Clinical Pathology & Clinical Bacteriology (For Medical Students and Practitioners) KN Sachdev
INDEX
A
Acquired immunity 351
artificially acquired 352
active 352
passive 352
naturally acquired 352
active 352
passive 351
non-specific 351
specific 351
Adrenal glands 226, 235
normal structure 226
tumours of 235
adrenocortical 235
medullary 236
Adrenocortical hyperfunction
Cushing's syndrome 229
adrenogenital syndrome 231
Adrenocortical insufficiency 232
hypoaldosteronism 235
primary 232
primary acute 233
primary chronic 233
secondary 234
Agammaglobulinaemia
Bruton type 384
Agglutination reaction 335
mechanism of 336
widal test 336
AIDS 396
aetiopathogenesis 388
AIDS-related complex 390
diagnosis of 396
epidemiology 392
high risk groups 389
transmission 394
Albuminuria
causes of 17
Alpha-globulin 80
Anaemia types
diagnosis of 209
aplastic anaemia 212
chronic anaemia 212
iron deficiency anaemia 209
megaloblastic anaemia 210
Anaphylactic reaction
generalised 369
local 369
Anaphylaxis
antibodies involved in 370
in vitro models of 370
target tissue cells of 370
Antibodies 325, 326, 346
biosynthesis of 346
production 325
theories of 325
clonal selection 325
template 325
theories 346
selective 346
template 346
types of 326
Anticoagulants 202
Antigen 324, 359
antibodies 325
bacterial 359
immunocyte 325
immunogen 324
types of 324
complete antigen 324
hapten 324
pro-antigen 324
Antigen-antibody reaction
immune mechanisms 326
Ataxia telangiectasia 384
Australia antigen 165
Autoimmune diseases 365, 367
classification of 367
pathogenesis of 365
Autoimmunity 363
altered antigen 364
mechanisms involved in 363
sequestrated antigens 364
B
B toxoid 355
B-lymphocyte 330
Bacteria
motility 297
flagella types 298
Bacteriological examination 318
blood 319
blood culture 320
clot culture 320
pus 322
sputum 322
stool 321
throat swab 322
transport media 323
for cholera vibrio 323
for enteric bacilli 323
urine 318
BCG vaccine 355
Bence Jones protein tests 18
Benedict's test 19
Benzidine test
method 36
principle 36
Bile salts
bile pigment 23
significance of 23
Bilirubin metabolism 91
Bleeding disorders 191
Bleeding time 192
Blood 129, 143, 169, 179, 400
analysis 143
film 169
examination of 170
glandular fever 172
leucocytosis 170
leucopenia 171
white cells count 175
Giemsa's stain 169
Leishmania donovani bodies 179
malaria 179
microfilariae 179
parasites 179
products 400
transfusion
reactions of 129
delayed complications 129
immediate complications 129
Blood grouping
method of 128
Blood sugar
estimation 144
methods for 144
Bone marrow aspiration 199
contraindications 200
indications 199
method 200
Brucella agglutination test 337
Bun
clinical relevance 152
postrenal 153
prerenal 153
renal 153
C
Carbohydrate metabolic tests
tolerance test 63, 64
galactose 63
glucose 64
Carbohydrate metabolism
coagulation factors 67
Carcinoid syndrome 27
Carwardine saccharometer 440
Cell types producing mediators of CMI 379
Cell-mediated
‘type 4’ reactions 366
hypersensitivity 375
immune response 327
Cells 30
casts 33
cylindroids (sp) 34
epithelial cells 33
types 33
erythrocytes 33
leucocytes 30
Cellular immunity
mediator of 376
Cellulose membrane filters 312
Ceruloplasmin 70
Cholesterol
estimation of 151
in plasma 152
Chronic
granulocytic disease 386
hemolysis 212
diagnosis of 212
mucocutaneous candidiasis 383
Cirrhosis
causes of 68
Clinical haematology
blood films 166
staining of 166
Clot retracion study 193
Coagulation time 191
Cold agglutination test 340
Colour index 188
mean corpuscular haemoglobin (MCH) 189
Complement activation 357, 360, 362
alternate pathway of 360
biologic consequences of 362
pathway of 357
CSF 113, 123
chemical examination of 119
composition of 123
cytologic examination of 118
differential count 118
leucocyte count 118
total count 118
investigations of 124
physical examination of 116
