Capillary Zone Electrophoresis in Humboldt Penguins (Spheniscus humboldti).

Electrophoresis is a useful diagnostic tool for detecting inflammation, including inflammation associated with infectious diseases (eg, aspergillosis in penguins). To our knowledge, reference intervals are not available for plasma proteins via electrophoresis in Humboldt penguins (Spheniscus humboldti). Therefore, preliminary reference intervals for blood plasma proteins measured by capillary zone electrophoresis were calculated for Humboldt penguins from a single zoological collection, and possible differences between the sexes and the ages of the birds were evaluated. Lithium heparinized plasma samples from 39 Humboldt penguins were analyzed. The following sex- and age-independent reference intervals were calculated: total protein 33.8-70.4 g/L, prealbumin 1.9-4.9 g/L, albumin 12.9-31.1 g/L, albumin: globulin ratio 0.7-1.7, α-globulins 4.5-11.6 g/L, ß-globulins 5.6-20.6 g/L, and γ-globulins 2.6-8.4 g/L. Male penguins had a significantly (P = 0.047) higher albumin: globulin ratio and lower percentage of ß-globulins (P = 0.015) in comparison with female penguins. Prealbumin (g/L) significantly (P = 0.021) decreased with increased age of the penguins. These results showed some differences between the sexes and ages of the penguins, which should be considered when interpreting the results. Further studies are needed to determine whether differences in other age groups or seasons exist, and also to evaluate which infectious diseases affect plasma proteins and how the reference values calculated here may deviate in ill penguins.

Clinical Chemistry and Haematology Values of a Captive Population of Humboldt Penguins (Spheniscus humboldti).

Reference intervals for clinical chemistry and haematology analytes are essential for the interpretation of blood results, but limited data are available for Humboldt penguins (Spheniscus humboldti) in zoological collections as well as in the wild. The aim of the present study was therefore to establish reference intervals for a series of clinical chemistry and haematology analytes in a single zoological population of Humboldt penguins. Furthermore, possible variations of the analytes influenced by the age and sex of the individuals were investigated. Lithium heparinized plasma and whole blood samples from 39 animals were analysed and statistically evaluated. The following clinical chemistry analytes were significantly higher in females: glutamate dehydrogenase (p = 0.0003), alanine transaminase (p = 0.0005), alkaline phosphatase (p = 0.0245), aspartate aminotransferase (p = 0.0043), creatine kinase (p = 0.0016), lactate dehydrogenase (p < 0.0001), inorganic phosphorus (p = 0.0223), and sodium (p = 0.0415). No significant differences between males and females were found for any haematology analyte. Cholesterol (p = 0.0233; -0.39), white blood cell count (p = 0.0208; -0.40), and absolute heterophiles (p = 0.0148; -0.42) had significant negative correlations with the age of the penguins. The results indicate that the factors of sex and age should be considered when interpreting the results of haematological and clinical biochemistry assays, and further studies are needed to determine whether there are more differences in other age groups or seasons.

Comparison of Plasma Trace Elements Between 2 Captive Humboldt Penguin (Spheniscus humboldti) Populations.

Environmental pollutants and their effect on wildlife health play an important role in the conservation of endangered species and can be clinically relevant in captive animals too. Data on relevant concentrations of trace elements in captive birds with no known exposures are rare. For this study, silver, arsenic, gold, barium, cadmium, cobalt, chromium, copper, iron, mercury, magnesium, manganese, molybdenum, thallium, selenium, and zinc were measured by inductively coupled plasma mass spectrometry in lithium heparinized plasma samples from 18 Humboldt penguins at 2 zoological collections in Germany. The results showed that the plasma concentrations of silver, arsenic, gold, cadmium, cobalt, chromium, mercury, and selenium differed significantly (P ≤ 0.05) between the 2 penguin collections. The results indicate that the location of the birds has a strong influence on the plasma trace element concentrations. Well water used in the enclosures was suspected to be associated with these significant differences. Trace elemental concentrations in feed (eg, marine fish) and contamination from enclosure construction materials may also play a role. This study could provide a basis for further comparative, biomonitoring, toxicity, and reference interval studies.

Establishment of a Real-Time PCR Assay for the Detection of Devriesea agamarum in Lizards.

