PATHOPHYSIOLOGY LAB - Hanbook of Pathophysiology Sevastre

Bogdan Sevastre
Handbook of Pathophysiology
Editura RISOPRINT
Cluj-Napoca • 2013
© 2013 RISOPRINT
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Descrierea CIP a Bibliotecii Naþionale a României
Director editurã: GHEORGHE POP

Consilier editorial: MIRCEA DRÃGAN
Design copertã: VOICHIÞA-MARIA CLINCI
Scientific referents:
IOAN MARCUS DVM, PhD – Professor

Department of Pathophysiology, Faculty of Veterinary Medicine,
University of Agricultural Sciences and Veterinary Medicine
Cluj-Napoca, Romania
PETER VAJDOVICH DVM, PhD, Dipl. of ECVCP – Associate Professor

Department and Clinic of Internal Medicine,
Faculty of Veterinary Science, Szent Istvan University,
Budapest, Hungary
ALINA E. PARVU MD, PhD – Associate Professor

Department of Pathophysiology, Faculty of Medicine
University of Medicine and Pharmacy Iuliu Hatieganu
Cluj-Napoca, Romania
Tiparul executat la:

S.C. ROPRINT S.R.L.
400 188 Cluj-Napoca • Str. Cernavodã nr. 5-9
Tel./Fax: 0264-590651 • roprint@roprint.ro
430 315 Baia Mare • Piaþa Revoluþiei nr. 5/1
Tel./Fax: 0262-212290
Content
Preface................................................................................................... 7
1. Laboratory Safety Rules .................................................................. 9

1.1. Physical Hazards. .................................................................. 9
1.2. Chemical Hazards................................................................10
1.3. Biological Hazards ...............................................................11
2. General Characteristics of Disease ..............................................13

2.1. The Influence of Disease on Animal Responsiveness
to environmental Demands.........................................13
2.2. Pathogenesis of Environmental Factors ..............................16
2.2.1. Local Action of the Heat: Thermal Burns.......................16
2.2.2. Local Action of the Cold: Frostbites...............................18
2.2.3. Experimental Photosensitization ...................................20
2.2.4. The Influence of General Status on Responsiveness
to Chemical Factors .......................................................22
3. Hemodynamic Disorders ...............................................................25

3.1. Peripheral hemodynamic disorders .....................................25
3.1.1. Hyperemia and congestion............................................25
3.2.2. Edema ...........................................................................25
3.2.3. Hemorrhage...................................................................28
3.2.4. Thrombosis ....................................................................29
3.2. Shock ...................................................................................31
4. Disease of Immunity.......................................................................35
4.1. Phagocytosis........................................................................35
4.2. Mechanisms of immune mediated injury ............................38
4.3. Inflammation ........................................................................40
4.3.1. General features of acute inflammation ........................40
4.3.2. Tissue Repair.................................................................42
4.3.2.1. Scar formation ............................................................42
4.3.2.2. Skin wound healing ....................................................44
4.3.3. Differentiation between exudate and transudate ..45
4.3.2. Histochemical identification of myeloperoxidase...........46
4.3. Fever....................................................................................48
5. Metabolic Disorders .......................................................................51

5.1. Carbohydrate metabolism....................................................51
5.2. The plasma proteins ............................................................57
5.2.1. Changes in total protein concentration..........................57
5.2.2. Electrophoresis ..............................................................61
5.3. Disturbances of acid/base balance......................................61
3
6. Hematologic Disorders ..................................................................67
6.1. Pathology of Red Blood Cells ..............................................67
6.1.1. Anemia...........................................................................67
6.1.2. Polycytemia ...................................................................72
6.2. Pathology of leukocytes.......................................................73
6.1.1. Characteristics and function of blood leucocytes ..........74
6.1.2. Interpretation of WBCs variations..................................78
6.1.3. Categories of hematological malignancies....................80
6.3. Pathology of hemostasis......................................................82
6.3.1. Stages of normal hemostasis ........................................82
6.3.2. Investigation of hemostasis ...........................................84
6.3.1. Blood clotting disorders .................................................86
6.3.1.1. Thrombocytopenia ...............................................86
6.3.1.2. Defective platelet function....................................87
6.3.1.3. Abnormal function of clotting factors....................88
7. Pathophysiology of the Heart........................................................91

7.1. Electrocardiography ............................................................91
7.1.1. Electrocardiogram of the isolated cardiac fiber .............92
7.1.2. The ECG leads. .............................................................93
7.1.3. Placement of the electrodes ..........................................96
7.1.4. Terminology ...................................................................96
7.1.5. The normal ECG............................................................97
7.1.6. Mean electrical axis .......................................................99
7.1.7. Main characteristics of ECG in dogs and cats.............100
7.2. Cardiac arrhythmias...........................................................101
7.2.1. Sinus rhythm abnormalities .........................................102
7.2.2. Disturbances of the stimulus generation (ectopia) ......104
7.2.3. Abnormalities of the conduction system (block) ..........110
8. Respiratory System ......................................................................113

8.1. Pulmonary Edema .............................................................114
9. Digestive System and Annex Glands .........................................115

9.1. Determination of acidity of gastric juice .............................115
9.2. Exploration of the exocrine function of the pancreas ........117
9.3. Differential diagnosis of jaundice .......................................119
10. Excretory System .......................................................................123

10.1. Main syndromes of renal disease ....................................123
10.2. Urine analysis ..................................................................124
10.2.1. Physical Examination of Urine...................................124
10.2.2. Microscopic Examination of Urine .............................126
10.2.3. Biochemical Examination of Urine.............................129
11. Endocrine System ......................................................................133

11.1. Hypocalcemic tetany........................................................133
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12. Nervous System..........................................................................137
12.1. Cerebellar syndrome (cerebellar ataxia) .........................137
12.2. Epilepsy ...........................................................................138
Annex I – Case presentation ...........................................................141

Case 1. Hemorrhagic chronic anemia (Ancylostomiasis) .........141
Case 2. Hemolytic anemia (Babesiosis) ...................................143
Case 3. Aplastic anemia (Feline infectious peritonitis) .............146
Case 4. Aplastic anemia (Hypothiroidism)................................148
Case 5. Polycythemia (Dehydration) ........................................150
Case 6. Polycythemia and lymphocytosis ................................152
Case 7. Leucopenia (Parvovirus Infection)...............................154
Case 8. Leukocytosis (Pneumonia) ..........................................156
Case 9. Pancreatitis..................................................................158
Case 10. Coumarin poisoning ..................................................160
Annex II – Laboratory techniques...................................................163
Hematology techniques ...................................................................163

II.1. Red blood cells ..................................................................163
II.1.1. Red blood cell count (RBCs)....................................163
II.1.2. Hemoglobin (HGB) ...................................................166
II.1.3. Hematocrit (HCT)/ Packed Cell Volume (PCV)........167
II.1.4. The Erythrocytes Indices..........................................168
II.1.5. The Reticulocytes Count ..........................................169
II.1.5. Abnormal Erythrocytes Morphology .........................170
II.1.6. Erythrocyte Sedimentation Rate (ESR)....................171
II.2. White Blood Cells...............................................................173
II.2.1. White Blood Cells Count (WBCs).............................173
II.2.2. Preparation and Staining of The Blood Smear.........174
II.2.3. The Differential Count ..............................................176
II.3. Investigation of hemostasis ...............................................178
II.3.1. Platelets Count .........................................................178
II.3.2. Bleeding time............................................................179
II.3.3. Prothrombin Time (Quick test) PT............................179
II.3.4. Activated Partial Thromboplastine Time (APTT)......180
Plasma biochemistry........................................................................182
II.1. Plasma glucose measurement .........................................182
II.2. Plasma total protein measurement (Biuret) .......................183
II.3. Dysproteinemia tests .........................................................183
II.4. Determination of plasma bicarbonate content ...................184
Annex III – Reference values ...........................................................187
References ........................................................................................191
5
Preface
Writing this new handbook of Pathophysiology is motivated by the

desire to synthetize and incorporate the new and relevant information
related to fundamental knowledge in veterinary science. In this
pathophysiology bock, I was driven by the same main goal like the other
authors before, to explain, in an accessible manner, the main
pathophysiologic mechanisms occurring in diseased animals.
In the past, the pathophysiology books designed for laboratory
activities were focused on animal experiments, to exemplify the
mechanisms and to provide the students with the practical skills they need.
In the actual view on animal protection requirements, this approach is no
longer acceptable; therefore, the laboratory content was adjusted
accordingly. Only experiments that do not involve any animals suffering or
distress, and do not influence the general health status were included. For
the rest they were replaced by videos and sometimes computer simulation.
The practical activities, essential to achieve the practical skills of
future veterinary practitioners, is done on biological samples originated
form naturally sick animals from clinic, this way the students not only get
used with techniques of hematology and plasma chemistry, but these
results are explained in context of pathologic condition which affect the
animal. Additionally significant analysis bulletins and blood smears are
available for case presentations that accomplish several chapters.
This laboratory bock is not only an attempt to adjust the
pathophysiology practical activities to the limitations of the laboratory
animal welfare. A second goal was to avoid excessive theorizing, hence to
make them more applied, useful for the future veterinary practitioners.
The Author
7
Laboratory Safety Rules
1. Laboratory Safety Rules
Hazards in Clinical Laboratory
In the clinical laboratory, tree types of hazards may be encountered:

physical, chemical, and biological.
1.1. Physical Hazards
Physical hazards are resent in ordinary equipment or surroundings.

Electricity is one major source of hazards; all electrical equipment
must be properly grounded, and used following the manufactures
instructions. Even for minor repairs such replacement of microscope bulbs
the electrical equipment must be disconnected from the power supply.
Overload circuits should be avoided, and extension cords should not be
used except for an emergency.
The most occurred open flames, are Bunsen and Teclu burners,
when they are in use care must be taken to ensure that loose clothing and
long hair do not catch fire. Do not let the burners on un survived even for
short time, in the end always switch them of, and then turn them on each
time is necessary. Flammable chemicals should be handled in fume hood
only, and stored in special ventilated areas. A correct type of extinguisher
must be available.
Laboratory equipment should be used according to manufacturer’s
instruction only, the centrifuge lid must always be latched before the
instrument is turned on, and when the centrifuge is turned off the lid should
not be opened until the rotor has come to a complete stop.
Only glassware that is free from chips and cracks should be used.
Broken glass should be cleaned with a brush and dustpan, not with bare
hands. Broken glass and sharp metallic instruments should be discarded
not into regular trashcans, but into rigid plastic containers, specially made
for this purpose.
1.2. Chemical Hazards
Chemicals present a wide range of hazards; they may be inflammable,

toxic, caustic, corrosive, carcinogenic or mutagenic. The chemicals must be
labeled; the label must contain data like substance in the bottle, concentration,
and eventually additional information the data of preparation and hazard
information, first aid, further medical treatment etc. Unlabeled will be never
used but they should be neutralized. It is strictly forbidden to taste or smell the
chemicals in the attempt to identify their content.
Caustic chemicals are strong acids and bases that are able to
induce severe skin burns. Fumes and vapors from these chemicals may
burn mucous membranes in the respiratory system. Potassium hydroxide,
sodium hydroxide, sulfuric acid, nitric acid and concentrated sodium
hypochlorite are examples of caustic chemicals. The chemicals that give
fumes should be handled in foam hood only.
Any chemical that gets in contact with the skin or eyes should
washed up immediately with the tap water for at least 5 minutes, than
emergency care should be provided according to the situation.
Toxic chemicals can be poisonous by skin contact or by respiratory
exposure. When handle toxic chemicals, the worker must use gloves to
protect skin from contact. If a chemical produce fumes it should be used in
fume hood only. The rule is available for all volatile chemicals.
Laboratory personnel must wear proper clotting, buttoned, knee length
and fluid resistant laboratory coat. Shoes should be comfortable and stable
10
Laboratory Safety Rules
and should have closed toes to protect against spills or sharp objects. Long
hear should be pulled back to prevent contact to open flames.
1.3. Biological Hazards
Biological hazards are always involved when biological samples are

handled, so each sample of blood, plasma, tissue should be considered as
potential contaminated. To prevent infection gloves must be worn when
working with the sample and hands must be washed with a disinfectant
soap after the gloves are removed. When a task has been completed, work
surface must be wiped with a disinfectant. Handle sharp object carefully,
like blades and needles.
The waste management
All chemicals and biological waste should be disposed of properly and

according to regulation. Actually they are several waste types: sharp waste
(needles, blade, broken glass, glass microtubes), that should be removed in
special containers (with rigid walls), stored safely and incinerated; biological
and chemical contaminate waste (syringe, tips, vacumtainers, eppendorf
tubes, gloves, cotton, animal tissue fragments, dead animals), that should be
placed into special bags and also incinerated, and regular waste (the syringe
protective cover paper) into the normal trash.
General laboratory rules
− Do not eat, drink, or apply cosmetics in the work area. Do not

touch the face and the mount.
11
− Wear a laboratory coat and close toe shoes. Pin long hear up. Do
not wear chains, bracelets, rings or other loose jewelry.
− Use gloves when handling hazardous chemicals and biological
specimens.
− Clean and disinfect the work area before and after laboratory
procedures.
− Wash hands after any laboratory procedures, after removing
gloves and any other time is appropriate.
− Do not use bare hands to pick up broken glass, use a brush and
dustpan.
− Wash immediately the skin and the eye (if chemicals or biological
samples are plashing on them) with large quantities of tap water.
− Report any event immediately to the supervisor.
12
General Pathophysiology
2. General Characteristics of Disease
Disease is an abnormal condition of an organism that impairs the

organism homeostasis and its ability to integrate into the environment.
Diseases always have an etiology, they may be caused by external
or internal factors, and they are accompanied by specific or unspecific
symptoms. Two animals have the same disease when they show similar
symptoms, involving the same mechanism and induced by the same
etiology.
A syndrome is the association of several symptoms that occur
together because of a common pathway. One syndrome might be found in
several disease (e.g. fever, anemia), while in many diseases several
syndromes might occur together (fever and anemia in babesiosis).
2.1. The Influence of Disease on Animal

Responsiveness to Environmental Demands
One common feature shared by many diseases is that the affected

animal is less able to respond effectively to the demands of its
environment, thus its ability to adapt is impaired.
Practical activities for students

In the present experiment one sick animal and a healthy one will be
evaluated, regarding the ability to responds to the demands of the
environment.
a) Behavioral investigation - open field test.
The open field test (OFT) is commonly used to measure general

locomotor activity and willingness to explore in rodents (qualitative and
quantitative). The open field is an arena with walls to prevent escape.
Commonly, the field is marked with a grid, and square crossings, rearing,
and time spent moving are used to assess the activity of the rodent. The
OFT is also often used to assess anxiety by including additional measures
of micturition and defecation, time spent in the center of the field, and the
first few minutes of activity. The test in association with other tests is often
used to assess the sedative or stimulant effects of pharmacological agents.
Perform the test in a simplified version as it follow:
- release the rat in the centum of an arena from which the animal cannot
escape, then observe the animal for 3 minutes and record:
- a) the time in which animal moves.
- b) the number of defecations and micturition
- c) the number of rearings on the back legs
b) Clinical investigation
Evaluate the general health status of animals, record the heart rate,
respiratory rate and internal body temperature. Additionally identify any
changes in clinical condition.
c) Hematological investigation
Draw out a blood sample from each animal and then determine the
complete blood count according to hematology techniques described in
Annex I.
Record all values in the tables below (Table 1):
14
Table 1. Disease related changes

a) Behavioral investigation (Open field test)
Rat Movement Defecations Micturitions Rearings
Time (sec) (nr.) (nr.) (nr)
Healthy
Diseased
b) Clinical investigation
Rat Hart rate Resp rate Temp ºC Other changes
(Beats/min) (Res/min)
Healthy
Diseased
c) Hematological investigation
Rat RBC HGB HCT MCV MCH MCHC
6
(10 /µl) (g/dl) (%) (fl) (pg) (g/dl)
Healthy
Diseased
Rat WBC N% E% B% M% L%
3
10 /µl
Healthy
Diseased
Control questions
1. What are the main characteristics of disease?
2. What are the consequences of disease on affected individual?
3. Which are the criteria used for disease diagnosis?
15
2.2. Pathogenesis of Environmental Factors
2.2.1. Local Action of the Heat: Thermal Burns
Heat effect General - hyperthermia – caloric shock

Local - burn
A burn is a type of injury to the skin caused by heat. Most burns

only affect the skin (epidermal tissue and dermis). Rarely deeper tissues,
such as muscle, bone, and blood vessels can also be injured. Managing
burns is important because they are common, painful and can result in
disfiguring and disabling scarring. Burns can be complicated by shock,
infection, multiple organ dysfunction syndrome, electrolyte imbalance and
respiratory distress. Large burns can be fatal, but modern treatments, have
significantly improved the prognosis. The severity of burn is influenced by
the intensity of heat and the contact time
Two main classification systems exist. The traditional system

divided burns in first-, second-, or third-degree. This system is however
being replaced by one reflecting the need for surgical intervention. The
burn depths are described as superficial, superficial partial-thickness, deep
partial-thickness, or full-thickness (Table 2).
Classification by degree:
Combustio erythematosa- First-degree burns are usually limited
to redness (erythema), a white plaque and minor pain at the site of injury.
These burns involve only the epidermis. Most sunburn can be included as
first-degree burns.
16
Combustion bullosa - Second-degree burns manifest as

erythema with superficial blistering of the skin, and can involve more or less
pain depending on the level of nerve involvement. Second-degree burns
involve the superficial (papillary) dermis and may also involve the deep
(reticular) dermis layer. Deep dermal burns usually take more than three
weeks to heal, in some cases severe hypertrophic scarring can result.
Table 2. Classification by thickness

Nomenclature Traditional Depth Clinical
nomenclature
Superficial first degree Epidermis Erythema,
thickness involvement significant pain,
lack of blisters
Partial second Superficial Blisters, clear
thickness – degree (papillary) fluid, and pain
superficial dermis
Partial second Deep (reticular) Whiter
thickness – degree dermis appearance or
deep fixed red
staining (no
blanching),
reduced
sensation
Full thickness third degree Epidermis, Charred or
Dermis, and leathery,
complete thrombosed
destruction to blood vessels.
subcutaneous
fat, scar
formation and
minimal pain.
Combustio gangrenosa - Third-degree burns occur when the

epidermis is lost with damage to the subcutaneous tissue. Burn victims will
exhibit charring and extreme damage of the epidermis (necrosis), and,
sometimes, hard scar will be present. These burns are not painful, as all
the nerves have been damaged by the burn and are not sending pain
17
signals; however, all third-degree burns are surrounded by first and

second-degree burns, which are painful.
Classification by thickness
A newer classification of "Superficial Thickness", "Partial Thickness"
(which is divided into superficial and deep categories) and "Full Thickness"
relates more precisely to the epidermis, dermis and subcutaneous layers of
skin and is used to guide treatment and predict outcome.
Burns can also be assessed in terms of total body surface area
(TBSA), which is the percentage affected by partial thickness or full
thickness burns (erythema/superficial thickness burns are not counted).
Burns of 15% (or greater) are potentially life threatening injuries (because
of the risk of hypovolemic shock) and should have formal fluid.
2.2.2. Local Action of the Cold: Frostbites
Cold effect General - hypothermia

Local - frostbite
Frostbite (congelatio in medical terminology) is the medical

condition where localized damage is caused to skin and to other tissues
due to extreme cold. Frostbite is most likely to happen in body parts
farthest from the heart and those with large exposed areas. The initial
stages of frostbite are sometimes called "frostnip".
At or below 0◦ C, blood vessels close to skin start to constrict. This
constriction helps to preserve core body temperature. In extreme cold, or
when the body is exposed to low temperatures for long periods, this
protective strategy can reduce blood flow in some areas of the body to
dangerously low levels. This lack of blood leads to the eventual freezing
18
and death of skin tissue in the affected areas. There are four degrees of
frostbite. Each of these degrees has varying degrees of pain.
Congelatio erythematosa First degree frostbite

This is called frostnip and this only affects the surface skin, which is frozen.
On onset there is itching and pain, and then, the skin develops white and
yellow patches and becomes numb. The area affected by frostnip usually
does not become permanently damaged because only the skin's top layers
are affected. Long-term sensitivity to both heat and cold can sometimes
happen after suffering from frostnip.
Congelatio bullosa Second degree frostbite

If freezing continues, the skin may freeze and harden, but the deep tissues
are not affected and remain soft and normal. Second degree injury usually
blisters 1-2 days after becoming frozen. The blisters may become hard and
blackened the deep frostbite results in areas of purplish blisters which turn
black and which are generally blood-filled.
Congelatio gangrenosa Third degree frostbite

If the area freezes further, deep frostbite occurs. The muscles, tendons,
blood vessels, and nerves will all freeze. The skin suffers necrosis, is hard,
feels waxy and use of the area is lost temporarily, and in severe cases,
permanently. Nerve damage in the area can result in a loss of feeling. This
extreme frostbite may result in fingers and toes being amputated if the area
becomes infected with gangrene.
Activities for students

Watch the movies and graphics that describe the types of thermal
burns and frostbites.
19
Control questions
1. Detail the classification of thermal burns.
2. Describe the anatomo-clinical characteristics of frostbites.
3. Detail the mechanism responsible for first-degree frostbite.
2.2.3. Experimental Photosensitization
Photosensitization is a clinical condition in which skin (areas

exposed to light and lacking significant protective hair, wool, or
pigmentation) is hyperreactive to sunlight due to the presence of
photodynamic agents.
Photosensitization requires three factors:

1. UV light,
2. the photodynamic pigment,
3. the process is intensified by the lack of melanic pigment -
albinism.
Molecules of photosensitizing agents present in the skin are
energized by light, than, when the molecules return to the less energized
state, the released energy generates reactive oxygen species (ROS). ROS
leads to mast cells degranulation and the production of inflammatory
mediators. Photosensitization can be difficult to differentiate clinically from
actual sunburn.
General consideration about UV activity
Sunlight is composed of infrared rays (700-20,000 nm), visible rays
(700-400 nm), and UV (400-100 nm). The most dangerous UV light is UVB
(290-320). Photodynamic chemicals may react with longer wavelengths.
The damage of the skin by UV light can be acute - sunburn
erythema, or chronic - solar dermatosis and neoplasia.
20
Photosensitization is often classified according to the source of the

photodynamic pigment. These categories are primary or type I
photosensitivity, type II aberrant endogenous pigment synthesis
photosensitivity, and type III or secondary (hepatogenous) photosensitivity.
Type I (primary photosensitization) occurs when the photodynamic
agent is absorbed either through the skin or from the GI tract unchanged,
reaching the skin in its native form. Example of primary photosensitizers
are hypericin (from Hypericum perforatum [St. John’s wort]) Species of
Trifolium spp., Medicago spp. (clovers and alfalfa), Erodium , Polygonum ,
and Brassica have been incriminated as primary photosensitizers.
Additionally, some coal tar derivatives, phenothiazine, sulfonamides, and
tetracyclines have induced primary photosensitivity.
Type II (photosensitivity due to aberrant pigment metabolism) is
known to occur in both cattle and cats. In this syndrome, the
photosensitizing porphyrin agents are endogenous pigments that arise from
inherited or acquired defective functions of enzymes involved in heme
synthesis. Bovine congenital erythropoietic porphyria and bovine
erythropoietic protoporphyria are the most commonly reported diseases in
this category.
Type III (secondary photosensitization) is when the photosensitizing
agent, phylloerythrin (a porphyrin), accumulates in the plasma due to
impaired hepatobiliary excretion. Phylloerythrin is derived from the
breakdown of chlorophyll by microorganisms present in the digestive tract.
Phylloerythrin, but not chlorophyll, is normally absorbed into the circulation
and is effectively excreted by the liver into the bile. Failure to excrete
phylloerythrin, due to hepatic dysfunction or bile duct lesions, increases the
amount in the circulation. Thus, when it reaches the skin, it can absorb and
release light energy, initiating a phototoxic reaction.
The clinical signs associated with photosensitivity are similar
regardless of the cause. Photosensitive animals are photophobic; they
21
develop erythema which is soon followed by edema. When exposure is

prolonged, serum exudation, scab formation and skin necrosis are marked.

To highlight the photosensitization on laboratory animals follow the
recorded experiment. An albino rat is previously inoculated 1 ml eosin
(primary photosensitization agent); then it is exposed at UV light along with
a control animal. 30 minutes later, the control animal shows no signs while
the eosin inoculated rat reveal hyperemia, photophobia and
hyperexcitability.
Control questions
1. What is the difference between photosensitization and solar
burn?
2. What factors are necessary for photosensitization
phenomena?
3. Describe the mechanisms involved in the each type of
photosensitization.
4. What are the main signs of photosensitization?
2.2.4. The Influence of General Status on Responsiveness to

Chemical Factors
The response on various external factors is highly influenced by the

activity of the nervous system. In the present study the toxic effect of
strychnine will be modulated by caffeine and phenobarbital.
Strychnine is highly toxic, excitant alkaloid used as poison in small
rodents. Strychnine is a neurotoxin. It primarily affects the motor nerves in
the spinal cord which control muscle contraction. Strychnine is an
antagonist of glycine and acetylcholine, which are known to be homologous
to the glycine receptor, which acts primarily as an inhibitor. Therefore, the
22
inhibiting effect of glycine is reduced, so nerve impulses are triggered with

lower levels of neurotransmitters. The common symptoms of strychnine
poisoning are: opisthotonus (hyperextension), trismus, and then
convulsions affect all the muscles, extension, dyspnea, cyanosis,
polypneea, oligopnea, the convulsions appear as crises triggered by small
stimulus like noise and light. Then the convulsions get more frequent; death
usually occurs due to asphyxia as respiration is affected by muscle spasm.
Self-consciousness is maintained until the dead.
Caffeine is a central nervous system and metabolic stimulant. The
principal mode of action is as a nonselective antagonist of adenosine
receptors, brain adenosine acts to protect the brain by suppressing neural
activity.
Phenobarbital is included in barbiturates class. Barbiturates act as
central nervous system depressants and can therefore produce a wide
spectrum of effects, from mild sedation to total anesthesia. The principal
mechanism of action of barbiturates is believed to be their affinity for the
GABA receptor (the principal inhibitory neurotransmitter in the nervous
system).

