Publications by authors named "Natali Bauer"

58 Publications

Evaluation of reticulocyte hemoglobin content (RETIC-HGB) for the diagnosis of iron-limited erythropoiesis in cats.

Vet Clin Pathol 2020 Dec;49(4):557-566

Small Animal Clinic, Internal Medicine, Justus-Liebig-University, Giessen, Germany.

Background: Decreased reticulocyte hemoglobin content (CHr) (Siemens ADVIA 2120) reflects iron-limited erythropoiesis (ILE). RETIC-HGB (IDEXX ProCyte Dx) is a novel marker of ILE for veterinary use.

Objectives: We aimed to evaluate reference intervals (RIs) and the utility of RETIC-HGB and CHr in the diagnosis of feline ILE.

Materials And Methods: RIs were established in 59 healthy cats. Intra-assay coefficients of variation (CVs) and correlations between RETIC-HGB and CHr were assessed. Two hundred and seventy-five cats were classified as having ILE or not based on low plasma iron or low transferrin saturation along with anemia and/or altered RBC indices. CHr, RETIC-HGB, and serum amyloid A (SAA) were compared between the groups. The sensitivity and specificity of RETIC-HGB and CHr to diagnose ILE were analyzed to determine the RI lower limits.

Results: RIs for RETIC-HGB and CHr were 12.5-18.0 and 14.0-19.9 pg, respectively. The CV was 3% for both variables. RETIC-HGB and CHr were moderately correlated (r = 0.59) with a bias of -1.2 picograms (pgs). Twenty of the 275 cats were classified as having ILE. Compared with non-ILE cats, ILE cats had significantly lower median RETIC-HGB (14.3 vs 15.2 pg, P = .0046) and mean CHr (14.7 vs 16.5 pg, P < .0001) values and significantly increased median SAA (44.6 vs 2.3 µg/dl, P < .0001) values. Using the lower RI limits resulted in a low sensitivity and relatively high specificity to diagnose ILE in cats.

Conclusions: ILE was characterized by decreased CHr and RETIC-HGB; however, sensitivity was low. The moderate correlation between RETIC-HGB and CHr is likely due to species differences and different methodology.
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http://dx.doi.org/10.1111/vcp.12925DOI Listing
December 2020

Analytical performance and method comparison of a quantitative point-of-care immunoassay for measurement of bile acids in cats and dogs.

J Vet Diagn Invest 2021 Jan 28;33(1):35-46. Epub 2020 Oct 28.

Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-University, Giessen, Germany.

Point-of-care analyzers (POCAs) for quantitative assessment of bile acids (BAs) are scarce in veterinary medicine. We evaluated the Fuji Dri-Chem Immuno AU10V analyzer and v-BA test kit (Fujifilm) for detection of feline and canine total serum BA concentration. Results were compared with a 5th-generation assay as reference method and a 3rd-generation assay, both run on a bench-top analyzer. Analytical performance was assessed at 3 different concentration ranges, and with interferences. For method comparison, samples of 60 healthy and diseased cats and 64 dogs were included. Linearity was demonstrated for a BA concentration up to 130 µmol/L in cats ( = 0.99) and 110 µmol/L in dogs ( = 0.99). The analyzer showed high precision near the lower limit of quantification of 2 µmol/L reported by the manufacturer. Intra- and inter-assay coefficients of variation were < 5% for both species and all concentrations. Interferences were observed for bilirubin (800 mg/L) and lipid (4 g/L). There was excellent correlation with the reference method for feline ( = 0.98) and canine samples ( = 0.97), with proportional biases of 6.7% and -1.3%, respectively. However, a large bias (44.1%) was noted when the POCA was compared to the 3rd-generation assay. Total observed error was less than total allowable error at the 3 concentrations. The POCA reliably detected feline and canine BA in clinically relevant concentrations.
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http://dx.doi.org/10.1177/1040638720968784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756073PMC
January 2021

Extremely high canine C-reactive protein concentrations > 100 mg/l - prevalence, etiology and prognostic significance.

BMC Vet Res 2020 May 20;16(1):147. Epub 2020 May 20.

Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-University Giessen, Frankfurter Str. 114, 35392, Giessen, Germany.

Background: In human medicine, extremely high CRP (C-reactive protein) concentrations > 100 mg/l are indicators of bacterial infection and the need of antibiotic treatment. Similar decision limits for septic pneumonia are recommended for dogs but have not yet been evaluated for other organ systems. The aim of the retrospective study was to investigate the prevalence and evaluate dogs with CRP concentrations > 100 mg/l regarding the underlying etiology, the affected organ system and the prognostic significance.

