Normal hemoglobins of humans and animals. Determination of hemoglobin amount

LESSON 17. Obtaining blood, determining the erythrocyte sedimentation rate and blood clot retraction

The location of the lesson is a clinic and laboratory.

Purpose of the lesson:

Master the technique of taking blood from animals.

Learn to determine the erythrocyte sedimentation rate and blood clot retraction.

ESR indicators different types animals and deviations.

Blood clot retraction index values ​​in horses and large cattle and in pathology.

For getting large quantity blood is taken from cattle, sheep, goats, horses from jugular vein using bloodletting needles, in pigs - from the tail, caudal artery and cronial vena cava.

A small amount of blood can be obtained in animals from the blood vessels of the ear, in chickens from the comb or earrings.

Determination of erythrocyte sedimentation rate

according to Nevodov

0.04 g of sodium citrate powder is weighed into the erythrosediometer, then blood is drawn into it to “0” from the jugular vein (having previously prepared the field). Carefully invert the erythrosediometer until the powder is completely dissolved. If there is more than “0” blood, then the excess is sucked off with a pipette. Having placed the test tube in a stand, monitor the progress of the reaction every 15 minutes for an hour, then note it after 24 hours.

According to Panchenkov

After washing with a freshly prepared 5% solution of sodium citrate, the capillary is filled with the same solution to the P mark or division 50 and released onto a watch glass or into a test tube, then blood is drawn twice to the K mark or division. 100 and released onto the same watch glass or into a test tube, mixed and the mixture is taken without air bubbles into another capillary, washed with sodium citrate, which is placed in a stand and the progress of the reaction is observed. ESR is taken into account after 1 hour and expressed in millimeters.

In healthy animals, the indicators per hour are: for cattle - 0.5-1.5; in sheep - 0.5-1.0; in horses - 40-70; in pigs - 2-9; in dogs - 2-6; in chickens - 2-3 mm.

Determination of blood clot retraction

according to P. V. Kaimakov

Blood in an amount of 10 ml is drawn into a dry test tube, well washed and cleaned with alcohol and ether, and left for 24 hours at room temperature.

After 24 hours, the settled serum is aspirated with a pipette and its amount is measured in a measuring tube, after which the retraction index (RI) is determined by dividing the amount of serum by the initial volume of blood.

Example: 10 ml of blood and 5 ml of serum are taken

I.R. 10 = 0.5

I.R. in a horse ranges from 0.3 to 0.7, with an average of 0.53

In cattle it is 0.4-0.6.

Research results.

Erythrocyte sedimentation rate indicators:

after 15 minutes

in 30 minutes

in 45 minutes

After 1 hour

in 24 hours

Blood clot retraction index

Conclusion

Educational equipment, demonstration material, animals

Nevodov tubes or erythrosediometers

Panchenkov apparatus

Sodium citrate

5% sodium citrate solution

Test tubes with a diameter of 1.5 cm

1. Blood collection needles

2. Tincture Yoda

   Alcohol ether

   Tables with ESR indicators and IR in animals

Animals: Cattle, horses, piglets

VIII. Evaluation of the student's work by the teacher.

LESSON 18. Counting the number of red blood cells, determining hemoglobin in the blood and color index

The location of the lesson is a clinic, laboratory.

The purpose of the lesson is to master the methods of counting red blood cells and determining hemoglobin.

Current student survey on the following questions:

Indicators of red blood cells and hemoglobin in different animal species.

Their deviations under various physiological and pathological conditions.

Explanation of the teacher’s methodology for performing the work.

Execution of work by students.

Counting the number of red blood cells.

Blood is drawn into the mixer to the 0.5 or 1 mark. The end of the melangeur is wiped of blood, then physiological solution is drawn into the capillary to the 101 mark. This results in a dilution of 200 or 100 times. When the mixer is filled with blood and saline, the ends are pinched between the thumb and forefinger and shaken vigorously several times.

Before charging the chamber, the contents of the mixer are thoroughly mixed again and introduced under a cover glass. To ensure that the height of the chamber does not change, it is necessary to wipe the cover glass tightly until the so-called. Newtonian rings.

The charged chamber is placed under the microscope without immersion. Counting red blood cells begins with the square located in the left corner, then moves on to other squares.

You need to count in 5 large squares all the red blood cells lying inside the square and on its internal lines.

The counted red blood cells in five large squares are summed up and the content of the number of red blood cells in 1 cubic meter is determined. mm. blood according to the following formula: M. 4 000 . at

The number of red blood cells X is equal to the number of counted cells in five large squares M multiplied by 4,000 and the dilution degree y divided by 80 (the number of small squares).

The volume of one small square is 1/4,000 cubic meters. m., and therefore, to get a quantity of 1 cubic. mm. the number of red blood cells must be multiplied by 4,000.

The calculation must be made as follows: the number of red blood cells in 5 squares is multiplied by 10,000 when diluted by 200 and

by 5,000 when diluted by 100.

Tube method for counting red blood cells.

4 ml of saline solution is poured into a clean Florinsky test tube. Blood is drawn into a pipette from a Sali hemometer (0.02 ml), carefully blown into a test tube with saline solution, the pipette is washed, capped and mixed thoroughly. Dilution 1:200.

The blood taken into the test tube is shaken several times before filling the chamber. The chamber is filled using a Pasteur pipette or the end of a round glass rod, it is important that the entire surface on which the grid is applied is filled with liquid. The calculation is made in the usual way, taking into account dilution.

In connection with the transition to the use of the International System of SI units in clinical laboratory diagnostics, the workshop also presents new units along with the old physical units. For example: instead of expressing the number of erythrocytes in millions/μl and leukocytes in thousand/μl, the number of erythrocytes should be indicated in 10 12 l, and leukocytes in 10 9 l.

The number of erythrocytes in adult healthy animals in million/μl or 10 12 l;

KRS 4.5-7.5; sheep - 7.6-11.2; horses - 5.5-9.0; pigs - 4.0-7.5; dogs - 5.2–8.4; chickens - 2.5–5.0.

Determination of hemoglobin. Sali's method

Using a pipette, measure to the 10th division or to the circular mark with the number 2 g per cent into a graduated test tube. decinormal hydrochloric acid solution. Blood is drawn into a capillary up to 20 mm and carefully, after cleaning the end of the capillary, the blood is transferred into a test tube with a decinormal HCl solution so that there are no traces of blood left on the walls of the capillary, rinsed three more times with the same acid, trying not to foam the liquid.

The blood is hemolyzed and hydrochloric acid hematin is formed.

After 5-7 minutes, distilled water is added to the test tube. Bringing the coloring to standard.

The number corresponding to the fluid level along the lower meniscus in the test tube shows the amount of hemoglobin according to Saly or g\100 ml.

Determination of color index

The color index is calculated using the formula:

where CPU is the color index;

H1 - the average amount of hemoglobin is normal, g per 100 ml (or g\l);

H2-amount of hemoglobin in the animal under study, g per 100 ml (or g/l);

E1 - the average number of red blood cells is normal, million \ μl (or 10 12 \ l);

E2-number of erythrocytes in the animal under study, mdn\µl

(or 10 12 \l).

The color index of blood in healthy animals is: in cattle - 0.7-1.1; in sheep - 0.5-0.7; in horses - 0.8-1.2; in pigs - 0.8-1.0; in dogs - 0.8-1.2; in chickens - 2-3.

Determination results:

Red blood cells.

Hemoglobin.

Color index.

Conclusion.

Microscope.

Goryaev's counting chamber.

Mixer for red blood cells.

Saline.

Cover slips.

Hemoglobinometer Sali.

Distilled water.

Decinormal hydrochloric acid solution.

Pipettes graduated at 1 and 5 ml, pipettes from Sali's hemometer.

Tables with indicators of red blood cells and hemoglobin in animals.

Animals: Cattle, horses, piglets.

LESSON 19. Counting the number of leukocytes. Preparation and coloring of smears. Removal of hemogram, hematological and leukocyte profile

The location of the lesson is the clinic and laboratory of the department.

Purpose of the lesson:

Master the technique of counting leukocytes.

Acquire skills in preparing and painting smears.

Derive leukocyte formulas of different animal species.

Determine the leukocyte profile in cattle.

Current student survey on the following questions:

Indicators of leukocytes in different animal species.

Their changes depending on certain physiological and pathological conditions.

The concept of leukocyte formula and leukocyte profile.

Indicators leukocyte formula in cattle and horses.

Morphological changes in leukocytes.

Explanation of the teacher’s methodology for performing the work.

Execution of work by students.

Leukocyte count.

Blood is drawn into a mixer or melanger to the level of 0.5 or 1 (from the ear or citrated blood). Then a 2% solution is taken into the same melangeur acetic acid to mark 11, a 20-fold dilution is obtained. After thoroughly mixing, the first two drops are removed from the mixer and a medium-sized drop is introduced under a cover glass to the chamber (previously ground in). The criterion for glass rubbing into the chamber is the formation of Newtonian rings. The counting rule is that the charged chamber is placed under the microscope without immersion. Leukocytes are counted in 100 large squares (i.e. 1600 small). It is permissible to count 75 large squares (i.e. 1200 small ones).

a x 4000 x b

Formula for calculation: X =

where X is the required number of formed elements in 1 m 3;

a is the sum of the shaped elements counted in a certain volume of the chamber;

b-number of small squares counted;

c-blood dilution.

