Anemia symptoms and treatment in dogs. Clinical blood test - Nephrology Veterinary Clinic VeraVet. Home veterinarian

It is no secret to anyone what an important role blood plays in the body of all mammals. The quality and quantity of this invaluable liquid directly affects general state person or animal and the duration of his life.

In our article, we will look at what happens if anemia occurs in dogs, as well as how and with what it is treated. Possession of such information gives the owners the opportunity to track the pathology in their pets on early stages and in time to seek help from doctors.

Varieties of anemia

Anemia is otherwise called anemia, but this does not mean that the blood volume decreases during the disease. The amount of blood in this pathology remains unchanged, but its qualitative composition changes. This is expressed in a decrease in the amount of hemoglobin and is most often accompanied by a decrease in the number of red blood cells in the blood.

If we recall that the role of hemoglobin is to deliver oxygen to the cells of the body and remove from them carbon dioxide, it becomes clear how important it is for a dog to have a normal amount of this protein in the blood.

Anemia has a classification and is divided into several types:

1. Massive bleeding after injury or surgery causes a decrease in the number of red blood cells in the dog's body, resulting in posthemorrhagic anemia.
2. When exposed to certain substances or microorganisms, the destruction of red blood cells occurs and this is the cause of hemolytic anemia.
3. In the case when the dog's body does not have enough of its own resources for the formation of red blood cells, hypoplastic and aplastic anemia develops.
4. If a dog has regenerative anemia, then his body can restore the lost volume of red blood cells; with non-regenerative anemia, such restoration does not occur.

The cause of non-regenerative anemia, as well as post-hemorrhagic, can be heavy bleeding, after which the dog needs a lot of strength to heal the wound and replenish the supply of red blood cells.

The problem is that after an injury or surgery, the animal sometimes loses its appetite and, accordingly, a sufficient amount of nutrients ceases to enter its body. The dog does not have enough iron to "build" new blood cells, and there is such a kind of disease as hypochromic, that is, low-color anemia.

Any of these conditions are dangerous for dogs, so anemia should be treated under the supervision of an experienced veterinarian.

What causes the disease?

The reasons why anemia occurs are quite diverse, but it makes sense to consider the most common of them:

Less common causes of anemia include tumors affecting the red blood brain, infectious diseases, chronic kidney disease, exposure to radiation, and long-term use toxic medicines.

Manifestations of the disease

Signs of anemia are due to the fact that the supply of oxygen to the cells is disrupted in the dog's body. It must be taken into account that the symptoms of anemia are often mistaken for other diseases, for example. With the manifestation of anemia, the owner can observe such symptoms of the disease in the pet as:

1. Lethargy and depression.
2. Fast fatiguability, decreased activity.
3. Bad mood, lethargy.
4. Drowsiness.
5. Decreased appetite.
6. Paleness of the mucous membranes and small hemorrhages on them.
7. Arrhythmias and palpitations.
8. Temperature increase.
9. Traces of blood in feces and urine.
10. Shallow and rapid breathing.
11. An increase in the volume of the abdomen.

However, the presence of such features does not allow accurate diagnosis, since anemia has symptoms similar to other pathologies, and therefore the treatment of a dog should begin with instrumental studies.

Establishing diagnosis

The main diagnostic method for determining anemia is a clinical blood test. Based on this study alone, anemia can already be detected. It is more difficult to understand what caused the pathology, so the veterinarian may prescribe:

Despite a wide range of studies, sometimes it is not possible to determine exact reason anemia in a dog.

Treatment

The tactics of treating anemia in dogs is developed by a veterinarian based on data on what caused this pathological condition:

The right treatment balanced diet and good care will help your pet recover in a month and a half, and in the chronic form of anemia, therapy will be prescribed on an ongoing basis.

Prevention and feeding of a dog with anemia

There are no preventive measures that would help to completely protect the dog from anemia, but it is recommended to follow the classic rules for preventing diseases:

1. Timely vaccinate.
2. Visit the veterinarian regularly.
3. Make sure that the dog does not come into contact with toxic substances.
4. Coordinate the intake of any medications with your doctor.
5. Pay special attention to the diet. You need to feed the dog in a balanced way, i.e. include foods rich in vitamins and minerals in the menu. For these purposes, meat, buckwheat and liver, which contain a lot of iron, are well suited. When choosing industrial feed, carefully study its composition and give preference to feeds made from meat waste, as they contain more nutrients.
6. Make sure your pet eats regularly, at least 3 times a day. If the dog has poor appetite, you can try to feed her often and in small portions, and also try to find the food that she likes.
7. If necessary, you can correct the deficiency useful substances food additives. Pregnant and lactating bitches, which often have low hemoglobin, are recommended to give B vitamins and iron tablets. A doctor in a veterinary clinic will help you choose the right drug and calculate the dosage.

An anemic dog can live a long and fulfilling life if it is treated on time and strictly followed by the doctor's recommendations.

It is characterized by an autoimmune mechanism of development and acute hemolytic crises. The disease is more common in females, young or middle-aged.

Etiopathogenesis.
The disease develops as a result of damage to erythrocytes by autoantibodies with their transformation into spherocytes (increased density, thickness, spherical shape) and subsequent destruction in the reticuloendothelial system.
The destruction of spherocytes occurs mainly under the action of macrophages in the spleen, but there may be intravasal hemolysis.
As a result of massive hemolysis, free hemoglobin is released, a large amount of which the reticuloendothelial system is not able to convert into bilirubin, which is why excess hemoglobin is washed out with urine.

Pathogenesis.
In AIHA, erythrocyte destruction is accelerated by antibodies and/or by the fact that complement is fixed to the erythrocyte membrane.

Susceptibility: Dogs, Cats

Symptoms.
Sick animals become lethargic, their condition is depressed, the mucous membranes due to severe anemia have a porcelain appearance.
Concerns about severe shortness of breath (due to lack of oxygen), anorexia, vomiting and fever.
The spleen is greatly enlarged due to increased functional activity enlarged peripheral lymph nodes. The breakdown of red blood cells is evidenced by clinical signs: jaundice, dark brown bloody urine and black feces (inconsistently).

General clinic:
1. Abnormal size of retinal vessels;
2. Anorexia (lack of appetite, refusal to eat);
3. Auscultation of the heart: Tachycardia, increased heart rate;
4. Paleness of visible mucous membranes;
5. Fast fatigue during physical. load;
6. Hemorrhagic diathesis, impaired blood clotting;
7. Generalized weakness;
8. Hepatosplenomegaly, splenomegaly, hepatomegaly;
9. Hypodipsia, Adipsia;
10. Hyphema, blood in the anterior eye chamber, "black eye";
11. Darya, diarrhea;
12. Dehydration;
13. Dyspnoe (difficulty breathing, with an open mouth);
14. Icterus (Jaundice);
15. Bloody feces, hematochezia;
16. Fever, pathological hyperthermia;
17. Melena, black feces
18. Petechiae, ecchymosis;
19. Polydipsia, increased thirst;
20. Polyuria, increased volume of urination;
21. Reduced water intake,
22. Vomiting, regurgitation, emesis;
23. Heart murmurs
24. Dark colored feces;
25. Increased frequency respiratory movements, polypnea, tachypnea, hyperpnea; Lymphadenopathy; 26. Oppression (depression, lethargy);
27. Urinalysis: Hematuria;
28. Urinalysis: Hemoglobinuria or Myoglobinuria;
29. Urinalysis: red or brown urine;
30. Epistaxis, Rhinorrhagia, discharge of blood from the nose;

Laboratory research show severe anemia (1-2 million erythrocytes), decreased osmotic resistance of erythrocytes, reticulocytosis, hemoglobinuria, bilirubin, urobilinogenuria, excretion of stercobilin with faeces.

Diagnosis is based on anemia, characteristic detectable spherocytes, and positive reaction for treatment with prednisone.

Differentiate from other anemias (history, blood picture), kaagulopathy (impaired blood clotting) and systemic lupus erythematosus.

Diagnosis:
There is a positive Coomb's test to detect antibodies.
Often you can find competitive thrombocytopenia, which itself can cause hemorrhage.
Diagnosis per juvantibus. Improvement after steroid therapy helps the diagnosis.
In most cases, blood tests show signs of erythroid regeneration, such as polychromasia and reticulocytosis, but in some cases these are absent in dogs, a condition known as regenerative (non-regenerative) anemia.