Results: Prevalence of CRP > 100 mg/l was investigated in dogs presented between 2014 and 2015 and was 12%. For evaluation of etiology and organ systems, dogs with CRP > 100 mg/l presented between 2014 and 2016 were enrolled. Dogs were classified into 4 main disease categories, i.e. inflammatory, neoplastic, tissue damage or "diverse". Diseases were assigned to the affected organ system. If an organ classification was not possible, dogs were classified as "multiple". 147 dogs with CRP 101-368 mg/l were included and classified into disease categories: 86/147 (59%) with inflammatory etiology (among these, 23/86 non-infectious, 44/86 infectious (33/44 bacterial), 19/86 inflammation non-classifiable), 31/147 (21%) tissue damage, 17/147 (12%) neoplastic (all malignant) and 13/147 (9%) diverse diseases. The affected organ systems included 57/147 (39%) multiple, 30/147 (20%) trauma, 21/147 (14%) gastrointestinal tract, 10/147 (7%) musculoskeletal system, 8/147 (5%) respiratory tract, 7/147 (5%) urinary/reproductive tract, 6/147 (4%) skin/subcutis/ear, 6/147 (4%) central/peripheral nervous system and 2/147 (1%) heart. The disease group (p = 0.081) or organ system (p = 0.17) did not have an impact on CRP. Based on CRP, a detection of bacterial infection was not possible. The prognostic significance was investigated by determining the 3-months survival and hospitalization rate in a subgroup with known outcome. The 3-months survival rate was 46/73 (63%) while the majority 66/73 (90%) of patients was hospitalized.

Conclusions: CRP concentrations > 100 mg/l are occasionally seen in a clinic population. They indicate a severe systemic disease of various etiologies with guarded prognosis. Extremely high CRP concentrations do not allow a conclusion of the underlying etiology or an identification of bacterial inflammation.
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http://dx.doi.org/10.1186/s12917-020-02367-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237877PMC
May 2020

Haemostatic, fibrinolytic and inflammatory profiles in West Highland white terriers with canine idiopathic pulmonary fibrosis and controls.

BMC Vet Res 2019 Oct 29;15(1):379. Epub 2019 Oct 29.

Department of Veterinary Clinical Sciences, FARAH, University of Liege, Avenue de Cureghem 3, 4000, Liège, Belgium.

Background: Canine idiopathic pulmonary fibrosis (CIPF) is a progressive interstitial lung disease mainly affecting old West Highland white terriers (WHWTs). The aetiology of CIPF is currently unknown and pathogenesis poorly understood. A genetic basis is strongly suspected based on the breed predisposition. CIPF shares clinical and pathological features with human IPF. In human IPF, coagulation disorders favouring a local and systemic pro-thrombotic state have been demonstrated in association with disease severity and outcome. The aim of this study was to compare the systemic haemostatic, fibrinolytic and inflammatory profiles of WHWTs affected with CIPF with breed-matched controls (CTRLs). Additionally, data collected in both groups were interpreted with regard to the reference intervals (when available) to assess possible pro-thrombotic features of the WHWT breed that may be related to CIPF predisposition. A total of 14 WHWTs affected with CIPF and 20 CTRLs were included.

Results: WHWTs affected with CIPF had prolonged activated partial thromboplastine time in comparison with CTRLs (12.2 ± 0.9 s vs. 11.5 ± 0.7 s, P = 0.028), whereas results obtained in both groups were all within reference ranges. There was no significant difference between groups for the other factors assessed including plasmatic concentrations of fibrinogen, D-dimers concentration, antithrombin III activity, protein S and protein C activities, anti-factor Xa activity, activated protein C ratio, serum C-reactive protein concentration, and rotational thromboelastometry indices. Platelet count and plasmatic fibrinogen concentration were found to be above the upper limit of the reference range in almost half of the WHWTs included, independently of the disease status.

Conclusions: Results of this study provide no clear evidence of an altered systemic haemostatic, fibrinolytic or inflammatory state in WHWTs affected with CIPF compared with CTRLs. The higher platelet counts and fibrinogen concentrations found in the WHWT breed may serve as predisposing factors for CIPF or simply reflect biological variation in this breed.
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http://dx.doi.org/10.1186/s12917-019-2134-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819526PMC
October 2019

Clinical diabetes mellitus in association with diestrus-induced acromegaly in 2 bitches.

Tierarztl Prax Ausg K Kleintiere Heimtiere 2019 Jun 18;47(3):193-201. Epub 2019 Jun 18.

Department of Clinical Science and Services, Royal Veterinary College, University of London.

