When to suspect hemolysis?

We suspect hemolysis especially in regenerative anemia when blood loss has been excluded (Fig. 1).

In most cases, hemolytic anemia are severe and show stronger signs of regeneration than blood loss anemia. In case of hemolysis, erythrocytes are destroyed. In addition to primary IMHA (referred to as non-associative IMHA), a variety of underlying diseases can cause hemolysis (Fig. 2).

Which tests should be performed?

Requested material for the diagnosis of IMHA are listed in Fig. 3.

CBC/cytomorphology (blood smear):

Provides information regarding regeneration and is useful as a first overview, e.g. to detect infectious agents. Erythrocyte morphology can also provide important information regarding the cause of anemia. At Laboklin, a reticulocyte count is included in every canine and feline CBC. The CBC should be measured from EDTA blood as soon as possible after blood collection as the erythrocyte fragility of dogs with IMHA is increased.

Blood chemistry: Measurement of serum bilirubin and total protein to identify the cause of anemia can help to differentiate between hemolysis and hemorrhages.

Urinalysis: In some cases of hemolysis, bilirubinuria or hemoglobinuria may be present. Please note that in dogs – especially in male dogs – a weakly positive test result can be physiological.

Infectious agents to detect infectious agents such as babesia and haemotropic mycoplasma in acute phases, molecular analysis (PCR) is recommended. Additionally, pathogens such as ehrlichia and anaplasma should be excluded. For ehrlichia, an additional antibody titer determination is useful.

Tests to diagnose immune-mediated destruction

Spherocytes: The presence of so-called spherocytes (Fig. 4) in the blood smear can provide valuable information about an immunemediated destruction of the erythrocytes. The sample material required for their detection are two to three blood smears directly made in the practice.

Autoagglutination: By looking at the EDTA sample, one may notice the presence of an unspecific autoagglutination. This finding should be followed by a saline agglutination test and a Coombs’ test.

Coombs’ test: Suitable to detect the presence of autoantibodies on the surface of erythrocytes. Sample material: Fresh EDTA blood. If the sample is kept refrigerated, the test can be evaluated up to 5 days from collection.

FAQs on assessing the Coombs’ test

What is the principle of the Coombs’ test?

The Coombs’ test, also named DAT (Direct Antiglobulin Test), is testing for the presence of antibodies on the surface of the red blood cells (Fig. 5).

Different test methods are available on the market: gel test, strip test, flow cytometry test, the well-known microtiter plate test, and more. All the mentioned tests are adequate for the diagnosis of IMHA in humans and dogs. Some tests are also available on the market for cats and horses.

What is the principle of the microtiter plate method?

In veterinary medicine, the microtiter plate is the most commonly used method. The antiglobulin (Coombs’ reagent) is gradually diluted (1:2, 1:4, 1:8, 1:16, etc.), then washed erythrocytes are added and incubated for at least 30 minutes. If no reaction occurs, the blood cells will sediment at the bottom of the wells and only a tiny red point will be observed after incubation: the test result for this well is negative. If the test is positive, the added antiglobulins bind the erythrocytes, refraining them from falling to the bottom of the well. The erythrocytes cover the well completely, or almost completely. If only the two first wells (1:2 and 1:4) show positivity, the test is still considered negative, because these low titers are considerated unspecific. Titers from 1:8 are considerate positive. It is not rare to see titers above 1:1024 in IMHA.

What is a prozone effect?
The prozone effect is a phenomenon observed in highly positive samples. The high concentration of anti-erythrocyte antibodies inhibits the positive reaction. An example is pictured below (Fig. 6).

The EDTA-blood sample presents a blood clot, can the Coombs’ test overcome this?
Unfortunately no. The presence of intact red blood cells is mandatory for the performance of a correct Coombs’ test. However, it is important to know the difference between a blood clot and an agglutination. The blood clot is the result of the physiological coagulation, and can occur when the sample is not correctly mixed with EDTA-anticoagulant. The blood clot is irreversible. To avoid this: after sampling, gently repeatedly invert the sample. The agglutination can be caused by different factors. In most cases, the agglutination is reversible, e.g. by warming up the sample in your hand, or by washing the cells.

Autoagglutination in my sample: Can it still be tested?

The erythrocytes are washed prior to the Coombs’ test. Despite autoagglutination, it is therefore possible to perform a Coombs’ test on the majority of these samples. A rare exception: If autoagglutination persists after washing, the result of the Coombs’ test cannot be read.

My sample shows autoagglutination: Is a Coombs’ test necessary, or is the agglutination enough to make a diagnosis?

One must differentiate between persistent autoagglutination after washing and an autoagglutination before washing the red blood cells. In many cases, the agglutination before washing is not diagnostic. Persistent autoagglutination is however strongly suggestive for IMHA. Our laboratory report will inform you about the persistent autoagglutination in your sample.