Follow the recorded experiment. The experiment was carried out on
tree male Wistar rats (approx. 250g b.w.);
1 - the first, was inoculated i.m. with 0.3 ml phenobarbital (10%),
2 - the second was inoculated i.m. with 0.25 ml caffeine (25%),
3 - the last remained non inoculated yet.
20 minutes later, all three rats were inoculated with 0.5 ml
strychnine 0.2%.
Expected results: The caffeine and strychnine injected rat is the first
who exhibit the symptoms of strychnine poisoning, followed by those
injected by strychnine only. The caffeine preinjected animal dies, also, the
23
first, sun followed by the second animal. The phenobarbital was

responsible for deep narcosis, the animal was not affected by the
strychnine, and several hours later it wakes up and survived the strychnine
administration.
Control questions
1. What are the symptoms of strychnine poisoning?
2. How was influenced the evolution of strychnine poisoning by
caffeine?
3. Explain the antagonist effect of phenobarbital on strychnine
poisoning. Could be the phenobarbital the medication of choice
in strychnine poisoning?
24
3. Hemodynamic Disorders
3.1. Peripheral hemodynamic disorders
3.1.1. Hyperemia and congestion
Hyperemia is an active process resulting from increased blood flow

due to arteriolar dilatation-for example, at sites of early inflammatory stage
or in skeletal muscle during exercise. The affected tissue is redder and
warm because of presence of oxygenated blood.
Congestion is a passive process resulting from impaired venous

return from a tissue. It may occur systemically, as in cardiac failure, or it
may be local, resulting from an isolated venous obstruction. The tissue has
a blue-red color (cyanosis), especially as worsening congestion leads to
accumulation of deoxygenated hemoglobin in the affected tissues.
Congestion of capillary beds is closely related to the development of
edema, so that congestion and edema commonly occur together. In long-
standing congestion, called chronic passive congestion, the stasis of poorly
oxygenated blood causes chronic hypoxia, which can result in parenchymal
cell degeneration or death.
3.2.2. Edema
Edema signifies increased fluid in the interstitial tissue spaces. In

addition, depending on the site, collections of fluid in different body cavities
are variously designated hydrothorax, hydropericardium, or
25
hydroperitoneum (ascites). Anasarca is a severe and generalized edema

with profound subcutaneous tissue swelling. Edema may represent a stage
in inflammation stage or it may have non inflammatory causes.
In general, the opposing effects of vascular hydrostatic pressure
and plasma colloid osmotic pressure are the major factors that govern
movement of fluid between vascular and interstitial spaces. Normally, the
exit of fluid into the interstitium from the arteriolar end of the
microcirculation is nearly balanced by inflow at the venular end; a small
residual amount of excess in interstitial fluid is drained by the lymphatics.
Either increased capillary pressure or diminished colloid osmotic
pressure can result in increased interstitial fluid. As extravascular fluid
accumulates in either case, the increased tissue hydrostatic and plasma
colloid osmotic pressures eventually achieve a new equilibrium and water
reenters the venules. Excess interstitial edema fluid is removed by lymphatic
drainage, ultimately returning to the bloodstream via the thoracic duct; clearly,
lymphatic obstruction (e.g., due to scarring or tumor) can also impair fluid
drainage and cause edema. Finally, a primary retention of sodium (and its
obligatory associated water) in renal disease also results in edema.
The edema fluid occurring in hydrodynamic derangements is typically a
protein-poor transudate, with a specific gravity below 1.012. Conversely,
because of the increased vascular permeability, inflammatory edema is a
protein-rich exudate with a specific gravity that is usually over 1.020.
Factors responsible for non-inflammatory edema:
Increased Hydrostatic Pressure

A localized increase in intravascular pressure may result from
impaired venous return, for example, secondary to deep venous thrombosis
in the lower extremities with edema restricted to the affected leg.
26
Generalized increases in venous pressure, with resultant systemic

edema, occur most commonly in congestive heart failure affecting right
ventricular cardiac function. Although increased venous hydrostatic
pressure is important, the pathogenesis of cardiac edema is more complex.
Congestive heart failure is associated with reduced cardiac output and
therefore reduced renal perfusion. Renal hypoperfusion in turn triggers the
renin-angiotensin-aldosterone axis, inducing sodium and water retention by
the kidneys (secondary aldosteronism). This is intended to increase
intravascular volume and thereby improve cardiac output with restoration of
normal renal perfusion. However, if the failing heart cannot increase cardiac
output, the extra fluid load results in increased venous pressure and,
eventually, edema. Unless cardiac output is restored or renal water
retention reduced (e.g., by salt restriction, diuretics, or aldosterone
antagonists), a cycle of renal fluid retention and worsening edema ensues.
Although discussed here in the context of edema in congestive heart
failure, it should be understood that salt restriction, diuretics, and
aldosterone antagonists are also of value in the management of
generalized edema resulting from a variety of other causes.
Reduced Plasma Osmotic Pressure. This can result from

excessive loss or reduced synthesis of albumin, the serum protein most
responsible for maintaining colloid osmotic pressure. An important cause of
albumin loss is the nephrotic syndrome, characterized by a leaky
glomerular capillary wall and generalized edema. Reduced albumin
synthesis occurs in the setting of diffuse liver diseases (e.g., cirrhosis; or as
a consequence of protein malnutrition. In each case, reduced plasma
osmotic pressure leads to a net movement of fluid into the interstitial
tissues and a resultant plasma volume contraction. Predictably, with
reduced intravascular volume, renal hypoperfusion with secondary
aldosteronism follows. Unfortunately, the retained salt and water cannot
27
correct the plasma volume deficit, because the primary defect of low serum
proteins persists. As with congestive heart failure, edema precipitated by
low protein is exacerbated by secondary salt and fluid retention.
Lymphatic Obstruction. Impaired lymphatic drainage and

consequent lymph edema is usually localized; it can result from
inflammatory or neoplastic obstruction.
Sodium and Water Retention. These are clearly contributory

factors in several forms of edema; however, salt retention may also be a
primary cause of edema. Increased salt, with the obligate accompanying
water, causes both increased hydrostatic pressure (due to expansion of the
intravascular fluid volume) and diminished vascular colloid osmotic
pressure. Salt retention may occur with any acute reduction of renal
function,
3.2.3. Hemorrhage
Hemorrhage generally indicates extravasation of blood due to

rupture of blood vessels. Hemorrhage may be external or may be enclosed
within a tissue; the accumulation is referred to as a hematoma.
Types of hemorrhage.
Petechiae- minute (1- to 2-mm) hemorrhages into skin, mucous
membranes, or serosal surfaces - locally increased intravascular pressure,
occurs in low platelet counts (thrombocytopenia), defective platelet
function, or clotting factor deficiencies.
Purpuras - (3- to 5-mm) the same as petechiae and there is
vascular inflammation (vasculitis), or increased vascular fragility.
28
Ecchymoses -(1- to 2-cm subcutaneous hematomas) The

erythrocytes in these local hemorrhages are degraded and phagocytized by
macrophages; the hemoglobin (red-blue color) is then enzymatically
converted into bilirubin (blue-green color) and eventually into hemosiderin
(golden-brown).
3.2.4. Thrombosis
Trombosis is blood clotting inside the vascular wall, in living animals.

Three primary factors influence the predisposition to thrombus formation, the
so-called Virchow’s triad: (1) endothelial injury (or dysfunction), (2)
hemodynamic changes (stasis or turbulence of blood flow), and (3) blood
hypercoagulability.
Endothelial injury is the dominant influence and by itself can lead to
thrombosis. It is particularly important in thrombus formation in the heart
and arterial circulation, for example, within the cardiac chambers when
there has been endocardial injury (e.g., myocardial infarction or valvulitis),
over ulcerated plaques in severely atherosclerotic arteries, or at sites of
traumatic or inflammatory vascular injury. Endothelium does not need to be
denuded or physically disrupted to contribute to the development of
thrombosis; any perturbation in the dynamic balance of prothrombotic and
antithrombotic effects can influence local clotting events. Thus, significant
endothelial dysfunction may occur from the hemodynamic stresses of
hypertension, turbulent flow over scarred valves, or bacterial endotoxins.
Even relatively subtle influences such as homocystinuria,
hypercholesterolemia, radiation, or products absorbed from cigarette
smoke may be sources of endothelial injury and dysregulation. Regardless
of the cause, physical loss of endothelium leads to exposure of
subendothelial collagen (and other platelet activators), adherence of platelets,
release of tissue factor, and local depletion of PGI2 and PA. Dysfunctional
29
endothelium may elaborate greater amounts of procoagulant factors (e.g.,

adhesion molecules to bind platelets, tissue factor, PAI, etc.) and smaller
amounts of anticoagulant effectors (e.g., thrombomodulin, PGI2)
Alterations in normal blood flow. Turbulence contributes to arterial
and cardiac thrombosis by causing endothelial injury or dysfunction, as well
as by forming countercurrents and local pockets of stasis; stasis is a major
factor in the development of venous thrombi. Normal blood flow is laminar
such that the platelet elements flow centrally in the vessel lumen separated
from the endothelium by a slower-moving clear zone of plasma. Stasis and
turbulence therefore (1) disrupt laminar flow and bring platelets into contact
with the endothelium, prevent dilution of activated clotting factors by fresh-
flowing blood, retard the inflow of clotting factor inhibitors and permit the build-
up of thrombi, and promote endothelial cell activation, predisposing to local
thrombosis, leukocyte adhesion, and a variety of other endothelial cell effects.
Hypercoagulability generally contributes less frequently to
thrombotic states but is an important (and interesting) component in the
equation. It is loosely defined as any alteration of the coagulation pathways
that predisposes to thrombosis, and it can be divided into primary (genetic)
and secondary (acquired) disorders.
Primary (Genetic) - factor V mutations (Leiden), prothrombin
mutation, antithrombin III deficiency.
Secondary (Acquired) - Tissue damage (surgery, fracture, burns),
disseminated intravascular coagulation.

Watch the movies and graphics that describe the peripheral
circulatory disorders described above.
Control questions
1. Explain the difference between congestion and hyperemia.
30
2. Give some examples of disorders accompanied by hyperemia or

congestion.
3. What is edema?
4. Describe several pathological circumstances associated with
non-inflammatory edema.
5. What are the characteristics of the fluid accumulated in non-
inflammatory edema?
6. What are the main types of hemorrhage?
7. Explain the Virchow’s triad influence on thrombosis.
3.2. Shock
Shock, or "cardiovascular collapse," is disproportion between the

effective circulating blood volume and vascular volume, it the final common
pathway for a number of potentially lethal clinical events, including severe
hemorrhage, extensive trauma or burns, large myocardial infarction,
massive pulmonary embolism and microbial sepsis.
Regardless the underlying pathologic lesion, shock is associated
with hypotension responsible by hypoperfusion and, consequently, cellular
hypoxia and tissue acidosis.
According to etiology and pathogenesis, shock may be grouped into

several categories: cardiogenic, hypovolemic, septic, toxic, neurogenic and
anaphylactic.
Stages of shock
Shock is a progressive disorder that if it is uncorrected leads to

death and it tends to evolve through three general stages. These stages
31
have been documented most clearly in hypovolemic shock but are common
to the others forms as well:
a) nonprogressive stage (compensated) in which reflex compensatory
mechanisms are activated and perfusion of vital organs is maintained,
b) progressive stage (decompensate) characterized by tissue
hypoperfusion and onset of worsening circulatory and metabolic imbalance,
c) irreversible stage that sets in after the body has incurred cellular
and tissue injury so severe that even if the hemodynamic defects are
corrected, survival is not possible.
Nonprogressive phase of shock, - various neurohumoral

mechanisms help maintain cardiac output and blood pressure. These include
baroreceptor reflexes, release of catecholamines, activation of the renin-
angiotensin axis, antidiuretic hormone release, and generalized sympathetic
stimulation. The general effects are tachycardia, peripheral vasoconstriction,
and renal conservation of fluid. Coronary and cerebral vessels are less
sensitive to the sympathetic response and thus maintain relatively normal
caliber, blood flow and oxygen delivery to their respective vital organs.
Progressive phase - during which, there is widespread tissue

hypoxia. In the setting of persistent hypoxia, intracellular aerobic respiration
is replaced by anaerobic glycolysis with excessive production of lactic acid.
The resultant metabolic lactic acidosis blunts the vasomotor response;
arterioles dilate, and blood begins to pool in the microcirculation –
vasoplegia. Peripheral pooling not only worsens the cardiac output but also
puts endothelial cells at risk of developing anoxic injury with subsequent
disseminated intravascular coagulation (DIC). As consequence of tissue
hypoxia, vital organs are affected, and they start to fail.
32
Irreversible stage - widespread cell injury is reflected in lysosomal

enzyme leakage, further aggravating the shock state. Myocardial contractile
function worsens, in part because of nitric oxide synthesis. If ischemic bowel
allows intestinal flora to enter the circulation, endotoxic shock may also be
superimposed. At this point, the patient has renal insufficiency and marked
by a progressive fall in urine output as well as severe fluid and electrolyte
imbalances. The complete renal shutdown is due to acute tubular necrosis.
The downward clinical spiral inevitably culminates in death.
Clinical Course. The clinical manifestations depend on the

precipitating insult. In hypovolemic and cardiogenic shock, the patient
presents with hypotension; a weak, rapid pulse; tachypnea, hypothermia;
and cutaneous vasoconstriction, is responsible for the characteristic
coolness and pallor of skin in shock (although septic shock may initially
cause cutaneous vasodilation and thus present with warm, flushed skin).
Capillary refill time is also prolonged as sign of vasoplegia. The self-
consciousness is maintained in the non-progressive phase, latter, in the
progressive phase the patient may become confused.

Watch the movies and graphics that describe the main shock types
and stages described before.
Control questions
1. What is shock?
2. Which are the most significant functional changes occurring in
all shock types?
3. What are the shock types classified according to
pathophysiologic mechanism?
4. What are the main stages described in shock evolution? What
are their main characteristics?
33
4. Disease of Immunity
4.1. Phagocytosis
Phagocytosis (from Ancient Greek phagein - to devour, kytos - cell,

and –osis - process) is the cellular process of engulfing solid particles by
the cell membrane. Phagocytosis is a specific form of endocytosis involving
the vesicular internalization of solid (phagosome) such as bacteria, and is,
therefore, distinct from other forms of endocytosis such as the vesicular
internalization of various liquids.
Phagocytosis is involved in the nonspecific cellular mediated
immunity, it is a major mechanism used to remove bacteria, dead tissue
cells and small mineral.
Phagocytosis consists of three distinct but interrelated steps: (1)

recognition and attachment of the particle to the ingesting leukocyte; (2)
engulfment, with subsequent formation of a phagocytic vacuole; and (3)
degradation of the ingested material.
1. Recognition and attachment of leukocytes to most

microorganisms is facilitated by serum proteins generically called opsonins.
Opsonins bind specific molecules on microbial surfaces and in turn facilitate
binding with specific opsonin receptors on leukocytes. The most important
opsonins are immunoglobulin G (IgG) molecules (specifically the Fc portion
of the molecule), the C3b fragment of complement (and its stable C3bi
form), and plasma carbohydrate-binding lectins called collectins, which bind
to microbial cell wall sugar groups. In many cases, the binding of IgG is
responsible for triggering the activation of the complement cascade that

results in deposition of the C3b fragments on the targeted particle;
however, a number of stimuli (e.g., microbial surfaces) can directly induce
complement activation by an IgG-independent alternative pathway. The
corresponding receptors on leukocytes are the Fc receptor (FcR) for IgG,
the complement receptors 1, 2, and 3 (CR1, 2, and 3) for complement
fragments, and C1q for the collectins.
2. Engulfment - pseudopods are extended around the object,

eventually forming a phagocytic vacuole. The membrane of the vacuole
then fuses with the membrane of a lysosomal granule, resulting in
discharge of the granule's contents into the phagolysosome and
degranulation of the leukocyte.
3. Degradation is the final step in the phagocytosis, microbial killing,

it can be oxygen-dependent or oxygen-independent. Oxygen-dependent
degradation depends on NADPH and the production of ROS (reactive
oxygen species). Oxygen-independent degradation depends on the release
of granules, containing proteolytic enzymes and other antimicrobial
peptides which are present in these granules.
a. Oxygen-dependent degradation is accomplished largely by

reactive oxygen species. Phagocytosis stimulates an oxidative burst
characterized by a sudden increase in oxygen consumption, glycogen
catabolism (glycogenolysis), increased glucose oxidation, and production of
reactive oxygen metabolites. The generation of the oxygen metabolites is
due to rapid activation of a leukocyte oxidase, which oxidizes NADPH
(reduced nicotinamide adenine dinucleotide phosphate). In the process, it
converts oxygen to superoxide ion. The quantities of hydrogen peroxide
produced are generally insufficient to effectively kill most bacteria, this is
36
why the lysosomes of neutrophils (called azurophilic granules) contain the

enzyme myeloperoxidase (MPO), and in the presence of a halide such as
Cl-, myeloperoxidase converts H2O2 to HOCl· (hypochlorous radical). HOCl·
is a powerful oxidant and antimicrobial agent (NaOCl is the active
ingredient in chlorine bleach) that kills bacteria by halogenation, or by
protein and lipid peroxidation. Fortunately, NADPH oxidase is active only
after translocation of its cytosolic subunit to the membrane of the
phagolysosome; thus, the reactive end products are generated only within
that compartment. After the oxygen burst, H2O2 is eventually broken down
to water and O by the actions of catalase, and the other reactive oxygen
species are also degraded. The dead microorganisms are then degraded
by the action of the lysosomal acid hydrolases.
b. Oxygen-independent degradation other constituents of the
leukocyte granules are capable of killing bacteria and other infectious
agents. These include bactericidal permeability-increasing protein (causing
phospholipase activation and membrane phospholipids degradation),
lysozyme (causing degradation of bacterial coat oligosaccharides), major
basic protein (an important eosinophils granule constituent with potent
cytotoxicity for parasites), and defensins (peptides that kill microbes by
forming holes in their membranes).

Watch the movies and graphics that describe the phagocytosis.
Control questions
1. What is the function of phagocytosis in superior organisms?
2. Which are the main steps of apoptosis?
3. How is facilitated the recognition of antigen during phagocytosis
process?
37
4.2. Mechanisms of immune mediated injury

(Hipersensitivity reactions)
The hypersensitivity reactions are traditionally subdivided into four

types; three are variations on antibody-mediated injury, whereas the fourth
is cell mediated:
Type I disease results from IgE antibodies adsorbed on mast cells
or basophils; when these IgE molecules bind their specific antigen
(allergen), they are triggered to release vasoactive amines and other
mediators that in turn affect vascular permeability and smooth muscle
contraction in various organs.
Type II disorders are caused by humoral antibodies that bind to
fixed tissue or cell surface antigen and cause a pathologic process by
predisposing cells to phagocytosis or to complement-mediated lysis.
Type III disorders are best thought of as "immune complex
diseases"; antibodies bind antigens to form large antigen-antibody
complexes that precipitate in various vascular beds and activate
complement. The immune complexes and complement activation
fragments also attract neutrophils. Ultimately, it is the activated complement
and the release of neutrophilic enzymes and other toxic molecules (e.g.,
oxygen metabolites) that cause the tissue damage in immune complex
disease.
Type IV disorders (also called "delayed-type hypersensitivity") are
cell-mediated immune responses where antigen-specific T lymphocytes are
the ultimate cause of the cellular and tissue injury.
38
Type I Hypersensitivity (Allergy and Anaphylaxis)
Type I hypersensitivity is a tissue response that occurs rapidly

(typically within minutes) after the interaction of allergen with IgE antibody
previously bound to the surface of mast cells and basophils in a sensitized
host. Depending on the portal of entry, type I hypersensitivity may occur as
a local reaction that is merely annoying (e.g., seasonal rhinitis or hay fever)
or severely debilitating (asthma) or may culminate in a fatal systemic
disorder (anaphylaxis).
Type I reactions are mediated by IgE antibodies. The sequence of
events begins with an initial exposure to certain antigens (allergens). The
allergen stimulates the IgE production by B cells. IgE antibodies bind to
high-affinity Fc receptors expressed on mast cells and basophils; once the
mast cells and basophils are thus "armed," the individual is primed to
develop type I hypersensitivity. Re-exposure to the same antigen leads to
mast cell degranulation with discharge of preformed or primary mediators;
another parallel set of signals induces de novo synthesis and release of
secondary mediators such as arachidonic acid metabolites and cytokines.
A type I reaction may occur as a systemic disorder or as a local
reaction. Often this is determined by the route of antigen exposure.
Systemic (parenteral) administration of protein antigens or drugs
(e.g., bee venom or penicillin) results in systemic anaphylaxis. Within
minutes of an exposure in a sensitized host, itching, urticaria (hives), and
skin erythema appear, followed in short order by profound respiratory
difficulty caused by pulmonary bronchoconstriction and accentuated by
hypersecretion of mucus. Laryngeal edema may exacerbate matters by
causing upper airway obstruction. In addition, the musculature of the entire
gastrointestinal tract may be affected, with resultant vomiting, abdominal
39
cramps, and diarrhea. Without immediate intervention, there may be

systemic vasodilatation (anaphylactic shock), and the patient may progress
to circulatory collapse and death within minutes.
Local reactions generally occur when the antigen is confined to a
particular site by virtue of the route of exposure, such as skin (contact,
causing urticaria), gastrointestinal tract (ingestion, causing diarrhea), or
lung (inhalation, causing bronchoconstriction). The common forms of skin
and food allergies, hay fever, and certain forms of asthma are examples of
localized anaphylactic reactions.

Watch the movies and videos that describe the allergy.
Control questions
1. Which are the four types of hypersensitivity reaction?
2. What are the mediators of hypersensitivity reaction?
3. Which are the symptoms of systemic anaphylaxis?
4. Nominate several symptoms associated with local anaphylaxis.
4.3. Inflammation
4.3.1. General features of acute inflammation
Inflammation is a local, nonspecific; protective response intended

to eliminate the initial cause of cell injury. Inflammation accomplishes its
protective mission by diluting, destroying, or otherwise neutralizing harmful
agents (e.g., microbes or toxins). It then sets into motion the events that
eventually heal and reconstitute the sites of injury. Thus, inflammation is
also intimately interwoven with repair processes.
40
The inflammatory response includes 1) circulating cells, 2) plasma

proteins, 3) vascular wall cells, and 4) cells and extracellular matrix of the
surrounding connective tissue;
1) the circulating cells include bone marrow-derived
polymorphonuclear leukocytes (neutrophils), eosinophils, and basophils;
lymphocytes and monocytes; and platelets,
2) the circulating proteins include clotting factors, kininogens, and
complement components, largely synthesized by the liver,
3) the vascular wall cells include the endothelial cells in direct
contact with the blood, as well as the underlying smooth muscle cells that
impart tone to the vessels,
4) the connective tissue cells include sentinels to invasion such as
mast cells, macrophages, and lymphocytes, in addition to the fibroblasts
that synthesize the extracellular matrix and can proliferate to fill in a wound.
Inflammation is divided into two basic patterns.
Acute inflammation is of relatively short duration, lasting from a few
minutes up to a few days, and is characterized by fluid and plasma protein
exudation and a predominantly neutrophilic leukocyte accumulation.
Chronic inflammation is of longer duration (days to years) and is
typified by influx of lymphocytes and macrophages with associated vascular
proliferation and scarring.
Acute inflammation is the immediate and early response to injury
designed to deliver leukocytes to sites of injury. Once there, leukocytes
clear any invading microbes and begin the process of breaking down
necrotic tissues. This process has two major components:
Vascular changes: alterations in vessel caliber resulting in
increased blood flow (vasodilation) and structural changes that permit
plasma proteins to leave the circulation (increased vascular permeability)
41
Cellular events: emigration of the leukocytes from the

microcirculation and accumulation in the focus of injury (cellular recruitment
and activation).
This cascade of events in acute inflammation is integrated by local
release of chemical mediators.
They are followed by the five classic local signs of acute
inflammation: heat (calor), redness (rubor), swelling (tumor), pain (dolor)
and loss of function (functio laesa).
4.3.2. Tissue Repair
Stimuli that induce death in some cells can trigger the activation of
replication pathways in others; recruited inflammatory cells not only clean
up the necrotic debris but also elaborate mediators that drive the synthesis
of new extracellular matrix (ECM). Tissue regeneration may be achieved
throughout two different mechanisms:
1. Regeneration by parenchymal cells
2. Replacement by connective tissue (and formation of the scar)
Tissue healing involves a combination of both processes, controlled
by similar mechanisms including cell migration, proliferation, differentiation,
and matrix synthesis. Usually, regeneration of epithelium by parenchymal
cells requires an intact basement membrane (BM) matrix; if the ECM has
also been destroyed by an injury, tissues can heal only by connective
tissue generating a scar.
4.3.2.1. Scar formation
Fibrosis, or scar formation builds on the granulation tissue

framework of new vessels. The process of fibrosis occurs in three distinct
42
stages: inflammatory stage, emigration and proliferation of fibroblasts, and

scar remodeling.
Inflammatory stage – will be detailed discussed at the inflammation
chapter.
Emigration and proliferation of fibroblasts into the site of injury is
followed by deposition of ECM by these cells, and formation of granulation
tissue. Many of the growth factors, including platelet-derived growth factor
(PDGF), bFGF, and TGF-β, drive the recruitment and stimulation of
fibroblasts. The sources of these factors are the activated endothelium and
inflammatory cells (macrophages, mast cells, and lymphocytes).
Scar remodeling shifts in the composition of the ECM, it represents
a balance between ECM synthesis and degradation. The degradation of
collagens and other ECM components is accomplished by a family of
metalloproteinases (so called because they are dependent on zinc ions for
their activity). These enzymes are produced by a variety of cell types
(fibroblasts, macrophages, neutrophils, synovial cells, and some epithelial
cells), and their synthesis and secretion are regulated by growth factors,
cytokines, phagocytosis, and even physical stress Their synthesis is
inhibited by TGF-β and may be suppressed pharmacologically with
steroids. They are typically elaborated as inactive (zymogen) precursors
that must be first activated; this is accomplished by certain chemicals (e.g.,
HOCl·) or proteases (e.g., plasmin) likely to be present only at sites of
injury. In addition, activated collagenases can be rapidly inhibited by
specific tissue inhibitors of metalloproteinase (TIMPs), produced by most
mesenchymal cells. Collagenases and their inhibitors are spatially and
temporally regulated in healing wounds. They are essential in the
debridement of injured sites and in the remodeling of the ECM necessary to
repair any tissue defects.
43
4.3.2.2. Skin wound healing
This is a process involving both epithelial regeneration and the

formation of connective tissue scar and is thus illustrative of the general
principles that apply to wound healing in all tissues.
Primary union or healing by first intention is the healing of a
clean, uninfected surgical incision approximated by surgical sutures. The
incision causes only focal disruption of epithelial basement membrane
continuity and death of a relatively few epithelial and connective tissue
cells. As a result, epithelial regeneration predominates over fibrosis. The
narrow incisional space rapidly fills with fibrin-clotted blood; dehydration at
the surface produces a scab to cover and protect the healing repair site.
Latter, in 24 hours, neutrophils migrate toward the fibrin clot. Epithelial cells
from both edges begun to migrate and proliferate along the dermis,
depositing basement membrane components as they progress. The cells
meet in the midline beneath the surface scab, yielding a thin but continuous
epithelial layer. In day 5, neovascularization reaches its peak as granulation
tissue fills the incisional space. Collagen fibrils become more abundant and
begin to bridge the incision. The epidermis recovers its normal thickness as
differentiation of surface cells yields a mature epidermal architecture with
surface keratinization. In a week the leukocyte infiltrate, edema, and
increased vascularity are substantially diminished. After 1 month, the scar
comprises a cellular connective tissue, which is largely devoid of
inflammatory cells and covered by an essentially normal epidermis.
However, the dermal appendages destroyed in the line of the incision are
permanently lost.
Secondary union, or healing by second intention appears when
cell or tissue loss is more extensive, as in infarction, inflammatory
ulceration, abscess formation, or even just large wounds. As a result, there
is extensive ingrowth of granulation tissue from the wound margin, followed
44
in time by accumulation of ECM and scarring. Secondary healing exhibits

the phenomenon of wound contraction. Within 6 weeks, for example, large
skin defects may be reduced up to 5% - 10% of their original size, largely
by contraction. This process has been ascribed to the presence of
myofibroblasts, modified fibroblasts exhibiting many of the ultra-structural
and functional features of contractile smooth muscle cells.