Two intact bitches aged 9 and 11 years were referred due to chronic polyuria, polydipsia, vomiting, anorexia and progressive lethargy. On clinical examination, signs of tissue overgrowth (large paws, widened interdental space, pharyngeal stridor) were noticed. Diabetes mellitus (DM) was diagnosed in one dog (case 1) and diabetic ketoacidosis (DKA) in the other (case 2). There were increased IGF-1 values suggestive of hypersomatotropism. Progesterone values and pathological findings of the ovaries and uterus indicated diestrus. Diagnosis of diestrus-induced hypersomatotropism was made and ovariohysterectomy was performed in both dogs. Dog 1 also had multiple mammary neoplasms treated with bilateral mastectomy. Treatment resulted in diabetic remission in case 1 and improved glycaemic control in case 2. Overall, diestrus-induced hypersomatotropism is rare but should be considered in any intact diabetic bitch with acromegalic features. Ovariohysterectomy is recommended and associated with a fair to good prognosis.
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http://dx.doi.org/10.1055/a-0886-9360DOI Listing
June 2019

Prospective Comparative Quality Control Study of a Novel Gravity-Driven Hollow-Fiber Whole Blood Separation System for the Production of Canine Blood Products.

Front Vet Sci 2019 17;6:149. Epub 2019 May 17.

Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-University Giessen, Giessen, Germany.

The aim of this prospective study was to compare quality of blood products produced either by a novel gravity-driven hollow-fiber separation system (HF) or by centrifugation (C). Whole blood was obtained from 31 healthy non-greyhound canine blood donors and separated into fresh frozen plasma and packed red blood cells using either HF or C in a university teaching hospital. Red blood cell (RBC) count, albumin and fibrinogen concentration, prothrombin time (PT), activated partial thromboplastin time (aPTT) and coagulation factor activity (FV, FVIII), von Willebrand Factor (vWF), and antithrombin activity were assessed. Plasma obtained with the HF showed a significantly higher median PT (9.4 vs. 7.9 s, = 0.0006) and aPTT (14.9 vs. 13.1 s, = 0.0128) than plasma prepared with C. Lower albumin (21.7 vs. 23.5 g/l, = 0.0162) and fibrinogen (1.0 vs. 1.5 g/l, = 0.0005) concentrations and activities of FV (105 vs. 114%, = 0.0021) and antithrombin (104 vs. 117%, = 0.0024) were seen in blood products obtained with the HF. In contrast, vWF was not affected by the method of plasma separation. Compared to HF, RBC count as well as hematocrit were not significantly higher (8.0 vs. 8.9 10/l, = 0.1308; 0.57 vs. 0.62 l/l, = 0.0736) when blood products were prepared with C. In conclusion, higher quality of blood products especially regarding coagulation parameters and RBCs was achieved by using C compared to HF. Despite the statistical significances, however, the clinical relevance has to be further elucidated. Nevertheless, HF provides an alternative to produce blood products if a centrifuge is not available.
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http://dx.doi.org/10.3389/fvets.2019.00149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533853PMC
May 2019

[Hypermagnesemia of ionized magnesium in 199 dogs - A hospital population-based study on prevalence, etiology and prognosis with special emphasis on kidney disease and the measurement of total magnesium].

Tierarztl Prax Ausg K Kleintiere Heimtiere 2018 Dec 18;46(6):370-379. Epub 2019 Jan 18.

Objective: To investigate the prevalence and etiology of an increased concentration of ionized magnesium (iMg) in dogs and to evaluate its prognostic relevance.

Materials And Methods: From April 2009 to December 2013, serum electrolytes were measured in 9950 dogs using an ion-selective electrode. Inclusion criterion was an iMg concentration ≥ 0.68 mmol/l, whereby total Mg (tMg), potassium, ionized calcium, and micturition were also evaluated in this retrospective study. In the case of repeated measurements in an animal, only the initial measurement of the increased iMg concentration was considered. According to the etiology, patients were categorized in the diagnostic groups: azotemia, iatrogenic/medication associated, endocrine diseases, tissue damage, and unknown etiology of hypermagnesemia. The survival rate was analyzed and the iMg concentration was compared between the diagnostic groups. The correlation between iMg and tMg was assessed.

