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Clinical pathology laboratory report by KULA JILO 2016
Jimma University
College of agriculture and veterinary medicine
School of veterinary medicine
Clinical Pathology Laboratory Report
Submitted by: Kula Jilo
ID.NUMBER=05537/04
Submission date: 13-01-2016
Submitted To: mr.Eshetu (lab.technician)
January, 2016
Jimma, Ethiopia
Clinical pathology laboratory report by KULA JILO 2016
1
Laboratory Manual and Review on Clinical Pathology
Abstract
Clinical pathology is a subspecialty of pathology that deals with the use of laboratory methods
(clinical chemistry, microbiology, hematology and emerging subspecialties such as molecular
diagnostics) for the diagnosis and treatment of disease. Hematology studies the blood and
blood-forming tissues to evaluate presence of disease and assist in therapeutic interventions as
clinically indicated. Clinical chemistry (also known as chemical pathology and clinical
biochemistry) is the area of clinical pathology that is generally concerned with analysis of bodily
fluids. Some of the objectives of this manual are to identify the most important hematological
and functional pathological tests of vet importance, to diagnose different animal diseases by
confirming the pathological causes that constraint livestock production and to have knowledge
more about clinical pathology. Part one discusses about hematology which includes equipment’s
and reagents, blood collection sites and procedures, preparation method for working solution,
staining methods (staining procedures), hemoglobin determination, hematocrit determination
(PCV), total RBC count, total WBC count, differential leukocyte count, determination of ESR,
coagulation time determination, bleeding time, calculating red blood cell indices and blood
group and Rh factor determination. Part two deals with function tests which includes
determination of Aspartate Amino Trasferase (AST) and Glutamic OxalacetateTransminase
(GOT), determination of Alkaline Phosphtase (ALP), determination of creatinine, total protein
determination, urea determination, total and direct bilirubin determination, enzymatic kinetic
colorimeter test, liver function test, kidney function test, rumen function test and pancreatic
function test. In general, the outline of this laboratory manual deals with the basic
hematological procedures and clinical chemistry analysis.
Objectives of Clinical Pathology Practical Course
Objectives of this course are enabling students to:
a) Identify the most important hematological and functional pathological tests of vet
importance
b) Diagnose different animal diseases by confirming the pathological causes that
constraint livestock production in general
c) Have knowledge more about clinical pathology
d) Write lab report pertaining the tests carried out
Clinical pathology laboratory report by KULA JILO 2016
2
Equipment’s and reagents required for hematological blood examination.
 Hematocrit centrifuge
 Compound microscope
 Sahlis instrument
 Capillary tube
 Hematocrit reader
 Distilled water
 Haemocytometer pipette
 Haemocytometer counting
chamber
 WBC diluting fluid
 RBC diluting fluid
 Slides cover slip
 special cover slip
 ESR stand
 ESR tube
 Filter paper
 blood lancets
 lead pencils
 marker
 Giemsa stain
 Wright’s stain
 Vacutainer tubes(different
type)
 vacutainer needles
 syringes
 needle holder
 Gloves
 Blood samples
 Staining racks and others
Lab.2. smear preparation
Procedures:
Blood was collected from jugular vein of animal (cow) with EDTA Vacutainertube.Then
collected blood is transported to the laboratory and wet smear, thin smear and thick smear
were done respectively.
I. Wet blood smear preparation
1. A drop of blood was placed at the center of a clean slide
2. Covered with a clean, dry cover slip
3. Then examined under the microscope (40 × objective).
Result: blood cells structures observed and parasites checked
II. thin blood smear preparation
1. A drop of blood made on one end of glass slide
2. A drop of blood was evenly spreaded across a clean grease free slide using a
smooth edged spreader holding at angle of 45 degree
3. Then smear was dried by waving rapidly in the air
4. Then examined under the microscope (40 × objective).
Clinical pathology laboratory report by KULA JILO 2016
3
Result: different blood cells observed
III. Thick bloods smear preparation
1. A large drop of blood was put at the center of a clean dry slide
2. The drop was spreaded with corner of another slide to cover an area
of ½ an inch square
3. The smear is thoroughly dried in a horizontal position so that the
blood could not ooze to one edge to the film and protected from
dust, insects and direct sunlight.