Watch the movies and videos that describe the inflammation and
wound healing process.
Control questions
1. What are the functions of inflammatory reaction?
2. Describe the elements involved in inflammatory response.
3. What are the stages commonly described in acute inflammatory
response?
4. Enumerate the five classic signs of acute inflammatory reaction.
5. Which are the two mechanisms responsible for tissue regeneration?
6. What are the two main mechanisms involved in skin wound healing.
Point the main differences between them.
4.3.3. Differentiation between exudate and transudate

(Rivalta test)
Rivalta test is used in order to differentiate a transudate from an

exudate. The main difference between exudate and transudate consist in
protein and cell content (the proteins precipitate in acid environment).
Exudate is a result of vascular changes in inflammation
(vasodilation and increased vascular permeability); it is rich in protein of
more than 2.9 g/dL, (consequently specific gravity is more than 1.020). It is
also rich in inflammatory cells.
45
Transudate results because decreased colloid osmotic pressure, or

increased hydrostatic pressure; and it has the protein content of less than 2
g/dL (specific gravity less than 1.012) and low cell content
(see Edema and General features of inflammatory reaction).
1. Prepare 250 ml solution acetic acid 0.8% (introduce 2 ml glacial

acetic acid in a cylinder then add distilled water up to 250 ml).
2. Introduce the solution into a conical glass flask.
3. Add drop by drop the tested effusion, by using a Pasteur pipette.
If the drop dissipates, the test is negative, indicating a transudate. If the
drop precipitates, the test is positive, indicating an exudate.
Control questions
1. What are the differences between exudate and transudate?
2. Enounce several pathological conditions associated to formation
of exudate / transudate.
4.3.2. Histochemical identification of myeloperoxidase
Myeloperoxidase (MPO) is a peroxidase enzyme found within

circulating granulocytes (neutrophils, eosinophils and basophils),
monocytes, and some tissue macrophages. It is never found in
lymphocytes. MPO is stored within the azurophilic granule; it has a heme
pigment, which causes its green color in secretions rich in neutrophils, such
as pus and some forms of mucus. In the presence of a halide such as Cl-,
MPO converts H2O2 to HOCl· (hypochlorous radical). HOCl· is a powerful
oxidant and antimicrobial agent (NaOCl is the active ingredient in chlorine
46
bleach) that kills bacteria by halogenation, or by protein and lipid

peroxidation.
MPO is identified by copper method of Sato. The MPO release O

from hydrogen peroxide, which reacts to benzidine and results a
distinctively blue-colored derivative. Azurophilic granules stain in dark blue,
while the nucleus stains in red. The granulocytes, especially neutrophils
reveal a high number of azurophilic granules, the staining is present in
eosinophils and basophils, but not so intense, while monocites show low
intensity staining. No staining in lymphocytes is found.
Make a blood (or pus) smear

Fix on flame
Staining procedure
1. Cover the slide with cooper sulfate 0.5% - 1 min
Remove the solution, do not wash,
2. Cover the slide with benzidine solution (prepare immediately
before use: benzidine 0.2g, hydrogen peroxide 5% 2
drops, aqua dist. ad 100 ml). – 2 min
Caution !
Benzidine is carcinogenic substance avoid any contact to skin!
Rinse with aqua dist.
3. Cover with Safranin 1% - 2-3 min
Rinse and dry, than examine to immersion objective.
Control questions
1. What is the function of myeloperoxidase?
2. What is the distribution of the enzyme in blood WBCs?
3. Describe the principle of identification of MPO
47
4.3. Fever
Fever (pyrexia), is a nonspecific, systemic defense response, in

which body temperature is above the usual range of normal (hyperthermia),
because of the set point of the hypothalamic temperature-regulating center
becomes higher than normal, and followed by changes in plasma
biochemistry and WBCs count and differences.
It can be caused by infections, abnormalities in the brain or by toxic
substances that affect the temperature-regulating centers.
Resetting the Hypothalamic Temperature- Regulating Center in

Febrile Diseases—Effect of Pyrogens. Many proteins, breakdown
products of proteins, and certain other substances, especially
lipopolysaccharide toxins released from bacterial cell membranes, can
cause the set point of the hypothalamic thermostat to rise. Substances that
cause this effect are called pyrogens. Pyrogens released from toxic
bacteria or those released from degenerating body tissues cause fever
during disease conditions. When the set-point of the hypothalamic
temperature-regulating center becomes higher than normal, all the
mechanisms for raising the body temperature are brought into play,
including heat conservation and increased heat production. Within a few
hours after the set-point has been increased, the body temperature also
approaches this level.
Mechanism of Action of Pyrogens in Causing Fever—Role of

Interleukin-1. Some pyrogens, when injected into the hypothalamus, can
act directly and immediately on the hypothalamic temperature-regulating
center to increase its set-point. Other pyrogens function indirectly and may
require several hours of latency before causing their effects. This is true for
48
many of the bacterial pyrogens, especially the endotoxins from gram-

negative bacteria. When bacteria or breakdown products of bacteria are
present in the tissues or in the blood, they are phagocytized by the blood
leukocytes, by tissue macrophages. All these cells digest the bacterial
products and then release the substance interleukin-1—also called
leukocyte pyrogen or endogenous pyrogens into the body fluids. The
interleukin-1, on reaching the hypothalamus, immediately activates the
processes to produce the prostaglandins, mainly prostaglandin E2 (PGE2),
or a similar substance, which acts in the hypothalamus to elicit the fever
reaction. When prostaglandin formation is blocked by drugs, the fever is
either completely abrogated or at least reduced. In fact, this may be the
explanation for the manner in which aspirin reduces fever, because aspirin
impedes the formation of prostaglandins from arachidonic acid. Drugs such
as aspirin that reduce fever are called antipyretics.
Control questions
1. What are the main physiological, biochemical and hematological
changes associated with fever?
2. What is the role of PGE in fever associated symptoms?
3. What are the beneficial effects of fever?
49
Pathophysiology of Systems and Organs
5. Metabolic Disorders
5.1. Carbohydrate metabolism
Normal fasting plasma concentration is about 4-6 mmol/L (70-110

mg/dL) in monogastrics and 3-5 mmol/L (50-90 mg/dL) in ruminants. This is
plasma glucose concentration, other practices are to measure the glucose
in all blood without centrifugation, which gave values about 0.5 mmol/L less
than this (depending on PCV), because the erythrocyte glucose
concentration is significantly lower than plasma concentration.
Other units for measuring glucose are mg/100ml the transformation
is 1 mmol/L = 18 mg/dLml (C6H12O6 molecular weight = 180.16 g/mol).
Glucose measurements has a specific sample requirement – blood
must be collected into tubes containing fluoride (usually with oxalate as
anticoagulant), fluoride blocks glycolysis in red blood cells, thus preventing
consumption of glucose in sample which would otherwise be significantly
reduced within 30 minutes. Swift separation of non-fluoride plasma from the
red cells cans extent also the viability of samples if it is done as soon as the
blood was collected.
An adequately high plasma glucose concentration is essential for
normal brain function, and the body goes to quite elaborate lengths to
ensure that this is maintained. As result the glucose in the plasma at any
particular time may come from one or more sources, depending on the
current state of carbohydrate metabolism. In the post absorptive phase,
glucose is being transported from its site of uptake in the gut to the sites of
glycogen storage principally the liver and muscles. In the fasting animal
plasma glucose concentrations are maintained by mobilizing carbohydrates
from liver (hepatic glycogenolysis), but if starvation is prolonged proteolysis

(neoglucogenesis) became more important. There is a complex and tight
feedback and hormonal control over these pathways to ensure constant
plasma glucose. In normal circumstances, glucagon and growth hormone
are responsible for maintaining an adequate level, white in abnormal states
as prolonged fasting or stress, glucocorticoids and adrenaline are
important. Insulin is the only one responsible for decrease the
concentration of plasma glucose.
Hyperglycemia is less dangerous in short terms and it is common,
while, hypoglycemia is potentially life threatening because is less
commonly encountered.
Renal glucose threshold
The kidney normally allows the plasma glucose to be filtered, but is
reabsorbed in the proximal tube. However the reabsorptive capacity is
limited, and when glucose rises at 10 mmol/L (160-180 mg/dL) reabsorbtion
will be not complete and glucose will appear in urine – glycosuria. The
plasma glucose concentration above which glycosuria occurs is renal
glucose threshold. The finding of glycosuria means that plasma
concentration has been exceeded before the urine sample was collected.
Renal glycosuria is the condition when renal glucose threshold is below 10
mmol/L, it is seen in some normal individuals especially in neonates and
during pregnancy, and in proximal tubular defects (Fanconi syndrome). In
some cases of long standing diabetes mellitus, the renal glucose threshold
may rise, leading to the absence of classical polydipsia /polyuria symptoms
while plasma glucose is still uncontrolled. Other possible reason is renal
problem with the reduction in glomerular filtration rate. That way
measurement of urine glucose is not enough, it should be always followed
by measurement of plasma glucose.
52
Hyperglycemia
Non diabetic hyperglycemia
a. After a high carbohydrate meal – post absorptive peak, unusual
above than 7 mmol/L
b. Sprint exercise – adrenalin secretion – race horses and gray
hounds – approx 15 mmol/L
c. Stress – particularly severe acute stress, severe pain, restrained
animals, animals that just undergo a journey, animals under anesthesia –
adrenalin and glucocorticoids are involved 8-10, 15 mmol/L. Cats are
particular problem stress hyperglycemia may reach 20 mmol/L, difficulties
in differentiating from diabetes mellitus.
d. glucocorticoids increased activity – corticoid therapy, Cushing’s
diseases (in horses and cats can progress in diabetes mellitus). 6-8
mmol/L.
f. treatment with glucose containing fluids (administration of iv
glucoses should not be higher than renal threshold – osmotic diuresis).
Diabetes mellitus
Diabetes mellitus is a common condition in both dogs and cats, and
rarely in elderly horses. There are occasional reports of cases in other
species such as rabbits. It is due to relative insulin deficiency, and can be
divided in two categories.
Type I diabetes
In these patients there is essentially not enough insulin secretion. It
tends to occur in younger animals, due to autoimmune condition or
pancreatitis. This condition is often associated with severe ketoacidosis,
polyuria and polydipsia. It is uncommon in other animals than dogs.
53
Type II diabetes
In this condition insulin is present but is less efficient. Type II
diabetes might be primary and secondary.
Primary type II diabetes occurs in middle aged and older animals
often associated with obesity. As insulin is still present, these patients
present less severe forms of ketoacidosis. However, in practice only
minority patients tend to be controllable only by diet and oral
hypoglycemics, most of them require insulin.
Secondary type II diabetes mellitus occurs as a result of some other
hormonal factors causes resistance to insulin. In dogs, Cushing’s disease
may became a common cause of diabetes, and usually resolves with
successful treatment of the Cushing’s. Another common cause is in bitch
that becomes diabetic shortly after estrus (due to hyperglycemic effect of
progesterone). However gestational diabetes mellitus such as occurs in
women is not recognized in veterinary medicine. Cats are also susceptible
because of use of powerful anti-inflammatory steroids. In horses, in almost
all cases of diabetes mellitus are secondary to Cushing’s disease.
Diagnosis of diabetes mellitus

Urine glucose measurement is not a sensitive investigation tool;
blood sampling is easier and more precise tool. Diagnose of diabetes is
highly standardized by the WHO. This guideline is not applicable to
veterinary cases, but is useful to be aware of the protocol.
random plasma glucose: 5.5 mmol/L or less – non diabetic; 11.1
mmol/L or over – diabetic (two investigation in asymptomatic patients), in
between – investigate further.
fasting plasma glucose (15 hours fast) (70-110 mg/dL) less than 6.1
mmol/L – not diabetic, 7 mmol/L or more diabetic, in between – investigate
further.
54
oral glucose tolerance test (following overnight fast, at 2 hours after

oral glucose load 1g/kg anhydrous glucose) – less 7.8 mmol/L not diabetic,
11.1 mmol/L and over diabetic, in between – impaired glucose tolerance,
but not yet diabetes.
Additional investigation in diabetes mellitus

Glycated proteins - Glucose reacts with free amino groups on
virtually all proteins to form covalent glycated proteins. Extend of the
glycation depends on the half-life of the protein and the intensity and time
span of hyperglycemia (longer and higher the hyperglycemia the higher
quantity of glycated proteins). Glycation of structural proteins are
responsible for diabetic angiopathy. Glycated proteins useful for diagnosis
and monitoring of diabetic patients are fructosamine and glycated
hemoglobin.
Fructosamines are ketoamine compounds formed when glucose
reacts with amino groups on plasma proteins, mainly albumin. Normal non
diabetic animals are under about 300 µmol/L. Plasma fructosamine
concentration is useful to measure the average blood glucose
concentration over the lifespan of these proteins with is about 2 weeks. It is
useful for diagnosis and monitoring of diabetes, 400µmol/L is a reasonable
goal for a diabetic patient. In human diabetes monitoring is rarely used, it
has been replaced by glycated hemoglobin.
Glycated hemoglobin, once formed, stays within the red blood cells
for its lifetime, in dogs this translates to a measure of average plasma
glucose for the previous 3 mounts. Glycated hemoglobin methods have
been validated for dogs but the technique is more complex then
fructosamine measurement and the assay is not widely used.
Ketone bodies (acetone, acetoacetate and beta hydroxybutyrate) –
normal plasma levels of beta hydroxybutyrate concentration is under 1
55
mmol/L, and is often undetectable. Older methods of measurements were

sensitive only for acetone, but now beta hydroxybutyrate is more commonly
used. Ketosis develops because of deficiency of glucose passing through
the glycolytic pathway in the cells. Therefore, fatty acids are being utilized
as an alternative body fuel, but the lipid catabolism requires carbohydrates,
without carbohydrates lipid catabolism forms ketone bodies. They may
appear in two situations: diabetic ketoacidosis and absolute carbohydrate
deficiency.
Routine plasma biochemistry (to check liver and renal function) is
required.
Hypoglycemia
Hypoglycemia is responsible for symptoms similar to cerebral
anoxia (faintness, sometimes convulsing fits and coma). These symptoms
are visible when plasma glucose drops under 3 mmol/L, horses are more
tolerant (2 mmol/L).
Insulin induced hypoglycemia
over dosage of diabetic patients, or of the dog may fail to eat after
insulin was given.
insulinoma – a tumor of the islet cells of the pancreas, is not
uncommon in dogs. Unlike the humans, in dog these tumors are usually
malignant. Hyperinsulinism is commonly followed by obesity.
islet cell hyperplasia, nonmalignant, uncommon, but it may occur
following prolonged steroid exposure.
Fasting hypoglycemia
ketonemia / pregnancy toxemia in ruminants, consequence of the
routes of carbohydrate metabolism in these species. Hypoglycemia is
associated to late pregnancy (sheep) and early peek of lactation (cows) in
56
twin fetuses or high milk production. Hypoglycemia as prolonged starvation

does not occur in monogastrics.
b. hypoglycemia of acute illness
5.2. The plasma proteins
Normal total plasma protein is around 60-80 g/L (a little lower in dogs).
Normal albumin is around 25-35 g/L (dogs and cats values are
lower than large animals).
Plasma contains a mixture of proteins – albumin, globulins
(immunoglobulins and other proteins loosely grouped under this name),
enzymes, specific transfer proteins (transferrin), protein hormones and
clotting factors. Because this heterogeneity, molar concentration cannot be
given. Most are synthesized in liver from amino acids. All have different
specific functions, but as a group, they function to maintain the osmotic
pressure of the plasma. Only the largest are completely trapped into the
blood stream. There is also a secondary circulation of proteins (especially
albumin) out of the capillaries into tissue fluids then back into the blood
stream via lymph.
5.2.1. Changes in total protein concentration
Total protein is usually measured by the biuret method, but

refractometry is useful if an emergency result is required. Separation of the
protein fractions may be done by measuring albumin separately and
subtracting this from the total protein concentration result to give the
globulin concentration.
Other specific proteins generally known as acute phase proteins,
considered to be markers for acute inflammatory diseases, are alpha 1
57
antitrypsin, alpha 1 acid glycoprotein, C reactive protein (CRP), alpha 2

macroglobulin, ceruloplasmin, haptoglobin and fibrinogen.
Changes in total protein concentration may be relative and absolute.
Relative increase in protein changes in plasma protein levels means that
there are no changes in total amount of plasma protein, but in total amount
of water contained in blood, therefore plasma proteins are concentrated or
diluted. In that case the protein fractions are increase approximately in the
same percent. Absolute changes involve increased or decreased plasma
proteins levels, and involve dysproteinemia - significant changes among
protein fractions.
Relative increase in protein concentration occurs in relative water
deficiency. As proteins are not completely confined into the circulation is
theoretically a poorer measure than PCV, but in practice has certain
advantages. It is not affected by splenocontraction and in dogs has a
narrower range than PCV which makes easier to assess the dehydration in
one sample.
Absolute increase in protein concentration occurs in chronic
inflammatory and immune mediated diseases that can cause increases in
globulin fractions, particularly the alpha globulins. Paraproteinemia
(increased level of abnormal proteins) is almost always associated with a
malignancy, where a proliferation of a single clone of immunoglobulin
producing cells leads to an abnormally large amount of one single
immunoglobulin. This condition, which is most usually associated with this
finding, is a plasma cell (or multiple) myeloma, but occasionally
lymphosarcoma or leukemia is involved.
Relative decrease in protein concentration occurs in overhydratation
which is uncommon, but can be induced iatrogenically. Sometimes
diagnostic errors may be involved. Again, the protein fractions remain
unchanged.
Absolute decrease in protein concentration may have two main
reasons: excessive loss of proteins and decreased protein synthesis.
58
Excessive loss of proteins involve mainly the albumin fraction,

because they have the smallest molecular weight, so this conditions often
presents primarily as hypoalbuminemia.
a) Renal protein loss – nephrotic syndrome, glomerulonephritis,
amyloidosis. This is easily confirmed by testing urine for protein, but a
concurrent cystitis may sometimes confuse the issue, when measurement
of urine protein/creatinine ratio will clarify the situation. The albumin is
always markedly depressed but the globulin can often be normal or
somewhat raised (mainly in glomerulonephritis).
b. Intestinal protein loss (protein lousing enteropathy). In large
animals, horses, this is associates with heavy parasite burdens. In small
animals some conditions to consider are lymphoma, villous atrophy, colitis
and eosinophilic enteritis. In protein losing enteropathy both albumin and
globulins are depressed, and protein concentration is below 50 g/L even
25g/L. Most patients have diarrhea. If no diarrhea is found, intestinal protein
lost might be due to intestinal lymphoma.
c. Hemorrhage leads to hypoproteinemia in addition to the anemia.
This is the easiest way of assessing whether a regenerative anemia is likely to
be hemorrhagic or hemolytic (if the latter, plasma proteins will be not
decreased).
Burns
Decreased protein synthesis
a) Dietary protein deficiency
b) Malabsorption (pancreatic insufficiency, small intestine disorders)
c) Liver failure present primarily as hypoalbuminemia. As certain
types of liver disease can be associated with hyperglobulinemia when total
protein concentrations may be more or less normal. Liver failure and
hepatocelular damage are not synonymous, liver failure should not be
rejected as a cause of hypoalbuminemia just because liver enzymes are
59
not elevated. Specialized liver function tests such as bile measurement are
necessary and sometime liver biopsy is required.
d) Viral condition followed by immune suppression is responsible for
low plasma proteins with normal albumin level.
Fig. 1. Normal and pathologic electrophoregrams

(according to Kerr, 1989)
60
5.2.2. Electrophoresis
This technique allows more detailed appreciations of the various

globulin fractions. Protein fractions are separated on agarose or
polyacrylamide gel at pH 8.4-8.6. The dried out and stained gel is then
scanned by densitometer to produce the characteristic electrophoretic
trace. The numerous variations that can be recognizing in the trace have
led to many suggestion diagnostics. Any inflammatory response will
produce increases in the inflammatory proteins, and the pattern will be
changed (with the more prominent peeks moving towards the gamma
region) as the condition becomes more chronic (fig. 1).
The main application for the technique is where the elevated
globulin concentration is found and there is genuine doubt as to whether
this is a simply inflammatory proteins or paraproteinemia.
5.3. Disturbances of acid/base balance
The blood pH (normally approx 7.4) depends mainly by the

CO2/HCO3-,
(1) alkalosis (pH>7.44) may be respiratory alkalosis (in hypocapnia)
or metabolic alkalosis (elevated level of HCO3-).
(2) acidosis (pH<7.36) may be respiratory acidosis (in hypercapnia)
or metabolic acidosis (low level of HCO3-).
Compensation
The body's acid–base balance is tightly regulated. Several buffering

agents which exists, reversibly bind hydrogen ions and prevent change in
61
pH. Extracellular buffers include bicarbonate and ammonia, while proteins

and phosphate act as intracellular buffers. The bicarbonate buffering
system is especially key, as carbon dioxide (CO2) can be shifted through
carbonic acid (H2CO3) to hydrogen ions and bicarbonate (HCO3- ) as shown
below.
CO2 + H2O ↔ H2CO3 ↔ HCO3- + H+
Respiratory compensation is done almost instantly by changing the

rate of ventilation. This reduces the concentration of carbon dioxide in the
blood. For instance, if the blood pH drops too low (acidemia), the body will
compensate by increasing breathing, expelling CO2, and shifting the
following reaction such that less hydrogen ions are free - thus the pH will
rise back to normal. For alkalosis, the opposite occurs.
The kidneys are slower to compensate, but renal physiology has

several powerful mechanisms to control pH by the excretion of excess acid
or base. In responses to acidosis, tubular cells reabsorb more bicarbonate
from the tubular fluid; collecting duct cells secrete more hydrogen and
generate more bicarbonate. In responses to alkalosis, the kidney may
excrete more bicarbonate by decreasing hydrogen ion secretion from the
tubular epithelial cells.
Categories of acid / base disturbances
Respiratory alkalosis – occurs in hyperventilation (damage of

respiratory neurons, lack of oxygen supply in inspiratory air – hypobarism).
It is compensated by decreased reabsorption of HCO3- by the kidneys, but
the process needs some time.
62
Respiratory acidosis – occurs commonly in respiratory insufficiencies

(obstructive, restrictive, abnormal breading regulation). Respiratory acidosis
can be acute or chronic. Chronic respiratory acidosis is usually
compensated (normal pH) by increase absorption of HCO3- in the distal
renal tube.
Metabolic alkalosis occurs in

Vomiting – in vomiting the acidic stomach content is loss, so a high
amount of H+. Consequently, the correspondent HCO3- produced when HCl
is secreted in the parietal cells, is secreted into the pancreatic juice, and is
not neutralized by the HCl from the gastric juice. Thus, it is intensively
reabsorbed in the distal area of intestine resulting in metabolic alkalosis.
Hypovolemia stimulates Na+/H+ exchange in the proximal tubules and
forces increased HCO3- reabsorption by kidneys. This effect is further
amplified by the aldosteron (normally released in hypovolemia) which
stimulates H+ secretion in distal nephron.
Hormonal changes - Hyperaldosteronism may be responsible for
alkalosis without volume depletion. Hypoparathyroidism can lead to
metabolic alkalosis because normally parathyroid hormone normally inhibits
HCO3- absorption in proximal tubes.
Liver insufficiency – Normally the liver generate urea from two NH4+
and two HCO3-, in liver failure the urea synthesis is decreased resulting in
elevated levels of HCO3- and alkalosis.
Metabolic alkalosis should be compensated by hypoventilation;
however this compensation is not effective because it is limited by the need
for oxygen.
The main effects of alkalosis
- hypokalemia the cells release less HCO3-, depolarize less, and
release less K+ (K+ enters into the cells redistribution hypokalemia).
63
- fall in ionized calcium concentration – because of alkaline

environment the Ca2+ is bound to plasma protein. Because of large amount
of calcium is bound to HCO3- the phenomena is more severe in metabolic
alkalosis than in respiratory alkalosis. This leads to increased
neuromuscular excitability, heart arrhythmias and disturbances in neuronal
function.
Metabolic acidosis occurs in

Renal failure – (see acute renal failure)
Hormonal disturbances – hypoaldosteronism, normally aldosteron
stimulates H+ secretion in the distal tubes,
Loss of bicarbonate from the gut – diarrhea, vomiting of the intestinal
content,
Intense formation of organic acids – they are fully dissociated and are
responsible for significant acidosis. Two types of acids are involved in this
pathology (1) lactic acid and (2) ketone bodies.
(1) lactic acid – is the end product of anaerobic glycolysis (respiratory,
circulatory failure, effort, tumors)
(2) ketone bodies – (acetoacetic acid, beta-hydroxibutic acid)
accumulates in blood especially in increased fat mobilization (mainly
in intracellular glucose deficiency condition) (starvation, diabetes
mellitus).
Protein consumption generates large amount of acidic ions, resulting
from amino acids degradation, sulfur containing amino acids (methionine,
cystine, cysteine) as well as lysine and arginine generate H+. Normally the
kidney of carnivores are adapted to excrete large quantities of H+, therefore,
carnivores have the most acidic urine among animals.
Metabolic acidosis is compensated by hyperventilation, and
hypocapnia.
64
The main effects of acidosis

- hyperkalemia the cells release more HCO3- , depolarize more, and
release more K+ (K+ exits from the cells redistribution hyperkalemia).
Hyperkalemia induces cardiac arrhythmia, and possibly syncope. When
hyperkalemia is mild, ECG changes are manifested by reduction in size of
P wave and developed of peaked T waves. Severe hyperkalemia results in
widening of QRS complex.
- Acidosis is also responsible for brain neurons dysfunction resulting
in coma.
- demineralization of bones
65
6. Hematologic Disorders
6.1. Pathology of Red Blood Cells
6.1.1. Anemia
Anemia is defined as a reduction of the red blood cells mass below

normal limits. Anemia reduces the oxygen-carrying capacity of the blood,
leading to tissue hypoxia.
In practice, anemia is usually diagnosed based on a reduction in the

hematocrit (HCT) (%) or packed cell volume (PCV) (the ratio of packed red
cells to total blood volume), the hemoglobin concentration of the blood
(HGB) (g/dl) and total number of red blood cell (RBC count) (cells/µl) to
levels that are below the normal range.
Anemia is a syndrome, consequence of an underlying disease that
has produced increased erythrocytes destruction, increased erythrocytes
loss trough hemorrhage, decreased production of erythrocytes, or some
combination of these events.
Whatever its cause, when sufficiently severe, anemia leads to
certain clinical features. Clinical signs are related to decreased oxygenation
or compensatory mechanisms and may include pale mucous membranes,
lethargy, reduced exercise tolerance, increased respiratory rate, increased
heart rate. Specific clinical signs that are associated with RBCs destruction
(hemolysis) include splenomegaly, icterus, and darkly pigmented urine
resulting from hemoglobinuria or bilirubinuria. Non-specific clinical signs
may be found if the anemic animal has other systemic illness related or not
related with anemia, and they include anorexia, fever, lymphadenopathy

etc. Hypoxia can cause fatty change in the liver, myocardium, and kidney. If
fatty changes in the myocardium are sufficiently severe, cardiac failure can
develop and compound the tissue hypoxia caused by the deficiency of O2 in
the blood. With acute blood loss and shock, oliguria and anuria can develop
as a result of renal hypoperfusion. Central nervous system hypoxia can
cause headache, dimness of vision, and faintness.
The diagnostic of anemia is never enough to perform a proper
therapy, the establishment of anemia etiology is essential for proper
medical care.
There are many classifications of anemia, but three general

schemes are widely used:
− erythrocyte size and hemoglobin concentration
− bone marrow response
− pathophysiologic mechanism
a. Erythrocyte size and hemoglobin concentration
This classification of anemia requires the determination of RBCs

indices, the values that can be calculated based on HCT, HGB, and HCT
(see Annex I.1.4.).
They are:
Mean cell volume (MCV): the average volume of a red cell
expressed in femtoliters (fL)
Mean cell hemoglobin (MCH): the average content (mass) of
hemoglobin per red cell, expressed in picograms (pg)
68
Mean cell hemoglobin concentration (MCHC): the average

concentration of hemoglobin in a given volume of packed red cells,
expressed in grams per deciliter (g/dl)
Red cell distribution width (RDW): the coefficient of variation of red
cell volume (anysocytosis) (fl, or %)
Anemias are classified by erythrocytes volume (MCV) and amount
of the hemoglobin within erythrocytes (MCHC).
According RBCs size (MCV) the anemias may be normocytic,
microcytic, or macrocytic; according to degree of hemoglobinization,
(MCHC) - normochromic or hypochromic (hypercromic anemia does not
exist, however falsely increased MCHC occur in intravascular hemolysis,
hyperlipemias and the presence of Hentz bodies); and shape.
Microcytic anemias (and sometimes hypochromic when the
condition is very severe) are caused by disorders of hemoglobin synthesis
(most often iron deficiency), while macrocytic normocromic anemias occur
often in abnormalities that impair the maturation of erythroid precursors in
the bone marrow. In humans macrocytic anemias are associated with folic
acid and B12 vitamin deficiency (primary but most common secondary to
chronic gastritis and pancreatitis), but this condition does not occur in
animals. However macrocytic anemias are found in Giant Schnauzers as
consequence of an inherited condition that impairs the absorption of B12
vitamin (cobalamin); several breads may have hereditary macrocitosis
(Poodle), or stomatocytosis with macrocytosis (Alaskan malamute, Drentse
Patrijshound, Miniature Schnauzer) without clinical significance. In cats,
macrocytic anemias are related to Fe-LV infections. Normochromic,
normocytic anemias have diverse etiologies (chronic renal insufficiency,
cirrhosis, myelodysplasia anemia of chronic inflammatory disease);
Macrocytic hypochromic anemias may occur in highly regenerative
anemias because the younger RBCs are larger, but with lower hemoglobin
concentration. It is also associated with increased RDW (anysocytosis index).
69
In some of these anemias, specific abnormalities of red cell shape