(1) Background: Devriesea (D.) agamarum is a potential cause of dermatitis and cheilitis in lizards. The aim of this study was to establish a real-time PCR assay for the detection of D. agamarum. (2) Methods: Primers and probe were selected targeting the 16S rRNA gene, using sequences of 16S rRNA genes of D. agamarum as well as of other bacterial species derived from GenBank. The PCR assay was tested with 14 positive controls of different D. agamarum cultures as well as with 34 negative controls of various non-D. agamarum bacterial cultures. Additionally, samples of 38 lizards, mostly Uromastyx spp. and Pogona spp., submitted to a commercial veterinary laboratory were tested for the presence of D. agamarum using the established protocol. (3) Results: Concentrations of as low as 2 × 104 colonies per mL were detectable using dilutions of bacterial cell culture (corresponding to approximately 200 CFU per PCR). The assay resulted in an intraassay percent of coefficient of variation (CV) of 1.31% and an interassay CV of 1.80%. (4) Conclusions: The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods.

Detection of nidoviruses in samples collected from captive snakes in Europe between 2016 and 2021.

BACKGROUND: Nidoviruses are increasingly detected in various snake species worldwide, but much remains to be learned about their distribution and the factors influencing their epidemiology. METHODS: This retrospective study evaluated the results of routine nidovirus testing, by PCR, of 5210 swab samples from pet snakes from various European countries that were submitted to a commercial veterinary laboratory in Germany between 2016 and 2021. RESULTS: The overall detection rate was 19.96%. However, the detection rate varied significantly depending on the snake species (p < 0.0001), with the highest rate in Indian pythons (Python molurus) (42.24%). Rates also varied depending on the season of sample collection (p < 0.0001), with the highest rate in winter (24.46%), and the country of sample origin (p < 0.0001), with the highest rate in Austria (36.69%). The detection rate also decreased significantly (p = 0.0003) over the 6-year observation period, from 26.43% to 17.64%. LIMITATION: No information on clinical signs was available for most of the sampled snakes. CONCLUSION: The present study supplies new information on the distribution of python nidoviruses (subgenus Roypretovirus) in pet snakes in Europe and indicates a dynamic situation with possible changes in prevalence over time.

PLASMA VITAMIN AND MINERAL CONCENTRATIONS IN CAPTIVE GREATER FLAMINGOS (PHOENICOPTERUS ROSEUS) AS INFLUENCED BY DIET CHANGE.

The greater flamingo (Phoenicopterus roseus) is often maintained in managed care. Although values for plasma concentrations of minerals and vitamins have been published for this species, limited studies investigate the effects of diet changes on these values. Plasma concentrations of selected vitamins (A [and the provitamin A carotenoid ß-carotene] and E), minerals (Ca, Cu, Fe, K, Mg, Mn, Na, inorganic P, Se, and Zn), and total protein (TP) were measured in a zoo collection of greater flamingos in November 2018 (n = 48) and in November 2019 (n = 52), 30 d after a change from the summer diet, which was the same in both years, to different winter diets. Chloride, cholesterol, and triglycerides were only measured once in 2019. The nutrient profiles of the two complete diets (labeled and analyzed) differed considerably, especially for the canthaxanthin concentration. The plasma concentrations of ß-carotene (P < 0.0001), Cu (P < 0.0001), Fe (P < 0.0001), Mg (P < 0.0001), and Se (P < 0.0001) were significantly higher and vitamin E (P < 0.0001), Ca (P = 0.0014), Mn (P < 0.0001), Na (P = 0.0005), and TP (P < 0.0001) were significantly lower in 2018 than in 2019. Four minerals (Cu, Fe, Mg, and Zn) were measured using both spectrophotometry and inductively coupled plasma mass spectrometry, and all of these minerals showed significant (P < 0.05) differences between the two methods. Based on the results of the study, both the influence of diet and the detection method used for circulating nutrients must be considered when interpreting the plasma vitamin and mineral concentrations in greater flamingos.

Antibodies against Two Testudinid Herpesviruses in Pet Tortoises in Europe.