I have already started immunosuppressive treatment: Is it still meaningful to order a Coombs’ test?

The Coombs’ test can remain positive for up to 24 weeks after treatment despite immunosuppressive medication. A negative result, however, does not exclude IMHA.

I have already evaluated the blood smear under the microscope and found spherocytes: Do I still need to perform a Coombs’ test or can I already diagnose IMHA?

Spherocytes can be seen in small quantity in healthy dogs, and in moderate quantity in other diseases (zinc intoxication, poisoning, DIC…). However, five spherocytes per visual field at 100x magnification are considered supportive for IMHA. Hereditary spherocytosis is an important but very rare differential diagnosis. Please note that cats physiologically have round erythrocytes without central pallor, which makes the evaluation of spherocytes in cats unreliable.

I already tested my patient, the Coombs’ test titer was high, what does it mean?

High and low titers have the same meaning: the erythrocytes are coated with auto-antibodies. So far, there are no proof – in human and veterinarian medicine – that a higher titer correlates with stronger anemia, hemolysis or worse prognosis.

Dr Nadine Idalan, Dr Maria Brockmann

As with all skin problems, a thorough history is the most critical tool to arrive at a diagnosis in the cat. The preliminary report at the initial consultation takes a good 20 minutes and often already represents up to 70 – 90% of the diagnosis. It is often easier to reach a tentative diagnosis with an owner without a cat than with a cat without an owner.

The anamnesis for the work-up of a skin problem in the cat contains the same questions as in any other animal species and begins with signalment. Questions such as “Do other in contact animals or humans have skin lesions?” must never be forgotten, as they can provide important clues to a contagious disease such as dermatophytosis or ectoparasitic diseases. It is also essential to ask whether the cat is free-roaming because with a free-roaming cat, for example, a strict elimination diet is more or less impossible. Changes in the family environment (new baby, moving house, changes in the family members, renovation of the flat) can lead to marking in sensitive cats and psychogenic excessive grooming behaviour, which may result in alopecia and other skin lesions.

Information on pruritus is often misleading, especially in cats. Many cats do not show their pruritus in front of the owner; they hide to lick themselves, and the owner is convinced that the cat has spontaneously lost its hair. Inquiring about the occurrence of hair vomiting or problems with hairballs may help to identify excessive grooming behaviour. In addition, some cats may be found to have hair between their teeth when the mouth is inspected.

It is also important to differentiate whether the hair has fallen out (the whole hair, including the root, is missing) or whether the hair has broken off (the hair root is still present). In the first case, there is a disease with hair loss, either of hormonal origin (relatively rare in cats) or follicular infections such as dermatophytosis. In these cases, the hair is easily depilated. If the hair is broken off (self-induced alopecia), pruritus is usually present, the cat licks itself, and the hairs break off in the process. The problem is that owners hardly ever observe their cat licking itself, and in the anamnesis, they swear that the hair has fallen out. There are several ways to convince oneself and the owners that the alopecia is self-induced: use a magnifying glass to see the broken hair shafts or take a skin fold and see the hair stubble at the edge. A trichography can also be performed. Place hair samples from the lesion on a slide with a drop of oil and examine them under the microscope. The tip of the hair usually is nicely pointed; however, if the hair tip is blunt, then the hair has broken off by grooming (Fig. 1). The presence of fractures confirms a self-induced licking alopecia.

Cats are not small dogs!

It is necessary to distinguish between pruritic and non-pruritic skin diseases, allergic and nonallergic diseases, and the differential diagnoses of each dermatological reaction pattern. Cats often react with different skin reaction patterns, which have many differential diagnoses, at the same time, the same disease can manifest with a variety of reaction patterns (Fig. 8). Therefore, it is often more challenging to get to the bottom of the cause of the skin problem in cats than, for example, in dogs.

Regarding the aetiology of pruritus in cats, the following causes are possible including ectoparasites (Notoedres, Otodectes, Cheyletiella, Demodex, lice, hair lice, Trombicula), allergies and infectious diseases (bacterial infections, dermatophytosis, which,are usually not primarily pruritic). Pruritus may also occur as an associated sign in viral diseases (herpesvirus, poxvirus), immune-mediated diseases (pemphigus foliaceus, drug reactions, mural folliculitis, sebaceous adenitis) and cutaneous neoplasms (e.g. lymphoma).

As discussed alopecia is a common lesion in cats which is often mistakenly dubbed as hair loss. In most cases, pruritus causes the cat to lick and pluck out its hair. Symmetrical alopecia is commonly produced by the cat itself and is not hair loss in the true sense of the word; pruritus of allergic, ectoparasitic or psychogenic origin is a possible cause. On the other hand, alopecia is primary in dermatophytosis, demodicosis, alopecia areata, pyoderma and paraneoplastic alopecia, in which the skin also appears smooth and shiny.