Result: RBC observed
Lab. 3 Staining Dyes
 Dry powder as bulk and as pre weighted vials, Capsule Readymade
solution and most convenient, but often deteriorated from long storage.
3.1. Preparation of stain
 Conventional method
1. 0.5gm of dry Wright’s stain powder was placed in a mortar
2. 300ml of absolute methyl alcohol was added
3. The mixture and pour was grinded in dark bottle
4. The bottleshacked each day for about two weeks, and then filtered.
5. Aging of the stain took 3 weeks for use
 Accelerated method
1. 0.3gm of dry Wright’s stain powder was placed in a mortar and over
layed with 3ml of glycerol and grinded thoroughly
2. Rinsed with 100ml of absolute methyl alcohol and placed in dark
container
3. Mixed with magnetic stirrer for about 1 week without heat
4. then filtered and used
Clinical pathology laboratory report by KULA JILO 2016
4
3.2. Giemsa Stain
General consideration
Giemsa stain has various azure compounds with eosin and methylene blue. It is an
excellent stain for blood parasites and for inclusion bodies it stains red granules
well but neutrophilic granules and erythrocytes are poorly stained. Commercial
stock solution are recommended for purchase and are stable indefinitely
Preparation method for working solution:
1. one part of giemsa stock solution and nine part distilled water was
taken
2. And mixed before to two hours and used.
3.3. Leishmen’s stain
Preparation
1. 0.15gm of Leishmen’s stain powder was grinded with small amounts
of absolute methyl alcohol until an even suspension is obtained.
2. Then a total of 100ml methanol is added to produce complete
solution
3. Then Poured in to a dark bottle
4. and age for a few weeks prior to used
3.4. Wright’s-Giemsa stain (modified Wright’s stain)
Preparation of stain
1. 500mg of dry Wright’s stain powder and 50mg of Giemsa stain
powder were ground in a mortar with 100ml of absolute methyl
alcohol (acetone free)
2. Allowed to stand for 24-48 hrs. before used
3. Kept with well stoppered to prevent evaporation of alcohol or
absorption of water vapor
Commercial Wright’s – Giemsa stain are available
• Wright – Leishmen stain
• May – Grunwald –Giemsa stain
• Methylene blue stain
• Field’s stain
Clinical pathology laboratory report by KULA JILO 2016
5
• Prussian blue stain and others
3.5. Buffered distilled water
Preparation:
 5.47g monobasic potassium phosphate and 3.8g of dibasic sodium
phosphate were mixed with 1000ml of distilled water
Methods of staining (staining procedure)
a) Wright’s stain
1. The dried blood smear is covered completely with Wright’s stain and
allowed to stand for 1 to 3 minute
2. An equal amount of buffered distilled water or neutral water (pH 6.6
to 6.8 for most animals blood) was added
3. Blown gently and allowed the mixture to stand for 3 to 5 minutes.
4. The metallic scum with a stream of water from a wash bottle was
floated from the top.
5. Distilled water was used.
6. The stain wiped from the back of the slide with cleaning tissue
7. Standing the slide on end and waved gently in the air to dry
8. then preparation examined by placing a drop of oil on a slide
Advantage: It conserves stain, Methyl alcohol, and time
Disadvantage: More precipitate is likely to be present
Cell Cytoplasm Chromatin
Erythrocytes Yellow to pinkish Purple
Lymphocytes Blue Purplish red
Monocytes Light blue Purple
Eosinophils Yellow to brownish red rods Light purple
Basophils Dark purple granules
Hetrophils/Neutrophils Yellow to brownish red rods Light purple
Thrombocytes Grey-blue Purple
Table 2: Staining characteristics.