The volume of a small square is 1\100 mm 3, then in 1 mm 3 of blood there will be 400 times more of them.

Example: (blood dilution 20 times).

In 1600 small squares (i.e. 100 large) 150 leukocytes are counted, then in 1 m3 there will be:

150 x 4000 x 20

1600 = 150 x 50 = 7500

those. the sum of leukocytes counted in 100 large squares must be multiplied by 50.

Leukocyte count (tube method)

Pipette 0.4 ml of acetic acid solution into a clean test tube. Blood is collected with a pipette from a Sali-0.02 ml hemometer and carefully transferred to a test tube with 3% acetic acid. Dilution 1:20. The chamber is filled using a Pasteur or the end of a round glass rod; it is important that the entire surface of the mesh is filled with liquid. The calculation is made in the usual way, taking into account dilution.

The number of leukocytes in adult healthy animals (thousand µl or 10 9 \l); for cattle - 6.5-9.5; in sheep - 6.6-10.6; in horses - 7.0-12.0; in pigs - 6.7-23.0; in dogs - 8.5-10.5; in chickens - 20.0-40.0.

Derivation of leukocyte formula.

The counting of individual classes of leukocytes is carried out under an immersion microscope. In total, at least 100 cells should be counted in the smear, preferably 200. There are counting methods according to Meander-4-pole and Mukhin (three lines perpendicular to the smear in its center).

Blood leukogram of healthy animals, %

Neutrophils

Animals B E M Y P S L M

*) Pseudoeosinophils.

Determination of leukocyte profile.

Leukocytes are counted by class on the basis of 100 cells, the leukocyte formula is derived, after which recalculation is made according to the number of leukocytes in mm 3.

Example: if there are 60 lymphocytes per 100 counted cells, and the total number of leukocytes in 1 mm 3 is 10,000, then according to the formula:

100-60 10,000 x 60

10,000- X X = 100 = 6,000

Then other classes are recalculated.

The received data is marked with a dot in the corresponding column of a special form.

By connecting the points with a line, a graphic image of the leukocyte profile is obtained. It makes it possible to determine whether the animal has relative or absolute leukocytosis.

Research results.

Indicators of leukocyte formula and pathological

changes in leukocytes.

I. Leukocytes (thousand\ µl):

basophils%;

eosinophils%;

myelocytes%;

• stab %;

  segmented %;

  lymphocytes%;

  monocytes %;

  Turkic cells%.

Pathological changes:

anisocytosis;

poikilocytosis;

Determination of leukocyte profile.

Determination of hematological profile

Conclusion

Educational equipment, demonstration material, animals:

Microscope

Goryaev's counting chamber

Melangers for leukocytes

Ground cover glass.

2-3% acetic acid solution, tinted with methylene blue.

Laboratory test tubes with rubber stoppers

Pipettes graduated to 1 and 5

Pipettes from Sali's hemometer

Slide (clean, degreased)

Ground glass

Romanovsky-Giemsa paint

Distilled water

Enameled baths

Immersion oil

Tables with indicators of the leukocyte formula, leukocyte and hematological profile.

VIII. Evaluation of student work by teacher.

LESSON 20. Determination of total calcium and inorganic phosphorus in blood serum

The purpose of the lesson is to learn how to determine the amount of calcium and inorganic phosphorus in blood serum and give clinical assessment research results.

Current student survey on the following questions:

The physiological role of calcium in the animal body.

Indicators of total calcium in blood serum in various types of farm animals and their changes.

The physiological role of phosphorus in the animal body.

Indicators of inorganic phosphorus in blood serum in farm animals and their changes

Explanation of the teacher’s methodology for performing the work.

Execution of work by students.

Determination of calcium in blood serum using the de-Ward method.

Place 0.5 ml of blood serum into one small centrifuge tube and 0.5 ml of distilled water into the other. Then 0.5 ml of a saturated aqueous solution of ammonium oxalate is added to each test tube.

After 30 minutes, the tubes are centrifuged for 15 minutes. The serum is sucked off, 1-2 ml of distilled water is added to the sediment and centrifuged again for 3-5 minutes.

After this, the liquid is sucked off, and 0.3 ml of sulfuric acid diluted 1:2 with water is added to the precipitate. The mixture is heated in a water bath to 50-65% and titrated with a centinormal permanganate solution (KMnO4) until a pink color appears, which persists for 2 minutes.

The amount of calcium in milligram percent is calculated using the formula: (a - k) x 2 x 0.2 x 100, where

a is the amount of centinormal permanganate solution used to titrate the serum;

k is the amount of permanganate consumed for the control titration of distilled water;

0.2 - the amount of calcium in milligrams corresponding to 1 ml of a centinormal permanganate solution;

2 - multiplier for calculating the amount of calcium in 1 ml of blood serum;

100 is a multiplier for calculating the amount of calcium in milligram percent in 100 ml of blood serum.

The amount of total calcium (mg per 100 ml) in cattle is 10.1-12.5; in horses - 12.0-15.5; in pigs - 10.5-12.5; in birds - 11.0-15.0.

Determination of inorganic phosphorus in blood serum

according to Ammon and Ginsberg (modified by S.A. Ivanovsky).

Add 3 ml of distilled water, 1 ml of test serum and 1 ml of 20% trichloroacetic acid solution into a centrifuge tube and mix well. After 5 minutes, centrifuge at 3,000 rpm. within 15 min. (or filter through a benzene filter). Then 2.5 ml of transparent centrifugate (or filtrate), 0.5 molybdenum reagent, 1 ml of 1% ascorbic acid solution and 6 ml of distilled water are poured into the test tube. At the same time, 3 ml of a working standard solution of phosphorus, 0.5 ml of molybdenum reagent, 1 ml of 1% ascorbic acid solution and 5.5 ml of distilled water are added to another test tube (standard). After 10 minutes, the liquids in both test tubes are colorimeterized using a photoelectric colorimeter using a green filter in cuvettes 10 mm wide.

The calculation is made using the formula:

Ex x 0.05 x 100 Ex x 10

X = Ek x 0.5 Ek, where

X is the amount of inorganic phosphorus in the test blood serum, mg per 100 ml;

Ex - optical density of the sample with serum centrifugate;

Ek is the optical density of the sample with a standard phosphorus solution (standard);

0.05 - amount of phosphorus in the volume of working standard phosphorus solution taken for analysis, mg; 100-coefficient for listing the amount of phosphorus per 100 ml of blood serum.

The blood serum of adult animals normally contains the following amount of inorganic phosphorus (mg per 100 ml); for cattle - 5.0-6.5; in pigs - 7.7-9.5; in horses - 5.1-6.0; in poultry - 5.6-8.0; in sheep - 4.5-7.5.

Results.

Amount of calcium (mg%).

Amount of inorganic phosphorus (mg %).

Conclusion.

Training equipment, demonstration material.

Reagents: saturated aqueous solution of ammonium oxalate,

centinormal solution of potassium permanganate, sulfuric acid diluted 1:2, 20% solution of trichloroacetic acid; molybdenum reagent (prepared by mixing a solution consisting of 5 g of ammonium molybdate and 60 ml of distilled water with a solution prepared from 15 ml of concentrated H2 SO4 and 25 ml of distilled water); 1% solution of ascorbic acid in 0.1 N HCl solution; basic standard solution of phosphorus (4.394 g KN 2 PO 4) dried to constant weight over HSO in a desiccator and distilled water up to 1 l); working standard solution of phosphorus (2 ml of stock solution and distilled water up to 100 ml + 20 ml of 20% trichloroacetic acid solution; 3 ml of solution contains 0.5 mg of phosphorus; distilled water).

Test tubes.

Graduated pipettes.

Benzene filters.

1. Photoelectric colorimeter.

2. Blood serum.

   Water bath.

   Chemical thermometer.

   Centrifuge.

Evaluation of student work by teacher.

LESSON 21. Determination of acid capacity and carotene in blood serum

The location of the lesson is the laboratory of the department.

Purpose of the lesson:

Master the methodology for studying the acid capacity of blood.

Learn to determine carotene in blood serum and give a diagnostic assessment of test results.

Current student survey on the following questions:

What is acid tank and normal indicators acid capacity in various types of farm animals.

Types of acid-base balance disorders.

The physiological role of carotene, its content in animals in normal conditions and in pathology.

Explanation of the teacher’s methodology for performing the work.

Execution of work by students.

Determination of reserve alkalinity of blood serum

according to I.P. Kondrakhin.

Glassware: paired flasks, pipettes.