Diagnostic algorithm:
- non-immunological tests: Total blood count (CBC), reticulocyte count, blood morphology, blood biochemistry and urinalysis.
A provisional diagnosis can often be made when a blood smear shows the appropriate erythrocyte morphology and other causes of anemia have already been ruled out.
- immunological tests: direct antiglobulin test (also known as direct Coomb's test). They determine antibodies or complements on erythrocytes. The test is positive in 60-70% of cases from dogs with AIHA.
Tests with polyspecific agents are also used, with the help of which immunoglobulin G, M or C3 (IgG, IgM and C3) are determined on the surface of erythrocytes. The spectrum of their activity is much wider than the direct Kuumbs test:
- Coomb's test, using separate, specific reagents for IgG, IgM and C3;
- Direct enzyme-linked antiglobulin test (Direct enzime-linked antiglobulin test) that quantifies the level of IgG, IgM and C3 on the surface of erythrocytes;
- Papain test (Papain test), which modifies the erythrocyte membrane in such a way that makes it more sensitive to agglutination and thus makes it easier to identify incomplete antibodies (incomplete antibodies);
- radioimmunoassay (radioimmunoassays), which quantifies the level of IgG associated with red blood cells.

TREATMENT, DEVELOPMENT AND PROGNOSIS

The course of the disease in the form of acute crises and rarely latent.

The forecast is doubtful.

Treatment: principles:
Immediately begin drip intravenous administration of prednisolone at a dose of 2 mg/kg.
If at the same time anemia decreases, then the dose is reduced to 0.5-0.25 mg / kg within a week.
In case of poor tolerability of prednisolone or ineffectiveness of therapy, immunosuppressive therapy with azathioprine at a dose of 1-2 mg/kg is additionally connected without risk to hematopoiesis.
Avoid blood transfusion during an acute crisis. Relapses after remission are rare. If they occur, then a splenectomy should be done.

Treatment: schemes and doses:
Detailed treatment described by Van Pelt and Stewart and Feldman.
It is divided into 3 categories:
- treatment of the described causes (if they are known)
- supportive therapy:
- immunosuppressive therapy:

Glucocorticoids are the main drugs of choice.
Prednisolone is usually recommended at a dose of 2-4 mg / kg / day, in two doses (1-2 mg / kg).
Dexamethasone sodium phosphate 0.5 mg/kg (which is approximately equipotent to 4 mg/kg prednisolone) may be given, followed by prednisone to continue treatment.
The PCV (total cell volume) should be monitored every day in the hospital until it begins to increase, at which time the animal is released and treatment continues at home.
The PCV is monitored weekly until it is completely normal, and then once a month to ensure that the animal is well tolerated by the prescribed corticoid treatment.

- Cyclophosphamide (Cyclophosphamide)- a cytotoxic alkylating agent that can be used in cases with acute intravascular hemolysis, autoaglutination, or cases resistant to corticoid monotherapy. Cyclophosphamide can be given orally at a dose of 50 mg/m2 every other day. If vomiting is caused by oral administration, cyclophosphamide can also be used as an intravenous bolus at a dose of 200 mg/m2. Side effects include myelosuppression, gastroenteritis, and the possibility of hemorrhagic cystitis.

- (Azathioprine) Azathioprine (Azathioprine). It can be used as monotherapy at a dose of 2 mg/kg/day per os for the first 7-10 days, then the dose can be reduced by 1 mg/kg/day. Side effects include myelosuppression, gastroenteritis, pancreatitis, and elevated liver enzymes.
Note: There is no difference in efficacy between azathioprine and cyclophosphamide.

- Danazol (Danazol)- a synthetic androgen that increases T-suppressor cells, reduces the binding of Immunoglobulin G (IgG) to the erythrocyte membrane (RBC) and reduces the number of Fc receptors in macrophages. Side effects: fatigue, lethargy, masculinization and mild elevation of liver enzymes. The main recommended dose of danozol when used together with glucocorticoids is 10 mg/kg/day. The dose of danazol can be reduced when remission occurs.

Intravenous human gamma globulin is used to treat dogs with AIHA.
Other treatments include cyclosporine, splenectomy (removal of the spleen), and plasmapheresis.
Cyclosporin may help to inhibit T-helper cells (T-helper cells).
Splenectomy may also be beneficial because the spleen is the primary site of phagocytosis of immunoglobulin G (IgG) coated erythrocytes (RBCs).
Plasmapheresis removes circulating anti-erythrocyte antibodies from serum, but is expensive and not readily available.

Forecast.
The average mortality rate for dogs with AIHA is 20-40%, but 80% can be reached in some cases.

J-L. PELLERIN, C. FURNEL, L. SHABAN

Autoimmune hemolytic anemia (AHA) is the most commonly detected type of autoimmune disease in dogs and cats (Person J.M., Almosni R, Quintincolonna F, Boulouvis H.J., 1988). In dogs, primary AGA occurs as a result of an autoimmune disease. Severely flowing secondary AGA of a non-traumatic nature is also often found (Squires R., 1993).

AGA is one of the most characteristic classic examples of autoimmune diseases. Therefore, autoantibodies are involved in the pathogenesis of AGA (Miller G., Firth F.W., Swisher S.N., Young L.E., 1957). In humans, the specificity of target antigens has been identified: there are autoantibodies for the blood group antigen (Person J.M. et al., 1988).

AGA in humans was first discovered in 1945 using an anti-globulin test called the Coombs method. Miller G. et al. (1957) first reported AGA in a dog.

AGA has also been identified in mice, guinea pigs, horses (Miller G. et al., 1957; Taylor FG.R., Cooke B.J., 1990), cattle (Dixon P.M. et al. 1978; FengerC.K., et al., 1992), sheep, pigs, dogs and cats (Halliwel R.E.W., 1982).

DEFINITION

The term "anemia" refers to a decrease in the concentration of hemoglobin in the circulating blood below 12 g per 100 ml in dogs and below 8 g per 100 ml in cats, which is accompanied by a decrease in oxygen transport.

AHA is defined as acquired severe hemolysis associated with

Table 1. Classification of AGA (PMC = direct Coombs method),

ny with the presence of immunoglobulins on the surface of erythrocytes and sometimes in the blood serum, the action of which is directed towards the antigenic determinants of the patient's erythrocyte membrane (Appendix 1).

AGA is characterized by two main criteria:

1. diagnosed with a blood test;

2. autoantibodies are detected using direct method Coombs.

Among hemolytic anemias of an immune nature, there are secondary anemias that develop after allogeneic immunization, due to an infectious process or drug sensitization, as well as AGA itself, sensu stricto (in the strict sense of the word). Alloimmunization is very rare in dogs and cats.

CLASSIFICATION

AGA is classified according to immunological and clinical features.

Criteria

The clinical picture, laboratory results, pathogenesis, prognosis and treatment of AGA largely depend on the type of immunopathological process.

The immunological classification of AGA is based on the class of antibodies (IgG or IgM) and their functions - agglutinating or sometimes hemolytic.

The AGA classification includes five main classes (Table 1). Cold agglutinins are defined as agglutinating antibodies detected at +4°C. They always belong to the IgM class.

Impact on prognosis and therapy

AGA occurs most often in dogs and is caused by the action of autoimmune IgG both together with complement and separately (Cotter S.M., 1992).

1. If IgG is expressed on the surface of erythrocytes in association with complement or without its participation (class I and III), then this disease is mainly idiopathic in nature with an acute and transient course. The clinical picture of the disease is characterized by the progressive development of hemolysis, sometimes proceeding severely and with remissions. This IgG-associated primary AGA responds well to corticosteroid treatment and is generally not associated with secondary AGA due to any comorbidities. According to Klag etcol. (1993), among 42 dogs tested, 74% tested positive for IgG and negative for complement. Such AGAs are generally classified as class III.

2. If we are talking about IgM antibodies (classes II, IV and V), then the disease responds worse to corticosteroid therapy, often has a secondary nature (oncological, in-

Table 2 Diseases associated with SAGA in dogs and cats (according to Werner L).

* Diseases caused by peri- or intra-erythrocyte agents may be responsible for the development of immune-mediated hemolytic anemia without autoantibodies, which may occur secondarily and be complicated by the development of true AGA.

infectious disease or some other autoimmune disease). Such AGAs can be detected directly or indirectly through the presence of C3b and IgM during elution or washing.

The prognosis of AGA associated with C3b and IgM is more questionable compared to IgG.