(best appreciated through visual inspection of peripheral smears) provide
an important clue as to the cause.
b. Bone marrow response
An anemia is classified either regenerative or nonregenerative

based on the number of circulating reticulocytes (immature erythrocytes).
The reticulocytes are immature erythrocytes that still maintain the
ribosomes and mitochondria. As it nears the final stages of maturity, the
nucleus undergoes degeneration and it is extruded from the cell, and the
organelles supporting the synthetic and metabolic events are removed. The
last step of maturation involves the loss of the ribosomes and mitochondria
during a 1-2 days period. To enumerate the reticulocytes, a stain is applied
to erythrocytes causing aggregation of their residual organelles. This
results in visible clumped granular material that can be seen
microscopically. The aggregation is referred as reticulum, hence the name
reticulocytes.
Stains that can be used are brilliant cresyl blue and new methylene
blue.
The reticulocytes concentration is most useful in dogs and cats, and
it has some applications in cattle. It is not used in horses because, in
horses, the bone marrow almost never releases reticulocytes into
circulation. Cats have two types of reticulocytes, aggregate reticulocytes
(similar to those found in other species, they lose the reticulum in 12
hours), and aggregate reticulocytes, which result from the previous ones
(cells that contain small granules and they became erythrocytes in 12
days). The most relevant are the aggregate reticulocytes.
70
The values can be express in percentage of total RBCs, or number

of reticulocytes/µl. The transformation can be performed according to
formula: RBC/ µl x reticulocytes % = reticulocytes/ µl.
The normal values are
Dogs 0-1%, 0-60,000cells/ µl
Cats 0-0.4% 0-15,000cells/ µl (aggregate reticulocytes)
Cows 0% (but they appear in intense regenerative anemias)
Horses do not release reticulocytes into the blood stream, neither in
intense regenerative anemias.
However, a normal value referees to an individual, which is not
anemic. A “normal” value found in an anemic patient is not normal
anymore, but it is a sign of nonregenerative anemia. This may be due to
deficiency in the reticulocytes response, or defective erythropoiesis.
Absolute increases in reticulocytes concentration indicate a
regenerative anemia, with extra-marrow cause (hemorrhage or hemolysis).
Reticulocytosis is more intense in hemolytic than in extern hemorrhagic
anemias.
Reticulocytosis became visible after 48-72 hours, (2-3 days) after
the occurrence of anemia, with the maximum intensity at 7 days. The
promptest response is found in dogs and pigs, cats are intermediary, and
the lowest is found in cattle.
c. Pathophysiologic mechanism
The pathophysiologic classification of anemia is based on the

mechanisms involved in anemia.
Non regenerative anemia (decreased erythropoiesis) is classified
according to whether neutrophil and platelet production are decreased or
not, and the severity of reduction of erythropoiesis. The diminished RBCs
71
production may be caused by an intrinsec (primary) marrow disorders such

as myelofibrosis, myelodysplasia, or by extrinsic (secondary) disorder.
Secondary disorders include nutritional deficiencies, chronic renal disease,
endocrine disorders (hypotiroidism), chronic inflammatory diseases,
infectious agents, immune mediated destruction of erythrocyte precursors,
and toxics.
Regenerative anemia is caused by hemorrhage or hemolysis.
Hemorrhage may be external or internal, acute or chronic. Common etiology of
acute hemorrhage include trauma, vascular rupture, various haemostatic
disorders (thrombocytopenia, warfarin toxicosis, disseminated vascular
coagulopathy). Chronic blood loss is due to ulcers or tumors, mainly located in
the digestive tract, or infestation with hematophagous parasites.
Hemolysis may be intravascular or extravascular, it may be caused
by increase red blood cells fragility (hereditary, nutritional), infections
(babesia, mycoplasma), immune mediated mechanism. Extra vascular
hemolysis occurs in the spleen or liver, where the erythrocytes are
intensively phagocytized macrophages.
6.1.2. Polycythemia
Polycythemia denotes an abnormally high concentration of

erythrocytes, with a corresponding increase in the packed cell volume
(hematocrit), and hemoglobin concentration. The term polycythemia implies
all blood cells, while erythrocytosis referees to increase of red blood cells
only. However, not all the authors make this difference.
Polycythemia is relative - hemoconcentration due to decreased
plasma volume, or erythrocyte redistribution and absolute when there is an
increase in the total red cell mass.
72
Relative polycythemia - Dehydration results from deprivation of

water, prolonged vomiting or diarrhea, excessive use of diuretics, or body
fluid shifts. Erythrocytes redistribution refers to splenic contraction in highly
excitable animals like cats and horses.
Absolute polycythemia is primary when it results from an intrinsic
abnormality of hematopoietic precursors and secondary when the red cell
progenitors are responding to increased levels of erythropoietin. The most
common cause of primary polycythemia is polycythemia vera, a
myeloproliferative disorder associated with mutations that lead to
erythropoietin-independent growth of red cell progenitors rarely found in
animals, but it was reported in dogs and cats exceptionally in horses and
cattle.
Secondary polycythemia is found in two pathological circumstances
when hypoxemia triggers the erythropoietin synthesis and when the
erythropoietin is produced in large quantities without hypoxemia.
Generalized hypoxemia and hypoxia is found in animals suffering
chronic circulatory and respiratory insufficiency. It is diagnosed by detecting
decreased oxygen saturation and increased erythropoietin level.
Increased erythropoietin level without hypoxemia is found in
patients with renal tumors (that may induce renal hypoxia), paraneoplastic
syndromes (non renal tumors that produce erythropoietin or erythropoietin-
like substances). The erythropoietin levels are very high, blood oxygen
saturation normal or slowly decreased.
6.2. Pathology of leukocytes
The leucocytes of blood (WBCs), normally consists of granulocytes

(neutrophils, eosinophils and basophils), lymphocytes and monocytes.
73
The granulocytes and monocytes flow in one direction only; new

cells are produced in the bone marrow and are released into the circulating
blood, and, after a brief stay in the blood they pass into the tissue. In
contrast the lymphocytes move back and forth between the circulating
blood and the lymphoid tissue.
Origin of blood leucocytes
All blood cells have a single origin represented by multipotential

hematopoietic stem cells; the early differentiation is in two types of cells
common myeloid progenitor and common lymphoid progenitor.
Neutrophilic granulocytes and monocytes have a common

progenitor (a cell does not have characteristic morphology). The
recognizable stages of neutrophilic granulocytes are: myeloblast,
promyelocyte, myelocyte, metamyelocyte, band and segmented
granulocytes. A separate progenitor cell colony stimulating factor (CSF)
exists for eosinophils and basophiles, witch mature in the marrow
throughout similar stages described for neutrophilic granulocytes, the
common stages are the myeloblast and promyelocyte. Myeloblast,
monoblast and promonocyte are the precursors of the monocytes.
6.1.1. Characteristics and function of blood leucocytes
Neutrophil (or PMN) stains a neutral color, is usually segmented into

two to five lobs, each connected by a strand. The cytoplasm is pale pink,
and contains fine pink granules. The younger (immature) stage of the
neutrophil is called a band cell (stab). The nucleus is like a curved sausage.
In generalized toxemic conditions, toxic neutrophils can be seen in
74
circulation with foamy vacuolated cytoplasm and sometimes scattered blue

granules.
The granule of neutrophils have different functions, primary
granules have powerful lytic enzymes and myeloperoxidase. Primary
granules are released intracellulary after phagocytosis and also escape
extracellular and attack the surrounding tissue. The secondary granules
translocate materials to cell surface during migration and activation. The
neutrophilic granulocytes have the function to engulf and kill bacteria that
have penetrated the skin and mucosal barriers.
Eosinophils have a high affinity for eosin. The nucleus is usually
divided into 2-3 lobes and stains purple. The cytoplasm is pink, and filled
with large red-orange granules. Eosinophils normally make up around 3%
of circulating leucocytes, and they accumulate at sites of invasion of tissue
by parasites and at the sites of allergic reactions. They reflect the release of
chemotactic molecules from activated tissue mast cells. Eosinophilic
granules contain major basic protein and eosinophilic myeloperoxidase;
upon their release they can damage the larva parasites that are to big to be
ingested by phagocytic cells. They can also induce tissue damage in
allergic reactions. Eosinophilia associated to parasitic and allergic disease,
mastocytoma, while eosinopenia with bacterial invasion and high level of
glucocorticoids (corticoid therapy, stress, Cushing syndrome).
Basophiles are blue; the nucleus is segmented ad stains as purple.
It contains large blue black granules. They are the less numerous
leukocytes, and they remain in blood stream for only hours, their tissue fate
is unknown. Tissues contain mast cells which resemble basophiles in
possessing blue black granules but they have a round rather than a
segmented nucleus. Although blood basophiles are not precursors of tissue
mast cells, both apparently arise from a common marrow precursor cell.
Tissue mast cells act against parasites and fungi, they poses surface
75
membrane receptors for the Fc fragment of IgE, and this provides a

mechanism from concentrating IgE in the tissue on mast cell surface with
the antigen binding sites of the immunoglobulins free to react with the
antigen binding sites. The consequence of antigen antibody binding
reaction is degranulation of the mast cells; the granule contents include
chemotactic materials for eosinophils. The same mechanism is involved
type I hypersensitivity – anaphylaxis.
Monocytes are the precursors of the macrophages and related
phagocytic cells. The nucleus is large, the nucleus is oval or in a horseshoe
shape, the cytoplasm is gray blue. They spend only 24 hours in circulation,
in tissue they undergo further differentiation to form a variety of
mononuclear phagocytic cells, some of which survive for many days to
months (histiocytes – wandering macrophages; fixed macrophages –
spleen, liver bone marrow, lymph nodes, alveoli; osteoclasts; microglial
cells). The macrophages accumulate at the site of chronic inflammation and
often differentiate further to form epithelioid cells or multinucleated giant
cells.
Macrophages perform diverse and important functions like:
phagocytosis of microorganisms, blood clearance, processing of the
antigen for presentation to lymphocytes (APC – Antigen Presenting Cells),
and secretion of cytokines like IL 1 and TNF.
Lymphocytes might be small or large; the nucleus is smooth, in
round or oval shape, and stains purple. The cytoplasm is basophilic and
varies in amount. They consists primarily of two classes of cells T
lymphocytes – involved in cellular immune response and B lymphocytes –
participates in humoral immune response, it is the precursor of the antibody
forming cell of the body – the plasma cell. A third class of lymphocytes, the
natural killer (NK) diverges from the T cell lineage at an early stage, and not
requires thymic conditioning. NK attacks target cells without prior
sensitization by the antigen on the target cell.
76
6.1.2. Interpretation of WBCs variations
WBCs react independently, so important for interpretation is not the

percentage of certain type of cell, which depends on how many of other cell
types present, but the absolute number of that in circulation. This is easily
calculated from the total WBCs count, or provided directly by automated
devices.
Neutrophilia – increased number of circulating neutrophils over 10

x10 /L in monogastrics or about 4 x106/L in ruminants. This can occur in
6
several ways:
− A shift from the marginal pool (pseudo neutrophilia), occurs in
acute stress, exercise, adrenalin response.
− Steroid effect acts by decreased migration out of the blood
vessels and increased mobilization from the bone marrow. Intense
synthesis is found in chronic effect.
− Response to infection – in acute infections neutrophilia is
accompanied by an increase in immature neutrophils, this is
called shift to the left. The term refers to the appearance of
immature neutrophils in circulation and refers to an index known
as “Arneth index” or “Schilling index”, a table of blood morphology
in with the immature cells appears to the left. Left shift with
neutrophilia is known as regenerative left shift, while associated to
normal or decreased neutrophils number is a degenerative left
shift.
− Neoplasia (Myeloid/granulocytic leukemia)
Neutropenia – decreased number of neutrophils in circulation under
4 x10 /L in monogastric animals or 1x106/L in ruminants.
6
78
− Viral infections (less common than in humans)

− Increased destruction – autoimmune neutropenia (rare)
− Increased movement into the marginal pool – shortly after
endotoxin ingestion
− Increased demand without compensatory inflow – acute or chronic
(cattle are more common affected).
− Decreased bone marrow production (along anemia and
thrombocytopenia) – radiation poisoning, cytotoxic anticancer
drugs, other drugs, toxins etc.
Eosinophilia – increase in the circulation of eosinophils (higher than
1 x10 /L normally 0.5 x106/L (3%) – higher over night)
6
− Allergy (hypersensitivity reactions)

− Parasitism
− Tissue injuries (skin, lungs, gastrointestinal tract, uterus – tissues
that contain mastocites)
− Mast cell tumors – Mastocytoma
− Estrus, pregnancy and early parturition in bitches
Eosinopenia – decreased number of eosinophils (below 0.1 x106/L)

− Effects of glucocorticoids (they neutralize histamine and prevent
the mast cell degranulation therefore the eosinophils are not
released from the bone marrow). High cortisol levels are found in
stress, Cushing’s syndrome, corticoid therapy.
Basophilia – increased numbers of circulating basophiles, above 0.5

x106/L.
− Hypothyroidism – sometimes
− Sometimes accompanies eosinophilia in horse and cats.
79
Monocytosis – increased number of circulating monocytes above

0.5 x106/L
− Chronic inflammatory condition
− Few days after the start of an acute inflammatory condition
(transitory)
− In dogs it accompanies neutrophilia
Monocytopenia – occurs in acute steroid response in cats, horses
and cattle.
Lymphocytosis – increased number of circulating lymphocytes (over
9 x106/L in ruminants and 6 x106/L in monogastrics). In horses the
lymphocyte number decreases with age. They does not normally increase
in viral infections (they percent increases because of decrease in
neutrophils). More common they are increase in malignancies.
Lymphopenia decreased number of circulating leucocytes (below 1
x10 /L in monogastric and 3 x106/L in ruminants). It reflects a decrease in
6
lymphopoiesis.
− Steroid effect
− Immunosuppressant viral infection
− Some malignancies (acute leukemias, lymphosarcoma)
6.1.3. Categories of hematological malignancies
Leukemias are unregulated proliferation in the bone marrow of a cell

of hematopoietic origin. The leukemic cells overgrow and replace normal
elements in all areas of hematopoietic marrow, and sometimes invade
extramedullary sites like spleen, liver, and for lymphocytic leukemia lymph
nodes. The leukemias are classified by cell type, often leukocytes are
involved, but occasionally megakaryocytic and erythroid lineage is involved.
Usually leukemia involves only a cell series.
80
The leukemias are classified on the basis of two major criteria: the
degree of cell differentiation and the cellular lineage.
- the degree of cell differentiation share leukemias into acute
leukemias (blasts of the cells of early differentiation) and chronic leukemias
(differentiated cells). In untreated acute leukemia the peripheral blood WBC
count might be high (with a plenty o immature cells) or low (in the leukemic
proliferation depress WBC production). In untreated chronic leukemia the
WBC count in almost always high.
- the cellular lineage involved in proliferation divide leukemias in
myeloid leukemias (when common mieloid progenitor is involved) and
lymphocytic leukemias (involving the common lymphoid progenitor).
Lymphomas are neoplasms of B or T lymphocytes of the peripheral
lymphoid tissues. Lymphomas often arise in a lymph node, but sometimes
they begin at extra nodal sites like submucosa of the stomach, intestine
and the skin. Latter the neoplastic cells invade locations like spleen, liver,
gastrointestinal tract and bone marrow, and possible other internal organs.
Monoclonal gammopathies are the proliferation of a single clone of
activated B lymphocytes or plasma cells and, consequently, they secrets
large amounts of immunoglobulin molecule or of a light chain of an
immunoglobulin molecule. The whole immunoglobulin molecule will be
found in large concentration in the plasma, where, because all of its
molecules have the same charge and molecular weight, it appears as
narrow protein band or a “spike” in the gamma globulin region on serum
protein electrophoresis. Light chain molecules are not retained in plasma,
but are excreted into the urine as a protein with distinctive properties
(Bence Jones). Two of the monoclonal gammopathies are considered as
hematological malignancies Waldenstrom’s macroglobulinemia – (a
proliferative disorder of B cells), and multiple myeloma (a malignant
proliferation of plasma cells).
81
Myeloproliferative disorders form a group viewed as non malignant,

including Polycythemia vera, myelofibrosis (fibrosis of the bone marrow
associated with proliferation of megakariocytes), essential throbocythemia
and myeloid metaplasia.
6.3. Pathology of hemostasis
Hemostasis is the cessation of blood loss from a damaged vessel.

Tree stages of hemostasis are described: primary hemostasis, secondary
hemostasis (coagulation) and definitive hemostasis.
6.3.1. Stages of normal hemostasis
Platelets immediately form a plug at the site of injury; this is called

primary hemostasis. When the endothelium is damaged, the normally-
isolated, underlying von Willebrand Factor (vWF) is exposed to blood and
recruits Factor VIII, collagen, and other clotting factors. Circulating platelets
bind to collagen with surface collagen-specific glycoprotein Ia/IIa receptors.
Activation of platelets is amplified further by the products of oxidation of
arachidonic acid by the cyclooxygenase pathway (PGH2 and its metabolic
product within the platelet – thromboxane A2). In platelet aggregation an
important factor is thrombin. Aspirin, witch inactivates the enzyme
cyclooxiygenase prevents the synthesis of platelet PGH2 and limits the
collagens ability to activate the platelets. In normal patients aspirin prolongs
bleeding time slightly because the normal generation of thrombin.
Conversely, although hemophilic patients cannot generate normal amounts
of thrombin, his bleeding time is normal or slightly prolonged because
collagen and PGH2 suffices to initiate platelet activation. If both thrombin
82
induced activation and collagen induced activation the bleeding time is

substantially increased. This is the case when a hemophilic patient or a
patient receiving the anticoagulant heparin, is giving aspirin.
Secondary hemostasis (Coagulation) is a complex process by which
blood forms clots. Secondary hemostasis occurs simultaneously: proteins in
the blood plasma, called coagulation factors or clotting factors, respond in a
complex cascade to form fibrin strands, which strengthen the platelet plug.
Intrinsic Pathway Extrinsic

Pathway
XII
XIIa Hageman
XI XIa IV Ca2+
VI – does not exist

Cristmas IX IXa Hem B Thromboplastin
III
IV Ca2+
IV Ca2+
Antihemophilic VIII VIIIa Hem A

VIIa VII Proconvertin
Stuart Power X Xa X Common Pathway
XIII Fibrin
Stabilization
Proaccelerin (Leiden) V Va Factor
IV Ca2+
XIIIa
Antitrombin Protrombin II IIa
Heparin Fibrinogen I Ia Fibrin. mono. Ia Fibrin. poli.

63
Fig. 2. Simplified version of secondary hemostasis (Coagulation)
The coagulation cascade of secondary hemostasis has three

pathways which lead to fibrin formation. These are the intrinsic pathway,
the extrinsic pathway, and common pathway (fig. 2).
Definitive hemostasis – Eventually, the vascular walls are healed
and blood clots are reorganized and resorbed by a process termed
83
fibrinolysis. The main enzyme responsible for this process (plasmin) is

regulated by various activators and inhibitors.
The defects of hemostasis include excessive bleeding and
intravascular thrombosis.
6.3.2. Investigation of hemostasis
Excessive bleeding can result from

1. increased fragility of vessels,
2. platelet deficiency or dysfunction,
3. derangement of coagulation, alone or in combination.
The common laboratory tests used in the evaluation of hemostasis

are focused on investigation of primary hemostasis and investigation of
secondary hemostasis. They are:
a) Investigation of primary hemostasis

Platelet counts. These are obtained on anticoagulated blood using
an electronic particle counter or manual techniques. The reference range is
100 × 103 to 800 × 103 platelets/μl (depending on the species, horses have
the lowest concentration and cats have the highest one). Counts well
outside this range should be confirmed by a visual inspection of a
peripheral blood smear, since clumping of platelets can cause false
“thrombocytopenia” during automated counting, and high counts may be
indicative of a myeloproliferative disorder, such as essential
thrombocythemia. A normal platelet count is usually associated to a
concentration of 5 to 10 platelets / oil immersion field (magnification x1000).
84
Tests of platelet function.

Bleeding time, measures the time taken for a standardized puncture
to stop bleeding, the wound can be created to the hairless area like lip,
gum, and nasal planum. If the coagulation factors are affected, the plug still
forms. It has some value but is time-consuming, difficult to perform well,
and not a good predictor of bleeding during hemostatic stresses such as
surgery.
Platelet aggregometry - tests of platelet aggregation, which
measures the ability of platelets to aggregate in response to a variety of
chemical stimuli.
b) Investigation of secondary hemostasis (coagulation)

Prothrombin time (PT). – Quick test. This test assesses the extrinsic
and common coagulation pathways (vit K deficiency and hepatopathies).
The clotting of plasma after addition of an exogenous source of
tissue thromboplastin (e.g., brain extract) and Ca2+ ions is measured in
seconds. Thromboplastin is a standardized Ca-thromboplastin reagent
obtained rabbit brain highly sensitive to coagulation factors: I (fibrinogen), II
(prothrombin), V, VII and X.
Sometimes it is heparin insensible; the reagent contains a heparin
neutralizing agent, in that case it cannot be used to assess the effect of
heparin therapy.
Activating Partial Thromboplastin Time (APTT). This test assesses
the intrinsic and common clotting pathways (A hemophilia, B hemophilia,
lupus erythematosus when associated with antiphospholipid antibody
syndrome, vit K deficiency and hepatopathies)
The clotting of plasma after addition of kaolin, cephalin, and Ca2+
ions is measured in seconds. Kaolin activates the contact dependent factor
85
XII, and cephalin substitutes for platelet phospholipids. Prolongation of the

PTT can be due to deficiency or dysfunction of factors V, VIII, IX, X, XI, or
XII, prothrombin, or fibrinogen, or to interfering antibodies to phospholipids.
6.3.1. Pathologic conditions associated with blood clotting disorders
6.3.1.1. Thrombocytopenia
Reduction in platelet number constitutes an important cause of

generalized bleeding. A count below 100,000 platelets/μL is generally
considered to constitute thrombocytopenia. However, spontaneous
bleeding does not become evident until platelet counts fall below 20,000
platelets/μL. Platelet counts in the range of 20,000 to 50,000 platelets/μL
can aggravate post-traumatic bleeding. Bleeding resulting from
thrombocytopenia is associated with a normal PT and APTT
It hardly needs reiteration that platelets are critical for hemostasis,
since they form temporary plugs that stop bleeding and promote key
reactions in the coagulation cascade. Spontaneous bleeding associated
with thrombocytopenia most often involves small vessels. Common sites for
such hemorrhages are the skin and the mucous membranes of the
gastrointestinal and genitourinary tracts. Most feared, however, is
intracranial bleeding, which is a threat to any patient with a markedly
depressed platelet count.
The many causes of thrombocytopenia can be classified into four
major categories
Decreased platelet production results from conditions that depress
marrow output generally or may affect megakaryocytes somewhat
selectively. This can be induced by drugs (estrogen toxicosis in dogs and
86
ferrets), toxins (stahibotriotoxicosis), infectious agents (Ehrlichia canis in

dogs), myelophthisis, etc.
Increased platelet destruction occurs in immune thrombocytopenia
(idiopathic thrombocytopenia). It was described mainly in dogs and horses,
the platelet destruction is caused by antibodies to platelets or, less often,
immune complexes that deposit on platelets.
Increased platelet consumption is associated with Disseminated
Intravascular Coagulation (DIC) in which unbridled, often systemic, platelet
activation reduces platelet life span. Hemorrhage does not induce the
platelet count less than 100,000/ μL.
Sequestration. The spleen normally sequesters some the body's
platelets, but this can rise when in splenomegaly (hypersplenism),
producing thrombocytopenia. In dogs hemangiosarcoma (more commonly
located in the spleen) is responsible not only by anemia but also
thrombocytopenia.
Dilution following blood transfusion (dilutional thrombocytopenia) is
a consequence of prolonged blood storage. The number of viable platelets
decreases; thus, plasma volume and red cell mass are reconstituted by
transfusion, but the number of circulating platelets is relatively reduced.
6.3.1.2. Defective platelet function
Qualitative defects of platelet function can be inherited or acquired.

Acquired platelets defects. Platelet function can be inhibited in several
pathologic circumstances:
- large doses of nonsteroidal anti-inflammatory drugs (NSAIDs)
- the presence of abnormal proteins (paraproteins in multiple
myeloma)
- anti-platelets antibody
87
Inherited platelets defects.