Results: The prevalence of increased iMg concentration was 2.0 % (199/9950). The most prevalent causes were azotemia (80/199; 40 %), followed by iatrogenic hyper-magnesemia (37/199; 19 %), tissue damage (21/199; 11 %), and endocrine diseases (12/199; 6 %). In 49/199 cases (25 %), the etiology of hypermagnesemia was unknown. An additional hypercalcemia was evident in 24/199 dogs (12 %) and 58/199 (29 %) dogs displayed hypocalcemia. In 64 % of the dogs (51/80) with azotemia, underlying renal disease was present, from which 53 % (27/51) exhibited hyperkalemia. Moreover, 37 % (19/51) of the animals with renal disease displayed anuria/oliguria, 59 % (11/19) of which were hyperkalemic. Overall, 83/199 (42 %) of the dogs died, whereby 48/83 (58 %) belonged to the azotemia group. The iMg concentration did not differ significantly between the groups (p = 0.15). There was a poor correlation between iMg and tMg (r = 0.28).

Conclusion: An increased iMg concentration is rare and indicative of a severe disease in the majority of cases. Particularly in patients with acute renal disease, hypermagnesemia can be associated with increased mortality. The tMg concentration does not reflect the iMg concentration.
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http://dx.doi.org/10.1055/s-0038-1677389DOI Listing
December 2018

Coagulation parameters in hyperthyroid cats before and after radioiodine treatment compared with healthy controls.

J Feline Med Surg 2019 12 20;21(12):1134-1140. Epub 2018 Dec 20.

Small Animal Clinic Hofheim, Hofheim, Germany.

Objectives: The aim of the study was to describe the coagulatory state of hyperthyroid cats before and after successful radioiodine therapy (RIT) compared with healthy age-matched controls, using classical coagulation parameters and thromboelastogram (TEG) as a global assessment method. The differences in coagulation activity after RIT, depending on the thyroid hormone (normal vs low total thyroxine [T4]) state, were also evaluated.

Methods: Fifteen hyperthyroid cats and 10 healthy age-matched controls were recruited. Hyperthyroid cats that remained hyperthyroid 14 days after RIT were excluded. Haematology, biochemistry, T4, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and TEG were assessed in control cats and hyperthyroid cats before and 7 and 14 days after RIT. Two weeks after successful RIT, further comparisons were made between cats with normal T4 vs those with low T4.

Results: Fourteen days after successful RIT, 7/15 cats had normal T4 and 8/15 had low T4. Thrombocytosis was noted in 6/15 cats after treatment. Fibrinogen was significantly higher ( <0.001) and PT shorter ( <0.01) in the hyperthyroid cats compared with the healthy controls and these changes persisted after RIT. Persistent increases in fibrinogen, PT, TEG maximal amplitude and TEG clot rigidity, reflecting clot stability, after RIT primarily occurred in the cats with normal T4. TEG-K (time until preset amplitude of 20 mm is reached) and alpha (α) angle reflected impaired fibrin cross-linking ability prior to RIT, which significantly increased after therapy ( <0.05).

Conclusions And Relevance: Based on some of the coagulation parameters, cats with hyperthyroidism showed hypercoagulable tendencies, which were mildly increased after RIT, possibly due to transient radiation-induced thyroiditis.
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http://dx.doi.org/10.1177/1098612X18820145DOI Listing
December 2019

[Non-epitheliotropic B-cell lymphoma with atypical spindle cell morphology in a Weimaraner dog].

Tierarztl Prax Ausg K Kleintiere Heimtiere 2018 Apr 4;46(2):119-125. Epub 2018 May 4.

The case report describes a very rare cutaneous non-epitheliotropic B-cell lymphoma in an 11-year-old male Weimaraner dog, which presented with multiple cutaneous proliferations. In addition to numerous cytoplasmic fragments of lymphatic cells, the cytological examination of a fine-needle aspirate taken from a skin nodule revealed a population of pleomorphic plump to spindle-shaped cells with round to oval nuclei, fine chromatin structure, moderate amounts of a lightly basophilic cytoplasm and predominantly indistinct cell boundaries. These findings suggested a possible cutaneous spindle-cell lymphoma, thus a biopsy was taken. In the histopathological examination, the cells displayed no epitheliotropism and immunohistochemically they were positive for multiple B-cell markers. In addition to the rarity of cutaneous non-epitheliotropic B-cell lymphomas per se, the special feature of this case is the atypical spindle-cell morphology of the lymphatic cells. A spindle-cell variant of cutaneous B-cell lymphoma has to date only been described in human medicine.
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http://dx.doi.org/10.15654/TPK-170254DOI Listing
April 2018

[Thrombocytopenia in horses].

Tierarztl Prax Ausg G Grosstiere Nutztiere 2018 Apr 4;46(2):73-79. Epub 2018 May 4.

Objective: The retrospective study aimed to evaluate the prevalence of true thrombocytopenia and ethylenediaminetetraacetic acid (EDTA)-induced pseudothrombocytopenia in horses and ponies and to assess the diagnostic and prognostic utility of true thrombocytopenia.

