size with the help of iris diaphragm) affects the NA of condenser
  8. Nosepiece: Fixed nosepiece is attached to the lower end of the tube and revolving nosepiece is mounted under it. The revolving nosepiece carries the objective lenses of different magnifying power.
  9. Lenses: The total magnification produced by compound microscope is the product of magnification caused by objective lens and eyepiece.
    • Eyepiece: It is fitted into the top of the body tube. In case of binocular microscope there are two eyepieces. One of the eyepieces (the pointer eyepiece) has a pin mounted in it, which is used for indicating any cell in the field of view. Each eyepiece has 2 lenses one mounted at the top and other at the bottom.
    • Objective Lenses: There are three spring-loaded objective lenses fitted with a revolving nosepiece.
      • – Low Power Objective 10x, magnifies image 10 times
      • – High Power Objective 40x, magnifies image 40 times
      • – Oil Immersion objective 100x, magnifies image 100 times
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Low Power objective lens 10X
  • Magnifies image 10 times
  • Used for initial focusing
  • NA (0.3) is less than that of condenser
  • To achieve maximum focus NA should be matched to that of condenser so the condenser should be fully lowered and the light has to be reduced by keeping the iris diaphragm slightly open
  • Total magnification achieved is 100 times (with 10x eye piece).
 
High Power objective lens 40X
  • Magnifies image 40 times
  • Used for broad view of blood films and RBC counting etc
  • NA (0.65) is almost close to that of condenser
  • Condenser should be slightly raised and iris diaphragm partially opened to achieve maximum focus
  • Total magnification achieved is 400 times (with 10x eyepiece).
 
Oil immersion lens 100X
  • Magnifies image 100 times
  • Oil immersion objective is used for detailed morphological examination of blood films and to obtain greater details of an object
  • NA of oil immersion lens (1.30) is more than that of condenser
  • Condenser should be placed at highest position and iris diaphragm should be fully opened
  • Total magnification is 1000 times (with 10X eye piece)
  • Its use requires special oil called immersion oil, which is placed between objective lens and slide. Commonly used immersion oils are cedarwood oil with Refractive Index (RI) of 1.51, and glycerin RI = 1.30. Liquid paraffin can also be used. Lens has to be immersed in oil during use. If we don't immerse the objective lens in immersion oil, there will be air between the slide and oil, the light will pass from glass slide (a denser medium) into thin layer of air between slide and oil immersion objective (a rarer medium, RI = 1.0). It will get refracted away from normal. So when light rays emerge from the slide, many of them will be refracted away from small aperture of oil immersion objective and very few will enter it, which will result in a faint image. If this air could be removed by a transparent liquid medium having same RI as that of glass slide and objective lens there would be no such refraction and enough light would enter the objective thus giving a clear and bright image. This purpose is achieved by using cedarwood oil, which has same refractive index (RI) as that of glass, i.e. 1.51.
 
Par Focal System
Objective lenses are so constructed that when one lens is in focus, other lenses are also more or less in focus. Thus switching over from one lens to another requires only a turn or two of fine adjustment screw to bring the objective into sharp focus. This arrangement of lenses is called Par focal system.
  1. Coarse adjustment screws: They are mounted on the sides of handle by double side micrometer mechanism. On rotating one, other screw is also rotated. They can be used to raise or lower the stage of microscope quickly to obtain an approximate focus.
  2. Fine adjustment screws: Two screws are mounted close to coarse adjustment screws by double side micrometer mechanism. They are used for exact sharp focusing.
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  3. Illuminating system: Compound microscope is a bright field light microscope where viewed objects look dark or colored contrasted against a lighted background. It uses white light which can be either external or internal.
    • Internal source: Electrical lamp is attached at the base of microscope directly under the stage.
    • External source: It can be in the form of electric lamp or direct sunlight. Microscopes using external source of light have mirror attached at the base to reflect light into condenser. Mirror has two surfaces; plane used for oil immersion objective and distant light source (direct sunlight) and concave for close source of light, e.g. electrical lamp and high power objective.
We require different degrees of illumination when using a microscope. The clarity of image depends upon optimal amount of light available. Raising or lowering the condenser and opening or closing the diaphragm can also alter illumination. A proper combination of two has to be selected under different conditions. In general less illumination is required for viewing a clear, unstained object, and more illumination is required for viewing a stained preparation
 