Skin lesions in the cat are often reaction patterns, but they do not represent specific diseases.

1. Miliary dermatitis: many small miliary scabs are scattered over the body; they are felt rather than seen. This reaction pattern is often seen in flea allergy, but all other allergies, ectoparasites and dermatophytoses can also trigger it (Fig. 2).

2. Self-induced ulcers of the head and neck: as the name suggests, these are self-inflicted injuries to the head and neck of the cat. It is not uncommon for the cat to scratch so severely that it injures itself and causes deep, bleeding ulcers. This reaction pattern is often seen in food allergies but also other allergies, ectoparasites and dermatophytoses (Fig. 3).

3. Self-induced symmetrical alopecia: the difference between hair loss and self-injury has already been discussed above. In this reaction pattern, patches of alopecia, sometimes symmetrical, appear on various body parts. Areas usually affected are the abdomen, limbs and occasionally the back. The skin is generally free of other lesions (Fig. 4).

4. Eosinophilic granuloma complex (EGC):

A. Eosinophilic ulcer (indolent/rodent ulcer): the lesion is ulcerated and necrotic, located on one or both sides of the upper lip and is usually not pruritic or painful. Eosinophilia is rarely present in the blood or tissues (Fig. 5).
B. Eosinophilic plaque: a pruritic, welldemarcated, slightly raised, rounded, exudative lesion that usually occurs on hindlimbs, abdomen or within the groin area (Fig. 6). Secondary bacterial infections aggravate the pruritus. Eosinophilic granulocytes are found in cytology.
C. Eosinophilic granuloma. This form has different presentations and locations.
a. Linear granuloma. An elongated, hard, palpable elevation on the caudal hindlimb.
Rarely on the chin, corner of the mouth, pinna, and paws (Fig. 7).
b. Pharyngeal granuloma: may be allergic or idiopathic (Fig. 5).
c. Eosinophilic granuloma of the chin, also called “fat chin”.

The reaction patterns mentioned above can occur together in the most diverse combinations. The differential diagnoses apply to all reaction patterns: allergic diseases (to flea bites, food and environmental allergens or feline atopic skin syndrome (FASS)), ectoparasitic diseases and dermatophytosis (Fig. 8). The cause must be identified and treated accordingly. If the cause is allergic, symptomatic treatment with glucocorticoids, cyclosporine or antihistamines should be applied. Indolent ulcers, eosinophilic plaques, miliary dermatitis and self-induced head and neck ulcers experience improvement of secondary infection with antibiotic treatment.

Unquestionably, the best therapy for allergic processes is allergen avoidance. It is possible in flea bite allergy dermatitis (continuous flea treatment of the affected animal and all accompanying animals) and food allergy (elimination diet, provocation diet and lifelong diet with tolerated components). In the case of environmental allergy (pollen, house dust and storage mites, moulds), allergen avoidance would not only be highly time-consuming and expensive but, in most cases, not even possible. For many cats, allergen-specific immunotherapy (ASIT, hyposensitization) offers an effective treatment option; the success rate is between 60-78%. ASIT is recommended as a lifelong therapy, as experience shows that recurrence is often expected within 1-2 years after treatment discontinuation.

Turtles and tortoises are popular pets which can become very old when kept properly. Apart from the commonly kept European species, such as Hermann’s tortoise (Testudo hermanni) (Fig. 1), experienced owners also enjoy keeping exotic species like radiated tortoises (Astrochelys radiata) or leopard tortoises (Stigmochelys pardalis). Clinical signs often appear very late and are only very non-specific in chelonians. Thus, in addition to the standard clinical examination and imaging, laboratory testing is the most important diagnostic tool for providing an accurate diagnosis quickly and at an early stage. This is particularly important in late summer for animals from temperate zones, as a detailed diagnosis is recommended before brumation so that a “rude awakening” or no awakening at all during and after brumation can possibly be avoided. The most helpful laboratory tests include haematology and biochemistry, parasitology, molecular biology and serology.

Blood testing in chelonians

Lithium heparin blood is best suited for blood testing in chelonians (Fig. 2). Clinical chemistry is particularly important for monitoring organ function, as hepatic or renal impairments can be dangerous for the animals, especially during brumation.
Depending on the species, gender, season and temperature, there may be changes in the individual clinical chemistry parameters that need to be taken into account. Contamination with lymph causes reduced protein levels, liverassociated enzymes, uric acid levels and a shift in electrolytes. Trauma during blood collection, for example after several unsuccessful venipuncture attempts, can lead to an increase in alkaline phosphatase and creatine kinase. Considering the wide range of normal values in some species, it is advisable that blood testing is also carried out on healthy animals, so that it is easier to compare and interpret the results if the animal is ill.