Common immunological disorders

In the same patient, it is often necessary to observe a large number of different antibodies in combination.

taniya with anti-erythrocyte autoantibodies. Canine AGA is especially common in association with systemic lupus erythematosus (SLE) or autoimmune thrombocytopenia. In the latter case, we are talking about Evans syndrome.

Evans Syndrome (E. Robert, Amer., 1951) [Eng. Evans "syndrome]. Si. syndrome de Ficher-Evans. Association of an autoimmune disease with thrombocytopenic purpura. Rarely occurs in humans, has a dubious prognosis.

Sometimes AGA is observed in association with autoimmune dermatosis, characterized by the presence of a depot of IgG and complement at the level of the dermoepidermal junction (Hasegawa T. et al., 1990). Anti-erythrocyte autoantibodies are a factor in an extensive immunological disorder even in the absence of a clinical picture of the disease.

Clinical classification

Immunological classification must be in conflict with strict clinical classification, as it contrasts idiopathic AGA with secondary AGA. Autoimmune hemolytic anemia, characterized by the presence of warm antibodies (IgG), corresponds to "idiopathic", while AGA associated with the persistence of cold antibodies (IgM) - "secondary".

Idiopathic AGA

In primary or so-called idiooptic AGA, no comorbidities are noted. In dogs, the incidence of idiopathic AGA is 60-75% of cases. In cats, this disease is rare, as they are dominated by secondary AGA due to an infectious disease caused by the leukemia virus (FeLV) (Jackon M. L et al., 1969).

Secondary AGA

In 25-40% of cases in dogs and 50-75% in cats, AGA is associated with other diseases. AGA precedes, accompanies or follows another disease, sometimes without special clinical symptoms(Table 2). The prognosis and effectiveness of treatment depends on the underlying cause of AGA.

Secondary AGA in cats is mainly associated with FeLV infection or haemobartonellosis (Haemobartonella felis).

The frequency of detection of IgM on erythrocytes in cats significantly exceeds IgG, while IgG autoantibodies predominate in dogs. The higher content of IgM antibodies in cats compared with dogs explains the predominance of the autoagglutination reaction.

SYMPTOMS OF THE CLINICAL PICTURE OF THE DISEASE AND THE RESULTS OF THE LABORATORY STUDY

In humans, a high positive correlation was found between the signs of clinical, hematological, and immunological manifestations of AGA (Stevart A.F., Feldman B.F., 1993).

Clinical symptoms

AGA manifests itself at any age, but most often they are observed from 2 to 7 years. The season also affects (Klag A.R., 1992), since 40% of AGA cases are detected in May-June. In humans, an increase in the incidence of AGA was also found in spring period(Stevart A.F., Feldman B.F., 1993).

Sex and breed are not factors predisposing to this disease.

The onset of the disease may be progressive or sudden. AGA is characterized by a combination of five pathognomonic symptoms:

1) loss of strength, lethargy (86%)

2) pallor of mucous membranes (76%)

3) hyperthermia

4) tachypnea (70%)

5) tachycardia (33%).

The top three reasons to visit a veterinarian are: Brown color urine, anorexia (90%) and loss of strength (Desnoyers M., 1992). Hepatomegaly and splenomegaly are not always detected (25% of cases), a similar trend is noted for lymphadenopathy (Stewart A.R, Feldman B.F., 1993).

Prostration and sometimes lethargy are also observed. Jaundice, slight or absent (50% of cases).

Petechiae and ecchymosis (bruising) are observed only in cases where thrombocytopenia occurs. According to Klag A.R. et al. (1993) moderate or severe thrombocytopenia was observed in 28 of 42 dogs (67%).

The intensity of anemia can vary and depends on 2 factors:

1) degree of hemolysis,

2) compensatory ability of the bone marrow.

The intensity of anemia in primary AGA is more pronounced than in secondary.

Quite rarely, when cold agglutinins (IgM) are detected, more often in idiopathic AGA, anemia is generally moderately expressed, with separate episodes of intensification.

Cyanosis and necrosis of the terminal parts of the body (ears, fingers, tail, nose) that can evolve into gangrene, sometimes with a fatal outcome, are the most pathognomonic signs in this disease (Vandenbusshe P. et al., 1991).

Drawing. 1. Coombs method: agglutination reaction.

Table 3. Norms of general biochemical analysis in domestic carnivores (according to Crespeau).

Appendix 3

All these injuries in dogs and cats are associated with circulatory disorders caused by agglutination of red blood cells in the peripheral capillaries, where the body temperature is much lower than that of its visceral part.

Clinical blood test

In the presence of AGA, the number of erythrocytes falls below 5,000,000/ml. The hematocrit is greatly reduced (up to 8-10%), a similar picture is observed for hemoglobin (up to 4 g/100 ml). Note normocytic, normochromic and sometimes macrocytic anemia (Jones D.R.E. et al., 1992, 1991, 1990).

Attention is drawn to the presence of small colored spherocytes (photo 1), and sometimes neutrophilia is noted (Desnoyers M., 1992).

Sometimes we state phagocytosis of erythrocytes by monocytes. AGA in dogs is more often regenerative (Appendix 2). The total number of reticulocytes varies from 20 to 60%. In 30% of dogs, mild reticulocytosis (1-3% of reticulocytes) is noted, in 60% it is moderate or severe (more than 3% of reticulocytes). Weakly regenerative and regenerative AGA have been described in dogs (Jonas L.D., 1987). Currently, these forms of the disease are being detected more and more often.

Blood chemistry

All dogs have marked bilirubinuria (brown urine) with urobilinuria, as well as hyperbilirubinemia (not conjugated). Jaundice is present in approximately 50% of cases. Increased hemoglobinemia is sometimes accompanied by hemoglobinuria, but intravascular hemolysis is less common (10% of 42 dogs) (Klag A.R. et al., 1993). At the same time, a significant decrease in haptoglobin and serum iron, while uricemia (uric acid in the blood) increases in 50% of cases. With the development of the disease, the indicators vary, sometimes the changes are prolonged or interrupted with subsequent relapses.

METHODS OF IMMUNOLOGICAL DIAGNOSIS

Direct Coombs test

This method in the diagnosis of AGA is a priority (Person et al., 1980).

Principle

The Coombs test is an immunological method that detects the presence of non-agglutinating antibodies due to the action of xenogenic (from another species) anti-immunoglobulin serum that provokes agglutination. With the help of only one direct Coombs test, a diagnosis is made for this disease. IN clinical practice this method is used for humans, dogs and cats.

The principle of operation of the direct Coombs test, or the so-called special specific antiglobulin test, is based on the effect of sensitization of erythrocytes with the help of immunoglobulin or complement fixed on their membrane, or due to both of them together (Stewart A.R, 1993).

The mechanism of the proposed method is to use species-specific "anti-antibodies" or specific antiglobulins to create bridges between antibodies that cover the surface of erythrocytes (Fig. 1).

At the first stage, polyvalent antiglobulins are used, directed against all serum globulins.

Table 4. Interpretation of the results of the direct Coombs method (according to Cotter).

Classification

The following reagents have been developed for humans: anti-lgG, anti-IgM, anti-lgA, and anti-C3.

For dogs, one polyvalent antiglobulin is used in routine diagnostics, sometimes three antiglobulins: one polyvalent and two specific - anti-lgG and anti-C3 (Jones D.R.E., 1990).

With the help of specific reagents, it was found that most often erythrocytes are sensitized only by IgG alone (AGA type IgG), or IgG in combination with complement (AGA mixed type), in particular with C3d expressed (present) on the erythrocyte membrane.

Sometimes erythrocyte sensitization is caused by complement alone (AGA of the complement type). This type of anemia is associated exclusively with the action of IgM, since IgM in the Coombs test usually elutes spontaneously during the washing process. In this case, after washing at 37°C, only C3d remains on the surface of the erythrocytes.

IgM can be detected through anticomplement using the Coombs method, or using the same method, but carried out in the cold, in which IgM is not eluted during the washing process. We are talking about IgM cold agglutinins, when at +4°C in dogs spontaneous agglutination can be observed.

IgA antibodies are extremely rare.

Each antiglobulin has species-specific properties. Setting up the Coombs reaction with feline erythrocytes means the need for timely preparation or purchase of an antiglobulin reagent for this type of animal. Kits designed for this test in humans or dogs are not suitable for cats.

In domestic carnivores, AGA detected with cold antibodies are much less common than with warm ones.