Clinical pathology laboratory report by KULA JILO 2016
6
b) Giemsa stain method
1. Place dried blood smear in a coplin jar containing absolute methyl
alcohol for about 3 minute to fix the smear
2. Drain off the alcohol and allow the slide to dry
3. Transfer the slide to a second coplin jar containing fresh stain and
allow staining for 16 to 60 minute. 60 minute staining period is used
when blood parasite is suspected. 12 to 18 hrs for inclusion bodies
4. Wash the stain with running/tap water, dry and examine under oil
immersion microscope
c) Leishman’s stain method
1. the air dried blood film flooded with Leishman’s stain and stayed for
1 to 2 minute to fix
2. the stain diluted on the smear with double the volume of buffered
distilled water and stain for 5 to 15 minute
3. Blew on the mixture gently
4. Washed with distilled water until the film has a pinkish tinge
5. the back of the stain/the slide Wiped and allowed to dry in upright
position
Lab.4 and 5 Hemoglobin Determinations
Objective:
 To determine the amount of hemoglobin present in 100 ml of blood of
a given sample.
Significance
a. It serves as an index of blood condition of the animal.
b. If the hemoglobin [Hb] content falls below the normal levels, it indicates
anemia, or pregnancy (physiological).
c. If it increases to the normal value, it indicates polycythemia, decrease in
O2 supply, heart disease, emphysema etc.
Method: Acid hematin method
Requirements: Sahlis instrument, blood sample
Procedure
1. 0.1N HCl (1%) was taken into central graduated tube up to mark 2.
Clinical pathology laboratory report by KULA JILO 2016
7
2. The blood sucked exactly up to mark 20 (20 μl) with the help of sahlis
pipette.
3. The blood transferred from pipette to central graduated tube of the
hemometer.
4. Then mixed well with the help of stirrer or rod and allowed it to react
for two minute.
5. The distilled water was added drop by drop until the color matches
with the standard comparator tube and mixed well.
6. After the color matched it taken out and recorded the values on the
side as gm/100ml and or in percentage.
7. Repeated 5 to 6 times and the average value was taken
Normal value:
Bovine 8 - 15gm/dl (11) Equine 11 -19gm/dl (14)
Ovine 9 – 15gm/dl (11.5) Feline 8 – 15gm/dl (12)
Caprine: 8-14gm/dl (11) Canine 12 – 18gm/dl (15)
Hematocrit Determination (PCV)
Aim: To determine the hematocrit value for a given blood sample.
Principle: Blood compartment is separated into three parts using capillary tube in
a hematocrit centrifuge.
Significance
• Packed Cell Volume (PCV) = erythrocyte mass; anemia when PCV falls dawn.
• Buffy coat; white to gray layer above PCV. It will give number of WBC (0.5mm
to1.5mm).Leukopenia or leukocytosis.
• Plasma content: usually about 55%, Yellowish in color. Degree of yellowness
indicates icterus (jaundice).
Requirements: Hematocrit tube, hematocrite centrifuge, hematocrit reader and
sealer.
Procedure
• The blood was filled in to a micro hematocrit tube (3/4th) and it sealed with
sealer.
• The filled hematocrit tube in a hematocrite centrifuged at 2000 rpm for 4-5
minutes.
• Then the value (the tube) with hematocrit reader was read and recorded.
Normal value
• Bovine 37 - 55 % (45%)
• Ovine 24 - 50 % (38%)
• Caprine 24 - 50 % (40%)
• Equine 32 - 55 % (42%)
• Swine 32 - 50 % (42%)
Clinical pathology laboratory report by KULA JILO 2016
8
Lab.6 and 7
Total Count of RBC
Objective: To enumerate the total count of RBC/cumm of a given blood sample.
Method: Hemocytometry method
Significance
• It performs some functions such as transportation of O2 and CO2
• A decrease in RBC accounts for less hemoglobin i.e., anemia
• An increase in RBC is referred as Polycythemia
Requirements: Hemocytometer, cover slip, microscope, RBC diluting fluid,
Haeyem’s solution or Physiological saline 0.85% Nacl.
Procedure
1. the blood is taken in to RBC pipette up to 0.5 marks
2. The RBC diluting fluid was drawn up to mark 101.
3. The pipette was rotated between thumb and other fingers with finger eight
(8) movements. This gives a dilution of 1:200.
4. the counting chamber of hemocytometer and cover slip was cleaned
5. cover slip placed in position over the counting chamber by gentle pressure
6. A drop of blood on to the counting chamber was expeled by holding the
pipette at an angle of 45º.
7. hemocytometer allowed for 2-3 min to settle down the RBC in counting
chambe
8. Counted less than 40 × microscope objective
9. Cells touched the left and top side lines were not counted.
10.Cells touching the bottom right side lines were not counted.