Reagents:

0.01 N NaOH solution (caustic soda);

5% H2SO4 solution;

1% alcohol solution of phenolphthalein;

0.01 N solution of H2SO4 (sulfuric acid).

Progress of the study. 0.5 ml of serum (or blood plasma) is added to one half of the flask; blowing out the remaining liquid from the pipette is not allowed; it is tightly closed with a stopper. Take 2 ml of 0.01 N sodium hydroxide solution into the second half of the flask and close it with a stopper. Then open the first half of the flask and add 1 ml of a 5% sulfuric acid solution to the blood serum located there and quickly close it with a stopper. Using rotational movements, thoroughly mix the serum with the acid. During the reaction, mixing is repeated 3-4 times.

Add 2 ml of 0.01 N sodium hydroxide solution to the control tube and close it tightly with a stopper. Take 1 ml of a 5% sulfuric acid solution into the second half of the paired flask and close it with a stopper. Before closing the flask holes, the stoppers are moistened with distilled water.

For greater accuracy, each serum sample is tested in two paired flasks. The control experiment is carried out in three double flasks.

After 4-6 hours (up to 12 hours is acceptable), the flasks are opened. In which there is a solution of sodium hydroxide, add one drop of a 1% alcohol solution of phenolphthalein. Mix (red color appears). The liquid in the flask is then titrated with 0.01 N sulfuric acid until completely discolored, which occurs at pH 8. Titration should be carried out carefully and at the same speed in all samples and controls.

The calculation is carried out according to the formula:

X = (a - 6) x 0.224 x 200 = (a - 6) x 44.8

where: X is reserve alkalinity (in percent by volume) CO;

a-amount of 0.01 N sulfuric acid solution used for titration of the test sample, ml;

b-amount of 0.01 N sulfuric acid solution used for titration of the test sample, ml;

0.224-factor of conversion of 0.01 N solution of sulfuric acid to CO for this reaction;

200-coefficient for recalculating the amount of blood serum (plasma) taken for analysis (0.5 ml per 100 ml.)

Normal indicators blood acid capacity (mg per 100 ml); Cattle-460-540, sheep-460-540, goats-380-520, horses-500-600, pig-500-600, dogs-450-550.

Determination of carotene in blood serum

(according to V.F. Koromyslov and L.A. Kudryavtseva)

Add 1 ml of blood serum and 3 ml of 95% ethyl alcohol into a test tube, mix thoroughly with a glass rod, add 6 ml of petroleum ether, shake vigorously for 2 minutes and carefully pour 0.5 ml of distilled water along the wall of the test tube, leave to stand until clear separation aqueous and organic phases. After this, 4.5 ml of carotene extract is carefully poured off and transferred to a cuvette.

Colorimeterize using a photoelectrocolorimeter in 1cm cuvettes with a blue filter against petroleum ether (gasoline). At the same time, a working standard solution of potassium dichromate is colorimeterized (5 ml of distilled water is mixed with 5 ml of basic potassium dichromate).

The calculation is made according to the formula: A

where: X is the amount of carotene in serum, mg per 100 ml;

A-optical density of the test sample;

B-optical density of the working standard solution

dichromate;

1.248 is the coefficient for transferring carotene in mg per 100 ml of blood serum.

Research results.

1. Indicators of acid capacity of blood serum.

Conclusion.

Educational equipment, demonstration material, animals.

0.5% solution of chemically pure sodium chloride prepared in neutral bidistilled water.

Santin normal solution of hydrochloric acid.

The indicator is a 1% aqueous solution of alizarin rot paint.

Petroleum ether.

Working standard solution of potassium dichromate.

Distilled water

Test tubes

1. Blood serum from various animals

   Photoelectric colorimeter

All vessels used for neutralization are pre-treated with a chromium mixture.

VIII. Evaluation of student work by teacher.

LESSON 22 . Determination of bilirubin and total protein

in blood serum

The location of the lesson is the laboratory of the department.

The purpose of the lesson is to learn how to determine in serum blood bilirubin, total protein and provide a clinical assessment of the study results.

Current student survey on the following questions:

The importance of testing blood serum for the content of bilirubin for differentiating various forms of jaundice.

Indicators of total serum protein in healthy animals and their changes.

Explanation of the teacher’s methodology for performing work

Execution of work by students.

Qualitative determination of bilirubin in blood serum

(according to Van den Berg)

Take 2 small plugs. In the first, 0.5 ml of the test serum is added and 0.3 ml of a mixture of diazo reagents is added; in the second - 0.5 serum, 0.5 ml of 96 0 alcohol and 0.3 ml of a mixture of diazoreagents (alcohol is added to the second test tube to destroy the bond of indirect bilirubin with blood globulin).

The contents of the test tube are stirred with a thin, clean stick. Pink coloring in the first test tube indicates a direct reaction, in the second test tube it indicates an indirect reaction.

Quantitative determination (according to Bokalchuk)

For multiple dilutions of serum, take 6 small test tubes and pour 0.5 ml of physiological sodium chloride solution into each, with the exception of the first one, then add 0.5 ml of the test serum into the first and second test tubes.

The serum in the second test tube is thoroughly mixed with saline, after which 0.5 ml of the mixture is transferred to the third test tube and also mixed thoroughly, 0.5 ml of the mixture from the third test tube is transferred to the fourth test tube, etc. The remainder (0.5 ml) from the last test tube is poured into a rinse cup. The result is a dilution:

Tube no. 1 2 3 4 5 6

Dilution rate 1 2 4 8 16 32

Then 0.5 ml of absolute (or 96 degrees of alcohol) is added to each test tube, shaken and a suspension of protein in an alcoholic extract of bilirubin is obtained. 0.5 ml of a mixture of diazo reagents is poured into the liquid, which has become cloudy due to protein precipitation, and the contents of the test tubes are carefully mixed with a glass rod. As a result of the reaction, the serum takes on a pink color. The end of the reaction is considered to be dilution, which gives a subtle pink color. By multiplying the power

dividing by 0.016, the amount of bilirubin in 1 ml of blood serum is determined, and by multiplying it by 100, the amount of bilirubin in milligram% in 100 ml of serum. In the first test tube this amount will be 1.6 mg%, in the second - 3.2, in the third - 6.4, in the fourth - 12.8, in the fifth - 25.6 and in the sixth - 51.2 mg%.

Determination of bilirubin in blood serum (according to Kazakov)

Eight clean and dry test tubes are placed in a rack. Then 0.5 ml of the test serum is added to each test tube and the contents are mixed. From this test tube, using a pipette, take 0.5 ml of the mixture and transfer it to the second test tube, from the second 0.5 ml of liquid is transferred to the third, etc. From the last (eighth) test tube, 0.5 ml of the mixture is removed into the drainer. In this way, serum dilutions that are multiples of two are obtained. Then add 1 ml of a 20% trichloroacetic acid solution to each test tube and shake thoroughly. After this, the contents of the tubes are poured into pre-prepared filter paper funnels, which should be marked in accordance with the blood dilution. The funnels are left to dry at room temperature. The reaction is recorded the next day. The end of the reaction is considered to be the dilution at which a clearly visible green color remains on the filter.

You can use the table for calculations.

Tube No. Dilution degree Amount of bilirubin

in 100 ml of serum

Determination of total protein in blood serum

refractometric method

The refractive index of light rays (refraction) is a characteristic value for various substances. There is a relationship between the concentration of a substance (in this case, proteins) and the degree of refraction, which is used to determine proteins in blood serum.

To heat the prisms, water is passed through rubber tubes to establish a constant temperature of + 20 0. Before work, fold back the upper part of the measuring head. Apply one drop of the test serum to the surface of the measuring prism with a glass rod and carefully close the head.

By observing through the eyepiece of the telescope and rotating the handwheel on the left, the boundary between light and shadow is found. Use the handwheel on the right to remove the colored border. Then, using the handwheel on the left, the interface is precisely aligned with the reticle crosshairs and a reading is taken on the refractive index scale. The protein content in the serum under study is calculated using a special table.

Refractive indices of blood serum and the corresponding amounts of total protein.

Indicators of total protein in blood serum in healthy animals (g per 100 ml); for cattle – 6-8.5; in sheep - 6-7.5; in pigs - 6.5-8.5; in horses - 6.5-7.8; in dogs - 5.9-7.6; in chickens it is 4.3-5.9.

Research results.

Amount of bilirubin (mg%)

Amount of total protein (g%)

Conclusion.

Educational equipment, demonstration material, animals.

1. Reagents:

sulfanilic acid-1g; hydrochloric acid(specific weight 1.19) - 10 g;

distilled water - 200.0;

sodium nitrate - 0.5 g, distilled water - 100 ml.

Before the study, prepare a mixture based on 10 ml of the 1st reagent 0.3 or 0.6 ml of the 2nd reagent.

Test tubes.

Graduated pipettes

1. Refractometer IRF-22

   Rinse cups

   Eye droppers

   Alcohol ether

   Blood serum

   Physiological solution of table salt.

   96 0 alcohol.

   20% trichloroacetic acid solution

   Filter paper

VIII. Evaluation of student work by teacher.

Postpartum bovine hemoglobinuria (Haemoglobinuria puerperalis) is a form of hemolytic anemia in cows; It is recorded, as a rule, during the winter - stall period in highly productive cows of 5-7 years of age in the first 5 weeks after calving and occurs with symptoms of severe hemoglobinuria. This disease is especially common in areas where there is a lack of phosphorus in the soil.