Kidney diseases

Uric acid, the major end product of protein and purine metabolism in terrestrial species, is the main indicator of kidney disease. There are strong increases in various kidney diseases, gout, renal dysfunction due to bacteraemia and septicaemia as well as in kidney necrosis due to nephrotoxic drugs such as aminoglycosides and sulphonamides. Feeding animal-based food, as it is physiological in some aquatic turtle species, can also lead to an increase in uric acid. Lowered levels are associated with hepatic diseases. Urea only plays a minor role in terrestrial species, as the levels remain within the normal range for a long time even if uric acid levels are high. In aquatic species, urea also is an excretory product of protein metabolism, which is why it plays a greater role in the diagnosis of kidney diseases. In kidney diseases, blood phosphate levels increase as well, but hyperphosphataemia can also be caused by excessive dietary intake, hypervitaminosis D and haemolysis. In young animals, phosphate levels as well as calcium and alkaline phosphatase are physiologically increased as a result of bone growth.

Hepatic diseases

GLDH (glutamate dehydrogenase) is the main indicator of liver cell damage. It naturally occurs in the mitochondria of liver cells and is only released into the blood when the cells are destroyed. Other enzymes also found in liver cells are ALT (alanine amino transferase), AP (alkaline phosphatase) and AST (aspartate amino transferase). However, these enzymes are not only found in the liver, but also in other organs of the body, so they are not very specific and have to be interpreted in combination with other blood test results and physiological conditions. Liver function parameters are substances that are naturally synthesised by the liver and are decreased or increased if the liver function is disturbed. In this context, especially bile acid should be mentioned, though it can also be affected by feeding, bile duct obstruction and dehydration. Other functional parameters are the protein fractions in the blood, which are, however, also influenced by food intake and renal function. For diagnosing steatosis, it may be useful to determine the triglyceride and cholesterol levels in the blood, as these are usually elevated in this context. However, it should be noted that they are physiologically elevated in females during yolk formation (vitellogenesis).

Haematology

By determining the level of haematocrit, anaemia or dehydration can be diagnosed. A haematological analysis can also provide important information on inflammation. Shifts in cell count are less pronounced in reptiles than in mammals and are therefore more difficult to interpret. However, bacterial and parasitic infections as well as stress can lead to an increase in heterophilic granulocytes, too. Eosinophilic granulocytes are increased in parasitic infections and when the immune system is stimulated. An increase in lymphocyte count occurs during wound healing, inflammation as well as parasitic and viral infections. Changes in the morphology of individual cells can also indicate infections. When looking at the smear, parasites or inclusions due to viral or bacterial infections can be diagnosed as well, but they should not be confused with artefacts caused, for example, during the preparation or drying of the smear. Species, gender, age and physiological condition of the animal also influence the total count and the ratio of the different leucocytes. It is, however, also important to note that physiologically there can be strong seasonal fluctuations in cell distribution.

Parvoviruses play a significant role as infectious agents in various species. They are small, nonenveloped viruses with a linear, single-stranded DNA genome. They are very stable in the environment and can lead to high animal losses, especially in larger facilities such as animal shelters and breeding kennels (Muzyczka and Berns, 2001; Decaro and Buonavoglia, 2012).

According to the current state of knowledge, the family Parvoviridae comprises three subfamilies, with the subfamily Parvovirinae covering the genera which are important for vertebrates. Within this subfamily, there are currently 10 genera: Amdoparvovirus, Artiparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus, Loriparvovirus, Protoparvovirus and Tetraparvovirus.

The viruses that are most relevant for dogs and cats are found in the genera Bocaparvovirus and Protoparvovirus (ICTV, 2022). Some important parvoviruses and their clinical pictures in dogs and cats are presented below.

Canine parvoviruses

Protoparvovirus in dogs
The best-known protoparvovirus is canine parvovirus 2 (CPV-2), which was identified in the 1970s as the main cause of viral enteritis in dogs (Cooper et al., 1979). The virus became endemic worldwide and several virus strains are now known to cause disease, especially in young dogs (Hoelzer and Parrish, 2010; Decaro et al., 2020).

Feline asthma belongs to the feline atopic syndrome (FAS), including other allergic diseases such as feline atopic skin syndrome (FASS). FASS presents with skin reaction patterns such as miliary dermatitis, eosinophilic granuloma complex, self-induced alopecia and head/neck pruritus. This nomenclature of feline allergic diseases has only recently been established. Feline asthma is a chronic disease with an eosinophilic inflammatory reaction to inhalant allergens. This reaction affects the bronchioles and leads to spontaneous and reversible bronchoconstriction, which manifests as acute dyspnoea or chronic cough and expiratory dyspnoea (1).