Execution technique

Blood for analysis (Appendix 3) must be taken with an anticoagulant (citrate or EDTA - ethylenediaminetetraacetic acid). It is extremely important that the medium in the tube contains a calcium chelating agent. In a blood sample, it provokes non-specific fixation of complement on erythrocytes in vitro and leads to a false positive reaction. That is why heparin is not used as an anticoagulant.

After thorough washing (three or five centrifugations from 5 min at 800 g to 5 min at 1500 g), the test sample of the suspension is ADJUSTED to 2% concentration. Direct Coombs reaction is recommended to be carried out as soon as possible after taking the material, preferably within 2 hours. The blood sample should be stored at 37°C. After incubation for one hour at 37°C with different serial dilutions of three antisera, the sample is kept at room temperature (1-1.5 hours). The results of the reaction should be taken into account visually in the wells of microplates placed on the Cahn mirror, or using a microscope (x100).

In parallel, it is necessary to carry out negative controls:

1. 2% suspension of the patient's erythrocytes in the presence of isotonic NaCl solution to test the ability of the tested erythrocytes to spontaneously agglutinate in the absence of antiglobulins. According to Desnoyers M. (1992), autoagglutinins are responsible for spontaneous autoagglutination both at 37°C (class I) and at 4°C (class IV). In cats, erythrocyte autoagglutination is common (Shabre B., 1990). Dilution of blood in an equivalent volume of isotonic NaCl solution eliminates this artifact due to the dissociation of tube-shaped erythrocytes, without adversely affecting true autoagglutinins (Squire R., 1993).

2. Mixing 2% RBC Suspension healthy dog(control animal) with species-specific serum antiglobulin allows you to check the quality of the antiserum.

If clinical symptoms suggest IgM-mediated AGA, then the clinician may request a conventional Coombs test at 37°C as well as a cold Coombs test at 4°C to detect cold-active antibodies (types IV and V) (Vandenbussche P. et al., 1991).

This test is not suitable for cats. The fact is that many normal cats have non-agglutinating antibodies that become active at lower temperatures and are detected using a direct Coombs test at 4 ° C. In this species, the method of indirect haemagglutination at 4°C should be used.

Discussion

The laboratory diagnosis of AGA is almost entirely based on the direct Coombs method in combination with a complete blood count. The interpretation of a positive reaction in the Coombs test is not difficult.

If the detected antibodies belong to the IgG class, then it is very likely that the identified anemia is of autoimmune origin.

The significance of detecting a positive result in the IgG + complement Coombs test in mixed type AGA requires discussion, since there is no complete certainty that complement is fixed on the complex formed by IgG with erythrocyte membrane antigens.

It turned out that it is even more difficult to establish the reliability of erythrocyte sensitization in AGA, detected using a positive Coombs test in the formulation of a reaction to “pure complement”.

It is possible that some of the Coombs' complement tests correspond to temporary fixation antigen-antibody complexes that rapidly elute from the surface of erythrocytes.

Differentiate AGA from true hyperhemolysis according to the following features: increased reticulocytosis, unconjugated hyperbilirubinemia, etc. Sometimes the Coombs test gives a false positive or false negative result (Table 4). This is quite rare (about 2% of cases), but a negative reaction to the Coombs test can occur with true AGA, especially if the number of fixed immunoglobulins is insufficient (less than 500 per erythrocyte).

The clinical symptoms of AGA are in many ways similar to piroplasmosis, which is very common in France. This requires the clinician to systematically perform the Coombs test in the case of hemolytic anemia in the absence of a positive response to classical treatment, when an animal becomes ill with piroplasmosis, even if persistence of piroplasms in the blood is established, because this disease can be simultaneously accompanied by AGA.

Elution

If using the Coombs method it is possible to determine which class the sensitized antibodies belong to, then elution makes it possible to determine their specificity. Elution at high temperature with ether or acid allows you to collect a pool of antibodies and test them on a panel with erythrocytes of the appropriate type using the indirect Coombs method (Person J.M., 1988).

Table 5. Doses of cytotoxic agents used immunosuppressive drugs and possible toxic effects.

This is mainly done in human medicine, where there are panels with typed erythrocytes.

In animals, acid elution is of particular importance when a false-positive reaction of specific antibodies to an antigen artificially fixed on the surface of erythrocytes is suspected. If the eluate obtained from the erythrocytes of a dog suspected of having a disease does not give an agglutination reaction with a pool of erythrocytes obtained from dogs with various groups blood, then we are talking about AGA (Tsuchidae tal., 1991).

Indirect Coombs Method

Its principle is to detect the presence of free autoantibodies in the blood serum against erythrocytes.

The blood of a sick dog must be collected in a clean, dry test tube and centrifuged. The test serum is incubated in the presence of erythrocytes, washed three times and obtained from a healthy dog ​​of the same blood type as the sick animal. The level of free autoantibodies in serum is often very low, since all the antibodies present are tightly fixed on the surface of red blood cells. In 40% of cases, the amount of free antibodies is insufficient to obtain a positive reaction in the indirect Coombs method (Stevart A.R, 1993).

MECHANISMS OF ERYTHROCYTE DESTRUCTION

AGA belongs to the group of autoimmune diseases for which the role of autoantibodies in pathogenesis has been clearly and convincingly demonstrated.

It is the binding of autoantibodies to specific antigens on the erythrocyte membrane that is responsible for the decrease in their life expectancy, which is mediated by three cytotoxic mechanisms: 1) phagocytosis; 2) direct hemolysis with the participation of complement; 3) antibody-dependent cellular cytotoxicity.

Extravascular erythrophagocytosis

In most cases, phagocytosis of erythrocytes by macrophages is observed. Erythrocytes sensitized by autoantibodies are destroyed after opsonization by macrophages of the spleen, liver, and, to a lesser extent, bone marrow. Bilirubinemia, as well as the presence of urobilin and bilirubin in the urine, prompt the clinician that extravascular hemolysis is occurring (Chabre B., 1990).

Minor differences in pathogenesis are present between the two RBC graveyards.

Extravascular erythrophagocytosis may be associated with intravascular hemolysis.

Complement mediated intravascular hemolysis

Destruction of erythrocytes in the circulatory system is a rather rare phenomenon (in 15% of dogs), which is observed exclusively in acute hemolytic anemia, or in acute complications developed in the chronic course of the disease (classes II and V).

This is due to the complete activation of complement along the classical path from C to CD on the surface of the same erythrocytes. As a result, the erythrocyte membrane is destroyed and their constituents (mainly hemoglobin) are released into the circulating blood, which leads to hemoglobinemia and hemoglobinuria.

This is observed only when fixing autoantibodies to complement with a pronounced hemolytic effect: the role in hemolysis is now well established for IgG and IgM. Only these forms of an autoimmune disease can be accompanied by icterus or subicterism.

Cytotoxicity of cells caused by antibodies

K-cells (killer cells or killer cells) have receptors for the Fc fragment of the IgG molecule, with the help of which they are fixed on the surface of sensitized erythrocytes and cause their death through direct cytotoxic effects.

Recently, the role of this third mechanism in the development of AGA has been well established, but not yet completely defined.

As with other autoimmune diseases, the degree of autoimmune disorders is not always directly proportional to the severity of the manifestation of the process.

Short term forecast

The short-term prognosis is unfavorable only in 15-35% of cases. Clinical improvement after adequate therapy is observed, according to different authors, in 65-85% of patients.

An increase in hematocrit and reticulocytosis against the background of a decrease in spherocytosis are positive prognostic criteria.

Mortality in dogs is significantly increased under the following circumstances: poor regeneration (moderate or insufficient reticulocytosis), low hematocrit (below 15%), blood bilirubin concentration above 100 mg/l.

Long term forecast

The long-term prognosis is less favorable in terms of possible complications. Usually you have to be content with the fact that recovery is achieved only in 30-50% of cases.

The prognosis of secondary AGA depends mainly on the underlying disease and its possible complications.

Most often observed pulmonary thromboembolism and disseminated intravascular coagulation (Cotter S.M., 1992). In rare cases, complications are noted in the form of lymphadenitis, endocarditis, hepatitis or glomerulonephritis, which can be fatal (Stewart A.F., Feldman B.F, 1993).

In class III disease, the prognosis is most often favorable. In cats, the prognosis is poor, since the disease is often associated with infection caused by one or another retrovirus (feline leukemia virus, FeLV; feline immunodeficiency virus, VIF) (ChabreB., 1990).