Material And Methods: In 3592 patients (2008-2015) hematological data (ADVIA® 2120, Siemens) were reviewed. Inclusion criteria were platelet counts < 90 x 109/l (EDTA-blood) and/or < 84 x 109/l (citrate blood). Thrombocytopenia was classified as true, EDTA-induced, and questionable. Patients with true thrombocytopenia were assigned to nine groups according to their history and four groups depending on the main diagnosis (inflammatory disorders, neoplasia, non-inflammatory intestinal disease, others). The frequencies of diagnoses were compared to the overall clinic population.

Results: Thrombocytopenia was diagnosed in 123/3592 patients (3.4 %) and classified as true in 60/123 (49 %), EDTA-induced in 6/123 (5 %), and questionable in 57/123 (46 %) of cases. In true thrombocytopenia, the most common reasons for referral were lethargy (23/60, 38 %), fever (19/60, 32 %), and colic (17/60, 28 %). In these patients inflammation, neoplasia, non-inflammatory intestinal disease, and others were diagnosed in 25/60 (42 %), 11/60 (18 %), 10/60 (17 %), and 14/60 (23 %) of cases, respectively. Compared with the overall clinic population, there was an increased frequency of neoplasia (18 % versus 1 %). The mortality rate was significantly higher at 38 % in comparison with the overall population. Remarkably high mortality rates of 32 % and 82 % were observed in patients with inflammatory and neoplastic diseases, respectively.

Conclusion And Clinical Relevance: True thrombocytopenia is relatively rare in horses with internal medical conditions and should be verified by measurement in samples with citrate as anticoagulant. Thrombocytopenia is rarely the primary reason for referral and is a negative prognostic factor in neoplastic and inflammatory diseases.
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http://dx.doi.org/10.15653/TPG-170429DOI Listing
April 2018

Evaluation of a novel quantitative canine species-specific point-of-care assay for C-reactive protein.

BMC Vet Res 2018 Mar 20;14(1):99. Epub 2018 Mar 20.

Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-University Giessen, Frankfurter Str. 126, 35392, Giessen, Germany.

Background: Species-specific point-of-care tests (POCT) permit a rapid analysis of canine C-reactive protein (CRP), enabling veterinarians to include CRP in clinical decisions. Aim of the study was to evaluate a novel POCT for canine CRP (Point Strip™ Canine CRP Assay) run on a small in-house-analyzer (Point Reader™ V) using lithium heparin plasma and to compare assay performance to an already established canine CRP assay (Gentian Canine CRP Immunoassay) run on two different bench top analyzers serving as reference methods (ABX Pentra 400, AU 5800). Linearity was assessed by stepwise dilution of plasma samples with high CRP concentrations. Limit of quantification (LoQ) was determined by repeated measurements of samples with low CRP concentrations. Coefficient of variation (CV) at low (10-50 mg/l), moderate (50-100 mg/l), and high (100-200 mg/l) CRP concentrations was investigated as well as possible interferences. Method comparison study was performed using 45 samples of healthy and diseased dogs. Quality criteria were fulfilled if the total observed error (TE = 2CV% + bias%) was below the minimal total allowable error of 44.4% (TE ). Additionally, a reference range (n = 60 healthy dogs) was established.

Results: Linearity was present at CRP concentrations of 10-132 mg/l (≙ 361 mg/l CRP with reference method) with a LoQ set at 10 mg/l. At moderate to high CRP concentrations, intra- and inter-assay CVs were ≤ 8% and ≤ 11% respectively, while CVs ≤ 22% and ≤ 28% were present at low concentrations. No interferences were observed at concentrations of 4 g/l hemoglobin, 800 mg/l bilirubin and 8 g/l triglycerides. Method comparison study demonstrated an excellent correlation with both reference methods (r = 0.98 for ABX Pentra 400; 0.99 for AU 5800), though revealing a proportional bias of 19.7% (ABX Pentra 400) and 10.7% (AU 5800) respectively. TE was 26.7-31.9% and 16.7-21.9% and thus < TE. Healthy dogs presented with CRP values ≤11.9 mg/l.

Conclusions: The POCT precisely detects canine CRP at clinically relevant moderate and high CRP concentrations. The assay correlates well with both reference methods. Due to the bias, however, follow-up examinations should be performed with the same assay and analyzer.
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http://dx.doi.org/10.1186/s12917-018-1415-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859731PMC
March 2018

[Magnesium in dogs and cats - physiology, analysis, and magnesium disorders].

Tierarztl Prax Ausg K Kleintiere Heimtiere 2018 Feb 21;46(1):21-32. Epub 2018 Feb 21.