Pathogenesis

Asthma is a relatively common inflammatory disease of the lower respiratory tract (according to literature, it affects 1 – 5% of the feline population). Affected cats are predominantly young, with the average onset age of symptoms between 0.5 and 4.5 years (2, 3). The similarity of feline asthma to human asthma is very strong, making the cat a model for human studies. Much of the information on this disease‘s pathogenesis, diagnosis, and therapy have been derived from these allergen-induced experimental models. Feline asthma is caused by a type 1 hypersensitivity reaction to aeroallergens (house and storage dust mites, pollen, fungal spores, animal dander). It leads to activation and differentiation of allergen-specific TH2 cells, which trigger an inflammatory reaction and IgE production. After sensitisation, repeated inhalation of allergens causes clinical signs. IgEs bound to mast cells, and basophil granulocytes are activated by the allergen and lead to degranulation, an inflammatory cascade is triggered, and eosinophil granulocytes migrate to the lungs (4). These immunological reactions lead to chronic airway inflammation, dominated by eosinophils in the long term.
Thickening of the airway epithelium, metaplasia and mucosal damage occurs. Hyperexcitability and obstruction of the bronchi lead to valve function and thus to so-called “air trapping”: air can no longer escape during expiration, and life-threatening bronchospasm can be triggered (3).

Clinical signs

Clinical signs of feline asthma are acute or chronic. A cat with asthma may present with acute respiratory signs such as dyspnoea, mouth breathing, hyperpnoea, tachypnoea, collapse and pale or cyanotic mucous membranes. Chronic signs include dyspnoea, expiratory wheezing breath sounds, chronic coughing (often misinterpreted as retching) and exercise intolerance. In some cats, the only clinical sign is a chronic cough. Exercise intolerance usually occurs in young and active cats. On auscultation, expiratory wheezing and cracklin breath sounds are typically heard (2, 3).

Acute-phase proteins (APP) are an important component of the body’s own innate immune system. The determination of individual APP is used in routine diagnostics to detect and monitor inflammatory reactions. However, it is important to be aware of species-specific differences.

APP are protein molecules which are produced in increased quantities – mediated by cytokines – when there is an inflammatory reaction. They are mainly produced by the liver. Their serum level already increases only a few hours after exposure to a noxious agent, often even prior to the onset of clinical signs or before changes can be seen in the blood count. They are therefore suitable biomarkers for inflammatory reactions. The level of APP often increases proportionally to the degree of inflammation. In turn, it rapidly decreases again after the noxious agent has been eliminated. APP are therefore also suitable for monitoring the course of an inflammatory reaction. The main noxious agents which trigger a reaction are:

• bacterial and viral infectious diseases
• aseptic inflammatory reactions
• autoimmune diseases
• traumata
• neoplasia

More than 30 different APP are known. Most APP belong to the fraction of alpha or beta globulins. Their concentration may not always be high enough to be reflected by electrophoresis. Furthermore, there may be an overlap with other protein components. It is therefore even more important to be able to routinely identify individual APP that are particularly conclusive. Depending on the extent of the increase, “positive APP” are categorised as follows: major APP, moderate APP and minor APP. In routine diagnostics, especially the major APP are important. Their serum level is very low in healthy animals, but they increase by 10- to 100-fold within a few hours of exposure to a noxious agent and decrease rapidly after the inflammation has subsided.
In contrast, a certain level of moderate or minor APP can also be detected when in good health, but they rise more slowly and not as much (up to 10-fold at most) and decline much more slowly. Thus, their significance in routine diagnostics is lower.

“Negative APPs” are another form. Their level decreases during an acute-phase response of the body. The best-known negative APP, which is regularly determined in routine diagnostics, is albumin. During an acute-phase response, the liver reduces albumin production by an average of about 10 to 30% in favour of the production of “positive APP”. Albumin can be used as a negative APP in all animal species.

There are considerable species-specific differences in positive APP. Table 1 provides a brief overview of the APP that are currently most often used in routine diagnostics for the respective animal species.
Their functions vary between proteins and are very complex. The individual APP usually have several tasks, thus regulating the immune response. For example, they activate the complement cascade and facilitate phagocytosis and lysis of bacteria (CRP – C-reactive protein). Leukocytes can be chemotactically attracted and their adhesion in the inflammatory area can be promoted (SAA – serum amyloid A). Moreover, anti-inflammatory processes are triggered to counteract the inflammatory reaction (CRP, SAA). Free haemoglobin can be bound and transported to the liver for reuse. For one, it prevents iron loss, and secondly, it removes available iron resulting in a bacteriostatic effect (Hp – haptoglobin). The spread of the cause of the inflammation can be contained through the formation of a fibrin network (Fb – fibrinogen).
It is impossible to imagine routine diagnostics without APP. Even though they cannot give any information on the site or the cause of the inflammatory reaction, they are still of great use for many diagnostic tasks and are particularly suitable for

• the diagnosis of subclinical and chronic diseases
• the early detection of inflammatory reactions
• therapy monitoring
• monitoring the healing process
• monitoring post-operative convalescence.