The most common is von Willebrand disease, which is strictly
related to platelet function. It is associated with a deficiency of von
Willebrand factor (vWF), a protein synthesized by the endothelial cells and
megakaryocytes. vWF is present at the level of endothelial cells and in
plasma connected with factor VIII. vWF is required for the platelet adhesion
to the collagen in the basement membrane exposed in the injured vascular
wall. Consequently the clot formation is impaired. According to plasma
levels of vWF three types were distinguished, each with its own
characteristics. It is has a variable severity beginning to benign to severe
disease, and it affects a large number of dog breeds like Welsh corgi,
Pinscher, German shepherd, Golden retriever, Poodle, Pointer, Scottish
terrier, etc. vWF was also found in rabbits, cats, horses and swine.
6.3.1.3. Abnormal function of clotting factors
Inherited or acquired deficiencies of virtually every coagulation

factor have been reported as causes of bleeding diatheses. Unlike the
petechial bleeding seen with thrombocytopenia, bleeding due to
coagulation factor deficiencies most commonly manifests as large post-
traumatic ecchymoses or hematomas, or prolonged bleeding after a
laceration or any form of surgical procedure. Bleeding into the
gastrointestinal and urinary tracts, and particularly into joints
(hemarthrosis), is common.
a) Acquired coagulation deficiencies

Vitamin K deficiency – is relatively common in both large and small
animals. Primary deficiency is rare, because the colic bacteria in healthy
animals are able to produce enough vitamin K. Moreover, normal diet
88
contains usually enough vitamin K. The main causes of vitamin K

deficiency are administration of vitamin K antagonists or antibiotics that
destroy the normal gut flora.
The vitamin K antagonists inhibit the reduction of oxidized vitamin K
back to active form. The most important antagonist are coumarin and
indanedione-type rodenticids. The vitamin K is required for the synthesis of
many clotting factor in the liver (II, VII, IX, X), but it is not involved directly
as clotting factor. The effect of vitamin K therapy is visible in four hours for
intravenously administration, and 12 hours if the vitamin K is injected
intramuscularly. For very severe cases plasma transfusion or blood
transfusion are required.
Severe liver diseases are also responsible for coagulopathy,
because almost all clotting factor are produce in liver.
Amyloidosis is associated with factor X deficiency because the
factor in retained by the amyloid matrix.
Autoimmune antibodies directed against clotting factors is rare, but
should not be excluded.
b) Inherited coagulation deficiencies

Hemophilia A (Factor VIII Deficiency)
Hemophilia A is the most common hereditary disease in domestic
animals. It was described in sheep, dogs, cats and horses. It is caused by
mutations in factor VIII, which is an essential cofactor for factor IX in the
coagulation cascade. Hemophilia A is inherited as an X-linked recessive
trait and thus affects mainly males and only homozygous females.
Hemophilia A exhibits a wide range of clinical severity that
correlates well with the level of factor VIII activity. The varying degrees of
factor VIII deficiency are largely explained by heterogeneity in the causative
mutations. Mutations permitting some active factor VIII to be synthesized
89
are associated with mild to moderate disease. The disease in such patients
may be modified by other genetic factors that influence factor VIII
expression levels, which vary widely in normal individuals.
In all symptomatic cases, there is a tendency toward easy bruising
and massive hemorrhage after trauma or operative procedures. In addition,
“spontaneous” hemorrhages frequently occur in regions of the body
normally subject to trauma, particularly the joints, where they are known as
hemarthroses. Recurrent bleeding into the joints leads to progressive
deformities that can be crippling. Petechiae are characteristically absent.
Patients with hemophilia A typically have a prolonged APTT and a normal
PT. These tests point to an abnormality of the intrinsic coagulation
pathway. Factor VIII–specific assays are required for diagnosis
Hemophilia B (Christmas Disease, Factor IX Deficiency)

Severe factor IX deficiency produces a disorder clinically
indistinguishable from factor VIII deficiency (hemophilia A). It was described
in dogs and cats. This should not be surprising, given that factors VIII and
IX function together to activate factor X. A wide spectrum of mutations
involving the gene that encodes factor IX is found in hemophilia B. Like
hemophilia A it is inherited as an X-linked recessive trait and shows
variable clinical severity. As with hemophilia A, the PTT is prolonged and
the PT is normal.
90
7. Pathophysiology of Heart
7.1. Electrocardiography General characteristics
Electrocardiography (ECG) represents the trans-thoracic

interpretation of the electrical activity of the heart, over time, captured and
externally recorded by skin electrodes.
An electrocardiograph is a voltmeter (galvanometer), a device that
measures the difference in electric potential between two different points;
consequently, the electrocardiography is recording these changing potential
differences.
0 0
-1 +1
-1 +1
B
A
++++++ -- +++++
++++++ -- +++++
0 0
-1 +1
-1 +1
C D
- - - +++ - - - - - + +
- - - +++ - - - 0- - + +
0 -1 +1
-1 +1
E F
- - - +++
- - - - - - - - - +++
- - - - - - 0
-1 +1
++++++
A ++++++
Fig. 3. Depolarization / repolarization process into isolated cardiac

fiber
7.1.1. Electrocardiogram of the isolated cardiac fiber
To understand better, how depolarization/ repolarization process is

reflected into the electrocardiogram, first the generation of a diagram will be
explained using an isolated cardiac fiber model (fig. 3).
A - the fiber is polarized, positive electric charges are placed to the
exterior, and there are no differences between the two extremities; the
indicator show a value of zero and on the diagram is recorded as an
isoelectric line.
B - the fiber starts to depolarize, negative electric appear to the left
extremity, and the indicator show an increasing value while the diagram is
recorded a ascendant line.
C - the fiber is half depolarized, the indicator show an maximum
value while the diagram recoded the maximum point.
D - the fiber is almost depolarized, positive electric charges appear
to the right extremity, and the indicator show an decreasing value while the
diagram is recorded a descendant line.
E - the fiber entirely depolarized, the indicator show zero value while
the diagram is again a isoelectric line.
F - the fiber starts to repolarize, positive electric charges appear first
to the left extremity, because they were the first which suffer the
depolarization process. The indicator show an increasing value while the
diagram, but with negative value. The diagram is recording a line, which is
symmetrical but negative compared to depolarization line.
In the end, the fiber is fully repolarized, the situation is similarly to
the initial stage described at paragraph A. Then the cycle is repeated again.
92
7.1.2. The ECG leads
The animal body has a very good electrical conductivity; therefore, the
heart electric activity can be easily recorded by placing the electrodes on the
skin. The electrodes placed directly on the skin are the exploratory electrodes.
The aspect of the ECG varies according to the position of the exploratory
electrodes, as depolarization vectors project differently on the different
exploratory axes. In other to use ECG as a diagnostic method, the recorded
electrocardiograms should be similar and comparable; this is why conventional
application points for exploratory electrodes are established. This results in
twelve standardized leads. A lead is an electrocardiogram measured between
two electrodes, corresponding to a certain exploratory axe.
Lead 1
R L
Lead 2 Lead 3
Right
Left
Foot
F
Fig. 4. Einthoven’s triangle
The first three leads were established by Einthoven, he placed three

electrodes on human body one on the right shoulder, one on the left
shoulder and one on the pubis, resulting the Einthoven’s triangle (fig. 4).
Later, the electrodes were replaced on the right arm, left arm and
respectively on the left foot. This were the first three leads, respectively
93
Lead 1 : right arm (RA) – left arm (LA), Lead 2: right arm (RA) – left leg
(LL), Lead 3: left arm (LA) – left leg (LL). They are bipolar leads because
both electrodes involved are exploratory electrodes (placed on the skin and
therefore with variable electric potential).
The leads described above have a disadvantage, they only
measure the difference between the two exploratory electrodes, no one of
them has a stable, known electric potential for reference.
An artificial zero potential point was created by connecting together
all exploratory electrodes plus the null electrode placed on the right leg.
This conventional point is known as Wilson’s central terminal (fig. 5). The
other leads are the connections between the Wilson’s central terminal and
exploratory electrodes. They are unipolar leads because only one electrode
is exploratory. Unipolar leads are named by V (Volt). The most common
unipolar leads are: VR right arm (RA) – Wilson’s central, VL right arm (RA)
– Wilson’s central, VF left leg (LL) - Wilson’s central. In practice, unipolar
leads are not used as described previously, but Goldberger’s augmented
leads are used instead. Augmentation is the amplification of the
electrocardiographic signal by removal of the direct connection between
Wilson’s central terminal and the exploratory electrode in use. This way the
augmented unipolar leads of the frontal plane are aVR, aVL, and aVF.
The connections used to create the leads of the frontal plane are
described in fig. 6. The leads should not be confused with the wires and
electrodes applied on the animal body. On the animal are connected four
wires, three active on right arm, left arm, left leg and one null electrode for
right leg. The ECG clips remain attached to the animal; the different leads
are created by switching the connections inside on the ECG device.
The transversal plane can be also investigated by unipolar leads, by
applying the exploratory electrodes in certain point on thorax, and
connecting them to Wilson’s central terminal. These are unipolar leads of
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the transversal plane V1, V2, V3, V4, V5, and V6. These leads are mainly
used in human cardiology.
VR VL
VF
Fig. 5. Wilson's central terminal
- + - +
Lead I Lead II - Lead III +
RA LA RA LA RA LA
RL LL RL LL RL LL
+ + - +
Lead aVR
- - Lead aVL Lead aVF
RA LA RA LA RA LA
RL LL RL LL RL LL
Fig. 6. The leads of frontal plane (bipolar and augmented unipolar)
95
7.1.3. Placement of the electrodes
The connector used to connect the ECG cable to the animals skin is
known as electrode (Table 3). In human cardiology, adhesive electrodes
are used, but generally, they are not suitable for animals which have a hairy
coat. Instead, pediatric limb electrodes can be used in veterinary
cardiology, because they can be fixed with a bandage. For animals, the
most widely used are the crocodile clips. They provide a good connection,
and are resistant to movement, but they can be painful if the clip is too
strong.
Table 3. ECG cable color-coding

European United States
Right arm (RA) Red White
Left arm (LA) Yellow Black
Left leg (LL) Green Red
Right leg (RL) Black Green
7.1.4. Terminology
Wave - deflection (positive or negative) – a wave is characterized

by length (sec), amplitude (mV), and shape.
Segment - isoelectric line between two waves – an segment is
characterized by length (sec).
Interval - distance between two points of ECG) – and interval is
also represented by length (sec)
Complex (QRS) – include the waves Q, R and S. ORS complex is
characterized by length (sec), amplitude (mV), and shape, other
characteristics are also consider as will be detailed below.
96
7.1.5. The normal ECG
The sinoatrial (SA) node is the structure responsible for the heart
rhythm it is named the pacemaker. The rate of SA node is increased or
decreased by the influence of vegetative nervous system. The sympathetic
system increases the rate while the parasympathetic decreases the rate.
The electrical discharge for each cardiac cycle starts in the SA
node. Depolarization spreads thought the atrial muscle cells. the
depolarization wave then spreads trough the atrio-ventricular (AV) node,
but it does relatively slower, creating a delay. Conduction passes the AV
ring (from the atria into the ventricles) thought a narrow pathway called the
bundle of His. This then divides in the ventricular septum into left and right
bundle branches to the left and right ventricles. The left bundle branch
divides further into anterior and posterior fascicles. The conduction tissue
spreads into the myocardium as very fine branches called Purkinje fibers.
P wave - The SA node is therefore the start of the electrical
depolarization wave. This depolarization wave spreads trough the atria in
the same way as the ripples in the water created by dropping a stone into it.
As the parts of the atria nearest the SA node are depolarized this creates
an electrical potential difference between depolarized this creates an
electrical difference between depolarized atria and parts not yet
depolarized.
If negative electrode is placed at the bases of the heart and the
positive one is placed in the apex the depolarization wave will move manly
from the negative electrode to the positive one which will result in a positive
deflection.
97
ORS complex
Q wave (before R wave) – the first part of the ventricles to
depolarize is the ventricular septum, with a small depolarization wave that
travels from the apex to the bases of the heart and result in a negative
deflection.
R wave – the most part of the ventricles is depolarized, this created
a depolarization wave to the apex of the heart resulting in large positive
deflection.
S wave (after R wave) – in the end of the depolarization of the
ventricles, the only remaining parts are the areas located to bases of the
ventricles. The depolarization of these remaining areas results in formation
of a small negative wave.
T wave- the ventricular repolarization phase creates a potential
difference between the apex and the bases of the heart, this result in a
deflection named T wave. In human ECG, normally T wave is positive
because the repolarization process occurs highly organized, in opposite
direction to depolarization. Consequently, the shape, direction, amplitude
are very useful indicators of myocardium status in humans. However, in
dogs and cats it can be positive, negative or biphasic, and they have limited
diagnostic value.
ECG measurements
Electrocardiography is recorded on milimetric paper, made on small

squares one mm each. Five small square form one large square of five
mm. The electricity is set at 0.1 mV/ mm, and the speed 25 mm/s (0.2 s/ 5
mm; 0.04s/mm).
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7.1.6. Mean electrical axis
Mean electrical axis (MEA) is the general direction of the heart's

depolarization wave front (or mean electrical vector) in the frontal plane.
When refer to mean electrical axes of the heart we consider the
ventricular axes, the average direction and magnitude of QRS. Calculation
of MEA is of highly importance in human cardiology, in animals because of
a very high physiologic variation, MEA is of less importance. Understanding
how is calculated MEA provides a better understanding of the principle of
ECG recording. In clinics, the calculation of MEA is rarely done, because,
many ECG devices calculated automatic the MEA.
Electrical axis - the general direction of the heart's depolarization

wavefront (or mean electrical vector) in the frontal plane.
L1 - 90°
- +
- -
L2 L3 180° 0°
+ +
Equilateral triangle method

+ 90°
Fig. 7. Equilateral triangle method
Many methods are available, one of the easiest and exact method is
the triangulation (fig. 7). The method uses two leads, usually L1 and L3.
99
The net amplitude of QRS complex is measured for each lead (the negative
and positive value of each wave is added). The value is than transfer to the
equilateral triangle as shown in fig . Then, draw a perpendicular line from
the maximum point of each projection, and, where the two lines represent
the direction of the MEA from the central point (the point that the bisectors
meet).
7.1.7. Main characteristics of ECG in dogs and cats
The main characteristics of ECG in dogs and cats are detailed

below:
Dog ECG characteristics

according to Tiley, Esential Canine and Feline Electrocardiography, CV Mosby, London 1979
P wave – Lenght max 0,04 sec

- Amplitude max 0,4 mV
P-R interval - Lenght 0,06 – 0,13 sec
QRS complex - Lenght 0,05 sec (small breads) 0,06 sec (large breads)
- Amplitude max 2.5 mV (small breads) 3.0 mV (large breads)
S-T segment - depression max 0,2 mV

- elevation max 0,15 mV
T wave - pozitiv, negativ or biphasic
max ¼ from R
Q-T interval - 0,15 – 0,25 sec (depending to heart rate)
Electrical axis - +40% - + 100%
100
Cat ECG characteristics

according to Tiley, Esential Canine and Feline Electrocardiography, CV Mosby, London 1979
P wave – Lenght max 0,04 sec

P-R interval - Lenght 0,05 – 0,9 sec
QRS complex - Lenght 0,04 sec

S-T segment - isoelectric

T wave - pozitiv, negativ or biphasic
max 0,3 mV
Q-T interval - 0,12 – 0,18 sec (depending to heart rate)
Electrical axis - 0% - + 160%
7.2. Cardiac arrhythmias
ECG is a useful tool to diagnose multiple heart dysfunctions. The

ECG abnormalities are largely classified as 1) changes of heart rhythm
(arrhythmias) and 2) changes in waves morphology.
Arrhythmia (dysrhythmia) means an abnormal heart rate. According
to the mechanism involved, arrhythmias are classified as sinus rhythm
abnormalities (the depolarization occurs normally but in an abnormal rate),
disturbances of the stimulus generation (abnormalities associated with
ectopia) and abnormalities of the conduction system (block) (the
transmission of the depolarization wave is impaired).
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7.2.1. Sinus rhythm abnormalities
The sinus rhythm originates from SA node which is the dominant

pacemaker of the heart. It generates the normal heart activity clinically
expressed by the normal heart sound, first the systolic sound
corresponding to closing of atrio-ventricular valves, then diastolic sound
corresponding to the closing of the semilunar valves.
Sinus tachycardia – the SA node generates the depolarization at

an increased rate, higher the normal limit of the species. The rhythm is
regular but of the heart rate is increased. The ECG showed decreased
length especially at T-P segment. Sinus tachycardia physiologically occurs
in effort and acute excitement response and pathologic in fever,
hypertiroidism, anemia, hypovolemia myocarditis etc.
− ECG showed decreased length especially at T-P segment
Sinus bradycardia – the SA node generates the depolarization at a

decreased rate, below the normal limit of the species (fig. 8). The ECG
show increased length especially at T-P segment. Normally, it occurs in
giant bread dogs and dogs with athletic training. Pathologically it appears in
hypothytiroidism, hypothermia, increased vagal tone (pilocarpin, arecolin)
poisoning, B1 deficiency, anesthesia, digitalis, beta-blockers, and quinidine.
The ECG show increased length especially at T-P segment
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Fig. 8. Sinus bradycardia (human)
Sinus arrhythmia - the stimulus is normally, originated in SA node,

but the rate increases and decreases regularly or irregularly (fig 9).
Regularly sinus arrhythmia is normal in dogs and foals, because of
increased parasympathetic activity (vagal tone). It is associated with
respiration, heart rate increases during inhalation and decreases during
exhalation. In dogs, when sympathetic tone is increased, the regularly
tachycardia occurs. The ECG show a regularly irregular rhythm, the T-P
segment varies the most. Irregularly arrhythmia is not associated to
respiration and is pathologic in all species.
− ECG show a regularly irregular rhythm,
− the T-P segment varies the most.
103
Fig. 9. Normal dog ECG expressing sinus arrhythmia
7.2.2. Disturbances of the stimulus generation (ectopia)
Disturbances of the stimulus generation represent a category of

ECG changes induced by stimuli not originated into the normal conduction
system (SA node, AV node, Hiss fascicle etc.) but in other areas of the
myocardium. This condition is known as ectopia.
The ectopia results in range of heart rhythm dysfunctions as
extrasystole, paroxysmal supraventricular tachycardia, flutter, fibrillation
and escape.
Extrasystole is a premature complex followed by a longer repose.

The repose length is longer as short is the preceding extrasystole time;
therefore the distance between the previous contraction and the contraction
which follows the extrasystole is always double as compared to the normal
length between two normal complexes. Extrasystole accomplish several
pathological conditions like myocardial ischemia, hypertrophy, drug
poisoning (digoxin), ionic imbalances (calcium excess, magnesium and
104
potassium deficiency), increased sympathetic tone (adrenaline, stress, and

exhaustion), hyperthyroidism etc.
There are two main explanations for genesis of extrasystole (and
the other disturbances of stimulus generation): (1) the presence of ectopic
focus with enhanced instability of the membrane potential and (2) the re-
entry signaling.
(1) The ectopic focus (ectopic pacemaker) is a group of myocardial
cells with increased excitability, located outside the electrical conduction
system of the heart. The contraction initiated this way, do not follow the
normal route within the conduction system and result in abnormal ECG.
The increased excitability is done by the instability of membrane potential.
(2) The re-entry signaling occurs when a single impulse activates
the same group of cells (and consequently the entire heart) two or more
times. The phenomenon is caused by a non-homogenous state of
conduction in a cross section of the conduction pathway that results in a
unidirectional block. The anterograde block prevents the anterograde
conduction, while the retrograde conduction is possible. Consequently,
corresponding the myocardial areas are depolarized latter, form the
depolarization waves transmitted by the surrounding myocardial regions.
When they depolarize, the impulse is then transmitted anterograde trough
the region affected by the unidirectional block, the rich an area on the
conduction system, and they might generate a new cardiac contraction
resulting a extrasystole. If the process is repeated several times, it results
in two or three associated extrasystoles. The existence of an extra AV
conduction pathway is the basis of supraventricular tachycardia
(paroxysmal tachycardia). When the process is localized in the atria or in
ventricle, it results in atrial or ventricular flutter.
Extrasystoles are classified according to the origin of ectopia in
atrial extrasystole and ventricular extrasystole.
105
Atrial extrasystole – the ectopic focus is placed in atrial

myocardium. It results in supraventricular premature complexes (fig. 10).
- P wave might not be seen, when present, it has abnormal
morphology (non-sinus),
- P-R interval is shorter than normal.
- QRS morphology remains unchanged.
Fig. 10. Atrial extrasystole (human)
Fig. 11. Ventricular extrasystole (human)
106
Ventricular extrasystole – ectopic focus is placed within

ventricular myocardium. It results in ventricular premature complexes –
commonly found in dogs and cats (fig. 11). P wave is missing, QRS
morphology is abnormal. It is wider, the amplitude is higher, T wave is also
large and opposite in direction to QRS.
Supraventricular tachycardia (paroxysmal supraventricular

tachycardia) – is a repetition of more then three supraventricular premature
complexes (atrial extrasystoles). The heart rate is highly elevated up to
200/min, (occasionally 400/min). This condition should be differentiated by
sinus tachycardia.
Fig. 12. Atrial flutter (human)
Flutter is a rapid and regular electrical activity of the atria or

ventricles characterized by the absence of isoelectric lines, resulting in
specific saw teethes waves – the F ways (fig. 12).
Atrial flutter is a rare condition found in dogs but not found in cats.
General characteristics are:
107
− F waves are replacing the normal P waves, they have a typical

aspect of saw tenths
QRS complexes interferes among the F ways, in a conduction ratio
of 2:1 3:1.
− the rhythm can be regular or irregular (fig. 13)
Fibrillation is disorganized electric activity of the atria or ventricles.

The rhythm is entirely irregular; no specific waves can be distinguish. They
are known as f ways. In atrial fibrillation the rhythm of QRS complexes are
irregular, while in ventricular flutter they are totally absent.
Normally, the myocardial cells have a synchronized contraction; an
excitation wave spreads uniformly, in a stable front. The activated and the
non-activated muscle are clearly delimitated. The wave is moving forward
because the backward movement is prevented by the non-responsiveness
of the refractory myocardial cells. The activation wave ends, when all
myocardial cells became refractory. When an excitation wave passes an
asynchronized myocardium it reaches islets of refractory cells (which delay
their repolarization membrane stage) and splits in several smaller waves.
The previously refractory cells became responsive before all myocardium
depolarized completely and a vicious cycle starts. Isolated myocardial
fibers depolarized randomly resulting in a irregular contraction, the heart
wall loses the pumping activity.
Atrial fibrillation is common described in dogs and cats, in atrial

fibrillation depolarization waves occur randomly thought the atria,
consequently the contractions are inefficient, but the heart still have a
functional activity because the ventricular contraction.
− QRS complex have a normal morphology,
− R-R interval is irregular,
108
− no recognizable P waves,
− f waves (irregular, similar to muscle tremor artifact).
Fig. 13. Atrial fibrillation (human)
Ventricular fibrillation - In ventricular fibrillation, the heart became

ineffective, unable to pomp the blood effectively, it is a terminal event
associated to cardiac arrest.
− No recognizable waves or QRS complexes
− only f waves are present
Escape is one or two consecutive impulses generated in the same

or different pacemakers because of delay in the formation of transmission
of the normal impulses of the SA node. It is usually associated to
bradyarrhythmias. They are rescue bets, because they save the animal
from imminent death. If no escape rhythm is developed, the electrical
activity of the heart stops this is termed as asystole.
Junctional escapes are similar to atrial extrasystole (normal QRS
complex) while ventricular escapes are similar to ventricular extrasystole
(modified QRS complex). Continuous junctional or ventricular escapes are
usually seen in complete AV block.
109
7.2.3. Abnormalities of the conduction system (block)
The term block defines a delay or failure of the impulse propagation.

They are classified according to their anatomical site into sinus block, atrio-
ventricular block, bundle branch block and fascicular block. When consider
the severity of the conduction impairment three degree are distinguished
fist degree (the impulse is delayed), second degree (some impulses arrive
but some do not) and third degree (no impulses propagate within the
affected area).
Sinus block – is a condition in which the impulse generated in SA

node do not rich the atria. When the SA node faille to generate the
impulses, the condition is known as sinus arrest.
− there is missing complex in the ECG,
− if R-R interval is double then normal if only a contraction is
missing it suggests a sinus block
− when the pause is longer, but not an multiple of R-R interval, it
suggest a sinus arrest.
Atrio-ventricular block (AV block)

AV block represent a conduction deficiency between atria and
ventricles. It may be partial (first or second degree block), or complete (third
degree block)
First degree AV block – represent a delay in the conduction to AV

node.
- P wave and QRS complexes are normal
- P-R interval is prolonged (> 0.13 –dogs, > 0.9 cats)
110
Second degree AV block – represent a failure of some of the

impulses to reach the AV node.
− P wave not followed by QRS complexes (occasionally or frequent
depend on the gravity).
The second degree block can be differentiate into:
Mobitz type I - when the P-R interval increases in the previous
contractions and
Mobitz type II – when the P-R interval remains constant, and the
fervency of the block remains also constant.
Third (complete) degree AV block – represent a failure of all

impulses to reach the AV node. A second pacemaker takes the control on
the ventricles. The pacemaker can be AV node, or bundle branches
(producing a normal QRS complex) or Purkinje cells (producing a abnormal
QRS complex).
− P waves regular rate of SA node
− QRS T complexes (normal or abnormal) also regular but at much
slower rate (the rate of secondary pacemaker)
− P waves and QRS T complexes occur independently and
sometimes QRS complex hides the P waves.
111
Intra-ventricular conduction defects.

Blocks may occur on both left and right bundle branches and even
on the fascicles.
Bundle branch block (BBB) – is a failure or delay of impulse

propagation through the one bundle branch (fig. 14). Depolarization of the
non-affected ventricle occurs normally, but the depolarization of the
affected ventricle is delayed because it receives the depolarization impulse
from the myocardial cell tissue. Consequently, the complex is prolonged
and modified.
- QRS complex prolonged > 0.06 sec
- the mean cardiac axes switch to the right (right bundle branch
block) and left (left bundle branch block)
Fig. 14. Bundle branch block (human)
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8. Respiratory System
Based on pulmonary function tests, chronic noninfectious diffuse

pulmonary diseases can be classified in one of two categories:
(1) restrictive diseases, characterized by reduced expansion of lung
parenchyma and decreased total lung capacity and an expiratory flow rate
that is normal or reduced proportionately. The loss of functioning lung
tissue reduces the area of diffusion and impairs the gaze exchange.
(2) obstructive diseases (or airway diseases), characterized by an
increase in resistance to airflow due to partial or complete obstruction at
any level, from the trachea and larger bronchi to the terminal and
respiratory bronchioles. Pulmonary function tests show decreased maximal
airflow rates during forced expiration.
Breeding is also impaired by dysfunction of the respiratory neurons,
and the nerves and muscles involved in breeding process.
Consequences of respiratory insufficiency are hypoxemia, and
hypercapnia or hypocapnia.
Terms used for various breeding activities:
Normal breeding – eupnea
Changes in breeding amplitude:
hyperpnea – increased breading movement
hypopnea - decreased breading movement
Changes in breeding rate:
tachypnea – increased breading rate
bradypnea - decreased breading rate
Arrested breeding – apnea
Difficult breeding – dyspnea
Inability to breed – asphyxia
8.1. Pulmonary Edema
The most common hemodynamic cause of pulmonary edema is

increased hydrostatic pressure as occurs in left-sided congestive heart
failure.
Clinical pulmonary congestion and edema is characterized by
dyspnea, asphyxia, a foamy brownish secretion is expressed on nostrils
level.
Morphologically, pulmonary congestion and edema is characterized
by heavy, wet lungs, the interstitium and sometimes alveoli are filled with a
poor protein containing fluid (transudate).