Etiology. The causes of postpartum hemoglobinuria in cows have not been precisely established. Usually its appearance is associated with errors in feeding and maintenance, hypothermia and intoxication from the gastrointestinal tract, long-term feeding of large quantities of alfalfa, beet pulp, beetroot and its leaves to the cow with a lack of phosphorus and other mineral salts necessary for the animal in the diet (more often occurs after a dry summer).

Pathogenesis. A sick cow quickly develops hemolysis, accompanied by accumulation of hemoglobin in the plasma. Hemoglobin is utilized in small quantities in the liver and spleen, but the bulk of it is excreted in the urine, leading to the development of hemoglobinuria in the animal. Subsequently, the cow develops urobilin jaundice. A decrease in the ability of blood to absorb oxygen and carbon dioxide due to a lack of hemoglobin in red blood cells causes oxygen starvation and acidosis in the animal.

Destroyed red blood cells and hemoglobin dissolved in the blood become for the body of a sick cow foreign substances, cause manifestations of parenteral protein breakdown and are accompanied by an increase in body temperature and damage to parenchymal organs.

The adhesion and disintegration of red blood cells leads to blockage of blood capillaries, with the subsequent development of degenerative changes in tissues, especially intralobular necrosis in the liver. Liver dysfunction, developing hemolytic anemia causes serious violation everyone metabolic processes which ultimately lead to the death of the cow.

Pathological changes. When autopsying dead cows, we find pronounced anemia and yellowness of the tissues. The blood is liquid, colored Brown color. The liver, spleen and kidneys are enlarged in size and are in a state of fatty degeneration. When opening the liver, intralobular necrosis is noted. On the epicardium there are hemorrhages, degeneration of the heart muscle, and in the intestines there are hemorrhages.

Clinical signs. The first signs of postpartum hemoglobinuria in cows sometimes appear in connection with childbirth and are manifested by symptoms of depression, moderate fever (39-39.8 ° C, sometimes up to 42 ° C), the cow’s appetite decreases, becomes apathetic, symptoms of hypotension of the proventriculus appear, and dysfunction occurs intestines and decreased milk production. After the onset of the disease, after 1-2 days the cow’s urine becomes dark cherry color, transparent, syrupy and has an alkaline reaction. When examining urine, protein, hemoglobin, urobilin, and in some cases ketone bodies are found in it; in the sediment - breakdown products of red blood cells, cells renal epithelium, sometimes renal casts. When examining blood in the first days of the disease, we register a sharp decrease in the number of red blood cells (up to 1.2 - 1.5 million per mm³) and hemoglobin (up to 16% according to Sali). The color index is above one (1.15); osmotic resistance of erythrocytes is reduced; ESR is slightly accelerated. In smears from peripheral blood we find anisopoikilocytosis, polychromatophilia, reticulocytes (20: 1000), erythrocytes with basophilic puncture and individual normoblasts. The total number of leukocytes in most sick cows is normal or increases to 15 - 20 thousand per 1mm³. The leukoformula is characterized by neutrophilia (up to 72%) and a pronounced regenerative shift. We note thrombocytosis. With severe postpartum hemoglobinuria in cows, the number of leukocytes in the blood decreases to 4-5 thousand per 1mm³. In the blood serum we note increased content indirect bilirubin and the appearance of methemoglobin, severe hypophosphatemia, in some sick cows, a decrease in the level of carotene and alkaline reserve.

With postpartum hemoglobinuria in cows, a violation of the bone marrow puncture indicators occurs. At acute course disease, the number of nuclear elements in the bone marrow increases by 20-30%. The erythroblastogram is characterized by an increase in the percentage of young erythroblastic forms - proerythroblasts and basophilic erythroblasts, while the percentage of normoblasts decreases. At the same time, the number of granulophilocytes is 10 times higher than normal. At the same time with regenerative processes In the bone marrow of a sick cow, degenerative phenomena are observed, which are accompanied by poikilocytosis, anisocytosis, lumpy structure of the erythrocyte protoplasm and pyknosis of the nuclei of some erythroblasts.

Inadequate erythropoiesis maintains hemolysis of red blood cells and slows down the process of blood restoration in a sick cow.

When examining the myelogram, we note a decrease in the ready reserves of myeloblasts and reticuloendothelial cells.

When absolute neutrophilia changes to relative, neutrophils increase to the left, and lysis and vacuolization of the nucleus and protoplasm are detected among granulocytes.

During a clinical examination of a sick cow, we note pallor and moderate yellowness of the visible mucous membranes. During percussion we note an increase in the area of ​​hepatic dullness, and by deep palpation we establish liver tenderness. On auscultation of the heart, the heart sounds are intensified; on palpation, the heartbeat is pounding. The pulse is accelerated - up to 100-120 beats per minute. Breathing is difficult and rapid (30-40 or more per minute). Sometimes we smell acetone in the exhaled air. The stool becomes liquid, with mucus, and has putrid smell. The milk of some animals has a reddish color.

Flow acute illness. In severe cases, the illness may last for 3-5 days. With a favorable outcome of the disease, normalization of blood composition and improvement general condition sick cow occurs within 1-2 months. In cows that have previously suffered from postpartum hemoglobinuria, the disease may recur.

Diagnosis. The diagnosis of postpartum hemoglobinuria in cows is made based on the fact that:

• Mostly highly productive cows fall ill in the first weeks after calving;
• in sick animals, we establish hemolytic anemia with hemoglobinuria using laboratory methods;
• The disease is recorded most often during the winter period when animals are kept in stalls (from November to April).

Differential diagnosis. When diagnosing postpartum hemoglobinuria, the veterinarian must exclude hemosporidial diseases, poisoning with plant and mineral poisons.

Treatment. Owners should provide sick cows with warm, dry, draft-free housing. Poor quality feed (frozen, rotten, moldy, silage with a high content of butyric acid, etc.) is immediately excluded from the animal feeding diet. Translate to dietary feeding, consisting of feed rich in vegetable protein, carbohydrates and carotene, from minerals– provide sufficient phosphorus. As a mineral phosphorus supplement, we include disubstituted sodium phosphate in the diet at a dose of 50 g per day. To prevent the occurrence of degenerative changes in the kidneys of a sick cow, sick cows must be given skim milk or whey in the amount of 5-10 liters per day. To increase the body's resistance and normalize metabolic processes, we administer intramuscularly tetravit in a dose of 10 ml, selevit, etc.

To relieve acidosis and neutralize toxins formed in the forestomach, the cow is given 80-100 g of sodium bicarbonate orally in the form of a 5-10% solution twice a day for 3-4 days.

We administer intravenously 20-40% glucose solutions of 150-200 ml, 2 mg of strophanthin, 20-30 ml of metapyrine per approximately 500 kg of live weight. This treatment is carried out daily until there is a noticeable improvement in the general condition of the cow. To maintain liver function, methionine preparations, vitamins B12 and E can be prescribed. To maintain cardiac activity, subcutaneously inject 4-5 g of caffeine in a 20% solution 2-3 times a day for 3-5 days.

If there is a deficiency of phosphorus in the body, it is administered: parenterally (urzolit, 500 ml/500 kg body weight) or orally (50 g of disodium phosphate or 250 g of rachitin).

To stimulate hematopoiesis, we prescribe a transfusion of compatible blood (up to 4 liters) simultaneously with the administration of iron and copper preparations orally, and intramuscular iron dextrin preparations. During the recovery period, intramuscular injections of Campolon (0.05 - 0.08 ml per 1 kg of body weight) are prescribed with an interval of 8-14 days, Vitogepad.

Prevention. Prevention of postpartum hemoglobinuria in cows is based on a balanced diet of feeding deep-pregnant and fresh-calving cows. Diets must be complete in protein, carbohydrate, vitamin and mineral terms, contain a sufficient amount of phosphorus and its correct ratio with calcium. Cows in last month pregnancy should not be fed in large quantities sugar beets and its products. You need to add 150-200g of a mineral mixture containing a large amount of phosphorus to your diet every day. During the winter - stall period, animals should use active exercise.

Determination of hemoglobin content in the blood of animals is one of the most important and widespread indicators. To determine hemoglobin, hemoglobin derivatives are most often analyzed, formed during its oxidation and the addition of various chemical groups to the gene, leading to a change in the valence of iron and the color of the solution.

For routine laboratory research colorimetric methods are most preferred, as they are the cheapest, simplest and

fast in execution. Animal blood is a normal mixture of hemoglobin derivatives with different absorption spectra. When quantifying hemoglobin by colorimetric methods, the problem arises in choosing a reagent that would convert all hemoglobin derivatives into only one form before photometric analysis. The best methods for quantitatively converting hemoglobin into its derivatives turned out to be hemiglobin cyanide (HbCN), hemichrome (HbChr) and hemiglobinazide (HbN3), which, when photometrically, give the smallest determination error among other methods of analysis.

Increase: some forms of hemoblastosis, in particular erythremia, dehydration.

Decreased (anemia): various types of anemia, including due to blood loss.