Table 2 shows some examples of the diagnostic use of APP in some species. If the clinical signs of a patient are unclear or if blood count and clinical chemistry are inconclusive, we recommend serum protein electrophoresis as an additional screening test. In most cases, the pattern indicates a direction for further examinations, which then lead to the diagnosis. APP also appear as typical peaks here, SAA and Hp in the alpha-2 fraction, CRP and Fb in the beta-2 fraction (for a comparison of electrophoresis patterns, see figure 3, page 4). However, due to an overlap in electrophoresis with other proteins with similar physical properties, the relevant APP should be measured quantitatively by clinical-chemical determination. It should be noted that plasma protein electrophoresis can show further peaks, as plasma still contains the coagulation factors. Especially fibrinogen can make it difficult to interpret the beta-2 fraction. It must also be borne in mind that both corticosteroids and NSAIDs can influence the clinical-chemical determination as well as the presence of APP in an electrophoresis run. This should always be taken into consideration when interpreting the results.

Dr. Ruth Klein, Dr. Karin Friedrich

General information

The parameters we use to assess kidney function are so-called biomarkers. If the kidney does not work as it should, they remain in the blood. By looking at their levels, we can deduce the severity of renal dysfunction. Sounds simple, and it actually is.

Azotaemia: The increase of urinary excreted substances in the blood is called azotaemia. The typical biomarkers are urea and creatinine. However, the presence of azotaemia does not automatically mean that the kidney is affected. Only once we are sure that there is neither prerenal nor postrenal azotaemia, we call it kidney disease.

Uraemia: Uraemia is a term used to describe the clinical consequences of renal dysfunction resulting from retention of toxic metabolites, dysregulation of water and electrolyte balance as well as hormonal imbalances. Signs associated with uraemia include lethargy, weakness, dehydration, inappetence, vomitus and weight loss.

Thus, azotaemia and uraemia are not the same. Renal azotaemia indicates renal dysfunction.
Uraemia signifies that the patient’s quality of life is affected. The former is therefore important for the diagnosis of kidney disease, the latter for assessing the clinically relevant severity of the disease. The difference between azotaemia and uraemia explains, among other things, why some patients with chronic kidney disease (CKD) still have a rather good general condition even with high kidney values, while others with lower values already feel worse.
The traditional biomarkers may be indicators of the degree of renal dysfunction, but do not necessarily indicate what impact this has on the individual patient. For most uraemic toxins that make our patients’ lives difficult, there are no commercially available test methods.
An exception to this is indoxyl sulphate (see below).

In dogs and cats, increased urinary protein excretion is pathological and highly correlated with reduced survival of the respective animal. The excretion of small quantities of albumin < 1 mg/dl is considered physiological in dogs and cats.

A urine protein-creatinine ratio (UP/C) of > 0.4 in cats and > 0.5 in dogs is defined as proteinuria according to the guidelines of the IRIS (International Renal Interest Society). A distinction is made between prerenal, renal and postrenal proteinuria. Their various causes are shown in Table 1.

Urine protein electrophoresis has proven to be a useful tool to differentiate proteinuria. Here, the diluted urine is placed on a gel, the proteins are separated according to molecular weight and it is then possible to distinguish the type of proteinuria from the protein pattern.

Prerenal proteinuria

The excretion of light-chain antibody fragments, so-called Bence Jones proteins, indicates highly pathological prerenal proteinuria. They are produced if multiple myeloma is present and usually cause high-grade proteinuria.
In urine electrophoresis, the patterns of kappa monomers (25 kDa) and lambda dimers (50 kDa) are depicted (Fig. 1). Other common causes of prerenal proteinuria are hypertension, hyperadrenocorticism in dogs and hyperthyroidism in cats.

Tab. 1: Proteinuria: possible causes and diseases (according to: Harley and Langston, 2012)

Renal proteinuria

For one, this includes glomerulopathies in which high molecular weight proteins larger than albumin (> 70 kDa) can no longer be retained by the glomerular apparatus due to various defects.

Glomerulopathies are associated with a variety of infectious (e. g. ehrlichiosis, babesiosis and leishmaniasis in dogs; FIV, FELV and FIP in cats), neoplastic, parasitic, autoimmune and endocrine causes.

Furthermore, we distinguish tubulopathies in which the capacity or ability of the proximal tubule to reabsorb low molecular weight proteins (< 70 kDa) is exhausted.

These tubulopathies manifest as Fanconi syndrome in dogs, which is hereditary in Basenjis and caused by toxic factors in other breeds, e. g. after the administration of gentamicin (Fig. 2).