Magnesium is the second most abundant intracellular cation after potassium. It plays a vital role in almost every metabolic process in the body and is important for bone mineralization, muscle contraction and relaxation, and neuronal signal transduction. Because of its expanding role in intensivecare medicine, there has been a significant increase in knowledge during recent years regarding the functions of magnesium in the body, problems leading to magnesium disorders, and limitations of laboratory testing. Alterations of serum magnesium constitute one of the most prevalent electrolyte abnormalities in critically ill patients and can lead to lifethreatening complications. In addition to human literature, most of the information regarding the role of magnesium is derived from buiatrics. In recent years, a few studies in veterinary medicine have also started to consider the importance of magnesium in dogs and cats.
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http://dx.doi.org/10.15654/TPK-170556DOI Listing
February 2018

An update on clinical biochemical RIs of rabbits with special consideration for age, gender, and size.

Vet Clin Pathol 2018 Jun 5;47(2):233-245. Epub 2018 Mar 5.

Department of Animal Breeding and Genetics, Justus-Liebig-University Giessen, Giessen, Germany.

Background: Literature regarding the impact of age and breed size on clinical biochemical measurands in rabbits is scarce.

Objectives: We aimed to establish clinical biochemical measurand RIs for rabbits bred and kept under standard conditions and to evaluate the impact of gender, age, and breed size on the results using a Nova CRT8 analyzer (Nova Biomedical GmbH) and an ABX Pentra 400 analyzer (ABX Horiba, Axonlab).

Methods: Serum samples were available from 122 adult rabbits (56 males, 66 females) of 10 different breeds and crossbreds with a mean age of 264 ± 21 days. The impact of age was evaluated by sampling 48 rabbits at weaning (8 weeks of age) and when they reached adulthood.

Results: Significantly higher median values were obtained for ALT, glutamate dehydrogenase (GLDH), and potassium in adult males compared with adult female rabbits. Total bilirubin, cholesterol, creatinine, and urea were significantly higher in adult females than adult males. Juvenile animals at weaning revealed significantly higher median values for ALP, cholesterol, GGT, GLDH, glucose, phosphate, and triglycerides compared with their adulthood values. In contrast, lower median albumin, ALT, chloride, creatinine, globulin, ionized calcium, magnesium, potassium, total protein, urea, and calcium-phosphate ratios were seen at the time of weaning compared with adulthood values. Significantly lower median CK, creatinine, and ALT were found in dwarf/small rabbit breeds compared with intermediate/large breeds.

Conclusions: These RIs are especially useful for rabbit production and experimental studies. Age should be considered when evaluating clinical biochemical measurands. Creatinine, CK, and ALT are affected by organ mass.
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http://dx.doi.org/10.1111/vcp.12584DOI Listing
June 2018

Preliminary reference intervals and the impact of citrate storage time for thrombelastography in cats including delta and the velocity curve.

BMC Vet Res 2017 Nov 29;13(1):366. Epub 2017 Nov 29.

Department of Veterinary Clinical Sciences, Clinical Pathophysiology and Clinical Pathology, Justus-Liebig University Giessen, Frankfurterstraße 126, 35392, Giessen, Germany.

Background: Thrombelastography is a useful tool in assessment of hemostasis. Beside the traditional variables, the velocity curve and the variable delta have lately earned attention. The velocity curve provides knowledge about the speed of clot formation including information about thrombin generation. Delta, which only reflects enzymatic coagulation, allows the determination of the origin of hypercoagulability when compared to clot rigidity, a variable that reflects both platelet and enzymatic activity. The aim was to establish preliminary reference intervals for feline thrombelastography including the velocity curve variables and delta obtained after 60 min of storage including the assessment of coefficients of variation. Furthermore, the effect of citrate storage time (30 versus 60 min) on feline thrombelastography will be determined.

Results: Prolonged storage times significantly reduced reaction (R) (P = 0.019) and clotting (K) (P = 0.008) times, split point (SP) (P = 0.019) and time to maximum rate of thrombus generation (TMRTG) (P = 0.023) values whereas maximum rate of thrombus generation (MRTG) significantly increased (P = 0.040). Preliminary reference intervals: R (min): 2.7-18.1; K (min): 0.8-3.9; alpha (°): 27.6-75.2; maximum amplitude (mm): 18.5-62.5; clot rigidity (dyn/cm): 1.2-8.2; coagulation index: -4.6 - 2.6; SP (min): 2.4-15.4; delta (min): 0.3-3.1; thrombus generation (mm/min): 255.3-751.2; MRTG (mm/min): 4.0-19.3; TMRTG (min): 3.5-22.0; maximum rate of lysis (mm/min): 0.0-4.7 and time to maximum rate of lysis (min): 0.4-55.8.