A more cautious prognosis for diseases of classes II and V, accompanied by intravascular hemolysis.

The prognosis is doubtful in diseases belonging to classes I and IV and accompanied by autoagglutination (Hagedorn J.E., 1988). They are more likely to end in death.

According to Klag et col. (1992, 1993) the overall mortality rate is about 29%.

In any case, the prognosis should always be restrained and depend on the adequacy of the pharmacological correction of the condition.

AGA therapy can be carried out in various ways. Most general approach treatment is based on the elimination of the immunological reaction by prescribing immunosuppressants that suppress the formation of autoantibodies and the activity of macrophages responsible for erythrophagocytosis.

Immunosuppressants

Corticosteroids are the main component of therapy. They are used both as monotherapy and in association with danazol, cyclophosphamide or azathioprine (Cotter S.M., 1992; Squires R., 1993).

Corticosteroids

In high therapeutic doses and with long-term use, corticosteroids are the main drugs that provide the effect of immunosuppression. From the clinician's point of view, prednisone (Cortancyl N.D. per os), prednisolone, methylprednisolone (methylprednisolone hemisuccinate: Solumedrol N.D., i.v.) shock doses 2 to 4 mg/kg every 12 hours give the best results. You can also use dexamethasone or betamethasone at doses of 0.3-0.9 mg/kg per day (Stewart A.F., Feldman B.F, 1993).

If corticosteroid therapy is effective in AGA with warm autoantibodies (IgG) in 80-90% of cases, then in AGA with cold autoantibodies (IgM) its effectiveness is ambiguous. However, the data obtained should be evaluated very carefully. If corticosteroid therapy is ineffective, cytotoxic chemotherapy should be considered.

Shock corticosteroid therapy should be initiated as soon as possible after confirmation of the diagnosis of AGA by the direct Coombs method. Treatment should not be long: the duration varies on average from three to eight weeks. A longer course of corticosteroid therapy has little benefit but carries risks severe complications(iatrogenic Cushing's syndrome).

With maintenance therapy, corticosteroids are prescribed every other day in doses equal to half, a quarter, or one-eighth of the shock. The gradual withdrawal of drugs is carried out within two to four months after clinical remission. Some animals are completely off corticosteroids. Others continue to be treated at low doses throughout life to avoid relapses (Squires R., 1993).

In dogs with idiopathic AGA (IgG), the Coombs test remains positive throughout the course of the disease, including during corticosteroid therapy and clinical remission. When the reaction is negative in the direct Coombs method, the recurrence of the disease is quite rare. This is a very favorable prognostic criterion (Slappendel R.J., 1979).

In cats, corticosteroid therapy is combined with tetracycline antibiotics if haematological examination reveals haemobartenellosis (Haemobartenella felis), or for prophylaxis bacterial complications on the background of immunosuppression.

Corticosteroid therapy should not be given long-term in cats, especially in FeLV infections. The immunosuppressive effect of corticosteroids may enhance the already pronounced immunosuppressive effect of the virus. In cats with latent viral infection corticosteroid therapy can exacerbate the pathology and cause viremia.

If in the first 48-72 hours after the start of corticosteroid therapy it is not possible to achieve stabilization or improvement in hematocrit, then therapy should be continued. A marked increase in hematocrit may occur 3-9 days after initiation of therapy. If there is no improvement even after 9 days, then more powerful immunosuppressive drugs should be used.

Powerful immunosuppressants

Cyclophosphamide and azathioprine are two cytotoxic drugs (cytostatics) that are more potent immunosuppressants than corticosteroids (Table 5). They suppress the production of antibodies by B-lymphocytes (Squires R., 1993).

These drugs should be used only in the most severe cases AGA diseases: patients with autoagglutination (classes I and IV) or with intravascular hemolysis (classes II and V) (Hagedorn J.E., 1988). In severe cases, vigorous therapeutic measures are required. Be sure to inform pet owners about side effects drugs.

Cyclosporine (10 mg/kg, IM, then orally for 10 days) has been successfully used to treat complex recurrent cases of AGA that are not amenable to classical corticosteroid therapy (Jenkins TS. et al., 1986; Preloud P., Daffos L, 1989 ). Patients with autoagglutination (classes I and IV) require combination therapy (corticosteroids + cytostatics) to prevent relapses and achieve remission. However, larger trials are required to better understand how effective this combination is in AGA therapy.

Danazol

Danazol (an ethisterone derivative), a synthetic hormone of the androgen group, is increasingly being used to treat autoimmune diseases (Stewart A.F., 1945). Danazol reduces the production of IgG, as well as the amount of IgG and complement fixed on cells (Holloway S.A. et al., 1990).

The main mechanism of action of danazol is inhibition of complement activation and suppression of complement fixation on cell membranes(Bloom J.C., 1989). Danazol modulates the ratio of T-helpers and T-suppressors, which is disturbed in autoimmune thrombocytopenia (Bloom J.C., 1989). It can also reduce the number of receptors for the Fc fragment of immunoglobulins located on the surface of macrophages (Schreiber A.D., 1987).

The therapeutic dose for dogs is 5 mg/kg PO 3 times a day (Stewart A.R, Feldman B.F., 1993). The action of danazol (Danocrine N.D., Danatrol M.D.) increases slowly over one or three weeks and is manifested in the improvement of hematological parameters (Bloom J.C., 1989; Schreiber A.D., 1987). It is recommended to combine danazol with any corticosteroid (Stewart A.F., Feldman B.F., 1993). When the patient's condition stabilizes, the doses of corticosteroids are reduced, and treatment with danazol is continued for two to three months (Schreiber A.D., 1987). Danazol can cause a noticeable increase in muscle mass if used for more than six months.

Splenectomy

The purpose of splenectomy is to remove the spleen, which is the main organ of red blood cell destruction in the case of IgG-associated AGA. It is also the main organ of the lymphoid system producing circulating antibodies, in the particular case of autoantibodies. Successfully applied in humanitarian medicine, this operation probably cannot be so favorable for a dog and a cat (Feldman B.F. et al., 1985). It is completely useless in AGA associated with IgM, where the destruction of red blood cells mainly occurs in the liver. Moreover, this operation can exacerbate the latent course of babesiosis or hemobartonellosis. Thus, we propose to consider splenectomy only as an extreme option (Feldman V. Fetal., 1985).

Blood transfusion

Blood transfusion is generally contraindicated due to the possibility of hemolysis. Transfused erythrocytes are rapidly covered with autoantibodies, which leads to their mass rupture, and, consequently, aggravation of the hemolysis crisis. On the other hand, blood transfusion reduces normal bone marrow hematopoiesis. Therefore, it should be prescribed for the following indications: hemolytic crisis, hematocrit below 10%, or respiratory failure.

In practice, the indication for blood transfusion is a drop in the number of red blood cells below 2x106/ml in dogs and 1.5x106/ml in cats. Very short-term improvement is noted with intravenous corticosteroids. Plasmapheresis gives positive results in humans, but in animals its use is complicated by the low availability of instrumentation for cats (Matus R.E. et al., 1985).

adjuvant therapy

As with all anemias, adjuvant therapy is used: ferrous sulfate at the rate of 60-300 mg per day (Squires R., 1993), vitamin B12, calm environment, warmth, and then intravenous infusion sometimes forced breathing. It is especially important that patients with cold agglutinins be protected from exposure to too low temperatures. Prevention of thromboembolism and DIC in dogs at risk ( elevated level total bilirubin, condition after blood transfusion) consists in the early administration of anticoagulants: 100 IU / kg of heparin s / c every 6 hours during the exacerbation period (Klein M.K. et al., 1989).

Patient follow-up

This is of great importance. Control over the condition of patients can be carried out using the Coombs test: two months after the patient enters the acute phase of the course of the disease, then every 2-3 months during the transition to the chronic course. If the criteria for clinical and hematological assessment are normal, the Coombs test gives backlash, we can assume that the dog or cat has recovered. However, it is difficult to talk about true recovery or simple remission.

In this case, you should be extremely careful, since with a probability of 50% any of the above options is possible.

To clarify the true situation, it is necessary to continue monitoring the condition of the animal, systematically conduct blood tests (for example, once a month for six months, and then once every three months) and, at the slightest threat of relapse, resume corticosteroid therapy. As a rule, this is enough to normalize the clinical condition. The minimum dose of corticosteroids (0.05-1 mg / kg per day) every other day helps to restore blood counts to the physiological norm. In chronic or recurrent AGA, permanent administration of corticosteroids, whenever possible, at the lowest therapeutic dose is recommended.