11.Cell counted first left to right direction, then to vise verse.
*******THE END******

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Clinical Pathology Laboratory Report by Kula Jilo 2016

  • 1. Clinical pathology laboratory report by KULA JILO 2016 Jimma University College of agriculture and veterinary medicine School of veterinary medicine Clinical Pathology Laboratory Report Submitted by: Kula Jilo ID.NUMBER=05537/04 Submission date: 13-01-2016 Submitted To: mr.Eshetu (lab.technician) January, 2016 Jimma, Ethiopia
  • 2. Clinical pathology laboratory report by KULA JILO 2016 1 Laboratory Manual and Review on Clinical Pathology Abstract Clinical pathology is a subspecialty of pathology that deals with the use of laboratory methods (clinical chemistry, microbiology, hematology and emerging subspecialties such as molecular diagnostics) for the diagnosis and treatment of disease. Hematology studies the blood and blood-forming tissues to evaluate presence of disease and assist in therapeutic interventions as clinically indicated. Clinical chemistry (also known as chemical pathology and clinical biochemistry) is the area of clinical pathology that is generally concerned with analysis of bodily fluids. Some of the objectives of this manual are to identify the most important hematological and functional pathological tests of vet importance, to diagnose different animal diseases by confirming the pathological causes that constraint livestock production and to have knowledge more about clinical pathology. Part one discusses about hematology which includes equipment’s and reagents, blood collection sites and procedures, preparation method for working solution, staining methods (staining procedures), hemoglobin determination, hematocrit determination (PCV), total RBC count, total WBC count, differential leukocyte count, determination of ESR, coagulation time determination, bleeding time, calculating red blood cell indices and blood group and Rh factor determination. Part two deals with function tests which includes determination of Aspartate Amino Trasferase (AST) and Glutamic OxalacetateTransminase (GOT), determination of Alkaline Phosphtase (ALP), determination of creatinine, total protein determination, urea determination, total and direct bilirubin determination, enzymatic kinetic colorimeter test, liver function test, kidney function test, rumen function test and pancreatic function test. In general, the outline of this laboratory manual deals with the basic hematological procedures and clinical chemistry analysis. Objectives of Clinical Pathology Practical Course Objectives of this course are enabling students to: a) Identify the most important hematological and functional pathological tests of vet importance b) Diagnose different animal diseases by confirming the pathological causes that constraint livestock production in general c) Have knowledge more about clinical pathology d) Write lab report pertaining the tests carried out
  • 3. Clinical pathology laboratory report by KULA JILO 2016 2 Equipment’s and reagents required for hematological blood examination.  Hematocrit centrifuge  Compound microscope  Sahlis instrument  Capillary tube  Hematocrit reader  Distilled water  Haemocytometer pipette  Haemocytometer counting chamber  WBC diluting fluid  RBC diluting fluid  Slides cover slip  special cover slip  ESR stand  ESR tube  Filter paper  blood lancets  lead pencils  marker  Giemsa stain  Wright’s stain  Vacutainer tubes(different type)  vacutainer needles  syringes  needle holder  Gloves  Blood samples  Staining racks and others Lab.2. smear preparation Procedures: Blood was collected from jugular vein of animal (cow) with EDTA Vacutainertube.Then collected blood is transported to the laboratory and wet smear, thin smear and thick smear were done respectively. I. Wet blood smear preparation 1. A drop of blood was placed at the center of a clean slide 2. Covered with a clean, dry cover slip 3. Then examined under the microscope (40 × objective). Result: blood cells structures observed and parasites checked II. thin blood smear preparation 1. A drop of blood made on one end of glass slide 2. A drop of blood was evenly spreaded across a clean grease free slide using a smooth edged spreader holding at angle of 45 degree 3. Then smear was dried by waving rapidly in the air 4. Then examined under the microscope (40 × objective).