The principle of the hemiglobin cyanide method is based on the conversion of all forms of hemoglobin into one - hemiglobin cyanide. The conversion of hemoglobin to hemiglobin cyanide is carried out by its interaction with a transforming solution containing potassium ferricyanide, potassium cyanide, potassium dihydrogen phosphate and nonionic detergent. Potassium dihydrogen phosphate maintains a pH level at which the reaction takes place in 3-5 minutes. The detergent enhances the hemolysis of red blood cells and prevents turbidity associated with plasma proteins. Potassium ferricyanide oxidizes all forms of hemoglobin into methemoglobin, which forms hemiglobin cyanide with potassium cyanide, which has a reddish color, the color intensity of which is directly proportional to the concentration of hemoglobin in the sample.

The principle of the hemichrome method is based on the conversion of all forms of hemoglobin into one - hemichrome. When hemoglobin interacts with a transforming solution containing fatty acids with potassium ferricyanide or sodium dodecyl sulfate, it is converted into an oxidized low-spin form - hemichrome (HbChr), which has a reddish color, the color intensity of which is directly proportional to the concentration of hemoglobin in the sample.

In large-scale tests of the hemichrome method, it was shown that in the range of hemoglobin concentrations from 40 to 200 g/l, the calibration graphs of hemiglobincyanide and hemichrome represent a straight line emerging from the origin, and close slope angles of the straight lines indicate the comparability of both methods.

When determining hemoglobin by two methods, Akhrem A.A. et al. showed that the hemichromic method provides greater accuracy (and lower s). The authors suggest that SDS promotes the solubilization of membrane particles and prevents protein adsorption on the glass of test tubes and cuvettes, thereby ensuring high accuracy of analysis.

Comparative assessment The results of determining hemoglobin in the blood by two methods showed that the results are comparable, and the correlation coefficient of the methods is 0.99. Thus, the hemichrome method for determining hemoglobin in the blood has all the advantages of the hemiglobin cyanide method, which are complemented by the absence of highly toxic cyanides and other toxic substances in the transforming reagent.

Performance. Add 5 ml of transforming solution into dry, clean test tubes with a dispenser, add 20 µl of blood to them using a certified Sali pipette or a mechanical dispenser with all the precautions listed in the previous section. Mix the sample thoroughly using a microshaker or manually until a homogeneous solution is achieved. Keep the solutions at room temperature for the time specified in the instructions for the kit, and measure the optical density of the solutions on a verified, calibrated device in a cuvette that has zero absorption of distilled water relative to a similar control (with an optical path length of 10 mm). The color of solutions is stable for up to 5 hours or more, which allows measurements to be taken at any convenient time in this time interval. Calculate the hemoglobin content in the blood using the calibration graph or factor determined on this device. If all the listed conditions for preparing and conducting the analysis described above are met, the error in determining hemoglobin in the blood will not exceed ±2%. Let us briefly summarize the sources of possible errors in determining hemoglobin: the use of uncalibrated pipettes and imperfect dosing techniques for blood samples; the use of unverified equipment that does not provide a linear dependence of optical density on hemoglobin concentration in the required measurement range; instability of the device; lack of in-laboratory quality control; insufficient cleanliness of cuvettes, especially flow-through ones; errors in constructing calibration graphs and calculating factors; use of hemoglobin control solutions Low quality; operator errors, errors made in the preanalytical phase.

Hemoglobin (Hb) is a complex protein (chromoprotein) that colors red blood cells red and consists of the globin protein and four heme molecules. Heme is the active part and contains divalent iron; one molecule of heme is capable of attaching and donating one molecule of oxygen. Globin is a protein carrier of heme. Hemoglobin in the lungs attaches oxygen to itself, forming a fragile, easily dissociated compound - oxyhemoglobin (HbO2). Blood saturated with oxyhemoglobin (arterial) enters the body tissues, where oxyhemoglobin breaks down into reduced hemoglobin and oxygen. Reduced hemoglobin (deoxyhemoglobin) in tissues combines with carbon dioxide, also forming the fragile compound carbhemoglobin (HbCO2). Blood saturated with reduced hemoglobin and carbhemoglobin (venous) enters the pulmonary circulation. The fetal blood contains fetal hemoglobin (HbF), which can be significantly more saturated with oxygen than maternal hemoglobin. It is believed that fetal hemoglobin is synthesized in the liver, and hemoglobin in adult animals is synthesized in the red bone marrow. Hemoglobin easily combines with carbon monoxide (carbon monoxide), forming carboxyhemoglobin (HbCO), which loses its ability to carry oxygen. Already with only 0.04% carbon monoxide in the inhaled air, severe poisoning, and at a concentration of 0.1% - the death of the animal. In mild poisoning, carbon monoxide is gradually broken off, and hemoglobin regains its ability to bind and carry oxygen. When hemoglobin is exposed to strong oxidizing agents ( Berthollet's salt, hydrogen peroxide, aniline, etc.) a fairly strong compound of hemoglobin with oxygen is formed - methemoglobin (MtHb), in which divalent iron passes into the trivalent form. This compound firmly holds oxygen and cannot be released by tissues. When a large amount of methemoglobin is formed, the animal dies from suffocation. In animal husbandry practice, methemoglobin is formed when feeding animal feed containing large amounts of nitrates from the application of large doses of nitrogenous fertilizers to the soil. Qualitative determination of hemoglobin and its derivatives can be carried out using spectral analysis, and quantitative – by various calorimetric methods (Table 7.).

To determine the saturation of red blood cells with hemoglobin, determine color index or index g.

“Hb” in the animal under study x normal amount red blood cells

“Nb” is normal x the number of red blood cells in the animal being studied

Normally, this figure is 1 ± 0.15%

Myoglobin is a complex protein found in skeletal and cardiac muscles. Myoglobin can bind 14-15% of the total amount of oxygen. Myoglobin oxygen is used by muscles during their contraction, when blood flow in their capillaries decreases. When muscles relax, myoglobin reacquires oxygen. In significantly large quantities myoglobin is found in the muscles of marine mammals, which allows them to stay under water for a long time.

HEMOGLOBIN

Reasons for increased hemoglobin concentration:
1. Myeloproliferative diseases (erythremia);
2. Primary and secondary erythrocytosis;
3. Dehydration;

Reasons for decreased hemoglobin concentration:
1. Iron deficiency anemia (a relatively moderate decrease - up to 85 g/l, less often - more pronounced - up to 60-80 g/l);
2. Anemia due to acute blood loss(significant reduction - up to 50-80 g/l);
3. Hypoplastic anemia (significant reduction - up to 50-80 g/l);
4. Hemolytic anemia after a hemolytic crisis (significant decrease - up to 50-80 g/l);
5. B12 - deficiency anemia (significant decrease - up to 50-80 g/l);
6. Anemia associated with neoplasia and/or leukemia;
7. Overhydration (hydremic plethora).

Reasons for a false increase in hemoglobin concentration:
1. Hypertriglyceridemia;
2. High leukocytosis;
3. Progressive liver diseases;
4. Sickle cell anemia (appearance of hemoglobin S);
5. Myeloma (with multiple myeloma (plasmacytoma) with the appearance of a large number of easily precipitating globulins).

HEMATOCRIT

Reasons for decreased hematocrit:
1. Anemia of various origins (can decrease to 25-15%);
2. Increase in circulating blood volume (pregnancy, especially 2nd half, hyperproteinemia);
3. Overhydration.

Reasons for increased hematocrit:
1. Primary erythrocytosis (erythremia) (increases to 55-65%);
2. Erythrocytosis caused by hypoxia of various origins (secondary, increases to 50-55%);
3. Erythrocytosis in kidney tumors, accompanied by increased formation of eryropoietin (secondary, increases to 50-55%);
4. Erythrocytosis associated with polycystic kidney disease and hydronephrosis (secondary, increases to 50-55%);
5. Decrease in the volume of circulating plasma ( burn disease, peritonitis, repeated vomiting, maladsorption diarrhea, etc.);
6. Dehydration.
Fluctuations in hematocrit are normal.
The ability of the spleen to contract and expand can cause significant changes in hematocrit, especially in dogs.

Reasons for the increase in hematocrit by 30% in cats and 40% in dogs due to contraction of the spleen:
1. Physical activity immediately before taking blood;
2. Excitement before blood collection.
Reasons for a drop in hematocrit below the standard range due to enlargement of the spleen:
1. Anesthesia, especially when using barbiturates.
The most complete information is provided by simultaneous assessment of hematocrit and total protein concentration in plasma.
Interpretation of data for determining the hematocrit value and the concentration of total protein in plasma:

Normal hematocrit
1. Loss of protein through the gastrointestinal tract;
2. Prytheinuria;
3. Serious disease liver;
4. Vasculitis.
b) Normal concentration of total protein in plasma is a normal state.
1. Increased protein synthesis;
2. Anemia masked by dehydration.

High hematocrit
A) Low concentration total protein in plasma - a combination of “contraction” of the spleen with protein loss.
1. “Contraction” of the spleen;
2. Primary or secondary erythrocytosis;
3. Hypoproteinemia masked by dehydration.
V) High concentration total protein in plasma - dehydration.