Follow the recorded experiment, and notice the procedures and the
results. The experiment was carried out on one male Wistar rat (approx.
250g b.w.);
The animal was inoculated i.m. with 1 ml adrenalin (0.1%) (which
represents a very high dose able to overcome the heart adaptation ability,
consequently ending in left-sided congestive heart failure)
The animal was observed during symptoms up to death, and then, it
was subject to necropsy and gross examination.
Control questions
1. Explain the mechanisms of pulmonary edema.
2. Explain the relation between adrenaline vascular effects and
pulmonary circulation overload.
3. Explain the clinical and changes and necropsy lesions in relation
with the mechanisms of pulmonary edema.
114
9. Digestive System and Annex Glands
9.1. Determination of acidity of gastric juice
Gastric acid is a digestive fluid, formed in the stomach. It has an

acidic pH and consists mainly of hydrochloric acid (HCl) (around 0.5%), and
large quantities of potassium chloride (KCl) and sodium chloride (NaCl).
The acid plays a key role in digestion of proteins, by activating digestive
enzymes, and making ingested proteins unravel so that digestive enzymes
can break down the long chains of amino acids. Gastric acid is produced by
cells lining the stomach, which are coupled to systems to increase acid
production when needed. Other cells in the stomach produce bicarbonate
to buffer the acid, ensuring the pH does not go too low (i.e., acid reduces
pH). Additionally, cells in the beginning of the small intestine, or duodenum,
produce large amounts of bicarbonate to completely neutralize any gastric
acid that passes further down into the digestive tract. The bicarbonate-
secreting cells in the stomach also produce and secrete mucus. Mucus
forms a viscous physical barrier to prevent gastric acid from damaging the
stomach.
Gastric acidity
Free acidity: The acidity due to free HCl (not combined with other
substances) present in gastric juice.
Total acidity: The acidity due to free HCl, HCl bound to mucin, organic
acids, acid proteins and other acidic substances in gastric juice.
Normal Range
Free acidity (in humans): 18 - 25 mEq/L
Total acidity: in humans 52 mEq/L (1.9), horse 57 – 63 mEq/L (2-2.3 g/L),

cattle 33 mEq/L (1.2 g/L), calf 123 mEq/L
(4.5 g/L), sheep 57 mEq/L (2.1 g/L), pig 117 mEq/L (4.26 g/L), dog 137-164
mEq/L (5-6 g/L)
Clinical significance:
Hyperchlorhydria is the increase of free acidity of gastric juice more
than normal is commonly associated with duodenal ulcers.
Hypochlorhydria is the decrease of free acidity of gastric juice less than
normal.
Achlorhydria is the free gastric HCl concentration in zero. Achlorhydria is
divided in to true achlorhydria and false achlorhydria. In true achlorhydria the
secretion of HCl is completely absent; while in false achlorhydria free HCl in gastric
juice is neutralized by alkaline substances. Achlorhydria is seen in case of
autoimmune disorders, the use of antacids, Helicobacter pylori infection, pernicious
anemia, stomach cancer, etc.
Achylia gastrica, both HCl and pepsin are not secreted.
Estimation of free and total acidity of gastric juice

Principle: Free and total acidity of gastric juice is measured by titrating
against known concentration of NaOH using different indicators.
Reagents: NaOH (0.1 N solution), Phenolphthalein indicator, Topfer’s
reagent (dimethylamino benzene 0.5 % w/v in 95% ethanol)
Procedure:
Total acidity: Take 10 ml of gastric juice in a 100 ml conical flask. Add 2 - 3
drops phenolphthalein indicator. Titrate contents against 0.1 NaOH taken in a
burette. Stop the titration when you observe pale pink color (end point)(pH 8.2-10).
Record the NaOH titer value.
Free acidity: Take 10 ml of gastric juice in a 100 ml conical flask. Add 2 - 3
drops of Topfer’s reagent. Titrate the content against 0.1 NaOH taken in a burette.
116
Stop the titration when the color changes from red to orange-yellow color (pH 3.1-
4.4). Record the NaOH titer value.
Calculation:
Note: the neutralizing equation: NaOH + HCl = NaCl + H2O
1 mEq of NaOH (40 mg) = 1 mEq of HCl (36.5 mg)
NaOH 0.1 N, = 0.1 Eq/L, 0.1 mEq/mL.
Total acidity = titer value (ml)________ X 10 = _________mEq/L

Free acidity = titer value ( ml)________ X 10 = _________m Eq/L
Transformation in g/l: 1 Eq HCl 36.5 g, 1 mEq HCl 0.0365 g,

Ex: 52 mEq HCl /L = 52 x 0.0365 g HCl /L = 1.9 g HCl /L
9.2. Exploration of the exocrine function of the pancreas –

amylase determination
Amylase is concerned with the breakdown of dietary starch and

glycogen to maltose. It is mainly present in the pancreas and salivary
glands, and is unusually for a protein molecule, excreted by the kidney. Its
main clinical use is in the diagnosis of acute necrotizing pancreatitis. This is
essentially a canine (and human) disease associated with high fat diet,
obesity, in which the proteolytic enzymes leak from the cells and begin to
auto-digest the organ. Symptoms of acute abdominal pain and vomiting
may be mistaken for a small intestinal foreign body, and amylase (and
lipase) should always be checked.
117
Amylase is not reliable for investigating pancreatitis in cats. This is

mostly because other organs in the body produce these enzymes, such as
the stomach and small intestine. Also, these enzymes are excreted by the
kidney, and the presence of concurrent kidney disease (which is fairly
common in cats) can falsely elevate the serum amylase and lipase levels.
Cats express usually chronic pancreatitis. Slight to moderate non specific
increase may be seen in other acute abdominal disorders (including
intestinal obstruction), and in renal failure, but there are not of diagnostic
importance.
Estimation of plasma amylase level

(Wohlgemuth method)
Principle: the intensity of serum amylase activity is assessed by

iodometric method; the method measures the concentration of the enzyme
that cannot hydrolyze the starch at the uncolored maltose, in a time period.
Procedure: put 2 ml serum in the first test tube and in the others 1
ml NaCl 0.9% solution each. Transfer 1 ml serum from the first test tube in
the second one, mix gently than transfer further 1 ml in the third one, than
continue like that until the last one. Remove the last 1 ml mixed serum,
from the last test tube. In the end you will get a serial dilution of 1/1, 1/2,
1/4, 1/8, 1/16, 1/32,…1/2048. Ad 2 ml starch 0.1% in each test tube, then
maintain in the incubator for 30 min at 37C. In the end, cool down in tap
water, than ad 1-2 drops of iodine 0.01N each.
Results: The test tubes containing starch become blue, while

hydrolyzed starch (resulting in maltose content) reveal a yellow colure. The
inferior limit of enzyme action is represented by the last unstained (in blue)
118
test tube. The serum dilution of this test tube represents the amylase units /
ml serum. Ex. Test tube nr. 5 (1/16) represent 16 amylase units / ml.
Normal values: horse 16-32, cattle 16-32, sheep 8-16, pigs 64-128,
dogs 64-128.
9.3. Differential diagnosis of jaundice
Bilirubin is the end product of heme degradation. The majority of

daily production is derived from breakdown of senescent red cells by the
mononuclear phagocytic system, especially in the spleen, liver, and bone
marrow. Intracellular heme is oxidized to biliverdin, which is immediately
reduced to bilirubin. Bilirubin thus formed outside the liver is released and
bound to serum albumin. Hepatic processing of bilirubin involves carrier-
mediated uptake at the sinusoidal membrane, conjugation with one or two
molecules of glucuronic acid, and excretion of the water-soluble, nontoxic
bilirubin glucuronides into bile. Most bilirubin glucuronides are
deconjugated in the gut lumen by bacteria and degraded to urobilinogen.
The urobilinogen are largely excreted in feces (responsible for the normal
brown color of the feces). Approximately 20% of the urobilinogen formed
are reabsorbed in the ileum and colon, returned to the liver, and re-
excreted into bile. A small amount of reabsorbed urobilinogen is excreted in
the urine.
Normal plasma concentration is about 2 µmol/L (a little higher in
ruminants), but in horses it can be up to 50 µmol/L (significantly increased
when they are fasting). Total plasma bilirubin represent the total amount of
bilirubin in plasma, direct bilirubin is conjugated bilirubin (non linked to
albumin), and indirect bilirubin (unconjugated) is calculated by the
subtraction from the total.
119
Both unconjugated bilirubin and conjugated bilirubin (bilirubin

glucuronides) may accumulate systemically. There are two important
pathophysiologic differences between the two forms of bilirubin.
Unconjugated bilirubin is virtually insoluble in water at physiologic pH and
exists in tight complexes with serum albumin. This form cannot be excreted
in the urine even when blood levels are high. Normally, a very small
amount of unconjugated bilirubin is present as an albumin-free anion in
plasma. This fraction of unbound bilirubin may diffuse into tissues, and
produce toxic injury. The unbound plasma fraction may increase in
excessive extrahepatic production of bilirubin (ex. severe hemolytic
disease); reduced hepatocyte uptake and impaired conjugation. In contrast,
conjugated bilirubin is water-soluble, nontoxic, and only loosely bound to
albumin. Because of its solubility and weak association with albumin,
excess conjugated bilirubin in plasma can be excreted in urine. It occurs in
decreased hepatocellular excretion in biliary ducts (the bile is released in
blood capillaries); and impaired bile flow.
Estimation of plasma levels of conjugated and unconjugated

bilirubin (Clark van den Bergh reaction)
Principle: Clark Van den Bergh reaction is a chemical reaction used

to measure bilirubin levels in blood. More specifically, it determines the
amount of conjugated bilirubin in the blood. The reaction produces
azobilirubin.
Reagents and materials – serum, 3 test tubes, ethanol 95°, NaCl
0.9%, Diazo I Solution Diazo II Solution, Ehrlich Solution (prepared
immediately before use) 10 ml Diazo I + 0.25 ml Diazo II (Table 4).
120
Table 4. van den Bergh reaction (Procedure and results)
C .bilirubin control u. bilirubin

Serum 0.5 ml 0.5 ml 0.5 ml
Ehrlich Solution 0.5 ml - 0.5 ml
NaCl 0.9% 1.0 ml 1.5 ml -
Ethanol 95° - - 1 ml
Jaundice Van den Bergh Biliary pigments

c. b. u. b. urine feces
Hemolytic - + - +
Hepatic + + + +
Obstructive + - + -
121
10. Excretory System
10.1. Main syndromes of renal disease
The clinical manifestations of renal disease can be grouped into

well-defined syndromes. Some are peculiar to glomerular diseases, and
others are present in diseases that affect any one of the components.
Azotemia is a biochemical abnormality that refers to an elevation of
the blood urea nitrogen (BUN) and creatinine levels, and is related largely
to a decreased glomerular filtration rate (GFR). Azotemia is a consequence
of many renal disorders, but it also arises from extrarenal disorders.
Prerenal azotemia is encountered when there is hypoperfusion of the
kidneys (e.g., in hemorrhage, shock, volume depletion, and congestive heart
failure) that impairs renal function in the absence of parenchymal damage.
Postrenal azotemia is seen whenever urine flow is obstructed
beyond the level of the kidney. Relief of the obstruction is followed by
correction of the azotemia
Uremia (uremic syndrome) is azotemia associated with a
constellation of clinical signs and symptoms and biochemical abnormalities.
Uremia is characterized not only by failure of renal excretory function but
also by a host of metabolic and endocrine alterations resulting from renal
damage. Uremic patients frequently manifest the secondary involvement of
the gastrointestinal system (e.g., uremic gastroenteritis), peripheral nerves
(e.g., peripheral neuropathy), and the heart (e.g., uremic fibrinous
pericarditis).
Acute renal failure is dominated by oliguria or anuria (reduced or no

urine flow), and recent onset of azotemia. It can result from glomerular,
interstitial, or vascular injury or acute tubular injury.
Chronic renal failure, characterized by prolonged symptoms and
signs of uremia, is the end result of all chronic renal parenchymal diseases
Nephritic syndrome is due to glomerular disease and is dominated
by the acute onset of usually grossly visible hematuria (red blood cells in
urine), mild to moderate proteinuria, and hypertension.
Nephrotic syndrome, also due to glomerular disease, is
characterized by heavy proteinuria, hypoalbuminemia, severe edema,
hyperlipidemia, and lipiduria (lipid in the urine).
Renal tubular defects are dominated by polyuria (excessive urine
formation), and electrolyte disorders (e.g., metabolic acidosis). They are the
result of diseases that either directly affects tubular structure or cause
defects in specific tubular functions. The latter can be inherited or acquired
(e.g., lead nephropathy).
Urinary tract infection is characterized by bacteriuria and pyuria
(bacteria and leukocytes in the urine). The infection may be symptomatic or
asymptomatic, and it may affect the kidney (pyelonephritis) or the bladder
(cystitis).
10.2. Urine analysis
A routine urine analysis consists in tree parts: the physical,

microscopic, and chemical examinations.
10.2.1. Physical Examination of Urine
124
The physical examination of urine includes observing the color and

appearance, and measuring the specific gravity. It is the easier and the
quickest urine analysis and it can be performed while the urine specimen is
being prepared for other procedures. Physical characteristics are the best
observed immediately after voiding, before the sample is refrigerated,
refrigerated specimens should be allowed to rich room temperature before
the analysis is performed.
Urine odor – normally recently voided urine has a characteristic
aromatic and not unpleasant odor. Changes in the urine odor may be due
to dieses, diet, or the presence of microorganisms. In cystitis, or other
urinary tract infections, or if urine is allowed to remain unrefrigerated for a
few hours, bacteria may break down urea to form ammonia; the resulting
odor is similar to ammonia. The fruity odor is because the presence of
ketones (uncontrolled diabetes of ketosis in cattle or sheep).
Urine color – the normal color ranges from pale yellow, to straw, to
amber. Dilute urine samples are pale; more concentrated urine samples are
darker yellow or amber. Variation in color may be cause by diet,
medications, physical activity, and diseases. Red urine, if it is cloudy, may
be due to hematuria (the present of erythrocytes), while clear red urine may
be due to the presence of hemoglobin (hemoglobinuria), brown urine
occurred in myoglobinuria (the presence of myoglobin that follow to
rhabdomyolysis), in this case the urine turns to brown color. Yellow brown
or green brown urine is usually found in bilirubinuria (hepatic and post
hepatic jaundice).
Urine appearance – usually urine is clear (not in the horses that
normally show opalescent urine due to increased precipitated salts). The
cause of cloudy or turbid urine specimen becomes evident during the
microscopic examination. Fresh normal urine is clear immediately after
voiding, at room temperature, or at 4C it may became turbid or cloudy
125
(depending of the pH the cloudiness my be due to urates of phosphates). In

freshly voided urine turbidity or cloudiness may be because WBCs,
epithelial cells, mucus and bacteria.
Specific gravity – 1.005-1.030, with most samples falling between
1.010 – 1.025 – indicated the concentrations of substances as urea,
phosphates, chlorides, proteins, and sugars.
10.2.2. Microscopic Examination of Urine
Microscopic examination of the urine sediment may reveal

infections, trauma o urinary tract, the presence of abnormal crystals
(suggest a metabolic disorder). All urine specimens should be examined as
soon as possible to avoid cellular deterioration or multiplication of the
bacteria. 10 ml of well mixed urine is centrifuged at 1500 2000 rpm
(4000xg) no more, for 5 minutes. The supernatant is carefully poured off,
leaving 0.5 ml in the tube, than is resuspended by gently tapping the tip of
the tube. One drop of mixture is placed on the slide, then, covered by cover
glass.
Urine sediment refers to the solids that settle to the bottom of the
urine specimen after centrifugation, or when urine is allowed to stand
undisturbed. It may contain cells, microorganism, crystals, casts,
amorphous material. Urine sediment may be observed unstained or colored
to methylene blue.
Cells in Urine Sediment (high power field Ob - 40x)
RBCs – Size: 7-8 µm in diameter, pale or yellowish, smooth

biconcave disk, no nucleus or cytoplasmic granules. Normal: Less than 2
126
RBC/HPF (in females consider the genital tract), larger numbers –

hematuria.
WBCs usually neutrophils, larger than RBC, 10-12 µm in diameter,
contain nucleus and cytoplasmic granules. Normal: no more than 2
RBC/HPF. Can originate from any part of the urinary tract (glomerulus to
urethra) – large numbers – In inflammatory processes of the urinary tract -
pyuria.
Epithelial cells are large and flat, with distinct nuclei and much
cytoplasm, the most common are squamos epithelial cells. Bladder and
tubular cells are smaller.
Renal Tubular Epithelial Cells are slightly larger than WBC, flat,
cuboidal or columnar with one large round nucleus. They occur in tubular
damage, pyelonephritis, ATN, salicylate intoxication, transplant rejection.
Transitional Epithelial Cells are 2-4 times larger than leukocytes,
round, pear-shaped, tail-like projections with large round nucleus.
Squamous Epithelial Cells are large, flat, irregular-shaped cells
originated principally from the urethra and vagina.
Casts in Urine Sediment (high power field Ob - 40x)
Hyaline Casts are most frequently observed, they are colorless,

homogenous and transparent with rounded ends. This casts can be found
in normal urine, following physical exercise, and dehydration.
Waxy Casts are very high refractive index, yellow, gray or colorless,
smooth homogeneous appearance, short, broad with blunt or broken ends
cracked or serrated edges. They are found in severe chronic renal failure,
malignant hypertension, and diabetic nephropathy.
Granular Casts (and fine granular casts) are the second-most
common type of cast. They have large and coarse granules, and fine
127
granular casts show fine granules, gray or pale yellow. Granular and fine
granular casts can result either from the breakdown of cellular casts, or the
inclusion of aggregates of plasma proteins (e.g., albumin) or
immunoglobulin light chains. They are found in chronic renal disease; and
as with hyaline casts, can also be seen for a short time following strenuous
exercise.
Red Cell Casts are brown to tan. They occur in renal hematuria,
glomerular disease (acute glomerulonephritis, lupus nephritis, renal trauma).
White Cell Casts are found in infection and noninfectious renal
inflammation (e.g. acute pyelonephritis, interstitial nephritis and lupus
nephritis)
Epithelial Cell Casts are found in stasis and desquamation of renal
tubular epithelial cells following tubular damage and necrosis.
Fatty Casts (Oval Fat Bodies) show a “Maltese-cross” pattern under
polarized light. They are found in fatty degeneration of the tubular
epithelium in degenerative tubular disease.
Crystals and Amorphous Deposits in Urine Sediment in acid urine
Uric Acid has many different shapes, diamond, rhombic prism or

rosette, and yellow or brown color. They are very common and can be normal
occurrence. They might be associated with increased purine metabolism.
Calcium Oxalate are colorless, octahedral or envelope. They appear
in ingestion of oxalate-rich foods: spinach, rhubarb, tomatoes, garlic,
oranges, asparagus, high intake of ascorbic acid, and in ethylene glycol
poisoning.
Cystine crystals are colorless, refractile, hexagonal plates. Of
diagnostic importance they are in congenital cystinosis or cystinuria.
Leucine are oily, highly refractile, yellow or brown spheroids and
concentric striations. They are mainly occurred in severe liver disease.
128
Cholesterol crystals are large, flat, transparent and with notched

corners. They appear in excessive tissue breakdown, obstructed lymphatic
flow, nephritis and nephrotic conditions.
Tyrosine crystals are very fine, highly refractile needles, black,
yellow, in sheaves or clusters. They occur in severe liver disease
Sulfonamides are fan or sheaf of needles, eccentric binding, clear or
brown.
Crystals and Amorphous Deposits in Urine Sediment in

alkaline urine
Triple Phosphates (Ammonium Magnesium Phosphate) are

colorless prisms, 3-6 sides, oblique ends, and have coffin lids shape. They
can be found in normal urines, and in chronic urinary inflammation.
Ammonium Biurates are yellow-brown spherical bodies with long,
irregular spicules.
Calcium Phosphate is long thin, colorless needles, with one pointed
end, and arranged as rosettes or star. They can be found in normal urines
Microorganisms
Bacteria – round or rod shape structures
Yeasts – smaller than RBCs, are ovoid, budding and chains the
most common is Candida albicans. To distinguish between yeasts and
RBCs one drop of acetic acid is added, RBCs will lyse while yeasts will not.
Protozoa – Trichomonas vaginalis (in cattle)
Spermatozoa
10.2.3. Biochemical Examination of Urine
129
Protein – proteinuria – may have two mechanisms. First, it

indicates that the permeability the glomerulus is abnormally increased
(albumins are the first proteins that pass throughout the filtrating membrane
– albuminuria can be an indicator of damage to the kidneys). Causes of
albuminuria can be discriminated between by the amount of protein
excreted, the larger amounts are excreted in the nephrotic syndrome, far
less albumins are found in nephritic syndrome and in diabetic nephropathy.
Secondly, it may be caused by renal infections, when proteins are in
fact the desquamated endothelial and inflammatory cells.
Glucose – glycosuria - normally, urine contains no glucose because

the kidneys are able to reclaim all of the filtered glucose back into the
bloodstream. Glycosuria is nearly always caused by elevated blood glucose
levels (when the blood glucose level exceeds about 180 – 200 mg/dl), most
commonly due to untreated diabetes mellitus. Sometimes, glycosuria is due
to an intrinsic problem with glucose reabsorption within the kidneys
themselves, a very rare condition termed renal glycosuria. Glycosuria leads
to excessive water loss into the urine with resultant dehydration, a process
called osmotic diuresis, which leads to polyuria.
Ketone bodies – ketonuria – occur when there is carbohydrate

deprivation, such as starvation or diabetes mellitus, when the body relies
increasingly on the lipid degradation for energy, in the lack of available
glucose. The metabolism of fat proceeds in a series of steps: triglycerides
are hydrolyzed to fatty acids and glycerol; the fatty acids are hydrolyzed
into smaller intermediate compounds (acetoacetic acid, beta hydroxybutyric
acid, and acetone - collectively known as ketone bodies). Finally, the
intermediate products are utilized in aerobic cellular respiration; the process
requires the presence of glucose. In the absence of glucose they
accumulate and are responsible for ketosis, followed by metabolic acidosis,
130
which may lead to coma. Part of them are eliminated through urine,
acetone is removed by the lungs.
Bilirubin – bilirubinuria – is the detection of conjugated bilirubin in

the urine. The term biliuria refers to the presence of any bile pigment in the
urine. The cause of bilirubinuria is hepatocellular and mechanic jaundice, in
humans it is also found in some inherited conditions that affect bilirubin
synthesis.
Blood - hematuria – is the presence as intact RBC which indicates

bleeding or hemoglobinuria – is the presence of hemoglobin (due to
massive hemolysis). In this case RBCs are absent.
Calcium – hypercalciuria - is the condition of elevated calcium the

urine. Chronic hypercalciuria may lead to impairment of renal function,
nephrocalcinosis, and renal insufficiency.
Phosphate - phosphaturia is the hyperexcretion of phosphate in the

urine. This condition is divided into primary and secondary types. Primary,
hypophosphatemia is characterized by direct excess excretion of
phosphate by the kidneys, secondary causes, including all three types of
hyperparathyroidism cause hyper excretion of phosphate in the urine.
131
11. Endocrine System
11.1. Hypocalcemic tetany
Hypocalcemia is the presence of low serum calcium levels in the

blood. Ca2+ is necessary for the actin dissociation from myosin, if not
enough Ca2+, sarcomere contract, but fails to relax. It also leads to
hyperexcitability of peripheral nerves.
Cows, can suffer hypocalcaemia, referred to as milk fever (post-
parturient hypocalcemia) after parturition. This is due to a large calcium
demand and a slow response from the animal in terms of intestinal
absorption or bone resorption. The condition is also linked to high milk
production. The clinical manifestation is collapse and muscle spasms. In
typical cases, the cow's head is in a so-called self-auscultation position. It
will eventually enter a coma and can die.
In dogs it may be found in hypoparathyroidism, a condition in which
either parathyroid glands secretes subnormal amounts of PTH, or the
hormone secreted is unable to interact with target cells.
Hypoparathyroidism is present in smaller breads such as schnauzers, and
terriers. Idiopathic hypoparathyroidism is caused by diffuse lymphocytic
paratiroiditis, tumor invasion of the parathyroid gland, and damage during
thyroid surgery and trophic atrophy of parathyroid glands resulting from
long term hypercalcemia.
Latent tetany - increased neuromuscular excitability and tetany, the
animals are restless, nervous, ataxic, and have intermittent tremors of the
separate muscle groups. Tendon reflexes are hyperactive
Acute tetany – manifested by generalized tetany and convulsive

seizures, peripheral vasoconstriction, intensive sweating, mydriasis. Life
threatening complications are laryngospasm (asphyxia) and cardiac
arrhythmias (ECG changes include intermittent QT prolongation)
Calcemia drops to 4-6 mg/dl (normally 9 mg/dl), and
hyperphosphatemia is due to renal tubular reabsorption. In urine the
phosphates decreases, and calcium first increase, latter decreases.
As pH increases, caused by respiratory or metabolic alkalosis,
freely ionized calcium concentration decreases. Since a portion of both
hydrogen ions and calcium are bound to serum albumin, when blood
becomes alkalotic, bound hydrogen ions dissociate from albumin, freeing
up the albumin to bind with more calcium and thereby decreasing the freely
ionized portion of total serum calcium. This hypocalcaemia related to
alkalosis is partially responsible for the cerebral vasoconstriction that
causes the dizziness, fainting (syncope), and paraesthesia, sometime
tetany seen with hyperventilation.

Follow the recorded experiment, and note the observation in
laboratory notebooks. The experiment was carried out on one rabbit
(approx. 2500 g b.w.);
1. The rabbit was inoculated i.V. with 10 ml Na EDTA (3.8%) preheated at

approx 37C. The inoculation is stopped when the fist signs of
tetania appear. The needle is maintained inside the vein.
2. The signs are observed for few seconds,
3. The animal was inoculated i.v. (using the same needle) with 10 ml
calcium gluconate 10%
4 In the end of calcium administration, the clinical signs of hypocalcemia
disappear.
134
Control questions
1. Which are the main causes responsible for clinical cases of
hypocalcemia?
2. Which are clinical signs associated with hypocalcemia?
3. What emergency therapy should be applied to hypocalcemic
patients?
135
12. Nervous System
12.1. Cerebellar syndrome (cerebellar ataxia)
Ataxia (from Greek a- + -taxia [order], "lack of order") is a

neurological symptom that consists of gross lack of coordination of muscle
movements.
Cerebellar ataxia is due to dysfunction of the cerebellum. This
causes a variety of elementary neurological deficits, such as antagonist
hypotonia, asynergy, and dysmetria. How and where these abnormalities
manifest themselves depends on which cerebellar structures have been
damaged, and whether the lesion is bilateral or unilateral.
The most common cause is the cerebellar hypoplasia due to in
utero infection with parvoviruses (puppies canine parvovirus, kittens –
panlekopenia virus) and pestivirus (calf and piglets). Primary cerebral
neuronal degeneration is also found in dogs, cats, horses and cattle. It is an
inherited condition, in which animals are born normal, and cerebellum
degeneration starts at young age.

Follow the recorded experiment and write the observations in
laboratory notebooks. The experiment was carried out on one male Wistar
rat (approx. 250 g b.w.);
1 – the animals was subject to inhalator anesthesia
2. - the cerebellum was surgically destroyed
3. – the signs of cerebellar dysfunction were recorded immediately after
recovery from anesthesia, three days latter.
Control questions
1. What is the function of cerebellum?
2. What are the main symptoms associated to cerebellar
deficiency?
3. How can be explained the reduction of the severity of the
symptoms in the next days after surgery?
12.2. Epilepsy
Epilepsy (from the Greek epilepsía — "to seize") is a common

chronic neurological disorder characterized by seizures. These seizures are
transient signs of abnormal, excessive or synchronous neuronal activity in
the brain.
Most seizures last for a relatively short period of time, usually
around 3-5 minutes or less. In cases where the seizures are recurrent, the
length of time between seizures can be quite variable and may be hours,
days, weeks, or even months.
An epileptic seizure itself can be broken down into four stages
1. The Prodome - This stage can last from minutes to hours or
even days before the manifestation of the actual seizure activity. This stage
is typically characterized by changes in the dog's mood or behavior
2. The Aura – This stage is characterized by the presence of
hallucinations and illusions.
A hallucination is a perception in the absence of a stimulus, while
illusion, involves distorted or misinterpreted real perception. Hallucinations
can occur in any sensory modality — visual, auditory, olfactory, gustatory,
tactile, etc. Some dogs will begin pacing, licking, salivating, trembling,
vomiting, wandering aimlessly, hiding, whining or urinating. Other dogs may
138
exhibit stranger activities such as excessive barking and attempts to get an

owner's attention.
3. The Ictus- This stage is the actual seizure itself, it is either tonic
or/and tonic-clonic, generally lasting from 1-3 minutes. It is a period of
abnormal activity in which the most common symptoms are that the dog
may lose consciousness, gnash their teeth or appear to be chewing gum,
thrashing about with their head and legs, hyper salivation, paddling their
feet as if running as well as losing control of their bladders and bowels.
Some dogs only have partial seizures in which the twitching is
localized in one area. This could in the face, one leg, in the shoulder or
over the hips.
4. The post-ictal stage is the period of recovery after the
seizure. Again, the length can be quite variable.
Brain damage due to prolonged (more than 30 minutes) convulsive

seizures (status epilepticus) is not widely accepted in domestic animals, but
is commonly found in humans and well documented in experimental
models. Some authors report this in dogs. During seizures there is a
increase metabolic demand for glucose and oxygen by neurons, but
cerebral flow increases during seizures, however neuronal necrosis still
occurs, but the mechanism is still unclear.