CONCLUSION

When the clinical picture is sufficiently indicative, AGA can be diagnosed with only one direct Coombs method. But this applies only to a positive Coombs test in the presence of IgG (both with and without complement). In general, positive reactions with complement alone are common in dogs and rarely associated with severe hemolysis. If delivered provisional diagnosis, additional research is needed. As with all autoimmune diseases, non-specific disorders in the immune system can be caused by a variety of reasons.

Finally, all autoimmune diseases share similarities, with each representing a group of disorders that overlap to varying degrees. Often, one can observe the simultaneous or sequential manifestation of AGA and systemic lupus erythematosus, and AGA and rheumatoid arthritis, or AGA and autoimmune thrombocytopenia. If an immunological diagnosis reveals the presence of one of these autoimmune diseases, then it is imperative to look for others even in the absence of characteristic clinical symptoms. When AGA is associated with SLE or thrombocytopenia in a dog, or with FeLV infection in a cat, the prognosis is more uncertain compared with isolated idiopathic AGA.

magazine "Veterinarian" № 2003

No wonder blood was previously considered a liquid with magical properties: it warms, carries oxygen and nutrients, with any “malfunction” of the circulatory system, the entire body suffers. One of the most severe pathologies is hemolysis in dogs. In this case, the blood is actually split into constituent components, which leads to very unpleasant consequences ...

Hemolysis is the process of destruction of red blood cells. But often this term is used in a "wider" form and implies damage not only to erythrocytes, but also to other shaped elements blood. Of course, such a severe pathology never occurs overnight and “just like that”. Moreover, the process of hemolysis is characteristic of hemolytic. By itself, it does not occur (except in some cases).

Pathology is divided into two large varieties: intravascular or intracellular. With intracellular hemolysis, blood cells are destroyed directly in the organs of the reticuloendothelial system, and in most cases this is the norm. The fact is that in the spleen of an animal, erythrocytes are destroyed daily, the “service life” of which has come to an end. Pathological is much more often intravascular hemolysis, in which the breakdown of blood cells occurs directly in the general bloodstream. This results in an ejection huge amount bilirubin, which causes jaundice. And she, by the way, is very difficult for dogs to carry, cases of death are not uncommon.

But in mild cases, the owner, most likely, will not be able to guess at all that something is wrong with his pet. It is worse when the volumes of degraded erythrocytes exceed all conceivable limits: at the same time, the reticuloendothelial system can no longer cope with their processing, it develops (urine turns into “tomato juice”). The second dangerous consequence of this process is anemia. It's far more dangerous than people think.

Read also: The main causes of urinary incontinence in dogs

Because erythrocytes carry oxygen, with their lack in the bloodstream develops chronic hypoxia of all organs and tissues. In especially severe cases, this can lead to serious damage to the brain, since this organ is extremely sensitive to lack of oxygen! So at the first sign of it, you should immediately contact your veterinarian.

Predisposing factors

The causes of pathology are extremely diverse. For example, cases of hemolysis that develop in animals exposed to elevated or low temperatures (hyper- and hypothermia) are described. In addition, the destruction of red blood cells is fraught with exposure of the dog to radioactive materials. Of course, in the wild, this is unlikely to happen. It is much more likely that a sick pet has some kind of dangerous infectious disease. In particular, call the breakdown of blood can many pathogenic strains of the most common Escherichia coli.

Some act similarly viruses. In addition, one should not forget about congenital pathologies of the reticuloendothelial system and autoimmune diseases, in which the body itself begins to destroy blood, mistaking it for foreign tissue. In addition, hemolysis develops when incompatible blood is transfused, but this is not typical for our country: since we do not have pet blood banks, there is nothing to transfuse. Let's look at some of the predisposing factors in a little more detail.

So, first possible cause"decompositions" of the blood are the bites of poisonous snakes and insects, as well as poisoning with rat poisons, salts of heavy metals, etc. Snake bites are especially dangerous. True, hemolysis occurs only if the reptile venom has a hemolytic effect (gyurza, viper, efa, rattlesnakes, etc.).

What else can cause hemolysis?

Eat Great chance hemolysis in cases when your pet has been in contact with some dangerous "chemistry". In particular, this result can lead to: ether, chloroform, benzene and other compounds. Even in low concentrations, they cause the destruction of red blood cells, while being powerful carcinogens and contributing to the development of cancer. Hemolysis is not uncommon in liver diseases: bile acids, which enter the bloodstream with pathologies of this organ, are very aggressive towards erythrocytes, destroying their membrane.

Read also: Why is the dog scratching? Looking for dangerous symptoms

Clinical signs

With mild hemolysis, clinical signs are often absent. Everything changes when the number of red blood cells that are destroyed increases. The animal becomes lethargic, refuses to feed, may experience shortness of breath. The dog refuses to play, lies in one place for a long time. Soon you can notice a change in the color of urine: it becomes blood-red, while strongly resembling tomato juice. In the early days, the urine becomes only slightly pink, but subsequently turns very red. How else is blood hemolysis manifested in dogs?

If the pet does not receive any medical care, the process worsens, develops. All visible mucous membranes turn yellow, the skin becomes noticeably stiffer, and the degree of its elasticity decreases. One has only to collect a “handful” of skin with your fingers, and then let it go, the fold is smoothed out for a long time and reluctantly. The pet gets tired very quickly: even after a light walk, the dog’s tongue “falls out”, the dog breathes heavily and hoarsely. After that, he drinks quickly and very greedily.

The main function of erythrocytes is the transport of oxygen (O 2) by the cells of the body and carbon dioxide (CO 2) from the body, participation in buffer systems of hydrogen ions, maintaining vascular integrity and participating in coagulation processes.

Anemia is a decrease in the level of circulating red blood cells in the systemic circulation. Anemia in cats and dogs is not a primary diagnosis, but a secondary process that can develop for three reasons: 1. With a decrease in the production and release of red blood cells in the bone marrow; 2. With increased utilization of erythrocytes in the animal's body (ex. hemolysis); 3. With blood loss (external or internal). That is, the distinction between the exact type of anemia is based on the level of production of red blood cells (hematopoiesis) in relation to the rate of their destruction (hemolysis) or loss (external environment and also the third space). In some cases, anemia can develop due to a combination of various factors - a decrease in the production of red blood cells, a shortening of their life span and blood loss.

Anemia as such is often diagnosed at a veterinary clinic appointment (from 10% to 30% of patients), it may be accompanied by various clinical manifestations(eg, pallor of the mucous membranes, general depression, intolerance, exercise, heart murmurs, weakness in the filling of the pulse wave, hemoglobinuria, peak (pica) or proceed without external signs and can only be diagnosed in the laboratory. In fact, the definition of anemia as such and its exact specifications are determined in the clinical diagnostic laboratory, and the diagnosis of anemia is exclusively laboratory.

Anemias in animals are divided into two main classes - regenerative and non-regenerative. Regenerative anemia is a decrease in the number of red blood cells with the presence of a functional ability of the bone marrow to produce and release red blood cells. Non-regenerative anemia is a decrease in the number of red blood cells due to or in combination with a decrease in the ability of the bone marrow to produce and / or release red blood cells. Below are the main causes of anemia in cats and dogs encountered at a veterinary clinic.

Table. Typical causes of anemia in cats and dogs.

Below is one of the diagnostic approaches after detection in cats and dogs, it follows from the assessment of the severity of anemia, counting the number of reticulocytes, assessing the morphology of the brain blood smear and attempting morphological classification.

Assessing the severity of anemia

The first step in assessing anemia in animals in a veterinary clinic is to determine its severity. For this, indicators such as hematocrit (Hct), PCV, hemoglobin level and red blood cell count can be used. PCV - percentage of whole cells consisting of erythrocytes, is obtained using a microhematocrit tube and centrifuge. The hematocrit is calculated by automatic analyzers after determining the mean RBC size and RBC count. PCV and hematocrit are usually used interchangeably and serve as the basis for assessing the severity of anemia. An exception to this rule occurs in microcytic hypochromic anemia and macrocytic hypochromic anemia.

Table. Classification of anemia in cats and dogs according to severity, depending on the level of hematocrit.