appearance 117
pressure 116
physiology of 113
absorption 113
circulation 113
formation 113
routine examination of 114
specimen collection 114
Cytolytic or cytotoxic ‘type 2’ reactions 366
Cytotoxic reaction 373
D
Diabetes 279, 285, 280
diagnosis of 285
mellitus 274
aetiopathogenesis of 275
type I 277
type II 278
classification of 275
type I 280
type II 280
Diagnostic cytology 132
branches of 134
exfoliative 134
interventional 134
role of 132
Different analysis
blood samples for 143
Diphtheria bacilli
staining of 316
Albert’s method 316
Neisser's method 317
Diphtheria toxoid 356
Disinfection techniques 412
E
Early pregnancy 421
biological method 422
collection of urine 422
detection of 421
preparation of specimen 422
Effusions 142
Electron microscope 8
Endocrine pancreas 273
normal structure 274
Enzymes
estimation of 86
cholinesterase 88
transaminase 87
Erythropoietic porphyria 26
Exfoliative cytology 134
Exocrine pancreas
physiology of 93
carbohydrate splitting 94
fat splitting lipase 94
proteolytic enzymes 94
F
Faeces 37
chemical examination 39
occult blood tests 40
reaction 39
examination of 37
collection 37
principle 37
microscopic examination 38, 42
blood 39
colour 38
odour 38
Female genital tract 135
Fibrinogen 81
Filters
types of 310
Folin-Wu sugar tube 440
G
Gastric juice analysis 49
chemical examination 54
reagents 54
exfoliative cytology 57
gastric juice 49
tubeless methods 51
methods 52
principle 51
Gastrointestinal tract 141
Glycosuria
causes of 20
with hyperglycaemia 20
without hyperglycaemia 20
Gram's staining 299
observation 300
procedure 299
H
Haemagglutination inhibition (HAI) test 424
Haematuria
causes of 35
Haemocytometer 437
Haemoglobin
estimation of 180
Haemolytic jaundice 61
Health worker 411
Hepatic porphyria 26
Heterogenic antigen 358
HIV transmission
prevention of 405
health care settings 405
Hyperbilirubinuria
causes of 24
Hyperparathyroidism 268, 271
primary 269, 271
pseudo 272
pseudo-pseudo 272
secondary 270
tertiary 271
Hyperpituitarism 219, 221
hyperfunction 219
acromegaly 220
Cushing's syndrome 221
gigantism 220
hyperprolactinaemia 220
hypofunction 222
panhypopituitarism 222
pituitary dwarfism 223
Hypersensitivity
classification of 368
delayed 369
immediate 369
Hypobromite 436
I
Immunity
innate 348
Immunodeficiency disease
features 380
highly suspicious 380
moderately suspicious 380
with specific disorder 380
Immunoglobulins 328, 341
classification of 343
IgA 345
IgD 345
IgE 345
IgG 344
IgM 344
‘H’ chain 342
‘J’ chain 343
‘L’ chain 342
basic unit 341
structure of 341
Islet cell tumours 291
gastrinoma 292
insulinoma 291
multiple endocrine neoplasia 292
J
Jaundice 90
K
Ketone bodies
qualitative tests for 21
dip stick test 22
Gerhardt's test 22
Rothera's test 21
tablet test 22
Ketonuria
causes of 22
relevance of 22
L
Latex agglutination inhibition (LAI) test 424
Lipid metabolism
interpretation 84
Lipoidal test 99
Liver
attenuated vaccines 354
biopsy 68, 89
function tests
indications for 58
types of 59
Lymphocytes
mediators affecting 376
M
Macrophages
mediators affecting 376
Microscope
types of 3
compound 5
simple 4
N
Neutralisation tests 303
Dick test 304
Schick test 303
Schultz Charlton reaction 304
Non-protein nitrogen 155
O
Oral glucose tolerance test 147, 289
diminshed 147
increased 148
renal glycosuria 148
Ovarian tumour 139
P
Packed cell volume
method 184
Pancreatic juice
examination of 97
Parathyroid gland 267
Parathyroid tumours
parathyroid adenoma 273
parathyroid carcinoma 273
Pituitary adenomas 224
craniopharyngioma 226
solid ameloblastous areas 226
stratified squamous epithelium 226
granular cell tumour 226
Pituitary gland 216
chromophil cells 218
with acidophilic granules 218
normal structure 216
Pituitary tumours
hormonal effects 224
pressure effects 224
Plasma proteins 78
Platelet counts 184
method 184
direct 184