  • 4. Clinical pathology laboratory report by KULA JILO 2016 3 Result: different blood cells observed III. Thick bloods smear preparation 1. A large drop of blood was put at the center of a clean dry slide 2. The drop was spreaded with corner of another slide to cover an area of ½ an inch square 3. The smear is thoroughly dried in a horizontal position so that the blood could not ooze to one edge to the film and protected from dust, insects and direct sunlight. Result: RBC observed Lab. 3 Staining Dyes  Dry powder as bulk and as pre weighted vials, Capsule Readymade solution and most convenient, but often deteriorated from long storage. 3.1. Preparation of stain  Conventional method 1. 0.5gm of dry Wright’s stain powder was placed in a mortar 2. 300ml of absolute methyl alcohol was added 3. The mixture and pour was grinded in dark bottle 4. The bottleshacked each day for about two weeks, and then filtered. 5. Aging of the stain took 3 weeks for use  Accelerated method 1. 0.3gm of dry Wright’s stain powder was placed in a mortar and over layed with 3ml of glycerol and grinded thoroughly 2. Rinsed with 100ml of absolute methyl alcohol and placed in dark container 3. Mixed with magnetic stirrer for about 1 week without heat 4. then filtered and used
  • 5. Clinical pathology laboratory report by KULA JILO 2016 4 3.2. Giemsa Stain General consideration Giemsa stain has various azure compounds with eosin and methylene blue. It is an excellent stain for blood parasites and for inclusion bodies it stains red granules well but neutrophilic granules and erythrocytes are poorly stained. Commercial stock solution are recommended for purchase and are stable indefinitely Preparation method for working solution: 1. one part of giemsa stock solution and nine part distilled water was taken 2. And mixed before to two hours and used. 3.3. Leishmen’s stain Preparation 1. 0.15gm of Leishmen’s stain powder was grinded with small amounts of absolute methyl alcohol until an even suspension is obtained. 2. Then a total of 100ml methanol is added to produce complete solution 3. Then Poured in to a dark bottle 4. and age for a few weeks prior to used 3.4. Wright’s-Giemsa stain (modified Wright’s stain) Preparation of stain 1. 500mg of dry Wright’s stain powder and 50mg of Giemsa stain powder were ground in a mortar with 100ml of absolute methyl alcohol (acetone free) 2. Allowed to stand for 24-48 hrs. before used 3. Kept with well stoppered to prevent evaporation of alcohol or absorption of water vapor Commercial Wright’s – Giemsa stain are available • Wright – Leishmen stain • May – Grunwald –Giemsa stain • Methylene blue stain • Field’s stain
  • 6. Clinical pathology laboratory report by KULA JILO 2016 5 • Prussian blue stain and others 3.5. Buffered distilled water Preparation:  5.47g monobasic potassium phosphate and 3.8g of dibasic sodium phosphate were mixed with 1000ml of distilled water Methods of staining (staining procedure) a) Wright’s stain 1. The dried blood smear is covered completely with Wright’s stain and allowed to stand for 1 to 3 minute 2. An equal amount of buffered distilled water or neutral water (pH 6.6 to 6.8 for most animals blood) was added 3. Blown gently and allowed the mixture to stand for 3 to 5 minutes. 4. The metallic scum with a stream of water from a wash bottle was floated from the top. 5. Distilled water was used. 6. The stain wiped from the back of the slide with cleaning tissue 7. Standing the slide on end and waved gently in the air to dry 8. then preparation examined by placing a drop of oil on a slide Advantage: It conserves stain, Methyl alcohol, and time Disadvantage: More precipitate is likely to be present Cell Cytoplasm Chromatin Erythrocytes Yellow to pinkish Purple Lymphocytes Blue Purplish red Monocytes Light blue Purple Eosinophils Yellow to brownish red rods Light purple Basophils Dark purple granules Hetrophils/Neutrophils Yellow to brownish red rods Light purple Thrombocytes Grey-blue Purple Table 2: Staining characteristics.