Follow the recorded experiment. The experiment was carried out on
one male rabbit (approx. 2500 g b.w.); the animal was inoculated i.v. with 3
ml Pentylenetetrazol l (1%).
Pentylenetetrazol has been used experimentally to study seizure
phenomenon and to identify pharmaceuticals that may control seizure
susceptibility. The mechanism of pentylenetetrazol is not well understood,
pentylenetetrazol has the opposite effect as those of the sedatives
139
(diazepam, phenobarbital), and so, presumably, it binds to the GABA-A

receptor.
In response to pentylenetetrazol administration, the animal develop
a seizure crisis very similar to spontaneous crises of epilepsy.
The recovery is slow, and is supported by intravenous
administration of isotonic glucose.
Control questions
1. What is epilepsy?
2. What are the stages of epileptic seizure?
3. What are the main symptoms of ictus stage?
140
Case Presentations
Annex I – Case presentations
Case 1. Hemorrhagic chronic anemia (Ancylostomiasis)
Signalment: Canine, Basset hound, female, 9 weeks old
Anamnesis and symptoms: hemorrhagic diarrhea, pale mucous

membranes for several days long
Laboratory data
Hematology
Hct 8↓ %
Hb 2.5 ↓ g/dl
RBC 1.69 ↓ x106/ µl
MCV 50 ↓ fl
MCH 15.2 ↓ pg
MCHC 30.5 ↓ g/dl
Reticulocytes 17↑ %
RBC morphology: anysocytosis, polychromasia, erythroblasts
(metarubicites)
PLT 336 x103/ µl
WBC 44 ↑ x103/ µl
Seg 23.32(53%) x103/ µl
Band 10.56(24%) ↑ x103/ µl
Lymph 4.4 (10%) x103/ µl
Mono 3.08 (7%) x103/ µl
Eos 2.64 (6%) x103/ µl
Baso 0.0 x103/ µl
WBC morphology: normal
Other Tests
Fecal: Ancylostoma sp.
141
Discussions
Hemorrhagic anemia
Severe (<15% Hct), regenerative anemia (reticulocytosis and
anysocytosis) with no signs of hemolysis, indicate severe blood loss.
Metarubricyte release may accompany intense erythrocyte regeneration.
Low MCV and additionally low MCHC suggest bone marrow over
stimulation and consequently the presence of a population of small
hypochromic erythrocytes, the first signs of iron deficiency anemia.
Neutrophilic leukocytosis
Neutrophilia frequently is associated with hemorrhage. In this
puppy, neutrophilia may have resulted from a combination of hemorrhage
and release of chemotactic factors at the sites of mucosal damage by
hookworms.
Summary: The dog responded to a blood transfusion and administration of

antihelmintics.
142
Case Presentations
Case 2. Hemolytic anemia (Babesiosis)
Signalment: Canine, American Pit Bull terrier, female, 1 year old
Anamnesis and symptoms: Lethargy, fever, icterus, hematuria
Laboratory data
Hematology
Hct 19 ↓ %
Hb 6 ↓ g/dl
RBC 2.7 ↓ x106/ µl
MCV 71 fl
MCH 32 ↑ pg
MCHC 36 ↑ g/dl
Reticulocytes 32 ↑ %
RBC morphology: anysocytosis, polychromasia,
metarubricyte, erythrocytic parasites
PLT 36 ↓ x103/ µl
WBC 27.75 ↑ x103/ µl

Seg 17.74(64%) x103/ µl
Band 2.77(10%) ↑ x103/ µl
Lymph 2.77 (10%) x103/ µl
Mono 2.49 (9%) x103/ µl
Eos 1.94 (7%) x103/ µl
Baso 0.0 x103/ µl
WBC morphology: WBC toxic changes in neutrophils
Other Tests
PCR : Babesia gibsoni
143
Discussions
Macrocytic, hyperchromic, regenerative anemia
Regenerative anemia with icterus indicates hemolysis. Increased

MCH and MCHC are signs of presence of free hemoglobin in plasma -
intravascular hemolysis. Very low platelet count can be explained by
hypersplenism (part of affected RBCs are retained by the spleen – resulting
extravascular hemolysis).
Babesia gibsoni is an intracitoplasmatic protozoan parasite. Acutely,
it causes a hemolytic anemia and thrombocytopenia. B. gibsoni can be
transmitted between dogs by tick vectors, direct blood inoculation or
transplacental transmission. In most endemic areas, B. gibsoni is
transmitted by Hemophysalis ticks. Parasitemia is detectable in the acute
phase of infection, but is generally low. Parasites are small (1-2.5µm) and
pleomorphic. The most common shape is a signet ring, although elongated
forms are also seen.
Babesia sp multiply in the erythrocyte and disrupt the membrane
upon existing cell. Erythrocytes are destroyed within the circulation,
releasing hemoglobin into the plasma where it is removed by the liver or
excreted by the kidneys. Hemoglobinemia is usually detected by red
discoloration of plasma and an increase of MCHC and MCH.
Reticulocytosis of this magnitude also suggests hemolysis because
iron from lysed erythrocytes is readily reused. In contrast, iron is lost from
the body in hemorrhagic anemia and mobilization of storage iron is
accomplished more slowly; reticulocytosis is of lower magnitude.
Metarubricytes (nucleated red blood cells) may accompany intense
erythrocytic regeneration or may be prematurely released from the bone
144
Case Presentations
marrow following increased eritropoetin synthesis in response to anemia-

induced hypoxia.
Neutrophilic leukocytosis with left shift

Neutrophilia is observed commonly in hemolytic anemias.
Destruction of erythrocytes, like destruction of other tissues, may incite a
tissue demand for neutrophils.
Neutrophil toxic changes may be observed in association with
inflammatory response. The term toxic change is unfortunate, these
morphological changes are attributable to altered bone marrow production,
and these cells have normal function.
The interpretation of toxic change is that neutrophils are made
under condition of accelerated production that occurs part of the
inflammatory response.
145
Case 3. Aplastic anemia (Feline infectious peritonitis)
Signalment: Feline, European Shorthair, female, 8 year old
Anamnesis and symptoms: Anorexia, emaciation, lethargy
Laboratory data
Hematology
Hct 20 ↓ %
Hb 6.4 ↓ g/dl
RBC 4.39 ↓ x106/ µl
MCV 41 fl
MCH 12.9 pg
MCHC 31 g/dl
Retic 0.1 %
RBC morphology: normal
PLT 26 ↓ x103/ µl
WBC 10.94 x103/ µl

Seg 9.84(90%) x103/ µl
Band 1(1%) x103/ µl
Lymph 0.54 (5%) ↓ x103/ µl
Mono 0.43 (4%) x103/ µl
Eos 0.0 x103/ µl
Baso 0.0 x103/ µl

Serum Chemistry
Total Protein 13 ↑ g/dl
Albumin 1.7 ↓ g/dl
A/G ratio 0.15 ↓
Laparatomy
Granulomatous peritonitis
Other Tests
FIV negative
FeLV negative
146
Case Presentations
Discussions
Anemia of chronic (inflammatory) disease

A normocytic, normochromic, nonregenerative anemia with normal
erythrocyte morphology might be associated with chronic inflammation.
Erythrocytes do not have any morphologic changes, and a specific etiology
of the anemia is not indicated. Patients with inflammatory disease
frequently develop anemia, and this phenomenon is associated with altered
iron metabolism. Affected individuals have iron deficiency, but increase iron
stores in the mononuclear phagocyte system.
Hyperproteinemia.
The high serum protein concentration with low A/G ratio suggests
an increase in globulins, which is characteristic of chronic antigen
stimulation such as that occurring with FIP viral infection. Causes of
hypoalbuminemia could include decreased production during inflammation,
diminished production with cachexia, or renal loss due to proteinuria.
Lymphopenia.
Lymphopenia is recognized by an absolute lymphocyte count of less
than 1,500 lymphocytes/ µl of blood in adult cats. In this case the
lymphopenia is attributable to viral (FIP) infection.
147
Case 4. Aplastic anemia (Hypothiroidism)
Signalment: Canine, Caucasian Shepherd, male, 4 year old
Anamnesis and symptoms: Lethargy
Laboratory data
Hematology
Hct 32 ↓ %
Hb 11↓ g/dl
RBC 5.28 ↓ x106/ µl
MCV 66 fl
MCH 22.9 pg
MCHC 33 g/dl
Retic 0.5 %

PLT 256 x103/ µl
WBC 6.32 x103/ µl

Seg 2.79(44%) x103/ µl
Band 0.15(2.5%) x103/ µl
Lymph 2.8 (45%) ↓ x103/ µl
Mono 0.25 (4%) x103/ µl
Eos 0.28(4.5%) x103/ µl
Baso 0.0 x103/ µl

Test
Total T4 0.78 ↓ µg/dl

Total T3 104 µg/dl
cTSH 1.2 ↑ ng/ml
148
Case Presentations
Discussions
Normocytic, normochromic, nonregenerative anemia

Common laboratory abnormalities in hypothyroidism include
anemia, it occurs in approximately 50% of affected dogs. Thyroid hormones
involve in hemoglobin synthesis in adult and maturation of hemoglobin in
fetus and by affecting hematopoietic process, hypothyroidism results in
anemia through slowing the oxygen metabolism.
Decreased Total T4, and increased cTSH concentration are consistent with
early primary hypothyroidism. Total T3 concentration may remain within
reference range as peripheral tissues and the thyroid gland produce a
larger percentage of T3
149
Case 5. Polycythemia (Dehydration)
Signalment: Canine, Labrador, male, 4 years old
Anamnesis and symptoms: Diarrhea
Laboratory data
Hematology
Hct 59 ↑ %
Hb 21 ↑ g/dl
RBC 8.8 ↑ x106/ µl
MCV 66 fl
MCH 24 pg
MCHC 34 g/dl
PLT 236 x103/ µl
WBC 11.59 x103/ µl

Seg 8.22(71%) x103/ µl
Band 0.11(1%) x103/ µl
Lymph 1.85 (19%) x103/ µl
Mono 0.46 (4%) x103/ µl
Eos 0.57 (5%) x103/ µl
Baso 0.0 x103/ µl
Serum chemistry
Total protein 8↑ g/dl
Albumin 3.4↑ g/dl
A/G ratio 0.74
BUN 49 ↑ g/dl
150
Case Presentations
Discussions
Polycythemia
Polycythemia (hemoconcentration) can result from relative or
absolute increase in red cell mass. Relative polycythemia is more common
and may be caused by dehydration and splenic contraction, in excitement,
fear, or strenuous short-term exercise (epinephrine response). In this case,
polycythemia is the result of dehydration (the effects that are also reflected
by hyperproteinemia with normal A/G ratio)
Hyperproteinemia, hyperalbuminemia, hyperglobulinemia,, and

normal A/G ratio.
Hyperproteinemia is likely a relative increase in protein
concentration because of plasma water loss from dehydration. The normal
A/G ratio indicates that both globulin and albumin concentrations are
increased in the same proportion (typical for relative hyperproteinemia).
Prerenal azotemia
The increased BUN concentration indicates azotemia. An increased
BUN concentration with dehydration suggests decreased renal perfusion
with a decreased glomerular filtration rate.
151
Case 6. Polycythemia and lymphocytosis

(Excitement response)
Signalment: Canine, Mixt breed, male, 2 year old
Anamnesis and symptoms: routine testing for blood donation, after

hydrotherapy
Laboratory data
Hematology
Hct 57 ↑ %
Hb 21 ↑ g/dl
RBC 8.6 ↑ x106/ µl
MCV 65 fl
MCH 20 pg
MCHC 33 g/dl

PLT 341 x103/ µl
WBC 18↑ x103/ µl

Seg 11.16 (62%) x103/ µl
Band 0.18 (1%) x103/ µl
Lymph 5.4 (30%) ↑ x103/ µl
Mono 0.54 (3%) x103/ µl
Eos 0.72 (4%) x103/ µl
Baso 0.0 x103/ µl

Serum chemistry
Total protein 5.6 g/dl
Albumin 3.1 g/dl
BUN 21 g/dl
152
Case Presentations
Discussions
Polycythemia
Polycythemia results from splenic contraction occurring in fear, or
strenuous short-term exercise (epinephrine response). No increase in total
protein concentration excludes dehydration.
Lymphocytosis
In this cause, the lymphocytosis has, as the most plausible cause,
the excitement response. The segmented neutrophils are very close to the
upper limit (with no left shift), the total WBCs count has also mild elevated
values. Additionally the lymphocytes do not show very elevated values,
they are very close to the upper limit. The percentages are all confined
within normal range, which suggest a redistribution leukocytosis.
153
Case 7. Leucopenia (Parvovirus Infection)
Signalment: Canine, Yorkshire terrier, female, 4 months old
Anamnesis and symptoms: Anorexia, vomiting, melena, mucous

membrane pallor
Laboratory data
Hematology
Hct 30 ↓ %
Hb 8↓ g/dl
RBC 4.6 ↓ x106/ µl
MCV 66 fl
MCH 24 pg
MCHC 31 g/dl

PLT 195 ↓ x103/ µl
WBC 2.93 ↓ x103/ µl

Seg 1.85 (62%) ↓ x103/ µl
Band 0.08 (3%) x103/ µl
Lymph 0.63 (23%) ↓ x103/ µl
Mono 0.29 (10%) x103/ µl
Eos 0.05 (2%) x103/ µl
Baso 0.0 x103/ µl
WBC morphology: reactive lymphocytes
154
Case Presentations
Discussions
Leukopenia and neutropenia

Leukopenia is usually caused by severe neutropenia. Neutropenia
can be caused by decreased production of neutrophils, or overwhelming
tissue consumption of neutrophils. Neutropenia is probably multifactorial,
including decreased neutrophil production from viral destruction of
hematopoietic cells, an intense tissue demand for neutrophils because of
viral enteritis, and enterotoxemia from enteritis with a shift of neutrophils
from the circulating neutrophils pool to the marginal neutrophils pool.
Lymphopenia
Lymphopenia is a common finding in acute systemic infections,
especially viral disease. Parvovirus infects and destroys rapidly dividing cell
population including lymphocytes, hematopoietic cells, and intestinal crypt
epithelial cells. Lymphopenia also may be due to stress and transient
redistribution of lymphocytes. Recirculating lymphocytes may be entrapped
within lymph nodes, promoting antigen exposure and amplifying the
immune response.
Hemorrhagic anemia
Normocytic, normochromic anemia and clinical signs of hemorrhage
(melena) indicate blood loss. Medullar viral infection prevents the
regenerative response. Hemorrhage is also responsible for moderate
thrombocytopenia.
155
Case 8. Leukocytosis (Pneumonia)
Signalment: Canine, Labrador, female, 4 years old
Anamnesis and symptoms: fever, anorexia, lethargy, cough
Laboratory data
Hematology
Hct 40 %
Hb 14 g/dl
RBC 7 x106/ µl
MCV 66 fl
MCH 24 pg
MCHC 34 g/dl

PLT 651 ↑ x103/ µl
WBC 25.7 ↑ x103/ µl

Seg 16.1 (63%) ↑ x103/ µl
Band 3.08 (12%) ↑ x103/ µl
Lymph 3.34 (13%) x103/ µl
Mono 2.93 (12%) x103/ µl
Eos 0.0 x103/ µl
Baso 0.0 x103/ µl
WBC morphology: neutrophils toxic changes
Serum chemistry
Total protein 9.1 ↑ g/dl
Albumin 2 g/dl
A/G ratio 0.28 ↓
156
Case Presentations
Discussions
Leukocytosis, neutrophilia and left shift

Neutrophilia is largely responsible for the leukocytosis although,
monocytosis is also present. Neutrophilia with left shift (elevated numbers
of bads) suggest acute systemic inflammation (inflammatory leukogram)
commonly accomplish with fever.
Neutrophil toxic changes suggest bacterial infection or severe
inflammation. Toxic changes are an indication of disturbed maturation in
the bone marrow. In dogs, monocytosis commonly accomplishes
neutrophilia in either acute or chronic disease.
Thrombocytosis
Reactive thrombocytosis commonly accompanies inflammation and
fever (increased production of thrombopoietin by IL6 in liver).
Hyperproteinemia, hypergammaglobulinemia
Hyperproteinemia is accomplished by normoalbuminemia and
decreased A/G ratio therefore hyperglobulinemia is the most likely cause.
Hyperglobulinemia might be induced by acute nonspecific immune
response (acute phase proteins - alpha and beta globulins), or to antigenic
stimulation associated with increased antibody production (gamma
globulins).
157
Case 9. Pancreatitis
Signalment: Canine, Golden retriever, male, 11 years old
Anamnesis and symptoms: fever, anorexia, abdominal pain
Laboratory data
Hematology
Hct 32 ↓ %
Hb 10 ↓ g/dl
RBC 4.54 ↓ x106/ µl
MCV 71 fl
MCH 24 pg
MCHC 34 g/dl
Retic 0.7 %
PLT 78 ↓ x103/ µl
WBC 32 ↑ x103/ µl
Seg 25.6 (80%) ↑ x103/ µl
Band 3.84 (12%) ↑ x103/ µl
Lymph 1.92 (6%) x103/ µl
Mono 0.64 (2%) x103/ µl
Eos 0.0 x103/ µl
Baso 0.0 x103/ µl
WBC morphology: cytoplasmic basophilia and vacuolation

Serum chemistry
Amylase 4065 ↑ U/L
Lipase 1408 ↑ U/L
Calcium 8.9 ↓ mg/dl
ALP 300 ↑ U/L
ALAT 80 ↑ U/L
158
Case Presentations
Discussions
Inflammatory leukogram
The neutrophilia with left shift indicates a tissue retention of
neutrophils, compensated by increased leucopoiesis. The toxic changes in
neutrophils indicates systemic toxemia caused by pancreatitis with
disturbance of neutrophil maturation within the bone marrow.
Anemia (moderate) usually nonregenerative is a result of chronic

inflammatory disease.
Thrombocytopenia is due to disseminated intravascular coagulation (DIC)

triggered by release of digestive enzymes into circulation.
Hyperamylasemia and hyperlipasemia suggest a pancreatic origin of the

increased serum enzymatic activity.
In dogs, the common bile duct of the liver and the main pancreatic
duct enter the duoden in close proximity. Pancreatitis can be associated
with inflammation and partial obstruction of the common bile duct.
Increased ALP (alkaline phosphatase) activity is induced by
cholestasis.
Increased ALT (alanine aminotransferase) activity indicates
hepatocellular injury. Because retained bile in the canaliculi it exerts a lytic
effect on cell membranes.
Hypocalcemia is done by glucagon released from the damaged pancreas

can stimulate thyrocalcitonin. This secretion reduces blood calcium
concentration.
159
Case 10. Coumarin poisoning
Signalment: Canine, Caniche, male, 10 years old
Anamnesis and symptoms: hematuria, hemothorax
Laboratory data
Hematology
Hct 25 ↓ %
Hb 9↓ g/dl
RBC 3.99 ↓ x106/ µl
MCV 69 fl
MCH 23 pg
MCHC 33 g/dl
Retic 4.7 %
RBC morphology: anysocytosis, anisochromia
PLT 78 ↓ x103/ µl
WBC 15 x103/ µl
Seg 12.75 (85%) x103/ µl
Band 0.75 (5%) x103/ µl
Lymph 1.35 (9%) x103/ µl
Mono 0.3 (2%) x103/ µl
Eos 0.1(1%) x103/ µl
Baso 0.0 x103/ µl

Serum chemistry
Total protein 5↓ g/dl
Albumin 2.1 ↓ g/dl
A/G ratio 0.72
ALT 98 U/L
Other tests
Fibrinogen 270 ↑ mg/dl
APTT 36 ↑ sec
PT 31 ↑ sec
ACT 300 ↑ ….sec
160
Case Presentations
Discussions
Regenerative anemia
The decrease in hematocrit, hemoglobin, and RBCs count indicates
anemia. The erythrocyte indices reveal that the anemia is normocytic and
normochromic. The absolute reticulocytes count reveals increased
erythropoiesis.
Thrombocytopenia occurs due to excessive platelet consumption in

hemorrhage.
Hypoproteinemia, hypoalbuminemia, A/G ratio - normal

Hypoproteinemia is the result of concurrent loss of albumin and
globulins via hemorrhage. Because these proteins are lost together, the
A/G ratio remains within the reference interval.
Coagulation deficiency
Hemophilia A (factor VIII deficiency) and hemophilia B (factor IX
deficiency) are excluded because both APTT and PT are prolonged, only
the APTT would be prolonged in these hereditary disorders. Prolonged
ACT (activated coagulation time), APTT (activated prothrombin time), and
PT (prothrombin time) are consistent with acquired deficiency of the vitamin
K-dependent clotting factors. Coumarin rodenticide toxicosis is highly likely.
Liver disease, as a cause of coagulation deficiency, is unlikely because
ALT activity is within the reference interval.
161
Clinical Laboratory Techniques
Annex II – Laboratory techniques
Hematology techniques
II.1. Red blood cells
Routine hematology tests may be performed manually or by using

one of the many types of hematology analyzers. All laboratories which use
hematology analyzers must also have backup systems for performing
analyses in the case of instrument malfunction. It is recommended that the
personnel should be trained in manual techniques for the frequently
requested tests.
For hematology tests is required only fresh blood, collected from
veins or capillaries on anticoagulants that do not produce significant dilution
of the blood (Na EDTA). Other anticoagulants are less or not
recommended.
II.1.1. Red blood cell count (RBCs)
The blood dilution may be done by using a Potain pipette for red
blood cells (which has the reservoir 100 times larger than capillary tube
diameter). It may provide a dilution 1:100 or 1:200, according to the level of
aspirating blood (0.5 or 1).
The dilution is realized using the Hayem solution, which is isotonic
and maintain the erythrocytes shape and preserve them during the
counting time.
Erythrocytes’ counting is made in Burker-Turk chamber (fig. 15).

In the first place the slide is placed into the microscope and a
coverslip is placed on the counting network, the margins of the counting
camera should not be entirely covered by the coverslip.
Blood is aspirated in Potain pipette to 0.5, the excess blood is
removed using a piece of filter paper and then the Hayem solution is
aspirated until 101 sign on the upper part of the reservoir. When the pipette
is full is quickly brought into horizontal position, fixed between pollex finger
and index finger and mixed gently for 3 minutes. The first tree drops are
discarded (they not contain any blood), and one drop from the diluted blood
is placed in the area between the margin of the camera and the cover slip,
and they will fill the network throughout capillarity.
RBC / µL = n *5*10*200 = n * 10000

n = the erythrocytes count in 5 middle squares, 5 is the correction
because we count only the 1/5 part from an 1 mm2 area, 10 to transform
the results in cells / mm3, and 200 the dilution of the blood (1 mm3 = 1 µL).
164
Fig. 15. Burker-Turk chamber 1/25 mm2 squares are used to count
erythrocytes (red circles) (1); 1 mm2 squares (blue circles) are used
to count leucocytes (2)
Sometimes the erythrocytes are expressed in millions / mm3, 106/

mm3. The international units for erythrocytes are 106/µL or, more common
1012/L, however, regardless of the units; the absolute number remains the
same.
165
II.1.2. Hemoglobin (HGB)
The measurement of blood hemoglobin is one of the most common

laboratory tests. The hemoglobin test measures the oxygen caring capacity
of the blood, and the severity of the anemia. The hemoglobin test is simple
to perform, and easily standardized. It may be performed manually or by
using analyzers.
Manual methods of measuring hemoglobin concentration are based
on converting hemoglobin to a stable oxidized form (methemoglobin), by
adding chemicals as Drabkin’s reagent, or ammonium hydroxide. These
methods use a spectrophotometer.
Measurement of hemoglobin concentration using

ammonia hydroxide method
First, 5 ml ammonium hydroxide 0.4% is introduced into the test

tube and then a 20 µL pipette is filled with blood. The excess blood is
eliminated using a piece of filter paper, after that, the pipette content is
mixed with the ammonium hydroxide.
Immediately after, the sample is read at the spectrophotometer at
545 nm wavelength, using distilled water as blank.
HGB (g/dL) = E x 33
The international units for HGB is g/dL or g/L
166
II.1.3. Hematocrit (HCT)/ Packed Cell Volume (PCV)
The hematocrit (HCT) may be determined as packed cell volume

(PCV) or with automatically devices.
For PCV, usually capillary tubes are used. As compared to older
methods that use test tubes, the capillary tubes require much less blood.
The capillary tubes may be treated with heparin and they are suitable for
fresh blood samples, and without heparin tubes, and should be used for
blood collected on anticoagulants only.
The packed cell volume (PCV) can be determined by centrifuging
blood in a capillary tube (also known as a microhematocrit tube) at 10,000
RPM for five minutes. Before centrifugation the capillary tubes must be
closed at the two extremities into the flame, carefully. Self sealing tubes
also can be used. These have a dry plug that expands to form a seal when
the blood contacts it.
This separates the blood into layers. The volume of packed red
blood cells, divided by the total volume of the blood sample gives the PCV.
Because a tube is used this can be calculated by measuring the lengths of
the layers.
With modern lab equipment, the hematocrit is calculated by an
automated analyzer and not directly measured. It is determined by
multiplying the red cell count by the mean cell volume. The hematocrit is
slightly more accurate as the PCV includes small amounts of blood plasma
trapped between the red cells.
167
II.1.4. Erythrocytes Indices
The erythrocytes indices are values obtained by calculation using

the RBC count, the hemoglobin value, and the hematocrit. The indices are
also known as the mean corpuscular values or the mean cell values. The
indices consist of the mean corpuscular volume (MCV), the mean
corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin
concentration (MCHC). The indices are used to classify anemia.
Mean Corpuscular Volume (MCV) - is the volume of an average
erythrocyte and it is calculated by using the formula:
MCV = HCT/RBC x 10.
It is reported in femtoliters (fL) (former value was cubic microns).
Normal values indicate normocytosis. An elevated MCV value indicates
macrocytes, while a decrease value indicates microcytes.
Mean Corpuscular Hemoglobin (MCH) - estimates the average
weight of HGB in RBC and is calculated by using the formula:
MCH = HGB/RBC x 10.
The units are picograms (pg) (10-12g). It is not that useful in
classifying anemia alone, but becomes useful when correlate to other
indices.
Mean Corpuscular Hemoglobin Concentration (MCHC) – express
the concentration of hemoglobin in the RBCs in relation to there size and
volume, it is calculated by using the formula:
MCHC = HGB/HCT x 100.
The result is express in g/L or g/dL (the same as the HGB). The
MCHC is always higher than HGB, because all blood hemoglobin normally
is contained by RBCs. A normal value indicates normocromia, while a
168
decreased value indicates hypocromia. Hypercromia is very rarely found,

except in spherocytosis.
Note: some authors express HGB and MCHC in %, which means
g/100 ml, they consider 1 ml = 1 g, which is scientifically not correct, but
accepted in some laboratories).
II.1.5. Reticulocytes Count
The reticulocytes count is a method of estimating the number of

immature RBCs in circulating blood; therefore it is an indirect method of
estimating the rate of RBC production. The test is most commonly used to
differentiate a primary from a secondary anemia. RBCs are produced in
bone marrow and, after the maturation process, RBCs enter the blood
circulation. For the twenty-four hours in the circulation, they are still slightly
immature and can be undefined by the presence of RNA in the cytoplasm.
When an immature RBC is exposed to certain stains, the RNA forms
granular aggregates or filaments called reticulum, these cells are called
reticulocytes.
The staining technique is called vital staining procedure because
the cells are still living. Two common dyes used for vital stains are: new
Metilen Blue and Brilliant Cresyl Blue. Reticulocytes appear as blue-tinged
RBCs with dark bluish purple granules or filaments, while mature RBCs
appear uniformly bluish green.
Reticulocytes staining
1. Mix in ration of 1:1 (into an eppendorf tube) anticoagulated
blood (NaEDTA) and staining solution (Brilliant Cresyl Blue 1%
in NaCl 0.9%). Isotonic solution is required to preserve the
RBCs during staining procedure.
2. Mix gently and allowed them to stay for 2-3 minutes
169
3. Make a blood smear in a similar manner to those described for

the blood smears.
4. Air dry and examine microscopically using the oil immersion
objective.
The normal values are:
Dogs 0-1%, 0-60,000cells/ µl
Cats 0-0.4% 0-15,000cells/ µl (aggregate reticulocytes)
Cows 0% (but they appear in intense regenerative anemias)
Horses do not release reticulocytes into the blood stream, not even
intense regenerative anemias do.
Mice 0-11.3 %, Rats 0 – 4.6 %,
Guinea pigs 0 – 6%, Rabbits 1 – 6.3 %
Ferrets 2-14 %
Humans
Reference values Upper limits of normal
Adults 0.5 – 1 % 3%
Newborns 2.5 – 6.5 % 10%
II.1.5. Abnormal Erythrocytes Morphology
Associated with changes in total number erythrocytosis or anemia,

disorders that affect the RBCs may cause poikilocytosis – variation in the
shape of RBCs, anisochromia – non uniform color of RBCs because of non
uniform distribution of hemoglobin, or anisocytosis – variations in the size of
RBCs.
Anysocytosis -if RBCs are smaller than normal size are microcytic
(often occurred in iron deficiency, cooper deficiency), and RBCs larger than
normal are macrocytic (occur in dysfunction of maturation in bone marrow –
170
deficiencies in vitamin B12, folic acid). In several conditions large RBCs

and small ones may coexist.
Poikilocytosis – Stickle cells (drepanocytes), spherocytes,
eliptocytes, target cells, stomatocytes, schizocytes, and acanthocytes.
Anisochromia – a deficiency in hemoglobin, due to lack of iron or
another condition, causes of RBCs to have a large central area of pallor
with only a small outer rim of hemoglobin – hypochromic cells. RBCs that
are completely filed with hemoglobin are called hyperchromic.
RBCs inclusions – are Basophilic stippling and Howell-Jolly bodies.
Basophilic stippling is caused by fine granular remnants of RNA and other
basophilic substances left inside the RBC when it loses its nucleus. These
RBCs are also known as reticulocytes when stained with a vital stain. On a
Wright stained smear, the RNA remnants cause cells to have diffuse blue
color (diffuse basophilia), orange and blue mottled appearance
(polychromatophilia), or punctate fine and coarse granules (basophilic
stippling). Normally only occasional stippled cells will be present, an
increased number in peripheral blood smear can indicate certain toxic
conditions or increased production of RBCs. Howell Jolly bodies are
nuclear remnants in the RBCs after nucleus is lost in Wright stain, as
intense dark blue purple bodies inside the cells. Cabot rings are found in
certain anemias like as blue purple ring like structures. Nucleated RBCs or
erythroblasts are normally found in bone marrow only, but they can be seen
in peripheral blood in severe regenerative anemia. They might be found in
small numbers in newborns.
II.1.6. Erythrocyte Sedimentation Rate (ESR)
Erythrocyte sedimentation rate (ESR) is not specific for a particular

disease but is used as an indicator of inflammation or tissue injury. ESR is
based on the principle of sedimentation, the process of solid particles
171
settling the bottom of the liquid. To perform the manual ESR a sample of
anticoagulated blood is placed in a calibrated tube at standard dimensions
and placed in a vertical position. At the end, the distance at which the
erythrocytes have fallen from the plasma meniscus (at the zero mark) is
measured in mm and reported.
Factors affecting the ESR: properties of the plasma, properties of
the erythrocytes, and technical factors.
Plasma proteins – normally RBCs suspended in the plasma form
few aggregates or clusters. Therefore the mass of the RBCs is small and
the rate of sedimentation is slow. In abnormal blood RBCs form aggregates
called rouleaux, and clusters of cells are heavier than single cells and will
settle faster. The rouleaux formation is induced by increased levels of
inflammatory proteins like fibrinogen, acute phase proteins, gamma
globulins etc.
Erythrocyte factors – are size, shape and number. Macrocytic cells
sediment more rapidly than microcytic cells, because they are heavier.
Spherocytic cells and stickle cells have slow ESR (they are unable to form
rouleaux). ESR is faster in anemia and slower in polycythemia therefore the
HCT should be checked on samples that have elevated ESR to see if ESR
is due to inflammation or anemia.
Technical factors (in fact possible errors) are time spent between
blood sampling and determination (less than 2 hours), temperature (20C-
25C), sample mixing, tube vibration and tilting.
Westergren method – is performed by using the Westergren tube,

it is an accurate method, and it requires predilution of blood with sodium
citrate (3:1). The vial is filed with blood until the sign, than the Westergren
tube is inserted into the vial with a twisting motion until the tube reaches the
bottom of the vial. The blood column will be automatically zero, and any
blood in excess will overflow into a sealed reservoir. The vial containing
172
tube is placed in the sedimentation rack, and the distance at which the
erythrocytes have fallen is measuring using the gradation on the tube.
Normal ESR in domestic animal species
Horse 15’ 18±0.5 mm 30’ 49±1.4 mm, 1h 89±15 mm, 2h 118±1,0
mm
Table 5. Normal ESR in domestic animal species