indirect 185
Platelets aggregating factor 372
Polymorphonuclear leucocytes 376
mediators affecting 376
Porosities
grades of 311
types 311
Posthepatic jaundice 61
Primary immune deficiency states 381
Prothrombin ratio 193
Prothrombin time
principle 193
method 193
R
RBC 204, 207
diseases of 207
disorders of 204
RBC (Hayem’s) fluid 438
RBC classification
aetiological 207
impaired formation 207
increased removal of erythrocytes 207
kinetic 208
excessive destruction 208
inadequate production 208
morphological 207
Reagents’ preparation 447
Red blood corpuscles
total count of 187
Renal blood flow 108
Renal function tests 100
concentration test 100
creatinine clearance test 107
dilution test 102
inulin clearance test 107
phenolsulfophthalein test 102
advantage 104
limitations 105
urea clearance test 105
Resuscitation
mouth to mouth 409
Reticulocyte count 189,206
Rh incompatibility
foeto-maternal 130
Ryles tube 439
S
Sabin poliomyelitis vaccine 354
Secondary immune deficiency states 385
Sedimentation rates (ESR)
Westergren method 195
Wintrobe method 196
Semen analysis 109
chemical examination 112
fructose 112
method 112
microscopic examination 109
total spermatozoa count 110
viscosity 109
Serum albumin 79
Serum aminotransferases
determination of 160
Serum amylase 95
Serum bilirubin
estimation of 60
direct reaction 60
indirect reaction 61
Serum calcium
determination of 163
Serum creatinine
estimation of 161
Serum lipase 95
Serum phosphatases
estimation of 156
acid phosphatase 159
alkaline phosphatase 157
Serum proteins
estimation of 155
Serum uric acid
principle 162
SGOT 160
SGPT 161
Sickle cell anaemia 214
Single blood sugar estimation 289
Skin test 301, 305
delayed reaction type 305
negative reaction 305
positive reaction 305
important tests 301
objective of 301
principles of 301
Sputum 45
microscopic examination
bronchial casts 46
myelin globules 47
physical examination
colour 45
consistency 45
odour 45
Squamous cell carcinoma
nomenclature 139
Staining smears
papanicolaou method of 136
Sterilisation 306, 310, 412
fields guide to 412
control 310
Sterilising agents
classification of 306
methods 307
physical 307
Suction
mouth to mouth 409
in newborns 409
Swiss agammaglobulinaemia 382
Syphilis 413
complement fixation test 416
diagnosis of 413
Kahn's flocculation test 415
precipitation reaction for 414
Wassermann reaction test 418
T
T-lymphocyte 330
Tetanus toxoid 356
Thalassaemia 213
Thick films
staining of 168
Thymic alymphoplasia 383
Thymic dysplasia 383
Thyroid 240, 258
cancer 258
follicular carcinoma 261
medullary carcinoma 264
papillary carcinoma 260
undifferentiated carcinoma 266
giant cell carcinoma 267
small cell carcinoma 266
spindle cell carcinoma 267
gland 240
functional disorders 242
cretinism 244
goitre 254
Graves’ disease 251
hyperthyroidism 242
hypothyroidism 243
myxoedema 244
thyroiditis 246
normal structure 240
Tolerance test 98, 146
glucose 146
adrenaline test 151
glucagon test 150
insulin tolerance test 151
intravenous 148
vitamin A 98
Total eosinophil count 187
Toxic complex ‘type 3’ reactions 366
Toxic complex reactions 374
Transfer factor 378
Transient hypogammaglobulinaemia
of infancy 385
Transmission of HIV
prevention of 404
guidelines on 404
routes of 404
U
Urea 154
principle 154
increased values 154
Ureameter 436
Urinary amylase 95
deposits 30
preliminary tests of 30
Urinary tract
bladder irrigation 142
retrograde catheterisation 142
Urine 11, 28, 62
analysis 11
chemical examination 15
for albumin 15
heat and acid test 15
Heller's nitric acid test 16
collection 11
physical examination 12
preservation 12
for sugar 28
quantitative analysis of 28
urobilinogen 62
estimation of 62
Urinometer 436
Urobilin 25
V
Viral hepatitis 70
hepatitis A 71
hepatitis B 72
W
WBC (Turk’s) fluid 438
White blood cells variations
eosinophilia 176
×
Chapter Notes