Conclusion: Storage for 60 versus 30 min induces hypercoagulable tracings including the velocity curve, some of which variables (MRTG, TMRTG) might function as sensitive markers for changes in the coagulation activity. Because of the impact of citrate storage time on thrombelastography, reference intervals have to be established using a specific and constant storage time in each laboratory.
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http://dx.doi.org/10.1186/s12917-017-1278-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707899PMC
November 2017

Evaluation of reticulocyte hemoglobin content (RET-He) in the diagnosis of iron-deficient erythropoiesis in dogs.

Vet Clin Pathol 2017 Dec 9;46(4):558-568. Epub 2017 Oct 9.

Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-University, Giessen, Germany.

Background: Reticulocyte hemoglobin content provided by the Siemens ADVIA (CHr) is an established marker of iron deficiency. The IDEXX ProCyte Dx hematology analyzer now provides a similar variable, reticulocyte hemoglobin equivalent (RET-He).

Objectives: The objective was to evaluate RET-He and its diagnostic utility in dogs, and to calculate a cutoff value for diagnosing iron-deficient erythropoiesis (IDE). Furthermore, the prevalence of RET-He values below this cutoff value was established.

Methods: One hundred and seventy-one CBCs of healthy dogs were used to establish a RI. Stability of RET-He was evaluated by repeated measurements over 48 hours (n = 10). The 25-run coefficient of variation (CV) was calculated, and correlation and bias between measurements of RET-He and CHr were assessed (n = 190). A cutoff value for diagnosing IDE was calculated. The utility of RET-He in the detection of IDE was evaluated in 123 dogs. The prevalence of low RET-He values was assessed retrospectively in a multicenter study (2012-2014) under participation of 7 veterinary clinics.

Results: Reticulocyte hemoglobin equivalent with an RI of 22.2 to 28.6 pg was statistically stable over 48 hours (P = .10). The CV was 1.8%. A fair correlation (ρ = 0.74) between RET-He and CHr with a small bias of -0.6 pg was found. The cutoff value for diagnosing IDE was 20.9 pg (sensitivity: 85%; specificity: 99%). The prevalence of RET-He values below 20.9 pg was 10.3% (1084/10,553 dogs).

Conclusions: RET-He on the ProCyte Dx is a precise screening tool in dogs to detect iron-deficient erythropoiesis.
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http://dx.doi.org/10.1111/vcp.12547DOI Listing
December 2017

Nonregenerative immune-mediated anemia associated with a diffuse large B-cell lymphoma in a captive jaguar (Panthera onca).

Vet Clin Pathol 2017 Dec 13;46(4):597-604. Epub 2017 Sep 13.

Clinical Pathophysiology and Clinical Pathology, Department of Veterinary Clinical Sciences, Justus-Liebig-University, Gießen, Germany.

An 18-year-old male castrated jaguar (Panthera onca) was presented with anorexia and continuous bleeding from the oral cavity after a history of fighting with the partner animal. Clinical evaluation revealed ulcerating lesions on the gingiva and hard palate and a hematoma on the tongue. Computed tomography of the head and endoscopic examination of the esophagus and stomach were unremarkable. Hematology and clinical chemistry revealed severe nonregenerative anemia, mild thrombocytopenia, and moderate azotemia. Several PCRs for feline hemotropic mycoplasmas (Mycoplasma haemofelis, M heamominutium, M turicensis), Babesia felis, and Bartonella spp., as well as an FeLV antigen test were negative. The cytologic examination of a bone marrow aspirate was consistent with ineffective erythropoiesis, most likely due to immune-mediated destruction of the erythroid precursor cells. Prednisolone therapy was initiated (1.25 mg/kg/day), and the CBC returned to normal 16 days after the initiation of the therapy. Anemia relapsed after 4 months and severe splenomegaly was noted. A repeat bone marrow aspirate revealed active erythropoiesis in the presence of erythroid precursor phagocytosis suggesting an immune-mediated process. Splenic fine-needle aspiration and tissue biopsies were taken, and all findings including histology and immunohistochemistry were consistent with a diffuse large B-cell lymphoma (DLBCL). Five days later, the clinical condition deteriorated and the jaguar died. Histopathology following necropsy showed infiltration with neoplastic lymphoblasts in the spleen, liver, and abdominal lymph nodes. This case report describes a nonregenerative immune-mediated anemia associated with a DLBCL in a jaguar.
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http://dx.doi.org/10.1111/vcp.12542DOI Listing
December 2017

Osteocyte Regulation of Receptor Activator of NF-κB Ligand/Osteoprotegerin in a Sheep Model of Osteoporosis.