Low hematocrit
a) Low concentration of total protein in plasma:
1. Significant current or recent blood loss;
2. Excessive hydration.
b) Normal concentration of total protein in plasma:
1. Increased destruction of red blood cells;
2. Decreased red blood cell production;
3. Chronic blood loss.
c) High concentration of total protein in plasma:
1. Anemia in inflammatory diseases;
2. Multiple myeloma;
3. Lymphoproliferative diseases.

AVERAGE VOLUME OF ERYTHROCYTES

Macrocytosis (high MCV values) - causes:
1. Hypotonic nature of water-electrolyte balance disorders;
2. Regenerative anemia;
3. Non-regenerative anemia caused by a disorder of the immune system and/or myelofibrosis (in some dogs);
4. Myeloproliferative disorders;
5. Regenerative anemia in cats - carriers of the feline leukemia virus;
6. Idiopathic macrocytosis (without anemia or reticulocytosis) in poodles;
7. Hereditary stomatocytosis (dogs, with a normal or slightly increased number of reticulocytes);
8. Hyperthyroidism in cats (slightly increased with normal or increased hematocrit);
9. Newborn animals.

False macrocytosis - causes:
1. Artifact due to red blood cell agglutination (in immune system-mediated disorders);
2. Persistent hypernatremia (when blood is diluted with liquid before counting the number of red blood cells in an electric meter);
3. Long-term storage blood samples.
Microcytosis (low MCV values) - causes:
1. Hypertonic nature of the water-electrolyte balance disorder;
2. Iron deficiency anemia due to chronic bleeding in adult animals (about a month after their onset due to depletion of iron reserves in the body);
3. Iron deficiency nutritional anemia in suckling animals;
4. Primary erythrocytosis (dogs);
5. Long-term therapy with recombinant erythropoietin (dogs);
6. Disorders of heme synthesis - long-term deficiency of copper, pyridoxine, lead poisoning, drugs (chloramphenicol);
7. Anemia in inflammatory diseases (MCV is slightly reduced or in the lower normal range);
8. Portosystemic anastomosis (dogs, with normal or slightly reduced hematocrit)
9. Portosystemic anastomosis and hepatic lipidosis in cats ( slight decline MVC);
10. May be with myeloproliferative disorders;
11. Impaired erythropoiesis in English springer spaniels (in combination with polymyopathy and heart disease);
12. Persistent elliptocytosis (in crossbred dogs as a result of the absence of one of the proteins in the erythrocyte membrane);
13. Idiopathic microcytosis in some breeds of Japanese dogs (Akita and Shiba) - is not accompanied by anemia.

False microcytosis - causes (only when determined in an electronic counter):
1. Severe anemia or severe thrombocytosis (if platelets are included in the MCV calculation when counting using an electronic counter);
2. Persistent hyponatremia in dogs (due to shrinkage of red blood cells when diluting blood in vitro for counting red blood cells in an electronic counter).

AVERAGE CONCENTRATION OF HEMOGLOBIN IN RED CELLS
Mean erythrocyte hemoglobin concentration (MCHC)- indicator of saturation of erythrocytes with hemoglobin.
In hematology analyzers, the value is calculated automatically or calculated using the formula: MCHC = (Hb (g\dl)\Ht (%))x100
Normally, the average hemoglobin concentration in erythrocytes in dogs is 32.0-36.0 g\dl, in cats 30.0-36.0 g\dl.

Increased MSHC (extremely rare) - reasons:
1. Hyperchromic anemia (spherocytosis, ovalocytosis);
2. Hyperosmolar disturbances of water and electrolyte metabolism.

False increase in MSHC (artifact) - reasons:
1. Hemolysis of erythrocytes in vivo and in vitro;
2. Lipemia;
3. Presence of Heinz bodies in erythrocytes;
4. Agglutination of erythrocytes in the presence of cold agglutinins (when counted in an electric meter).

Decrease in MCHC - reasons:
1. Regenerative anemia (if there are a lot of stressed reticulocytes in the blood);
2. Chronic iron deficiency anemia;
3. Hereditary stomatocytosis (dogs);
4. Hypoosmolar disturbances of water and electrolyte metabolism.
False MCHC downgrade- in dogs and cats with hypernatremia (as the cells swell when the blood is diluted before being counted in an electronic counter).

AVERAGE CONTENT OF HEMOGLOBIN IN ERYTHROCYTE
Calculation of the average hemoglobin content in an erythrocyte (MCH):
MCH = Hb (g/l)/number of red blood cells (x1012/l)
Normally, in dogs it is 19-24.5 pg, in cats it is 13-17 pg.
The indicator has no independent significance, since it directly depends on the average volume of the erythrocyte and the average concentration of hemoglobin in the erythrocyte. Usually it directly correlates with the value of the average volume of erythrocytes, with the exception of cases when macrocytic hypochromic erythrocytes are present in the blood of animals.

The classification of anemia according to erythrocyte parameters has been accepted, taking into account the average erythrocyte volume (MCV) and the average hemoglobin concentration in the cell (MCHC) - see below.

NUMBER OF RED CYTES
The normal content of red blood cells in the blood of dogs is 5.2 - 8.4 x 1012/l, in cats 6.6 - 9.4 x 1012/l.
Erythrocytosis is an increase in the content of red blood cells in the blood.

Relative erythrocytosis- due to a decrease in the volume of circulating blood or the release of red blood cells from blood depots (“contraction” of the spleen).

Absolute erythrocytosis- an increase in the mass of circulating red blood cells due to increased hematopoiesis.

Erythrocytopenia is a decrease in the number of red blood cells in the blood.

Classification of anemia according to erythrocyte parameters, taking into account the mean erythrocyte volume (MCV) and the mean hemoglobin concentration in the cell (MCHC)

b) Macrocytic normochromic anemia:
1. Regenerative anemia (the average concentration of hemoglobin in the erythrocyte is not always reduced);
2. For infections caused by feline leukemia virus without reticulocytosis (usually);
3. Erythroleukemia (acute myeloid leukemia) and myelodysplastic syndromes;
4. Non-regenerative immune system-mediated anemia and/or myelofibrosis in dogs;
5. Macrocytosis in poodles (healthy mini-poodles without anemia);
6. Cats with hyperthyroidism (weak macrocytosis without anemia);
7. Folate deficiency ( folic acid) - rarely.

c) Macrocytic hypochromic anemia:
1. Regenerative anemia with noticeable reticulocytosis;
2. Hereditary stomatocytosis in dogs (often mild reticulocytosis);
3. Increased osmotic instability of erythrocytes of Abyssinian and Somali cats (reticulocytosis is usually present);

d) Microcytic or normocytic hypochromic anemia:
1. Chronic deficiency iron (months in adult animals, weeks in suckling animals);
2. Portosystemic shunts (often without anemia);
3. Anemia in inflammatory diseases (usually normocytic);
4. Hepatic lipidosis in cats (usually normocytic);
5. Normal condition for Japanese Akita and Shiba dogs (without anemia);
6. Long-term treatment with recombinant human erythropoietin (moderate anemia);
7. Copper deficiency (rare);
8. Drugs or agents that inhibit heme synthesis;
9. Myeloproliferative disorders with impaired iron metabolism (rare);
10. Pyridoxine deficiency;
11. Familial disorder of erythropoiesis in English springer spaniels (rare);
12. Hereditary elliptocytosis in dogs (rare).

PLATELET COUNT

Thrombocytosis is an increase in the number of platelets in the blood.