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1Clinical Pathology2

Microscope and MicroscopyChapter 1

 
MICROSCOPY
Modern medical training as well as clinical diagnosis utilises the facility of microscope (Fig. 1.1) to such an extent that today it has become an indispensable tool for a practitioner, student, teacher, research worker. Fine structural organisation could only be appreciated after the advent of microscope. Today, a range of different microscopes from simple magnifying lens to electron microscope are utilised for various purposes in the medical, biological and geological studies. Selection of the individual type of microscope depends upon the purpose of the investigator.
zoom view
Fig. 1.1: Monocular microscopy
4How microscopy has evolved and reached to its present minitext as since 1695 when Robert Hook made first compound microscope, the microscopy had undergone tremendous improvement to reach the present day perfection. However, certain types of use in medical and biological field are discussed here.
Before various types of the microscopes are described a word about microscopy is essential. Microscopy is a technique by which a small object is magnified and observed without compromising with the resolving power of the eye and the colour of the object. Microscopy works on the basic principles of optics. In electron microscopy, the same principles are transpolated by the use of a series of electromagnets used for converging or diverging the beam of electrons. Thus, broadly microscopy is of two types viz. Light Microscopy and Electron Microscopy. Light microscopy involves the use of either artificial composite light or monochromatic light or natural sunlight while electron microscopy involves the use of a beam of electrons which, by virtue of refraction/diffraction or absorbance through a series of converging/diverging electrostatic plate lenses or magnets, renders the view of the object as enlarged one. The efficiency of the instrument or system depends upon resolving power of the observer and the instrument and non-splitting of incident light into a diffuse hand of coloured light, which may occur on account of prismatic effect of the lenses. In this chapter only light microscopes are introduced. There are following types of light microscopes.
 
SIMPLE MICROSCOPES
Simple microscopes are of various types ranging from simple biconvex magnifying lens to a combination of two to three lenses. These work on the principle of simple biconvex lens. The object lies within the focus of single or composite lens and eye also remains close to the lens. The image formed is virtual, erect and at 10″ away from the eye. Physically we come across many a type, e.g., simple examination loop, simple magnifier, dissecting microscopes and binocular magnifier. A good simple microscope would have optics from chromatic aberration and spherical aberrations. The magnification of the simple microscope may range from 2× to 20×. In 20× magnification, simple microscope has to be too near the object and thus 5renders dissection impossible. Further, these simple microscopes do not give a three dimensional appreciation of the object in a way similar to most of the compound microscopes to be described later. They are of immense use in preliminary screening examination of the object and also in the event where dissection of, or effect of manocuring with the object has to be viewed.
 
COMPOUND MICROSCOPES
These are combinations of a double sets of lenses. One set of lens lies in the proximity of the object while the other is near the eye. Accordingly, the two sets are called objective and eye- piece. There are various types of compound microscopes to meet the requirements of diversified field of study.
  1. Monocular compound microscope with vertical tube
  2. Inclined monocular compound microscope
  3. Binocular compound microscope
  4. Trinocular compound microscope
  5. Phase contrast microscope
  6. Polarising microscope
  7. Ultraviolet microscope
  8. Ultra microscope
  9. Fluorescence microscope
  10. Interference microscope.
These various types differ either in their light sources or in their lenticular system, but they all have eye pieces and objectives in common. These days it is a fashion to refer to even electron microscopic structures either as ultrastructure or ultramicroscopic view of the object. In fact, ultramicroscope is quite different from an electron microscope. The former one is a light microscope while the later utilizes a beam of electrons. The ultra microscope is basically utilised for detecting the matter of particulate size and works on the principle of Tyndall effect. It incorporates double objective and a ray of right perpendicular to the optical axis in jet dark field. It is used to detect the size of the colloid particles.
 