Mixed proteinuria, in which both protein groups can be detected, mainly occurs in stages 2 – 4 of chronic kidney disease and in acute kidney injury because, in addition to glomerular dysfunction, it also causes renal tubular acidosis (Fig. 3).

Since treatment of proteinuria can vary greatly depending on the cause, it is essential to determine whether renal proteinuria is glomerular, tubular or mixed glomerular-tubular proteinuria.

Postrenal proteinuria

This is usually caused by lower urinary tract and reproductive tract disease, can be detected by examination of urine sediment and does not require differentiation by urine electrophoresis.

Small pets are now among the most popular domestic animals in Germany (5 million small pets in 5% of all households, according to a survey by the IVH, the German Industrial Association of Pet Care Producers, in 2020). The willingness of owners to take their pets to a vet in case of illness has increased. As a consequence, more samples are sent to the laboratory for analysis.

In 2020, for example, 797 samples from small mammals were sent to Laboklin GmbH & Co. KG for histopathological examination.

Most of the samples were from rabbits, closely followed by samples from guinea pigs (Fig. 1). Other species were grouped under the umbrella term “rodents”: rats (n=85; 10.7%), hamsters (n=26; 3.3%), gerbils (n=12; 1.5%), chinchillas (n=11; 1.4%), degus (n=8; 1.0%), mice (n=6; 0.6%), squirrels (n=3; 0.4%) and one alpine marmot (0.1%). Samples from ferrets (n=77) and hedgehogs (n=58) were not included in these statistics.

Below, the possibilities are discussed when and why a certain examination is useful and where there may be limitations.

Cytology

Cytology is a method used for the microscopic diagnosis of single cells. Fine needle aspirates (FNA) are suitable samples for examination. They may come from masses or even from organs. Other suitable samples are impression smears, e.g. from open wounds or scabs. Body cavity effusions can be analysed for their cell content, cell composition and the presence of pathogen structures.

Special stains are available as well. They are used, for example, to detect pathogen structures. Ziehl-Neelsen staining (ZN) detects acid-fast rods (e.g. mycobacteria). PAS reaction (periodic acid-Schiff reaction) is used, among others, to detect fungal elements. Prussian blue staining differentiates haemosiderin from other pigments through its blue colouration.

Advantages

Cytological examination is a micro-invasive procedure. As small mammals are often more sensitive to anaesthesia and surgical intervention than other domestic animals, cytology is sometimes preferred to surgical extirpation. It is a simple type of test that provides quick results. With this method, it is often already possible to distinguish between an inflammatory and a tumorous process (Fig. 2).

Disadvantages/limitations

Only positive cytological findings are conclusive. This means a tumour can only be diagnosed if tumour cells are found in the preparation. If no tumour cells are present, it is not possible to definitely rule out a tumour. The clinical history is also important for cytological evaluation. Especially in cytology, which is based on the analysis of single cells, information on the sampling site, the clinical picture and previous treatment is often essential. In order to ensure diagnostically conclusive results, sufficient cell material must be obtained. This can sometimes be complicated, e.g. if there is a cystic mass or if the cells are arranged in a dense cell cluster making aspiration of cell material difficult. Diagnosis is hampered by slides covered with cover glasses or adhesive tape (insufficient staining of the cells, formation of air bubbles). Smears that are too thick also make diagnosis difficult. In these cases, it is no longer possible to evaluate single cells. Furthermore, cell quality suffers as it cannot be guaranteed that cells are sufficiently fixed by air drying.

Histopathology

In contrast to cytology, histological examination involves the examination of a formalin-fixed tissue structure. It is thus possible to evaluate the organ structure (regular organ-specific or autologous growth in case of neoplasia). Masses, biopsies or organ samples from small mammals can also be sent in for examination (Fig. 3 and 4).

Advantages

It is possible to evaluate whether the underlying process is inflammatory or tumorous. If there is a mass, this type of examination cannot only clarify whether it is a neoplasm but also checks for completeness, i.e. the resection margins are evaluated. The analysis of skin punch biopsies is also gaining importance in small mammals. Sometimes, histopathology is the only way to make a conclusive diagnosis, for instance in sebaceous adenitis in rabbits. It can only be determined histologically whether sebaceous glands are present or not. To clarify an underlying infectious cause, a variety of further special stains can also be used in histopathology, e.g. PAS reaction, ZN stain, Warthin-Starry (WS) stain (detection of spirochaetes, e.g. Treponema paraluiscuniculi) and many more. Immunohistochemistry, which has become a standard test for dogs and cats, is, in some areas, also established for small mammals. For example, at Laboklin, the lymphocyte markers CD3 (T lymphocytes) and CD79α (B lymphocytes) have been validated for guinea pigs, rabbits and ferrets (Fig. 5a and b). In all three species, lymphoma is diagnosed quite frequently. Immunohistochemistry can then be used for further differentiation into B- or T-cell lymphoma.