Cow Dog Cat Rat
1 hour 1.2 mm 2 mm 4 mm 3 mm
2 hours 2 mm 4 mm 10 mm 5 mm
II.2. White Blood Cells
II.2.1. White Blood Cells Count (WBCs)
The blood dilution may be done by using a Potain pipette for white
blood cells (WBC) (which has the reservoir 10 times larger than the
capillary tube). It may provide a dilution of 1:10 or 1:20, according to the
level of aspirating blood (0.5 or 1). The dilution is done using the Turk
solution, that contains 3% acetic acid that lyses (destroys) the red blood
cells, so the WBC are easily seen at the microscope. WBC counting is
done in Burker-Turk chamber (see the RBC count). Blood is aspirated in
Potain pipette to 0.5, the excess blood is wiped using a piece of filter paper,
and after that Turk solution is aspirated until the sign 11 on the upper part
of the reservoir. When the pipette is full is quickly brought into horizontal
position, fixed between pollex finger and index finger and mixed gently for 3
minutes long. The first tree drops are discarded (they not contain any
173
blood), and one drop from the diluted blood is placed at the area between
the margin of the counting camera and the cover slip, and they will fill the
network throughout capillarity. The counting starts after 2-3 minutes, time
required for the cells to lay down, the counting is done within the 4 large
squares, placed in the corner of the chamber in the same way like the
RBCs.
The total number of cells is given by formula:
WBC / µL = average counted cells *200
10 to transform the results in cells / mm3, and 20 the dilution of the
blood (1 mm3 = 1 µL).
In Romania usually WBCs are express in 103/ mm3. The
international units for erythrocytes are 103/µL or, more common 109/L,
however regardless of the units; the absolute number remains the same.
II.2.2. Blood Smear Preparation and Staining
Examination of stained blood smear is a routine part of the complete

blood count (CBT). Stains are applied to the elements, so the formed
elements RBCs, WBCs, and platelets may be viewed, identified, and
evaluated using the microscope, as in a differential count. A properly
prepared blood smear enables the examiner to view the cellular
components of blood in a natural state as possible.
Blood smears must be prepared in a manner that alters relative
distribution and morphology of cells as little is possible. Must be paid
attention to specimen collecting and handling. Anticoagulated blood must
be well mixed before smears are made. Smears must be made within 2
hours of blood collection. Anticoagulants other than EDTA should not be
used, since they may alter the morphology or staining characteristics of the
cells. The preferred specimen for blood smear is capillary blood that has no
174
added anticoagulant, and of course applied directly on the slide before the
blood coagulates. A satisfactory smear specimen may also be made from
blood which has EDTA added to, within two hours from collection.
Slides used for blood smears must be free of any grease and dust.
Slides may be purchased already cleaned, or may be washed with soap
and water, rinsed thoroughly in hot water and then in distillated water,
dipped in 95% ethanol and polished with a lint-free cloth. Clean slides
should be handled by the edges only. Slides with frosted ends are preferred
be cause they can be easy labeled.
Making the smear

The smear is prepared by placing a small drop of well mixed blood
on the right end of a slide placed on a flat surface. The end of a second
spreader slide is placed at 30◦ angle in the front of the drop. The spreader
is then brought back into the blood drop until the drop spreads along three
quarters of the edge of the spreader slide. This should be performed in
smooth, quick sliding motion. As soon as the blood spreads along the edge,
the spreader is pushed to the left with a quick steady motion (avoiding the
pressure on the slide) to spread the blood in a thin film. Then the smear
should be dried as quickly as possible.
The smears should be stained immediately, if not they should be
fixed in methanol 30-60 seconds, and then allowed to air dray. This way
they can be stained at a later time.
Slides Staining – Dia Quick Panoptic method.

Dia Quick Panoptic staining solution set is used for hematological
and cytological staining. It contains three reactives: Dia fix (fixation), Dia
red (eozinophilic staining), Dia blue (basophilic staining).
The slide is dipped several times, in each staining solution, as
follows:
175
Dia fix – 5-10 times 20 seconds

Dia red – 3-13 times 20 seconds
Dia blue – 3 15 times 30 seconds
The intensity of the staining is adjusted by the number of the
immersions for each step.
In the end the slide is rinsed in distillated water, not in between the
staining steps, and then allowed to air dray.
II.2.3. Differential Count
Differential counts known as “diff”, is part of the CBC (complete

blood count), and consist in examination of leukocytes and classification of
cells into different types. “WBC count and diff” is a common laboratory
request. The differential procedure involved counting 100-200 WBCs on a
stained blood smear and recording how many of each of the five types of
WBCs is seen. Secondly, RBC and platelets are also evaluated.
The area of the smear to be examined is known as feathered edge,

where the cells do not overlap each other. For avoiding of counting the
same cell two times is important to evaluate the area in serpentine pattern,
from one exterior limit to other of the smear.
Cellular features evaluated in identification
Neutrophil (or PMN) stains a neutral color, is usually segmented into

two to five lobs, each connected by a strand. The cytoplasm is pale pink,
and contains fine pink granules. The younger (immature) stage of the
neutrophil is called a band cell (stab). The nucleus is like a curved sausage.
176
Eosinophil has a high affinity for eosin. The nucleus is usually

divided into 2-3 lobes and stains purple. The cytoplasm is pink, and filled
with large red-orange granules.
Basophil are blue, the nucleus is segmented ad stains as purple. It
contains large blue black granules.
Lymphocyte might be small or large; the nucleus is smooth, in round
or oval shape, and stains purple. The cytoplasm is basophilic and varies in
amount.
Monocyte is large, the nucleus is oval or in a horseshoe shape. The
cytoplasm is gray blue.
For the differential count, a table should be made, which include all
types of leucocytes. Each element is marked in the corresponding column,
by making a line. The elements should be grouped 10 lines quarters, to be
easier to count at the end of the counting (Table 6).
Table 6. WBCs differential count

Granulocytes Agranulocytes
Neutrophils Eosinophils Basophiles Lymphocytes Monocytes
Bands Polynuclear
neutrophils
These values represent percentages so it is necessary to correlate

them with the total WBCs count. If the percentage and the WBCs count is
known, the values of each element may be calculated, relative variation
(the changes in the percentage) have little signification while the absolute
values are important for diagnosis.
177
II.3. Investigation of hemostasis
II.3.1. Platelets Count
Due to agglutination, lysed platelets count is not easy to perform,

even that are various techniques available. They are two types of available
methods, the direct and the indirect ones.
Indirect method
The method uses the stained blood smear, and is realized counting
the platelets reported at 1,000 RBCs, than the RBCs are counted in the
hemocytometer as described above, then the platelet concentration is
calculated proportionally and express in 109/L or 103/µl
Direct method
The blood dilution may be done by using a Potain pipette for red
blood cells (with has the reservoir 100 larger then the capillary tube). It may
provide a dilution 1:100 or 1:200, according to the level of aspirating blood
(0.5 or 1).
The dilution is done by using a special solution (Na2EDTA 0.01g,
ammonium oxalate 1.00g, distillated water ad. 100ml). Mix the pipette with
diluted blood for 3 minutes, allow it to stay for 30 minutes (for the lyse of
RBCs), then mix again for 3 minutes.
Platelets are count in Burker-Turk hemocytometer in similar manner
to the RBCs (on five second degree squares). Then they are multiplied to
5x or 10x (10 or 20, depending to the dilution), and they are expressed in
109/L or 103/µl.
178
Reference values:
Dog 200-900 103/µl, Cat 300-700 103/µl, Cow 100-800 103/µl,
Horse 100-600 103/µl, Pig 200-500 103/µl, Sheep 250-750 103/µl,
Goat 300-600 103/µl, Rabbit 250-650 103/µl
II.3.2. Bleeding time
Bleeding time is a standardized in vivo test investigating primary

hemostasis. It is performed on mucosal membranes (Buccal mucosal
bleeding time - BMBT) in dogs and cats, while in large animals both
mucosal membranes and skin sites are used. Mucosal bleeding method is
considered more repeatable then skin bleeding.
To perform the BMBT, the upper lip is rolled out and fixed by a
gauze strip. A standardized single or double cut (5 mm x1 mm) is made on
mucosal surface of the upper lip by using a spring-loaded device. The
excess blood is wiped away by filter paper without touching the incision.
The end point is when no drops of blood are seen on filter paper. The
normal BMBT time is not longer than 4-5 minutes.
Prolonged BMBT is found in: thrombocytopenia, thrombopathia
(platelet dysfunction), severe anemia, vascular disease, aspirin therapy etc.
II.3.3. Prothrombin Time (Quick test) PT
Prothrombin time (Quick test) (PT) is used for the control of blood
coagulation disorders of the extrinsic pathway, for the surveillance of oral
anticoagulant therapy and to check the liver’s function of synthesizing
coagulation factors in hepatopathies. A heparin-neutralizing agent in the
179
reagent permits the assessment of plasma obtained from patients on

conventional heparin therapy (0.2-0.8 U/ml).
Composition – Technoplastin HIS (Heparin Insensible) is a

standardized Ca thromboplastine reagent obtained rabbit brain highly
sensitive to coagulation factors II, V, VII and X. In addition, the reagent
contains a heparin-neutralizing agent.
Test procedure
Preparation of plasma samples – Mix 9 parts venous blood and 1
part sodium citrate solution (0.11 mol/l), and centrifuge for 15 min at an
RCF of at least 2500. Keep the plasma at room temperature (up to 4
hours).
Performance of the test
The lyophilized reagent is reconstituted with the indicated volume of
distillated water at room temperature and it may be used immediately.
The substances are preheated at 37°C and are a mixture of 0.1 ml
plasma and 0.2 ml Technoplastin HIS, and after that start the stopwatch
and determinate the clotting time.
Normal time is 13-14 sec.
II.3.4. Activated Partial Thromboplastine Time (APTT)
The Activated Partial Thromboplastine Time (APTT), is used fro

screening procedure to detect abnormalities of intrinsic coagulation system.
It can be used to detect deficiencies of Factors II, V, VIII, IX, X, XI, and XII.
In addition it can be used to detect the Lupus anticoagulant and monitoring
of heparin therapy.
180
Composition – Dapttin TC is a standardized APTT reagent

consisting of kaolin and sulfatide as surface activators.
Test procedure
Preparation of plasma samples – Mix 9 parts venous blood and 1
part sodium citrate solution (0.11 mol/l), and centrifuge for 15 min at an
RCF of at least 2500. Keep the plasma at room temperature (up to 4
hours).
Performance of the test
The lyophilized reagent is reconstituted with the indicated volume of
distillated water at room temperature and the reagent should stay 10
minutes before use.
The CaCl2 25 mmol/L should be preheated to 37°C.
Pipetting scheme:
0.1 mL plasma sample + 0.1 mL Dapttin TC
Shake briefly and incubate for two minutes at 37°C.
+ 0.1 mL CaCl2 25 mmol/L
Start timing after the addition of CaCl2 solution and determine
the clotting end point.
Normal time is 29-42 sec.
181
Plasma biochemistry
II.1. Plasma glucose measurement (Roee and Goetze method)
The method is based on dehydration of glucose with sulphuric acid

resulting furfurolic compounds which give a green staining in reaction with
anthrone.
The reaction is done in three steps:
a. Protein removal stage – 0.2 ml (blood, plasma or serum) +
1.8 ml trichloroacetic acid. The samples are mixed and then centrifuged at
2500 rot/min 5 min.
b. Color reaction – 0.5 ml from supernatant + 5 ml anthrone
solution then keep the probe in water bath for 25 min. Concomitantly,
prepare a blank by using 0.5 ml trichloroacetic acid instead supernatant (it
contains no glucose)
c. Colorimetric method – The sample is read at Spekol
machine at 620 nm wavelength, as compared to blank (distilled water).
After that, the extinction is transformed in glucose concentration. The
transformation is performed using an etalon curve, prepared previously by
measuring the extinction of serial solution of known concentration of
glucose. The values are expressed in mg/100 ml
182
II.2. Plasma total protein measurement (Biuret)
In alkaline environment, the proteins react with copper sulfate and

results a violet complex.
The reaction is realized in two steps:
a. Color reaction – 0.1 ml (plasma or serum) + 4.9 ml Biuret
reactive then are left for 30 minutes at room temperature.
b. Colorimetric method – The sample is read at Spekol
spectrophotometer at 620 nm wavelength as compared to distillated water
and read on the calibration curve. The calibration curve is previously made
by measuring the extinction of protein solution (bovine serum albumin) of
already known concentration. The values are expressed in grams / 100 ml.
II.3. Dysproteinemia tests
Dysproteinemia tests despite their heterogeneity are based on a

simple principle. The albumins acts as stabilizations agents, while globulins
act as precipitating agents. In dysproteinemia the albumin: globulin ration is
imbalanced, in the most of the cases in favor of globulins. Consequently,
the serum precipitates more easily, faster and more intense in contact to
precipitating agents.
Takata Ara modified test

In one test tube mix 0.3 ml serum, 5 ml carbonate chloride solution
and 2 ml mercuric chloride solution. After 60 minutes, read the percentage
absorption at Spekol spectrophotometer at 660 nm wavelength as
compared to distillated water.
183
Results: 100% - 80% - negative,

80% - 70% - low positive,
70% - 50% - positive,
less than 50% - highly positive
Gross test
The serum reacts with Hayem solution, and in case of
dysproteinemia, the precipitation occurs easily.
In one test tube put 1 ml of serum, and then from one burette add
drop by drop Hayem solution, and record the moment of first precipitation
and the moment of irreversible precipitation of the serum. The normal
amount of Hayem solution required for the reversible serum precipitation is
1.5 ml, while for irreversible serum precipitation is 2.5 ml. The values under
this limit reveal dysproteinemia.
Kunkel test
In one test tube add 0.1 ml serum and 1.8 ml Kunkel reactive.
After 30 min the result is assessed visually the intensity of
precipitation. The intensity of dysproteinemia is correlated with the intensity
of precipitation.
II.4. Determination of plasma bicarbonate content
Principle: The bicarbonate anion is part of the blood buffer system

responsible for maintaining the normal blood pH at 7.4. The reference
range for serum is 22-28 mEq/l. The plasma concentration of the
bicarbonate can be assessed by titrimetric method. The principle of the
method consists in the addition in a serum or plasma sample with known
184
volume a fix amount of the hydrochloric acid. The acid will consume the
plasma bicarbonate, as shown in the following reaction:
NaHCO3 + HCl → NaCl + H2O +CO2
Then, the excess of acid will be neutralized with sodium hydroxide
by using a titrimetric method.
HCl + NaOH → NaCl + H2O
The difference between the acid added initially and the acid
neutralized in the second reaction represents the acid consumed by the
plasma bicarbonate.
Reagents: HCl (0.01N solution), NaOH (0.01N solution), blue bromtimol

indicator,
Procedure:
1 ml plasma (or serum) is introduced into an Erlenmeyer flask, and
then 5 ml HCl 0.01N and 2 - 3 drops bromothymol indicator are added. Prepare
then a control in which the hydrochloric acid solution is replaced by
distillated water. After mixing, the test sample is titrated against 0.01 NaOH taken
in a burette. Stop the titration when the color is similar to those of the control flask.
Record the volume of NaOH used.
Calculation:
The calculation is simplified by the fact that 1 mEq HCl consume 1
mEq of NaHCO3, and respectively 1 mEq of NaOH. Additionally both
solutions of HCl, and respectively NaOH have a similar concentration
respectively 0.01N (which means that we have 0.01 mEq /ml of each
solution), therefore, we can say that 1 ml sol of NaOH neutralize 1 ml
NaHCO3 .
NaHCO3 (mEq/ml) = 0.01 x [5 – vol NaOH 0.01N(ml)]
185
As the concentration of NaHCO3 is expressed in mEq/L, the value will be

multiplied to 1000. In conclusion, the final calculation formula will became:
NaHCO3 (mEq/L) = 10 x [5 – vol NaOH 0.01N (ml)]
186
Annex III Reference values
Table 7. Plasma protein concentration and electrophoretic fractions
days (D) Albumins Globulins % Total

weeks (W) % α1 α2 β γ proteins
mounts (M) g/100/ml
years (Y)
Dog 1-2 M 54-61 8-14 5-7 20-24 5-8 2.6-3.2
2-12 M 46-58 6-12 5-9 20-23 6-13 3.8-5.3
2-9 Y 46-56 4-8 5-11 18-21 10-17 4.2-5.2
over 9 Y 43-53 5-7 8-11 20-24 10-17 4.2-5.9
37-49 4-6 8-12 22-28 11-21 4.8-6.4
Cat 15 D-1 M 42-50 5-10 18-26 10-14 8-14 3.8-4.4
3 M -1 Y 35-47 5-6,6 17-23 9-13 15-20 5.5-7.0
2-11 Y 30-42 4-7 16-21 10-16 22-30 5.0-7.0
over 11 Y 27-36 4-6 18-23 10-14 22-34 6.2-7.0
Horse new born 65 32 3 - 3.7
la 5 D 37 36 25 2 5.2-5.5
la 8 D 33 14 23 28 6.8
la 15 D 37 6,4-19 16,5 21 4.4
2-5 Y 41-47 4-6 8-12 13-17 20-27 4.7-6.6
5-10 Y 35-41 4-6 9-12 15-20 26-33 5.8-7.0
10-15 Y 34-42 5-7 7-12 15-20 25-33 5.3-6.5
15-30 Y 32-40 5-7 8-14 19-22 20-28 5.0-6.2
42 Y 36.5 4.5 7.6 19.1 32.3 7.0
Cow 6 D -1 M 47-53 15-27 13-17 8-17 3.4-5.8
6 M -1 Y 35-43 13-16 11-14 29-33 5.5-6.5
1-2 Y 35-37 13-15 13-17 33-38 6.0-7.0
2-10 Y 30-40 12-16 9-13 31-44 7.0-7.6
Goat 5-7 M 43 15 11 7 24 5.3
2-7 Y 40-47 11-13 8-10 7-12 22-24 4.6-4.9
Sheep 3 W- 2 m 40-50 6-8 9-14 8-10 15-25 4.5-5.5
2-7 Y 41-45 7-9 10-15 9-11 20-28 6.0-6.5
Guinea pig 54.6 4.0 18,9 2.8 6.6 6.2
Syrian hamster 63.1 2.6 8.1 8.9 17.3 7.09
Rat 60 15 20 5 6.3
Mouse 48.0±3.97 18.5±7.5 19±7.5 14.5±11 60.2
Pop et all. 2002
187
Table 8. Hematological parameters of the main species of

domesticated and laboratory animals
Species RBC HGB HCT MCV MCH MCHC

6
(10 /µl) (g/dl) (%) (fl) (pg) (g/dl)
1
Dog 5.5-8.5 12-18 37-55 60-77 19.5-24.5 32-36
1
Cat 5-10 8-15 30-45 39-55 13-17 30-36
1
Cow 5-10 8-18 24-46 40-60 11-17 30-36
1
Horse 6-12 10-18 32-48 34-58 13-19 31-37
1
Pig 5-7 9-13 36-43 52-62 17-24 29-34
1
Sheep 9-15 9-15 27-45 28-40 8-12 31-34
1
Goat 8-18 8-12 22-38 16-25 5.2-8 30-36
1
Rabbit 5-8 10-17 33-50 58-67 17-24 29-37
2
Rat 6.5-9 13-16.5 41-51 52.6-64.4 16.5-21.3 30.2-34.6
2
Mouse 6.5-10 10-16 32.8-48 42.3-56 13.7-18 29.5-31
2
Hamster 2.7-12.3 13.4-19.2 48-57 64.8-77.6 - -
Guinea 4-6 10.5-15.3 34-48.3 75-91 - 28.2-33
2
pig
Ferret 2 6.8-9.8 14.8-17.4 42-55 42.6-51 13.7-16 30.3-34.9
1
Merck Veterinary Manual
2
Thrall AM et.all., 2006
188
Table 9. Reference values of white blood cells values in the main

species of domesticated and laboratory animals
WBCs N E B M L
Bands Mature
Dog1 103/µl 6-17 0-0.3 3-11.4 0.1-0.75 rare 0.1-1.3 1-4.8
% 0-3 60-70 2-10 3-10 12-30
1 3
Cat 10 /µl 5.5-19.5 0-0.3 2.5-12.5 0.1-0.75 rare 0-0.85 1.5-7
% 0-3 35-75 2-10 1-4 20-55
1 3
Cow 10 /µl 4-12 0-0.12 0.6-4 0-2.4 0-0.2 0.02-0.8 2.5-7.5
% 0-2 15-45 2-20 0-2 2-7 45-75
1 3
Horse 10 /µl 6-12 0-0.1 3-6 0-0.8 0-0.3 0-0.6 1.5-5
% 0-1 30-75 1-10 0-3 1-8 25-60
1 3
Pig 10 /µl 11-22 0-0.8 2-15 0-1.5 0-0.5 0-1 3.8-16.5
% 0-4 20-70 0-15 0-3 0-10 35-75
1 3
Sheep 10 /µl 4-12 0 0.7-6 0-1 0-0.3 0-0.75 2-9
% 0 10-50 0-10 0-3 0-6 40-75
1 3
Goat 10 /µl 4-13 rare 1.2-7.2 0.05-0.6 0-0.12 0-0.55 2-9
% 30-48 1-8 0-1 0-4 50-70
1 3
Rabbit 10 /µl 5-12.5 rare 1-9.4 0.05-0.5 0.05-0.9 0.05-0.5 1.6-10.6
% 20-75 1-4 1-7 1-4 30-85
2 3
Rat 10 /µl 7.3-12.6 0-0.02 1.25-3.7 0.04-0.3 0-0.03 0.05-0.4 5-9
2 3
Mouse 10 /µl 2.6-10 0-0.02 0.4-2 0-0.17 0-0.02 0- 1.3-8.4
Guinea
2 3
pig 10 /µl 8.2-14 0-0.01 1.35-3.6 0-0.69 0-0.02 0.06-0.56 5.5-10.5
1
Merck Veterinary Manual
2
Thrall AM and colab., 2006
189
References
References
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6th Edition, W.B. Saunders, Philadelphia, Pennsylvania, USA.
Dunlop RH, Malbert CH, 2004, Veterinary Pathophysiology, Blackweell
Publishing, Oxford UK
Estridge BH, Reynolds AP, Walters NJ, 2000, Basic Medical Laboratory
Technicques 4th Edition, Thomson Learning, London UK
Jackson MJ, 2007, Veterinary Clinical Pathology an Introduction,
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Hrapkiewicz K, L Medina 2007, Clinical Laboratory Animal Medicine – an
introduction. Third Edition. Blackwell Publishing Ames, Iowa USA
Keer MG, 2002, Veterinary Laboratory Medicine – Clinical Biochemistry
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Kerr and colab., 1989 Veterinary Laboratory Medicine 2nd Edition,
Blackweell Science
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Pathologic Basis of Disease 8th Edition, Saunders Elsevier,
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Latimer K.S. 2011, Duncan and Prasse’s veterinary laboratory medicine:
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Marcus I, 2011, Fiziopatologie tulburări func ionale i mecanisme
etiopatogenetice, Risoprint Cluj Napoca
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Vajdovich P, 2008, Free Radicals and Antioxidants in Inflammatory
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Bogdan Sevastre

Descrierea CIP a Bibliotecii Naþionale a României

Director editurã: GHEORGHE POP

IOAN MARCUS DVM, PhD – Professor

PETER VAJDOVICH DVM, PhD, Dipl. of ECVCP – Associate Professor

ALINA E. PARVU MD, PhD – Associate Professor

Tiparul executat la:

1. Laboratory Safety Rules .................................................................. 9

2. General Characteristics of Disease ..............................................13

3. Hemodynamic Disorders ...............................................................25

5. Metabolic Disorders .......................................................................51

7. Pathophysiology of the Heart........................................................91

8. Respiratory System ......................................................................113

9. Digestive System and Annex Glands .........................................115

10. Excretory System .......................................................................123

11. Endocrine System ......................................................................133

Annex I – Case presentation ...........................................................141

Annex II – Laboratory techniques...................................................163

Hematology techniques ...................................................................163

Annex III – Reference values ...........................................................187

Writing this new handbook of Pathophysiology is motivated by the

1. Laboratory Safety Rules

Hazards in Clinical Laboratory

In the clinical laboratory, tree types of hazards may be encountered:

1.1. Physical Hazards

Physical hazards are resent in ordinary equipment or surroundings.

1.2. Chemical Hazards

Chemicals present a wide range of hazards; they may be inflammable,

1.3. Biological Hazards

Biological hazards are always involved when biological samples are

The waste management

All chemicals and biological waste should be disposed of properly and

General laboratory rules

− Do not eat, drink, or apply cosmetics in the work area. Do not

2. General Characteristics of Disease

Disease is an abnormal condition of an organism that impairs the

2.1. The Influence of Disease on Animal

One common feature shared by many diseases is that the affected

Practical activities for students

The open field test (OFT) is commonly used to measure general

Table 1. Disease related changes

2.2. Pathogenesis of Environmental Factors

2.2.1. Local Action of the Heat: Thermal Burns

Heat effect General - hyperthermia – caloric shock

A burn is a type of injury to the skin caused by heat. Most burns

Two main classification systems exist. The traditional system

Combustion bullosa - Second-degree burns manifest as

Table 2. Classification by thickness

Combustio gangrenosa - Third-degree burns occur when the

signals; however, all third-degree burns are surrounded by first and

2.2.2. Local Action of the Cold: Frostbites

Cold effect General - hypothermia

Frostbite (congelatio in medical terminology) is the medical

Congelatio erythematosa First degree frostbite

Congelatio bullosa Second degree frostbite

Congelatio gangrenosa Third degree frostbite

Activities for students

2.2.3. Experimental Photosensitization

Photosensitization is a clinical condition in which skin (areas

Photosensitization requires three factors:

Photosensitization is often classified according to the source of the

develop erythema which is soon followed by edema. When exposure is

Activities for students

2.2.4. The Influence of General Status on Responsiveness to