When determining the severity of anemia, the interpretation of PCV and hematocrit should be based on the animal's hydration status, usually achieved by measuring total plasma protein on a refractometer. Dehydration (dehydration, hemoconcentration) is usually accompanied by a decrease in plasma volume and an increase in total protein, which falsely increases the animal's hematocrit (relative polycythemia). Dehydration can mask moderate forms of anemia, more often this occurs at the initial stages of diagnosis. Therefore, the hydration status of the animal must be normalized over a period of time, and only after that an assessment of the level of erythrocytes can be carried out (for example, in case of bleeding, this time can take up to 1-2 days). Hyperhydration, on the contrary, is accompanied by an increase general level plasma and a decrease in total protein, this phenomenon usually manifests itself in a false decrease in the level of erythrocytes, which also requires some correction. Some difficulties are noted in hypoproteinemia due to bleeding, bowel disease, glomerular disease and similar pathologies accompanied by protein loss.

In dogs, the tone of the spleen may play a role in hematocrit levels, and its rapid contraction may cause some changes in the distribution of red blood cells, because it contains up to 20% -30% of the mass of red blood cells. In cats, the spleen is not as actively involved in the circulation of red blood cells, and this phenomenon is rarely observed. The release of erythrocytes from the spleen develops quite quickly, and is normally observed in healthy animals. Less important for determining the level of erythrocytes is the relaxation of the spleen with the removal of erythrocytes from the circulating bed (pr. anesthesia).

There is some difference in the course of acute and chronic anemia, with the slow development of a decrease in the level of erythrocytes, the animal undergoes physiological adaptation to hypoxemia and they can live for weeks and months with minimal clinical manifestations (ex. hematocrit 5% -8% at chronic development anemia).

When determining the severity of anemia, one should be aware of age and breed differences in the level of red blood cells in animals, so in puppies aged 2 and 8 weeks of age, the hematocrit level usually corresponds to 28%, in beagles hematocrit usually has higher values ​​compared to the general population dogs.

It is difficult to assess the severity of anemia in methemoglobinemia when total weight hemoglobin of the animal is within the normal range, but there is significant reduction its ability to carry oxygen (ex. carbon monoxide poisoning).

Moderate anemias usually develop secondary to other non-hematological problems (ex. anemia of inflammatory diseases; neoplasia, renal and liver failure, endocrine pathologies), while the main diagnostic and therapeutic efforts should be directed to the search for primary pathologies. Severe and severe anemias often indicate a primary hematological disease and require active diagnostic and therapeutic actions.

Reticulocyte count

The primary judgment of an animal's erythropoiesis and bone marrow function is based on its ability to produce reticulocytes. Some difficulties may arise, and for a more complete assessment, blood smear analysis and bone marrow cytology are used.

Reticulocytes are immature red blood cells released in increased numbers from normal bone marrow in response to a decrease in the number of red blood cells. Reticulocytes contain residual intracytoplasmic organelles (ribosomes, RNA) where hemoglobin synthesis continues, when stained with new methylene blue (NMB) or brilliant cresyl blue (BCB), dark blue granules are determined by light microscopy.

Reticulocytes in cats are divided into two forms - aggregate and punctate, which reflects a significant difference in the rate of their maturation (in contrast to dogs). Initially, cats release aggregate reticulocytes, which very quickly (within 12 hours) mature to punctate forms. When evaluating a blood smear of cats, aggregate reticulocytes contain more than 12-15 points (drops) until it is impossible to determine their exact number, punctate reticulocytes of cats contain from 3-12 points (drops) of intracytoplasmic inclusions. Punctate reticulocytes mature quite slowly (10-12 days) in the cat's body to mature erythrocytes and accumulate in the bloodstream in significant quantities (compared to aggregate reticulocytes). Aggregate reticulocytes of cats are the best indicator the degree of regeneration (compared with punctate forms) and the absolute number of aggregate reticulocytes are a more significant indicator of regeneration than the corrected percentage of reticulocytes or the index of their production. Feline punctate reticulocytes reflect the overall regenerative response and are not usually counted.

None of the current automated analyzers is able to accurately determine the number of punctate feline reticulocytes, and manual counting must be relied solely on in assessing the regenerative response. They also have considerable difficulty in identifying the types of reticulocytes. According to the pattern of reticulocytes in cats, the time of development of anemia can be assumed, about 4 days from the moment of the onset of anemia in cats, aggregate forms of reticulocytes reach a peak, then their number decreases and the number of punctate reticulocytes continues to increase. Polychromatophiles in cats with a smear stained with Wright's stains usually reflect the amount of aggregate reticulocytes.

Dogs, like cats, first release aggregate reticulocytes, which very quickly pass through the punctate reticulocyte stage (up to 24 hours) and mature into mature erythrocytes. As a result of rapid maturation, primary dog ​​reticulocytes consist of aggregate forms, they contain significant masses of precipitated RNA with a small amount of punctured forms. Based on the foregoing, dog reticulocytes are not divided into two forms, due to the small number of punctate forms and their absence. clinical significance in the diagnosis of anemia. Most laboratories do not count RBCs as reticulocytes with only one or two individual granules, but include them in RBCs with granular artifacts or precipitates. Canine reticulocytes are roughly equal to the polychromatophiles seen with Wright's stains on smears, with different stains varying in their ability to show polychromatophiles.

In anemia of blood loss or hemolytic anemia, the bone marrow should increase the number of reticulocytes according to the severity of the anemia. The maximum production of reticulocytes is observed within 3-7 days from the onset of an acute episode of anemia. If on initial stage the number of reticulocytes is small, but there are other signs of regeneration in the blood smear (eg anisocytosis, polychromasia) - re-evaluation of reticulocytes is carried out (after 3-5 days). A reticulocyte count is performed at a hematocrit of less than 30 in dogs and less than 20 in cats. Below is a table for determining the degree of regeneration depending on the number of reticulocytes in the peripheral blood of dogs and cats.

Table. The degree of regeneration of cats and dogs depending on the number of reticulocytes.

Reticulocyte assessment uses absolute reticulocyte count per microliter of blood, reticulocyte index (RI), reticulocyte percentage (RP), and corrected reticulocyte percentage, or can be assessed by observing polychromatophiles on a blood smear (see below). Reticulocytes are larger than erythrocytes (ex. macrocytes) and have a lower concentration of hemoglobin than mature erythrocytes. Therefore, the description in a normal smear of an increased number of macrocytic hypochromic erythrocytes usually reflects reticulocytosis and a regenerative bone marrow response.

Percentage of reticulocytes by manual counting.

For microscopic definition reticulocyte count, blood with EDTA is mixed with vital dye. The most commonly used are the new methylene blue (1% NMB + 1.6% sodium oxalate anticoagulant) and brilliant cresyl blue (BCB; 1% saline solution). Blood and dye are mixed in appropriate proportions and incubated for 10 minutes for NCB or 30 minutes for BCB. Residual endoplasmic reticulum in reticulocytes appears as dark blue granules. The smear is dried and determined percentage of reticulocytes per 1000 non-nuclear erythrocytes. Mature erythrocytes are poor in RNA and do not stain. Nuclear erythrocytes are counted separately.

Manual counting is largely dependent on the operator, and variations occur compared to automatic counting. However, punctate reticulocytes of cats are not counted by any machine today, therefore, only manual method can provide relevant information. An appropriate assessment of the feline erythroid response is made by determining the percentage of punctate reticulocytes (10%, 25%, 50%, 75%, or 90%). Canine reticulocytes are defined as having more than 2 drops (dots). Canine reticulocytes may have several punctate reticulocytes, but dogs do not have as many punctate reticulocytes as cats, which can significantly change interpretation.

The percentage of reticulocytes in automatic counting.

The reticulocyte count is carried out by various brands of hematology analyzers, with varying degrees of sensitivity and reliability. A common mistake in interpreting reticulocytes is to conclude that the anemia is regenerative based on a slight automatic increase in reticulocytes. Once again, it should be said that the assessment of feline reticulocytes is exclusively the manual work of a laboratory assistant.

In anemia, the percentage of reticulocytes somewhat overestimates the strength of erythropoiesis. The absolute reticulocyte count is a more consistent indicator of a true regenerative response.

The absolute number of reticulocytes.

The absolute reticulocyte count serves as a better indicator of hematopoietic response than the percentage of reticulocytes. The bottom line is that in anemia, the number of mature erythrocytes is variously reduced, so the percentage of reticulocytes overestimates the hematopoietic response. The absolute reticulocyte count is calculated by multiplying the percentage of reticulocytes by the number of red blood cells, therefore it adjusts the percentage of reticulocytes to the severity of anemia.