Parts of a Compound Microscope
Eyepieces There are principally two types of eyepieces, viz. negative and positive.6
In case of negative eyepiece The focus is inside the lenticular system with diaphragm in the ocular. This cannot be utilised for simple microscopy, because the image is formed from objective at its diaphragm.
In case of positive eyepiece The focus is outside the lens and primary image is formed within the diaphragm. Accordingly, later one can be utilised for simple microscopy. Former one is also known as Hygenian eyepiece invented by Christian Hygens while later is also known as Ramsden eyepiece. Routinely, microscopes are supplied with eyepieces of the magnifications 6×, 10×, and 15×. In some microscopes, an additional eyepiece of 20× may also be provided. For Binocular microscope, these eyepieces are supplied in pairs.
 
Objectives
These are functionally of three types:
  1. In those which have not been corrected either for colour aberrations or spherical aberrations
  2. Achromatic objective which have been corrected for one colour
  3. Apochromatic objectives, those which have been corrected for two colours. From the use point of view there are two types: dry objectives and wet or immersion objectives. Magnifications of these objectives range from 3× to 100×. Dry lenses usually have magnification of the order of up to 45× while objectives with magnification above 60× are usually wet or immersion type. Wet or oil immersion types of objectives are better if have a double barrel, i.e., one sliding in the other on a spring mount, such construction adds to the safety of the slide and the objective itself.
 
Body
It consists of several parts, viz., body tube, stage, mechanical vernier, stage body proper with rank and pinion adjustment for coarse and fine adjustment and the stand. Body tube houses on the top, the draw tube while on the lower side it houses the nosepiece carrying a battery of objectives. Stage is a mechanical platform having a central hole for light to pass. The stage may be circular capable of rotatory motions coupled with vernier graduation in degrees of the movement or may be of fixed 7square type where movement of the entire stage is not needed. Body proper hinges along a joint on the tripod stand. The hinge joint enables the observer to tilt the microscope to adjust the proper illumination as well as observe angle. An angle of 40 degree is adequate enough to enable the observer to observe without any strain for prolonged periods. The stage has on its lower side a condenser with iris diaphragm attached with the body proper through another rack and pinion adjustment screw by which the condenser can be brought to desired heights below the stage. Such movements are necessary to adjust the amount of light needed for different objectives of varied magnifications. The body as such may be of two types, in one the stage is fixed and the body tube can be moved up or down while in other, the body tube is fixed and the stage is moved up or down. In any case these movements are controlled by rack and pinion operated fine and coarse adjustments.
 
Condensers
The purpose of the condensers is to bring the light rays from source of illumination to be focussed at the plan of the specimen. The concave mirror attached at the bottom of the instrument serves as a condenser for objective of low power. For high power objectives, this condenser is inadequate and a special substage condenser is required with which concave mirror should not be used. Plain mirror is used for directing the light beam to condenser. The substage condenser may be of many types. The two lens type condenser is uncorrected for spherical and chromatic aberrations while four lens condenser may be corrected one either for spherical or chromatic aberration or for both. There are several type of condensers meeting the specialised requirements, but for routine work the Abbe condenser with numerical aperture 1.20 or 1.25 gives satisfactory performance with objective of nearly all available power. To name a few others are Watson holoscopic condenser, Cardiod condenser, Dark field condenser and Rheinberg differential colour illumination condenser. As they are also not routinely used, they are discussed in brief in this chapter.
 
Vernier Stage
This is a detachable device of immense importance for scanning or examining desired and several fields of the object without 8repetition. It is fixed on the stage and has a spring finger which fixes the slide as if in a claw and the entire set can be moved in two directions over the stage perpendicular to each other on the horizontal plane of the stage. The slide can be moved with the help of two screws 25 mm forward and 75 mm sidewards on the stage proper. In medical microscopes, the vernier mechanical stage is attached at right working hand side and requires special care for operation, because it does not sustain the untender and hard use.
 