Causes:
I. Thrombocytopenia associated with decreased platelet formation (hematopoietic insufficiency).
a) purchased
1. Cytotoxic damage to the red bone marrow:
- cytotoxic antitumor chemotherapeutic drugs;
- administration of estrogens (dogs);
- cytotoxic drugs: chloramphenicol (cats), phenylbutazone (dogs), trimetoptim-sulfadiazine (dogs), albendazole (dogs), griseofulvin (cats), probably thiacetarsemide, meclofenamic acid and quinine (dogs);
- cytotoxic estrogens produced by tumors from Sertoli cells, interstitial cells and granulosa cell tumors (dogs);
- increased concentration of cytotoxic estrogens in functioning cystic ovaries (dogs).
2. Infectious agents:
 Ehrlichia canis (dogs);
- parvovirus (dogs);
 infection with feline leukemia virus (FLV infection);
 panleukopenia (cats - rarely);
- infection with feline immunodeficiency virus (FIV infection).
3. Immune-mediated thrombocytopenia with death of megakaryocytes.
4. Irradiation.
5. Myelophthisis:
- myelogenous leukemia;
- lymphoid leukemia;
- multiple myeloma;
- myelodysplastic syndromes;
- myelofibrosis;
- osteosclerosis;
- metastatic lymphomas;
- metastasizing mast cell tumors.
6. Amegakaryocytic thrombocytopenia (rare);
7. Long-term use of recombinant thrombopoietin;
8. Lack of endogenous thrombopoietin.
b) hereditary
1. Moderate cyclic thrombocytopenia with a wave-like decrease and increase in platelet production in gray collies with hereditary cyclic hematopoiesis;
2. Thrombocytopenia with the appearance of macroplatelets in Cavalier King Charles Spaniels (asymptomatic).
II. Thrombocytopenia caused by increased platelet destruction:
1. Immune-mediated:
 primary autoimmune (idiopathic) - idiopathic thrombocytopenic purpura (can be combined with autoimmune hemolytic anemia - Evans syndrome) - common in dogs, more often in females, breeds: cocker spaniels, toy and toy poodles, Old English and german shepherds;
- secondary for systemic lupus erythematosus, rheumatoid arthritis;
- secondary for allergic and drug-allergic;
- secondary in infectious diseases accompanied by the deposition of antigen-antibody-complement complexes on the surface of platelets (ehrlichiosis, rickettsiosis);
- secondary in chronic lymphocytic leukemia.
2. Hapten - associated with hypersensitivity to certain drugs (drug-toxic) and uremia;
3. Isoimmune (post-transfusion thrombocytopenia);
4. Infectious processes(viremia and septicemia, some inflammations).
III. Thrombocytopenia caused by increased platelet utilization:
1. DIC syndrome;
2. Hemangiosarcoma (dogs);
3. Vasculitis (for example, with viral peritonitis in cats);
4. Other disorders causing endothelial damage;
5. Inflammatory processes (due to damage to the endothelium or increased concentrations of inflammatory cytokines, especially platelet adhesion and aggregation factors);
6. Snake bites.
IV. Thrombocytopenia associated with increased platelet sequestration (deposition):
1. Sequestration in hemangioma;
2. Sequestration and destruction in the spleen with hypersplenism;
3. Sequestration and destruction in the spleen with splenomegaly (with hereditary hemolytic anemia, autoimmune diseases, infectious diseases, splenic lymphoma, congestion in the spleen, myeloproliferative diseases with splenomegaly, etc.);
4. Hypothermia.
V. Thrombocytopenia associated with external bleeding:
1. Acute bleeding (minor thrombocytopenia);
2. Massive blood loss associated with poisoning with anticoagulant rodenticides (severe thrombocytopenia in dogs);
3. When transfusion of platelet-depleted donor blood or red blood cells to animals that have suffered major blood loss.
Pseudothrombocytopenia can occur when automatic platelet counters are used to count platelets.

Causes:
1. Formation of platelet aggregates;
2. In cats, since their platelets are very large in size, and the device cannot reliably distinguish them from red blood cells;
3. In Cavalier King Charles Spaniels, their blood normally contains macroplatelets, which the device does not distinguish from small red blood cells.

NUMBER OF LEUKOCYTES

A decrease or increase in leukocytes in the blood can be due to individual species leukocytes (more often), and general while maintaining the percentage of individual types of leukocytes (less often).
An increase or decrease in the number of certain types of leukocytes in the blood can be absolute (with a decrease or increase in the total leukocyte content) or relative (with a normal total leukocyte content).
The absolute content of individual types of leukocytes per unit volume of blood can be determined by multiplying the total content of leukocytes in the blood (x109) by the content a certain type leukocytes (%) and dividing the resulting number by 100.

LEUKOCYTE BLOOD FORMULA

Cells Percentage of total leukocytes
Dogs Cats
Myelocytes 0 0
Metamyelocytes (young) 0 0 - 1
Band neutrophils 2 - 7 1 - 6
Segmented neutrophils 43 - 73 40 - 47
Eosinophils 2 - 6 2 - 6
Basophils 0 - 1 0 - 1
Monocytes 1 - 5 1 - 5
Lymphocytes 21 - 45 36 - 53
When assessing the leukocyte formula, it is necessary to take into account the absolute content of individual types of leukocytes (see above).
Shift to the left - change in leukogram with increasing percentage young forms of neutrophils (band-eating neutrophils, metamyelocytes, myelocytes).

Causes:
1. Acute inflammatory processes;
2. Purulent infections;
3. Intoxication;
4. Acute hemorrhages;
5. Acidosis and coma;
6. Physical overexertion.

Regenerative left shift- the number of band neutrophils is less than the number of segmented neutrophils, the total number of neutrophils is increased.
Degenerative shift to the left- the number of band neutrophils exceeds the number of segmented neutrophils, the total number of neutrophils is normal or leukopenia exists. Result increased need in neutrophils and/or increased destruction of them, leading to bone marrow destruction. A sign that the bone marrow cannot meet the increased need for neutrophils either short term (several hours) or long term (several days).
Hyposegmentation- a shift to the left, due to the presence of neutrophils that have condensed nuclear chromatin of mature neutrophils, but a different nuclear structure compared to mature cells.

Causes:
 Pelger-Huyne anomaly (hereditary trait);
 transient pseudoanomaly during chronic infections and after the administration of certain drugs (rare).

Shift left with rejuvenation- Metamyelocytes, myelocytes, promyelocytes, myeloblasts and erythroblasts are present in the blood.

Causes:
1. Chronic leukemia;
2. Erythroleukemia;
3. Myelofibrosis;
4. Metastases of neoplasms;
5. Acute leukemia;
6. Comatose states.

Shift right (hypersegmentation)- change in leukogram with an increase in the percentage of segmented and polysegmented forms.

Causes:
1. Megaloblastic anemia;
2. Kidney and heart diseases;
3. Conditions after blood transfusion;
4. Recovery from chronic inflammation(reflects the increased residence time of cells in the blood);
5. Exogenous (iatrogenic) increase in the level of glucocorticoids (accompanied by neutrophilia; the reason is a delay in the migration of leukocytes into the tissue due to the vasoconstricting effect of glucocorticoids);
6. Endogenous (stressful situations, Cushing's syndrome) increase in glucocorticoid levels;
7. Old animals;
8. Dogs with a hereditary defect in cobalamin absorption;
9. Cats with folate deficiency.

NEUTROPHILS

Neutrophilosis (neutrophilia)- an increase in the content of neutrophil leukocytes in the blood above the upper limits of normal.
May develop as a result of increased production of neutrophils and/or their release from the bone marrow; reducing the migration of neutrophils from the bloodstream into tissues; decreased transition of neutrophils from the marginal to the circulating pool.

A) Physiological neutrophilia- develops with the release of adrenaline (the transition of neutrophils from the marginal to the circulating pool decreases). Most often it causes physiological leukocytosis. It is more pronounced in young animals. The number of lymphocytes is normal (in cats it may increase), there is no shift to the left, the number of neutrophils increases no more than 2 times.

Causes:
1. Physical activity;
2. Convulsions;
3. Fright;
4. Excitement.
b) Stress neutrophilia - with increased endogenous secretion of glucocorticoids or with their exogenous administration. Causes stress leukocytosis. Glucocorticoids increase the yield of mature leukocytes from the bone marrow and delay their transition from blood to tissue. The absolute number of neutrophils rarely increases by more than two times compared to the norm, the shift to the left is absent or weak, lymphopenia, eosinopenia and monocytosis are often present (more often in dogs). Over time, the number of neutrophils falls, but lymphopenia and eosinopenia persist as long as the concentration of glucocorticoids in the blood remains elevated.

Causes:
1. Increased endogenous secretion of glucocorticoids:
- pain;
- prolonged emotional stress;
- abnormal body temperature;
- hyperfunction of the adrenal cortex (Cushing's syndrome).
2. Exogenous administration of glucocorticoids.
V) Inflammatory neutrophilia- often the main component of inflammatory leukocytosis. There is often a shift to the left - strong or slight, and the number of lymphocytes is often reduced.

Causes of extremely high neutrophilia (over 25x109/l) with high leukocytosis (up to 50x109/l):
1. Local severe infections:
- pyometra, pyoterax, pyelonephritis, septic peritonitis, abscesses, pneumonia, hepatitis.
2. Immune-mediated disorders:
- immune-mediated hemolytic anemia, polyarthritis, vasculitis.
3. Tumor diseases
- lymphoma, acute and chronic leukemia, mast cell tumor.
4. Diseases accompanied by extensive necrosis
- within 1-2 days after surgery, trauma, pancreatitis, thrombosis and bile peritonitis.
5. The first 3 weeks after administration of a toxic dose of estrogen (dogs, subsequently develop generalized hypoplasia or bone marrow aplasia and panleukopenia).

Leukemoid reaction of neutrophil type- a sharp increase in the number of neutrophil leukocytes in the blood (above 50x109/l) with the appearance of a large number of hematopoietic elements, up to myeloblasts. Resembles leukemia in the degree of increase in the number of leukocytes or in cell morphology.

Causes:
1. Acute bacterial pneumonia;
2. Malignant tumors with multiple metastases to the bone marrow (with and without leukocytosis):
- kidney parenchyma cancer;
- prostate cancer;
- breast cancer.