Corrected percentage of reticulocytes

The corrected reticulocyte percentage (CRP) does not require an RBC count, it is calculated from the formula: CRP - (percentage of reticulocytes) x (determined hematocrit) / (normal hematocrit). Normal hematocrit in dogs is 45%, in cats - 37%. A corrected reticulocyte percentage (CRP) >1% supports a regenerative response.

Reticulocyte index or reticulocyte production index (RI orRPI)

The Reticulocyte Production Index (RPI) is used in dogs to compensate for young reticulocytes (>24 hours maturation time) in response to severe anemia. This indicator is calculated based on the formula RPI = (percentage of reticulocytes) x (hematocrit: 45) x (1: maturation time). Maturing time is calculated based on: hematocrit 45% = 1 day, hematocrit 35% + 1.5 days, hematocrit 25% = 2 days, hematocrit 15% = 2.5 days. At RPI >1.0 = average regeneration, at RPI >3 = pronounced regenerative response.

Evaluation of the peripheral blood smear

Polychromasia

Polychromasia - an increase in the number of polychromatophiles in a blood smear, in dogs they are equivalent to reticulocytes and polychromasia reflects reticulocytosis quite well. Polychromatophiles are larger than mature erythrocytes and are more blue in color due to the presence of ribosomes. Polychromatophiles are defined as polystained cells with orange staining of the cytoplasm and blue staining of RNA. It is aggregate reticulocytes that, when treated with Wright's stains, look like polychromatophiles, their presence is suitable for assessing reticulocytosis in dogs, but is poorly applicable in cats due to the important role of punctate reticulocytes. The increase in polychromasia in the first 3-7 days after the development of anemia reflects the degree and strength of erthyropoiesis.

macrocytosis

A macrocyte is a larger erythrocyte, the size is determined by determining the average volume of an erythrocyte (MCV) or by a cytogram of erythrocytes. Reticulocytosis is the most common cause of macrocytosis, especially 3-5 days after the onset of anemia, but mature macrocytes may also be released during the period of accelerated erythropoiesis. One of the authors believes that macrocytosis, and especially the percentage of macrocytic hypochromic erythrocytes, is a more sensitive indicator of increased erythropoiesis in the regenerative response than polychromasia and reticulocytosis.

Other causes of macrocytosis - artificial swelling of red blood cells in EDTA when long-term storage, poodle congenital dyserythropoiesis, stomatocytosis, and .

Anisocytosis, erythrocyte distribution width, hemoglobin distribution width.

Anisocytosis is the variability in the size of red blood cells. The breadth of erythrocyte distribution is calculated automatically and determines the degree of anisocytosis. A common cause of increased anisocytosis is regenerative anemia with release of reticulocytes and macrocytes. Hemoglobin breadth numerically describes the variation in red blood cells based on hemoglobin concentration. An increase in latitude serves as an indicator that erythrocytes have increased variability.

Nuclear erythrocytes, basophilic granularity, Howell-Jolly bodies

Other hematologic findings in regenerative anemia typically include Howell-Jolly bodies, nucleated erythrocytes, and basophilic stippling. These changes are well defined in the smear, but are not quantitative and specific indicators of erythrocyte regeneration, unlike reticulocytes and macrocytic hypochromic erythrocytes. The number of nuclear erythrocytes is calculated in different ways (relatively, absolutely), but they do not in the best way reflect erythropoiesis in the bone marrow, can be observed in sepsis and neutropenia, spleen diseases, extracerebral hematopoiesis, heat stroke, sepsis, lead poisoning, hypoadrenocorticism, leukemia, and some pathologies of the bone marrow.

Basophilic stippling - More commonly associated with regenerative anemia and rarely a concern, it can also develop with lead poisoning but is not considered a specific and sensitive diagnostic tool for this poisoning in dogs (better determined by toxicological testing).

Howell-Joli bodies are small round nuclear residues in erythrocytes. They can be observed with increased erythropoiesis or with a decrease in their splenic removal. The feline spleen is less efficient at removing red blood cells, and therefore Howell-Jolie bodies are more common in cats than in dogs. Steroid treatment or Cushing's disease reduces phagocytic activity and the number of Howel-Joli bodies. Also, these bodies are noted in poodles with macrocytosis, can be multiple and dysplastic.

Below is a summary table of changes in the peripheral blood smear of cats and dogs with anemia.

Table. Morphological changes in erythrocytes in anemia of dogs and cats with clinical significance.

IMHA– immune-mediated hemolytic anemia

RBC indices

Mean cell volume (MCV) – the average size erythrocytes in femoliters (fL). This indicator can be calculated directly on the analyzer or based on the formula (hematocrit x 10) / (number of red blood cells x 10 6). The population of small erythrocytes is microcytes, the population of normal erythrocytes is normocytes, and the population of large erythrocytes is macrocytes.

Mean Hemoglobin Concentration (MCHC) calculates the ratio of hemoglobin concentration relative to hemactocrit. MCHC (g/dl) = (hemoglobin concentration x 100) / hematocrit. Depending on this indicator, erythrocytes can be normochromic, hypochromic and hyperchromic. Hyperchromic erythrocytes are not normally found, but may occur with hemolysis, lipemia, or the formation of Heinz bodies.

Red cell distribution width (RDW) is obtained on an automatic hematology analyzer. This indicator is associated with a change in the size of red blood cells, anemia with a large population of macrocytes or microcytes have an increased latitude.

Bone marrow examination (aspirate, biopsy).

This type of study is indicated in cases where there are no peripheral signs of regeneration (reticulocytosis, polychromasia) or when the degree of response does not correspond to the severity of anemia. At cytological examination bone marrow is an attempt to assess the function of the production of erythroid function - the norm, hypoplasia or hyperplasia.

Morphological classification of anemia.

Morphological classification is usually based on erythrocyte indices, and the decrease in the level of erythrocytes can be divided into the following types:

1. Macrocytic, hypochromic: regenerative anemia.

2. Normocytic, normochromic: non-regenerative anemia.

3. Macrocytic normochromic: early regenerative response, defective erythropoiesis, especially during infection.

4. Microcytic hypochromic: iron deficiency anemia

5. Microcytic normochromic: iron deficiency, portosystemic shunt.

Below are some features of these types of anemia.

Normocytic normochromic anemia

Normocytic normochromic anemia is noted as non-regenerative anemia when the bone marrow releases little or no macrocytic reticulocytes. Most erythrocytes in the smear are normocytic normochromic leftovers from previous production. Anemia due to hemorrhage or hemolysis with a development time of 1-2 days often precedes the regenerative response, it is also normocytic and normochromic and is classified as pre-regenerative.

Macrocytic hypochromic anemia.

Macrocytic hypochromic anemia usually indicates regenerative anemia with an elevated reticulocyte count. Reticulocytes are relatively larger (ex. macrocytic) than mature erythrocytes. Reticulocytes are hypochromic because they do not complete the synthesis of hemoglobin. One author believes that macrocytic hypochromic ribosome-deficient erythrocytes (not reticulocytes) are still a late regenerative response reflecting increased erythropoiesis.

Microcytic hypochromic anemia

Microcytic hypochromic anemia is often observed in iron deficiency, which prevents adequate production of hemoglobin. Erythrocytes are small with significant production of hemoglobin. Microcytosis and impaired iron metabolism are common in dogs with portosystemic shunt and liver atrophy. Microcytic hypochromic anemia associated with sideroblastic anemia is a type of myelodysplastic syndrome in dogs. The Japanese Akita, Shiba Inu, and some other breeds have smaller red blood cells.

Macrocytic normochromic anemia

Too many highlights for vitamin B12 or folate deficiency causing macrocytic hypochromic anemia came to veterinary medicine as a result of extrapolation from human medicine. Macrocytic normochromic erythrocytes in this deficiency are not observed in either cats or dogs, but are characteristic of humans and primates. In animals, macrocytosis, in addition to a regenerative response, is caused by pathologies such as infection and myelodysplastic syndrome not associated with vitamin B12 and folate deficiency.

Below is a summary table of characteristic laboratory changes in regenerative and non-regenerative anemia in cats and dogs.

Table. Regenerative and non-regenerative response to anemia in cats and dogs.

Valery Shubin, veterinarian, Balakovo