Herpesviruses are important pathogens of tortoises, and several serologically and genetically distinct virus types have been described in these animals. Virus neutralization testing is commonly used in Europe to determine previous infection with the two types most often found in pet European tortoises, testudinid herpesvirus (TeHV) 1 and 3. In this retrospective study, the results of serological testing for antibodies against each of these viruses in serum or plasma samples from 1728 tortoises were evaluated, and antibody detection rates were compared based on virus type, host species, year, season, and country of origin. Antibodies (titer 2 or higher) against at least one of the two viruses used were detected in a total of 122 (7.06%; 95% CI 5.95−8.37%) of the animals tested. The antibody detection rates differed significantly depending on the tortoise species (p < 0.0001) and the year of sampling (TeHV1 p = 0.0402; TeHV3 p = 0.0482) for both virus types. For TeHV1, antibody detection rates differed significantly (p = 0.0384) by season. The highest detection rate was in summer (5.59%; 95% CI 4.10−7.58%), and the lowest was in fall (1.25%; CI 0.53−2.87%). TeHV1 antibody detection rates did not differ significantly (p = 0.7805) by country, whereas TeHV3 antibody detection rates did (p = 0.0090). The highest detection rate, 12.94% (95% CI 7.38−21.70%), was found in samples from Italy. These results support previous hypotheses on the species' susceptibility to TeHV1 and 3 and the use of serology as a diagnostic test for the detection of herpesvirus-infected tortoises.

COMPARISON OF CLINICAL CHEMISTRY ANALYTES IN GREATER FLAMINGOS (PHOENICOPTERUS ROSEUS) AND AMERICAN FLAMINGOS (PHOENICOPTERUS RUBER) IN A ZOO IN EUROPE.

Reference intervals for clinical chemistry analytes are important for the interpretation of blood results, but reference intervals available in journal articles for specific flamingo collections are often based on a small number of individuals or are only available for a limited number of analytes. Differences in husbandry and nutritional conditions may impact reference intervals. The goal of this study was to establish reference intervals from a mixed population of greater (Phoenicopterus roseus) and American (Phoenicopterus ruber) flamingos when husbandry parameters are equal and to compare the results between the different species, sexes, and age groups in order to evaluate differences between these factors. Lithium-heparinized plasma samples from 93 animals were analyzed for the following: amylase, cholinesterase (CHE), bile acids (BA), total protein (TP), glutamate dehydrogenase (GLDH), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), uric acid (UA), inorganic phosphorus (P), total calcium (Ca), sodium (Na), and potassium (K). The results show many differences between the species and a few between the different age groups but no differences between males and females of either species. Amylase (P= 0.0010), AST (P= 0.0090), CK (P= 0.0011), and Ca (P= 0.0217) differed significantly between greater and American flamingos. The age group significantly impacted blood levels of CK (P = 0.0299) and LDH (P = 0.0165) in greater flamingos and BA (P = 0.0495), P (P = 0.0422), and Ca (P = 0.0006) in American flamingos. The results show the importance of species-specific reference intervals and the consideration of age-related variations in order to facilitate correct interpretation of blood results.

A testudinid herpesvirus 1 (TeHV1)-associated disease outbreak in a group of Horsfield's tortoises (Testudo horsfieldii).

In spring 2020, a severe disease outbreak with high morbidity and mortality was observed in a collection of 15 Horsfield's tortoises (Testudo horsfieldii). Affected tortoises showed upper respiratory- and gastrointestinal tract signs, including rhinitis and stomatitis. Testudinid herpesvirus 1 (TeHV1) and Mycoplasma spp. were detected by PCR in oral swabs of affected animals. Histological examination of one deceased animal showed intranuclear inclusion bodies typical for herpesvirus infections in liver, spleen and oesophagus. The virus was likely introduced into the collection 2 years earlier by a clinically healthy Horsfield's tortoise that was tested positive for TeHV1 by PCR.

Herpesviruses in Captive Chelonians in Europe Between 2016 and 2020.

Herpesviruses are important pathogens in tortoises and turtles, yet little is known about the epidemiology of these viruses. We analyzed herpesviruses detected by PCR in samples from captive chelonians in Europe according to virus strain, host species, year and season in which the animal was tested, and country in which the animal was kept. A total of 4,797 samples submitted to a diagnostic laboratory in Europe between January 2016 and December 2020 were evaluated. Of these, 312 (6.50%) were positive for herpesviruses. The types most commonly found were testudinid herpesvirus (TeHV)1 (143 positive, 45.83%) and TeHV3 (153 positive, 49.04%), but also included TeHV2 (1 positive, 0.32%), TeHV4 (3 positive, 0.96%), Terrapene herpesvirus 1 (7 positive, 2.24%), Trachemys herpesvirus 1 (2 positive, 0.64%), and three previously undescribed herpesviruses (0.96%). Herpesviruses were detected in chelonians in the families Testudinidae, Emydidae, Geoemydidae, and in the suborder Pleurodira. Among the species for which 100 samples or more were available, the highest proportions of positive samples (positivity rates) were found in samples from Horsfield's tortoises (Testudo horsfieldii) (14.96%), and radiated tortoises (Astrochelys radiata) (14.05%). Among tortoises (Testudinidae), viruses were most often detected in the spring, while in emydid turtles (Emydidae) they were most often detected in the summer. A comparison of the positivity rates according to country showed significant differences, with the highest rate in samples from Italy (16.01%). This study indicated possible differences in herpesvirus positivity rates depending on host species, virus strain, year of sampling, season, and country of origin. It provides useful information in further understanding fluctuations in infection rates as well as in helping to guide decision making for herpesvirus diagnostics in chelonian patients. It also provides evidence for the international dispersal of herpesviruses with their hosts through international trade.