Method of Using Microscope
  1. Place the slide on the fixed stage with object to be viewed over the central aperture.
  2. Start with low power objective. By rotating nosepiece bring it into position. It will come in place with a click sound.
  3. Make appropriate adjustments of condenser and diaphragm.
  4. Use coarse adjustment screw to bring the object into focus and fine adjustment screw for sharp focus.
  5. For examining the whole slide use the screw attached to mechanical stage for moving the slide sideways and to and fro.
  6. After seeing under low power objective lens, proceed to examine under high power by making appropriate adjustments of objective, condenser and iris diaphragm. Use fine adjustment for sharp focusing.
  7. For oil immersion objective, swing away the high power and put a drop of cedar wood oil over the slide.
  8. Bring oil immersion objective to position.
  9. Raise condenser to highest level and fully open the iris diaphragm.
  10. By using coarse adjustment screw and looking from the side carefully lower the objective till it just touches the oil drop.
  11. Lower the objective further down to the slide.
  12. Increase the gap between slide and objective to bring object into focus.
  13. Use fine adjustment screw for final focusing.
  14. Rack the microscope as the cells and their constituents are three dimensional structures and lie at different levels. It is important to continuously “rack” the microscope (keep rotating fine adjustment screw) once the specimen or object being viewed has been focused under any magnification. By racking they will come into and go out of focus alternatively. In this way no cell will be missed.
 
Precautions
  1. Eyepiece and objective should never be cleaned with paper tissue or gauze. They should be cleaned with lens paper only.
  2. Never touch the lenses with fingers.
  3. Remove oil from oil immersion objective immediately after use by wiping it with a clean lens paper.
  4. Use little of xylene to remove hardened oil if present.
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  5. Use of excess of xylene or benzol should be avoided. It loosens the cement material in which lenses are fitted.
  6. While changing position of objective it should always click into position.
  7. Always look from side while bringing down the oil immersion objective. Never lower the tube while looking through eyepiece.
  8. Make proper adjustments of condenser and diaphragm for each objective for proper illumination.
  9. Once a slide has been focused, use only fine adjustment for racking the microscope.
  10. Never tilt microscope when counting cells in counting chamber, or when using immersion oil.
  11. Carry microscope by holding its handle with one hand and keeping other hand under the base.
  12. After use always cover the microscope.
 
DISCUSSION
 
Sources of Error
 
Inability to Focus an Object
It can occur:
  1. If the object being viewed is not placed within the focal distance of the objective.
  2. If the slide carrying the object has been placed upside down on the microscope stage.
  3. If thick cover slip is placed over the object.
  4. If the object is covered with layer of dried oil or dirt.
 
Unclear Image Under Oil Immersion when it is Clear under Other Objectives can Occur
  1. If sticky/old immersion oil is used which will decrease visibility.
  2. If dried oil (lens left unclean from the previous use) is adhered to oil immmersion objective.
  3. If there is air bubble in immersion placed over the slide. It will produce dark shadow in the field of view.
 
Poor Illumination
Most commonly it is due to faulty combination of adjustment of the condenser, iris diaphragm, lamp or the type of mirror being used. Normally the combination should be:
Objective
Condenser Position
Diaphragm
Mirror
Low power (10x)
Lowest level
Slightly open
Concave
High power (40x)
Raised optimally to mid level
A little more open
Concave
Oil immersion objective (100x)
Raised fully to highest level
Fully opened
Plane
 
An Oval Field of View
This can occur if the objective lens being used is not properly clicked into position.
 
A Smudge in the Field can be due to
  1. Dirt on slide
  2. Dirty objective, if the smudge moves with the field.
 
DIFFERENT TYPES OF MICROSCOPES
Based upon the illuminating system there are different types of microscopes.
  • Bright field or Light Microscope: It uses white light either external or internal.
  • Dark field microscope: It is used for examination of unstained microorganisms. Condenser is darkened in the center and 9oblique light rays pass through the periphery illuminating the microorganisms
  • Fluorescent microscope: Object being viewed is attached to a fluorescent dye which glows when exposed to the light.
  • Electron microscope: It is used for the study of ultra structural details of the cells. By using electron beam of light the resolving power of the microscope is increased to 50,000 to 100,000 times and very small structure can be visualised.
  • Ultra microscope: Ultraviolet light is used which because of low wavelength gives higher magnification and the image can be viewed on the fluorescent screen.
WORK SPACE
  • Q1. According to the objective in the use what adjustments should be made in the position of condenser and diaphragm?
    Objective lens
    Position of condenser
    Position of diapragm
  • Q2. Give different conditions in which plane and concave mirrors are used.
    Type of mirror
    Condition
    Plane
    Concave
  • Q3. What is the reason for changing in the position of the condenser with the change in objective lens used?
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  • Q4. What precautions are taken when using oil immersion objective?
VIVA QUESTIONS
  • Q1. What is numerical aperture? Give its significance.
  • Q2. Define resolution.
  • Q3. What is Par focal system?
  • Q4. What is the principle of using immersion oil?
  • Q5. Name the fluids which can be used for working under Oil immersion lens.