  • 7. Clinical pathology laboratory report by KULA JILO 2016 6 b) Giemsa stain method 1. Place dried blood smear in a coplin jar containing absolute methyl alcohol for about 3 minute to fix the smear 2. Drain off the alcohol and allow the slide to dry 3. Transfer the slide to a second coplin jar containing fresh stain and allow staining for 16 to 60 minute. 60 minute staining period is used when blood parasite is suspected. 12 to 18 hrs for inclusion bodies 4. Wash the stain with running/tap water, dry and examine under oil immersion microscope c) Leishman’s stain method 1. the air dried blood film flooded with Leishman’s stain and stayed for 1 to 2 minute to fix 2. the stain diluted on the smear with double the volume of buffered distilled water and stain for 5 to 15 minute 3. Blew on the mixture gently 4. Washed with distilled water until the film has a pinkish tinge 5. the back of the stain/the slide Wiped and allowed to dry in upright position Lab.4 and 5 Hemoglobin Determinations Objective:  To determine the amount of hemoglobin present in 100 ml of blood of a given sample. Significance a. It serves as an index of blood condition of the animal. b. If the hemoglobin [Hb] content falls below the normal levels, it indicates anemia, or pregnancy (physiological). c. If it increases to the normal value, it indicates polycythemia, decrease in O2 supply, heart disease, emphysema etc. Method: Acid hematin method Requirements: Sahlis instrument, blood sample Procedure 1. 0.1N HCl (1%) was taken into central graduated tube up to mark 2.
  • 8. Clinical pathology laboratory report by KULA JILO 2016 7 2. The blood sucked exactly up to mark 20 (20 μl) with the help of sahlis pipette. 3. The blood transferred from pipette to central graduated tube of the hemometer. 4. Then mixed well with the help of stirrer or rod and allowed it to react for two minute. 5. The distilled water was added drop by drop until the color matches with the standard comparator tube and mixed well. 6. After the color matched it taken out and recorded the values on the side as gm/100ml and or in percentage. 7. Repeated 5 to 6 times and the average value was taken Normal value: Bovine 8 - 15gm/dl (11) Equine 11 -19gm/dl (14) Ovine 9 – 15gm/dl (11.5) Feline 8 – 15gm/dl (12) Caprine: 8-14gm/dl (11) Canine 12 – 18gm/dl (15) Hematocrit Determination (PCV) Aim: To determine the hematocrit value for a given blood sample. Principle: Blood compartment is separated into three parts using capillary tube in a hematocrit centrifuge. Significance • Packed Cell Volume (PCV) = erythrocyte mass; anemia when PCV falls dawn. • Buffy coat; white to gray layer above PCV. It will give number of WBC (0.5mm to1.5mm).Leukopenia or leukocytosis. • Plasma content: usually about 55%, Yellowish in color. Degree of yellowness indicates icterus (jaundice). Requirements: Hematocrit tube, hematocrite centrifuge, hematocrit reader and sealer. Procedure • The blood was filled in to a micro hematocrit tube (3/4th) and it sealed with sealer. • The filled hematocrit tube in a hematocrite centrifuged at 2000 rpm for 4-5 minutes. • Then the value (the tube) with hematocrit reader was read and recorded. Normal value • Bovine 37 - 55 % (45%) • Ovine 24 - 50 % (38%) • Caprine 24 - 50 % (40%) • Equine 32 - 55 % (42%) • Swine 32 - 50 % (42%)
  • 9. Clinical pathology laboratory report by KULA JILO 2016 8 Lab.6 and 7 Total Count of RBC Objective: To enumerate the total count of RBC/cumm of a given blood sample. Method: Hemocytometry method Significance • It performs some functions such as transportation of O2 and CO2 • A decrease in RBC accounts for less hemoglobin i.e., anemia • An increase in RBC is referred as Polycythemia Requirements: Hemocytometer, cover slip, microscope, RBC diluting fluid, Haeyem’s solution or Physiological saline 0.85% Nacl. Procedure 1. the blood is taken in to RBC pipette up to 0.5 marks 2. The RBC diluting fluid was drawn up to mark 101. 3. The pipette was rotated between thumb and other fingers with finger eight (8) movements. This gives a dilution of 1:200. 4. the counting chamber of hemocytometer and cover slip was cleaned 5. cover slip placed in position over the counting chamber by gentle pressure 6. A drop of blood on to the counting chamber was expeled by holding the pipette at an angle of 45º. 7. hemocytometer allowed for 2-3 min to settle down the RBC in counting chambe 8. Counted less than 40 × microscope objective 9. Cells touched the left and top side lines were not counted. 10.Cells touching the bottom right side lines were not counted. 11.Cell counted first left to right direction, then to vise verse. *******THE END******