Comparison of Capillary Zone Electrophoresis in Greater Flamingos (Phoenicopterus roseus) and American Flamingos (Phoenicopterus ruber).

Electrophoresis can be used to aid in the diagnosis of infectious diseases (eg, aspergillosis) in avian species. Reference intervals for blood plasma proteins of 2 different flamingo species (Phoenicopterus roseus and Phoenicopterus ruber) and their hybrids were calculated by capillary zone electrophoresis (CZE) and differences between these species, sexes, and age groups were evaluated. Lithium-heparinized plasma samples from 111 animals from a zoological collection were analyzed by CZE and statistically evaluated. Differences were only found between greater and American flamingos (P = .003) and between greater flamingos and hybrids (P = .001) in the γ-globulin fraction. Male greater flamingos showed significantly higher α-globulins (P = .022) and females higher total albumin by CZE (P = .037). In American flamingos, the percent total albumin (P = .017), total albumin (P = .025), prealbumin (P = .005), and albumin/ globulin (A/G) ratio (P = .008) were higher in females, and α- (P = .023) and ß-globulins (P = .021) were higher in males of the same species. The following parameters differed significantly between the age groups: γ-globulins (P = .048) in greater and α- (P = .021) and ß-globulins (P = .001) in American flamingos increased with increased age and percent total albumin (P = .002), total albumin (P = .024), and A/G ratio (P = .002) decreased with age in American flamingos. The results showed only small differences between the species, but greater differences between the sexes and ages, especially in American flamingos, which must be considered when interpreting laboratory results.

PLASMA CAPILLARY ZONE ELECTROPHORESIS AND PLASMA CHEMISTRY ANALYTES IN TORTOISES (TESTUDO HERMANNI, TESTUDO GRAECA) AND TURTLES (TRACHEMYS SCRIPTA ELEGANS, GRAPTEMYS SPP.) IN FALL.

Clinical health checks including blood testing before brumation in fall are an important tool in reptile medicine and help to reduce the risk of complications during brumation. Electrophoresis may be useful for the evaluation of liver cell function and the detection of antigenic stimulation. The goal of this study was to compare species-specific reference intervals for plasma chemistry analytes and capillary zone electrophoresis (CZE) for a variety of pet chelonian species in fall. Lithium heparinized samples were collected from 68 clinically healthy spur-thighed tortoises (Testudo graeca), 79 red-eared sliders (Trachemys scripta elegans), and 30 map turtles (Graptemys spp.) from September to November 2016 and 2017. During the same period, 128 equivalent samples were collected from Hermann's tortoises (Testudo hermanni) and the obtained data was used for comparison. Chemistry analytes were measured from plasma using an autoanalyzer and electrophoresis was carried out by CZE. Significant variations (P < 0.0001) between the species were found for several analytes including total protein, percent albumin, and albumin: globulin ratio, α-, ß-, and γ-globulin, alkaline phosphatase, glutamate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, bile acids, creatine kinase, urea, uric acid, inorganic phosphorus, total calcium, and potassium. The variations in albumin (g/l) measured by CZE were also significant (P = 0.0064). No significant variations were detected for sodium levels. The results demonstrate the importance of species-specific reference intervals and provide reference intervals for the plasma chemistry and capillary zone electrophoresis in three chelonian species.

CLINICAL CHEMISTRY, VITAMIN, ELECTROPHORESIS, AND HEMATOLOGIC ANALYTES OF BLACK-HEADED IBIS (THRESKIORNIS MELANOCEPHALUS).