Neutropenia- a decrease in the absolute content of neutrophils in the blood below the lower limit of normal. Often it is absolute neutropenia that causes leukopenia.
A) Physiological neutropenia- in dogs of the Belgian Tervuren breed (together with a decrease in total number leukocytes and absolute number lymphocytes).
b) Neutropenia associated with a decrease in the release of neutrophils from the red bone marrow (due to dysgranulopoiesis - a decrease in the number of precursor cells or impaired maturation):

1. Myelotoxic effects and suppression of granulocytopoiesis (without a shift in the leukocyte formula):
- some forms of myeloid leukemia, certain myelodysplastic syndromes;
- myelophthisis (with lymphocytic leukemia, some myelodysplastic syndromes, myelofibrosis (often associated with anemia, less often with leukopenia and thrombocytopenia), osteosclerosis, in the case of lymphomas, carcinomas and mast cell tumors);
- in cats, infections caused by feline leukemia virus, feline immunodeficiency virus (together with leukopenia);
- toxic effect on endogenous (hormone-producing tumors) and endogenous estrogen in dogs;
- ionizing radiation;
 antitumor drugs(cytostatics and immunosuppressants);
- some drugs (chloramphenicol)
 infectious agents - early stage of viral infection (infectious hepatitis and canine parvovirus, feline panleukopenia, Ehrlichia canis infection in dogs);
- lithium carbonate (delayed maturation of neutrophils in the bone marrow in cats).
2. Immune neutropenia:

- isoimmune (post-transfusion).

c) Neutropenia associated with redistribution and sequestration in organs:

1. Splenomegaly of various origins;
2. Endotoxic or septic shock;
3. Anaphylactic shock.

d) Neutropenia associated with increased utilization of neutrophils (often with a degenerative shift of the leukocyte formula to the left):

1. Bacterial infections(brucellosis, salmonellosis, tuberculosis);
2. Severe purulent infections (peritonitis after intestinal perforation, abscesses that have opened inside);
3. Septicemia caused by gram-negative bacteria;
4. Aspiration pneumonia;
5. Endotoxic shock;
6. Toxoplasmosis (cats)

e) Neutropenia associated with increased destruction of neutrophils:

1. Hypersplenism;
2. Severe septic conditions and endotoxemia (with a deregenerative shift to the left);
3. DIC syndrome.

f) Hereditary forms:

1. Hereditary deficiency of cobolamine absorption (dogs - together with anemia);
2. Cyclic hematopoiesis (in blue collies);
3. Chediak-Higashi syndrome (Persian cats with partial albinism - light yellow eyes and smoky blue fur).

In addition to the above cases, neutropenia can develop immediately after acute blood loss. Neutropenia accompanying non-regenerative anemia indicates chronic illness(for example, rickettsiosis) or a process associated with chronic blood loss.

Agranulocytosis- a sharp decrease in the number of granulocytes in the peripheral blood until their complete disappearance, leading to a decrease in the body’s resistance to infection and the development of bacterial complications.

1. Myelotoxic - develops as a result of the action of cytostatic factors, combined with leukopenia, thrombocytopenia and, often, anemia (i.e. pancytopenia).
2. Immune
- haptenic (idiosyncrasies to medicinal substances) - phenylbutazone, trimethoprim/sulfadiazine and other sulfonamides, griseofulvin, cephalosporins;
- autoimmune (with systemic lupus erythematosus, chronic lymphocytic leukemia);
- isoimmune (post-transfusion).

EOSINOPHILES

Eosinophilia - increased level of eosinophils in the blood.

Causes:

Eosinopenia is a decrease in the level of eosinophils in the blood below the lower limit of normal. The concept is relative, since they may not be normally present in healthy animals.

Causes:

1. Exogenous administration of glucocorticoids (sequestration of eosinophils in the bone marrow);
2. Increased adrenocorticoid activity (Cushing's syndrome primary and secondary);
3. Initial phase of the infectious-toxic process;
4. The patient’s serious condition in the postoperative period.

BASOPHILES

LYMPHOCYTES

Absolute lymphocytosis is an increase in the absolute number of lymphocytes in the blood above normal limits.

Causes:

1. Physiological lymphocytosis - increased content of lymphocytes in the blood of newborns and young animals;
2. Adrenaline rush (especially cats);
3. Chronic viral infections (relatively rare, often relative) or viremia;
4. Reaction to vaccination in young dogs;
5. Chronic antigenic stimulation due to bacterial inflammation(for brucellosis, tuberculosis);
6. Chronic allergic reactions(type IV);
7. Chronic lymphocytic leukemia;
8. Lymphoma (rare);
9. Acute lymphoblastic leukemia.

Absolute lymphopenia is a decrease in the absolute number of lymphocytes in the blood below normal limits.

Causes:

1. Increased concentration of endogenous and exogenous glucocorticoids (with simultaneous monocytosis, neutrophilia and eosinopenia):
- treatment with glucocorticoids;
- primary and secondary Cushing's syndrome.
2. Viral diseases(canine parvovirus enteritis, feline panleukopenia, canine distemper; infection with feline leukemia virus and feline immunodeficiency virus, etc.);
3. Initial stages of the infectious-toxic process (due to the migration of lymphocytes from the blood into tissues to foci of inflammation);
4. Secondary immune deficiencies;
5. All factors that can cause a decrease in hematopoietic function of the bone marrow (see leukopenia);
6. Immunosuppressants;
7. Irradiation of bone marrow and immune organs;
8. Chronic uremia;
9. Heart failure (circulatory failure);
10. Loss of lymphocyte-rich lymph:
- lymphangiectasia (loss of afferent lymph);
- rupture of the thoracic duct (loss of efferent lymph);
- lymphatic edema;
- chylothorax and chylascitis.
11. Violation of the structure of lymph nodes:
- multicentric lymphoma;
- generalized granulomatous inflammation
12. After stress for a long time, together with eosinopenia - a sign of insufficient rest and poor prognosis;
13. Myelophthisis (together with a decrease in the content of other leukocytes and anemia).

MONOCYTES

Monocytosis is an increase in the number of monocytes in the blood.

Causes:

1. Infectious diseases:
 recovery period after acute infections;
- fungal, rickettsial infections;
2. Granulomatous diseases:
- tuberculosis;
- brucellosis.
3. Blood diseases:
- acute monoblastic and myelomonoblastic leukemia;
- chronic monocytic and myelomonocytic leukemia.
4. Collagenoses:
- systemic lupus erythematosus.
5. Acute inflammatory processes (with neutrophilia and a shift to the left);
6. Chronic inflammatory processes (with normal level neutrophils and/or without left shift);
7. Necrosis in tissues (inflammatory or tumors);
8. Increase in endogenous or introduction of exogenous glucocorticoids (in dogs, together with neutrophilia and lymphopenia);
9. Toxic, supraspinal inflammatory or severe viral infections(canine parvovirus enteritis) - together with leukopenia.
Monocytopenia is a decrease in the number of monocytes in the blood. Monocytopenia is difficult to assess due to the low levels of monocytes in the blood in normal conditions.
A decrease in the number of monocytes is observed with hypoplasia and aplasia of the bone marrow (see leukopenia).

PLASMO CYTES

Reasons for the appearance of plasma cells in peripheral blood:

1. Plasmacytoma;
2. Viral infections;
3. Long-term persistence of the antigen (sepsis, tuberculosis, actinomycosis, autoimmune diseases, collagenosis);
4. Neoplasms.

ERYTHROCYTE SEDIMENTATION RATE (ESR)

Accelerate ESR:

1. Formation of coin columns and agglutination of red blood cells (the mass of settling particles increases) due to the loss of negative charge on the surface of red blood cells:
- increased concentration of some blood proteins (especially fibrinogen, immunoglobulins, haptoglobin);
- blood alkalosis;
- presence of anti-erythrocyte antibodies.
2. Erythropenia.
3. Reduced plasma viscosity.
Diseases and conditions accompanied by accelerated ESR:
1. Pregnancy, postpartum period;
2. Inflammatory diseases of various etiologies;
3. Paraproteinemia (multiple myeloma - especially pronounced ESR up to 60-80 mm/hour);
4. Tumor diseases (carcinoma, sarcoma, acute leukemia, lymphoma);
5. Connective tissue diseases (collagenosis);
6. Glomerulonephritis, renal amyloidosis, occurring with nephrotic syndrome, uremia);
7. Severe infectious diseases;
8. Hypoproteinemia;
9. Anemia;
10. Hyper- and hypothyroidism;
11. Internal bleeding;
12. Hyperfibrinogenemia;
13. Hypercholesterolemia;
14. Side effects medications: vitamin A, methyldopa, dextran.

Leukocytosis, increased ESR and corresponding changes in the leukocyte formula - reliable sign the presence of infectious and inflammatory processes in the body.

Slow down ESR:

1. Blood acidosis;
2. Increasing plasma viscosity
3. Erythrocytosis;
4. Marked changes in the shape and size of red blood cells (sickling, spherocytosis, anisocytosis - since the shape of the cells prevents the formation of coin columns).
Diseases and conditions accompanied by a slowdown in ESR:
1. Erythremia and reactive erythrocytosis;
2. Severe symptoms of circulatory failure;
3. Epilepsy;
4. Sickle cell anemia;
5. Hyperproteinemia;
6. Hypofibrinogenemia;
7. Mechanical jaundice and parenchymal jaundice (presumably due to the accumulation of bile acids in the blood);
8. Taking calcium chloride, salicylates and mercury preparations.