Coarse and Fine Adjustments
These are provided on either side of the ordinary fixed stage compound microscope or fixed body tube microscope. The coarse adjustment is identified by bigger size of the knob while fine adjustment has a smaller knob. With practice objects can be focussed very clearly with the aid of coarse adjustment only under ordinary high power objective while under oil immersion objective, the object could be brought to near focus. Fine adjustment in some microscopes is provided with either a vernier scale or the entire knob or drum divided into 100 or 50 equal divisions. By this graduation movements of body tube or the stage in the optical axis can be monitored in the tune of 0.5 to 2.0 microns. This device helps to measure/caliberate the thickness of the slide, coverslip, object (section thickness) or even the refractive index of the liquids.
 
Illumination
Routinely, day light illumination is used. When artificial light is to be used then the illumination is achieved with the help of microscopical lamps which may give adequate illumination. For better illumination special illuminators based on Nelson's principle of critical illumination of Kohler method of illumination are employed. On the later principle there is available “Pancratic Condenser” manufactured by Zeiss which has a built-in illuminating device for all powers of objectives.
 
ELECTRON MICROSCOPE
By substituting a beam of electrons instead of light rays, a deface of resolution of much greater deface, could be obtained. 9Since at the subjection of 50,000 volts; electrons exhibit a wavelength of 0.001 µ only, a resolving power of 0.00005 µ could be applied enabling the molecules to be seen thereby. The use of special hard planters, special value microtomes, and steel knives cannot be used to cut these sections. Glass or diamond knives could be used instead.
 
Terminologies used in Microscopy
  1. Resolving power of the eye It indicates the capacity of eye to differentiate in between two points kept close to each other. Resolving power of the eye is highest for monochromatic yellow light (575 millimicron) next in order are green, red and blue. It also depends on the quantity of light. Optimum light intensity for maximum resolving power is light of 10 to 20 foot candles. Under best illumination eye can distinctly separate 1/2000th part of an inch.
  2. Magnification of the microscope Usually it is calculated by multiplying the magnifying power of the eyepiece with the magnifying power of the objective. But taking into consideration the resolving power of the eye only the magnification capable of separating 1/100th of an inch would be useful. Further magnification shall cause strain over the eye.
  3. Optical index It is an index of the usefulness of the objective and is calculated as follows OI = NA × 1000/objective magnification. OI = Optical index, NA = Numerical aperture. High optical index indicates high resolving power.
  4. Numerical aperture It is the product of the refractive index of the medium in front of the lens and sine of the half of the angular aperture. NA = n sine u, n = refractive index, u=half of the angular aperture. High numerical aperture indicates high resolving power of the lens system.
  5. Angular aperture The angle of the light rays taken in the objective from a point on the subject.
  6. Units Micron=0.001 mm, 1 millimicron=0.000001 mm (10−6 mm) etc.
    1 Angstrom (A) = 0.1 millimicron or 0.0000001 mm (10−7 mm)
Cover glass thickness = 0.17 = 0.18 mm
Mechanical tube length = 160 mm10
Additional magnifications due to:
a. Binocular head
=
1.5 times.
b. Camera
=
1.5 times usually.
 
Precautions in Handling Microscope
  1. Always clean all the lenses with fine linen cloth or tissue lens paper and if necessary with little xylene. Avoid excess use of xylene as it may dissolve the lens cement.
  2. Always focus first under low power then gradually increase power. When using oil immersion objective only cedar wood oil is to be used because it has same refractive index as that of glass and least dispersion of light rays occurs. If the oil has gone too thick it should be discarded. It should not be used after thinning it with xylene.
  3. Primary adjustments are to be made with coarse adjustment and then high power, and then if need be then oil immersion lens is to be used.
  4. When examination is done under low power the mirror could be concave and condenser slightly lower than the stage. But a low situation of condenser with plain mirror is preferred. When high power or oil immersion lens is used the condenser is kept on top situation and plain mirror is used.
  5. When oil immersion is used a drop of oil cover condenser is also desired but should only be used when being directed to do so.
  6. If the objectives and condensers are not par focal (same optical axis) then centering is achieved through the help of a mechanic.
  7. Each microscope should be thoroughly checked by mechanic at least once a year to ensure long trouble-free service.