The evaluation of laboratory blood analytes is an important tool for health assessment in avian medicine. Unfortunately, there is a lack of suitable reference values for many zoo bird species. The goal of the present study was to establish reference intervals for a wide range of blood analytes in healthy black-headed ibis (Threskiornis melanocephalus) kept in a zoologic garden. Lithium heparinized blood samples from 15 individuals were collected, and 18 different clinical chemistry analytes were measured. New are especially the minimum and maximum values for bile acid (2.38-49.4 µmol/L), cholinesterase (439-1260 U/L), vitamin A (0.65-1.70 mg/L), vitamin E (26.3-52.5 mg/L), and capillary zone electrophoresis (prealbumin, 10.2-23.4%; albumin, 35.9-44.1%; A/G ratio, 1.07-1.69; α-, 15.7-20.0%; ß-, 13.5-19.1%; γ-globulin, 6.08-11.3%). The measured values for clinical chemistry, vitamin, electrophoresis, and hematologic analytes are a basis for further studies and for diagnostics and clinical treatment in this ibis species.

PROTEIN ELECTROPHORESIS OF PLASMA SAMPLES FROM BOA CONSTRICTORS WITH AND WITHOUT REPTARENAVIRUS INFECTION.

Reptarenaviruses infect a variety of boid and pythonid snake species worldwide and have been shown to be the cause of inclusion body disease (IBD). Little is known about the correlations between virus infection and clinical disease, as well as the effects of viral infection on the immune system and the blood protein fractions. The goal of this study was to examine the differences in the plasma protein fractions in reptarenavirus reverse transcription polymerase chain reaction (RT-PCR)-negative and -positive tested snakes with and without clinical signs of disease. Blood from a total of 111 boa constrictors (Boa constrictor) was evaluated. Reverse transcription PCRs and H&E staining for inclusion bodies were carried out on each sample for the detection of reptarenavirus, and the plasma protein fractions were evaluated by capillary zone electrophoresis (CZE). Thirty four of the 111 evaluated snakes were positive by RT-PCR and 19 of the 34 showed clinical signs of disease. In comparison with IBD-negative healthy boa constrictors, the positive snakes with clinical signs had significantly lower albumin levels (P = 0.0052), lower A: G ratios (P = 0.0037), and lower α-globulin levels (P = 0.0073), while their γ-globulin levels were significantly higher (P = 0.0004). In the same comparison, clinically healthy arenavirus-positive boas showed only significantly lower α-globulin (P = 0.0124) and higher γ-globulin levels (P = 0.0394). The results of the present study indicate that reptarenavirus infection may influence plasma protein fractions in boa constrictors.

A comparison of thyroid hormone levels and plasma capillary zone electrophoresis in red-eared sliders (Trachemys scripta elegans) and map turtles (Graptemys spp.) depending on season and sex.

BACKGROUND: Thyroid hormones and capillary zone electrophoresis are rarely studied in reptile medicine. OBJECTIVES: The goal of this prospective study was to establish RIs for plasma proteins and thyroid hormones, total tetraiodothyronine (tT4), free T4 (fT4), total triiodothyronine (tT3), and free T3 (fT3), as well as iodine blood levels in red-eared sliders (Trachemys scripta elegans) and map turtles (Graptemys spp.). METHODS: Variables were measured over 1 ½ years to determine variations depending on sex and season, as well as any correlations between the parameters. A total of 131 blood samples from red-eared sliders and 51 blood samples from map turtles were collected from May 2016 to November 2017. The levels of tT4, fT4, and fT3 were measured using standardized autoanalyzer test systems; tT3 was measured by ELISA and iodine inductively coupled plasma mass spectrometry. Total protein was quantitated using the biuret method, and plasma protein fractions were measured using capillary zone electrophoresis. RESULTS: The results showed significant variations in thyroid hormone levels and plasma protein fractions depending on sex and season. Total T4 and fT4 concentrations were significantly (P < .05) correlated in both turtle species and sexes in all seasons. Thyroid hormone levels correlated with blood proteins in a few seasons, including a positive correlation between tT4 and fT4 in female red-eared sliders in fall and female map turtles in summer and fall. CONCLUSIONS: This study demonstrates the importance of considering species, season, and sex variations when interpreting thyroid hormone and plasma protein levels. It also shows that thyroid hormone levels are not always influenced by total protein and albumin.

Prevalence of inclusion body disease and associated comorbidity in captive collections of boid and pythonid snakes in Belgium.