Disadvantages/limitations

As in cytology, it is important to provide the clinical history, i.e. information on the sampling site, the clinical picture and possible previous treatment, in order to evaluate the submitted sample. Moreover, immediate fixation of the sample is required (4% neutral buffered formaldehyde ≙ 10% formalin). If it is not done or if the quantity is not sufficient, autolysis of the tissue will occur which limits getting a reliable evaluation. The formation of artefacts, such as freezing artefacts or heating artefacts due to thermosurgery, may also be a disadvantage. Specimens should be submitted from representative sites. Samples for histology shall not be too small, as otherwise the tissue structure cannot be evaluated. Skin punch biopsies in particular should have a minimum diameter (approx. 0.4 cm), if site and species permit (if not, alternative sampling, e.g. “shave biopsy”). Limitations also arise if a secondary lesion, e.g. inflammation, overlaps the primary process (tumour).

How would we diagnose without laboratory tests?
Whether it is blood count, pathogen detection or pathology – successful diagnostics without laboratory tests is hardly imaginable.
What is often not considered is how important it is what happens to the sample before it arrives at the laboratory. We want to make the best of your samples. Help us to do so!

The basics

The clinical history helps us to help you.
The diagnostic task is particularly important, especially with blood smears, cytology, histopathology and pathogen detection by culture. The more we know about the patient and the problem, the more precisely we can look for specific changes and interpret the findings for you.

Accurate laboratory values are obtained from the right material.
If a parameter is determined from the wrong material, it may become useless for diagnosis. The material you need is indicated on the submission form.
Please let us know what kind of sample you have sent. Plasma can be obtained from citrate, EDTA and heparin blood. Urine, plasma/serum and CSF look very similar. Correct labelling helps to avoid errors.

In a modern laboratory, many things are automated.
Incorrectly attached or missing barcodes on test tubes lead to additional time and effort. This takes up valuable time in the diagnostic process.

The type of sample packaging can be crucial for analysis and may have consequences for the colleagues in the laboratory.
You should avoid the following: closing syringes with cannulas instead of appropriate plugs, pathology samples in glass containers and faecal samples in gloves. There is a risk of injury, sample contamination and sample loss.

Courier transport is only permitted for correctly packaged and labelled samples.
Use the packaging materials provided (consisting of primary sample tube, secondary transport tube/ container, outer packaging). For liquid samples, there must be absorbent material between the primary and secondary tube/container, and tubes must be protected against breakage. For sending samples to Germany per air-freight, it is absolutely necessary they are packed and marked according to the EU-regulations: all sample tubes are to be packed in leek-proof protective secondary containers and then in rigid cardboard box with extensive cushioning material. The cardboard box should be marked with “UN3373 Biological substance Category B” label.
You can find more details in the Laboklin „Directory of Tests“.

Blood tests

The filling level of the tubes has significant influence on the measured parameters!
If there is too little blood in a tube, there is an excess of anticoagulant. This will, among others, influence cell morphology and lead to incorrectly prolonged coagulation values. Overfilling also causes problems (e.g. coagulation of plasma samples).

The order of the tubes during blood collection can have enormous consequences.
EDTA tubes should be filled last. Even the slightest contamination of the needle with EDTA can lead to significant errors when measuring calcium and potassium. If blood coagulation is needed, the first few drops of blood should either be discarded or another tube should be filled first.

The type of tube affects the laboratory workflow.
The small test tubes with hinged lids are popular. But they pose problems. For one, the lids often do not close completely, so that sample volume gets lost. And for another, the needles of the analysers cannot pierce them. The samples have to be pipetted by hand. This causes delays in the workflow.

The condition of the sample is decisive for how useful it is for diagnosis.
Ideally, the sample is cooled and protected from light until it is shipped. Attention should already be paid in the practice regarding the correct storage of the sample material. A blood sample which has been lying around all day at room temperature in full daylight will yield incorrect results in certain examinations (e.g. bilirubin). The error is in the detail!

Centrifugation and pipetting are tedious but necessary!
When whole blood is supplied, haemolysis and cellular metabolism will occur. This leads to incorrect measurements, possibly with far-reaching consequences for clinical decisions. Place the sample upright (do not transport it in a lying position at this point!) and separate the serum/plasma as soon as possible after collection (first allow serum to clot at room temperature). Make sure to also pipette off the supernatant for shipping. Merely centrifuging without transferring the supernatant is not helpful!

A blood smear is part of a blood count.
Even the short time of transport leads to fundamental changes in the cells. If you already prepare a smear in the practice, you fix the intact cells on the slide and thus give us the opportunity to make a good evaluation.