Inclusion body disease (IBD) is caused by reptarenaviruses and constitutes one of the most notorious viral diseases in snakes. Although central nervous system disease and various other clinical signs have been attributed to IBD in boid and pythonid snakes, studies that unambiguously reveal the clinical course of natural IBD and reptarenavirus infection are scarce. In the present study, the prevalence of IBD and reptarenaviruses in captive snake collections and the correlation of IBD and reptarenavirus infection with the clinical status of the sampled snakes were investigated. In three IBD positive collections, long-term follow-up during a three- to seven-year period was performed. A total of 292 snakes (178 boas and 114 pythons) from 40 collections in Belgium were sampled. In each snake, blood and buffy coat smears were evaluated for the presence of IBD inclusion bodies (IB) and whole blood was tested for reptarenavirus RNA by RT-PCR. Of all tested snakes, 16.5% (48/292) were positive for IBD of which all were boa constrictors (34.0%; 48/141) and 17.1% (50/292) were reptarenavirus RT-PCR positive. The presence of IB could not be demonstrated in any of the tested pythons, while 5.3% (6/114) were reptarenavirus positive. In contrast to pythons, the presence of IB in peripheral blood cells in boa constrictors is strongly correlated with reptarenavirus detection by RT-PCR (P<0.0001). Although boa constrictors often show persistent subclinical infection, long-term follow-up indicated that a considerable number (22.2%; 6/27) of IBD/reptarenavirus positive boas eventually develop IBD associated comorbidities.

REFERENCE INTERVALS FOR PLASMA CAPILLARY ZONE ELECTROPHORESIS IN HERMANN'S TORTOISES (TESTUDO HERMANNI) DEPENDING ON SEASON AND SEX.

Electrophoresis is a useful diagnostic tool for the detection of shifts in plasma protein fractions, which can be associated with a variety of diseases. This method is not often used in reptile medicine because there are only a few studies available and no reference intervals have been published for most species. The goal of this study was to establish reference intervals for plasma capillary zone electrophoresis in Hermann's tortoises (Testudo hermanni) under consideration of variations by season and sex. 251 samples from healthy adult Hermann's tortoises were collected from May 2016 to October 2017. Total protein was measured by the biuret method and albumin was measured twice by bromocresol green dye-binding method (BCG) and capillary zone electrophoresis (CZE) in order to compare the detection methods. The globulin fractions were analyzed by CZE. Seasonal variations were significant for total protein (P = 0.0055), albumin (P = 0.0322) and α-globulins (P = 0.0007). The variations between male and female Hermann's tortoises were highly significant for albumin (P < 0.0001), α-globulins (P < 0.0001) and ß-globulins (P < 0.0001). The differences between the measurements of albumin by CZE and BCG also differed depending on the sex and season. This study demonstrates that it is important to include the variations by sex and season when establishing reference intervals and interpreting results. Furthermore, it confirms the importance of using electrophoresis for the correct determination of albumin in tortoises.

Blood reference intervals for rabbits (Oryctolagus cuniculus) from routine diagnostic samples.

OBJECTIVE: The goal of this study was to establish reference intervals for blood parameters in rabbits based on data from routine diagnostic samples. MATERIALS AND METHODS: Data sets from 1679-2039 values for clinical chemistry and 937-1559 values for hematological parameters were evaluated using the statistical program Reference Limit Estimator 20151017. RESULTS: The following reference intervals were calculated for hematology: erythrocytes 4.37-7.43 × 1012 cells/l; hematocrit 0.28-0.48 l/l; hemoglobin 89.63-153.82 g/l; leucocytes 2.71-12.23 × 109 cells/l; neutrophils 0.87-7.82 × 109 cells/l; lymphocytes 0.36-6.58 × 109 cells/l; monocytes 0.08-1.71 × 109 cells/l; eosi nophils 0.07-0.19 × 109 cells/l; basophils 0.06-1.1 × 109 cells/l; thrombocytes 225.45-905.3 × 109 cells/l. Calculated intervals for clinical chemistries were: alkaline phosphatase 9.05-94.68 U/l; aspartataminotransferase 3.75-32.44 U/l; creatine kinase 1.63-559.53 U/l; γ-glutamyltransferase 2.5-14.46 U/l; glutamatdehydrogenase 0.68-14.78 U/l; fructosamin 248.08-501.43 µmol/l; bile acid 0.76-19.63 µmol/l; total protein 48.66-73.64 g/l; urea 2.63-10.28 mmol/l; creatinine 51.38-154.35 µmol/l; calcium 3.02-4.3 mmol/l; magnesium 0.66-1.51 mmol/l; phosphorus 0.54-2.18 mmol/l; sodium 132.61- 154.0 mmol/l and potassium 3.52-6.04 mmol/l. CONCLUSION AND CLINICAL RELEVANCE: The calculation of intervals based on a large number of routine diagnostic samples allows the establishment of labora tory specific reference intervals without the use of experimental animals.