Am J Pathol 2017 Aug 12;187(8):1686-1699. Epub 2017 Jun 12.

Experimental Trauma Surgery, Faculty of Medicine, Justus-Liebig University of Giessen, Giessen, Germany; Department of Trauma, Hand and Reconstructive Surgery, University Hospital of Giessen-Marburg, Giessen, Germany. Electronic address:

Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.
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http://dx.doi.org/10.1016/j.ajpath.2017.04.005DOI Listing
August 2017

Evaluation of a species-specific C-reactive protein assay for the dog on the ABX Pentra 400 clinical chemistry analyzer.

BMC Vet Res 2017 May 30;13(1):146. Epub 2017 May 30.

Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-University Giessen, 35392, Giessen, Germany.

Background: A canine-specific immunoturbidimetric CRP assay, Gentian Canine CRP Immunoassay) with species-specific controls and calibrators was introduced and recently evaluated on the clinical chemistry analyzer Abbott Architect c4000 as well as on the Olympus AU600. Aims of our study were 1) to independently evaluate the canine-specific CRP assay on the ABX Pentra 400 clinical chemistry analyzer in comparison to the previously validated human-based immunoturbidimetric assay (Randox Canine CRP assay) and 2) to assess the impact of different sample types (serum versus heparinized plasma) on the results. Imprecision, accuracy, interference and the prozone effect were determined using samples from healthy and diseased dogs (n = 278). The Randox Canine CRP assay calibrated with canine specific control calibration material served as a reference method. Additionally, the impact of the sample type (serum and lithium heparin) was evaluated based on samples of healthy and diseased dogs (n = 49) in a second part of the study.

Results: Linearity was present for CRP concentrations ranging from 4 to 281 mg/l. For clinically relevant CRP concentrations of 7-281 mg/l, recovery ranged between 90 and 105% and intra- and inter-assay CVs ranged between 0.68% - 12.12% and 0.88% - 7.84%, respectively. CV was thus lower than 12.16%, i.e. the desired CV% based on biological variation. Interference was not present up to a concentration of 5 g/l hemoglobin, 800 mg/l bilirubin and 10 g/l triglycerides. No prozone effect occurred up to 676 mg/l CRP. Method comparison study revealed a Spearman's rank correlation coefficient of r = 0.98 and a mean constant bias of 5.2%. The sample type had a significant (P = 0.008) but clinically not relevant impact on the results (median CRP of 30.9 mg/l in lithium heparin plasma versus 31.4 mg/l in serum).

Conclusions: The species-specific Gentian Canine CRP Immunoassay reliably detects canine CRP on the ABX Pentra 400 clinical chemistry analyzer whereby both serum and heparin plasma can be used. The quality criteria reached on the Abbott Architect c4000 and Olympus AU600 could be met.
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http://dx.doi.org/10.1186/s12917-017-1065-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450169PMC
May 2017

Canine reticulocyte hemoglobin content (RET-He) in different types of iron-deficient erythropoiesis.

Vet Clin Pathol 2017 Sep 16;46(3):422-429. Epub 2017 May 16.

Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-University, Giessen, Germany.

Background: Reticulocyte hemoglobin content (RET-He) is a diagnostic marker for iron deficiency (ID) in people and dogs.

Objectives: The aim of our study was to evaluate the clinical utility of RET-He in the diagnosis of different causes of iron-deficient erythropoiesis (IDE).

Methods: Canine CBCs were separated into 2 groups according to RET-He values, < 20.9 pg or ≥ 20.9 pg. Erythrocyte and reticulocyte variables were compared between dogs with decreased and normal RET-He values. Additional data for a subgroup of dogs were collected, and dogs with low RET-He values were categorized as having ID, inflammatory disorders (INFL), portosystemic shunt (PSS), miscellaneous diseases (MISC), or combinations of these diseases (ID+INFL, ID+PSS). Hematologic variables were compared between dogs of the different disease groups.

Results: Overall, 10.3% (1084/10,553) of canine CBCs showed decreased RET-He values. Significant differences between dogs with decreased and normal RET-He values were found for all erythrocyte and reticulocyte variables. The majority (68.9%, 747/1084) of dogs with low RET-He values was anemic; 28.9% (216/747) of those anemic dogs had microcytosis and hypochromasia. In the subgroup of dogs, 8.9% (205/2306) had low RET-He values. According to their diagnosed diseases, anemic dogs (138/205) could be categorized as ID (17/138; 12.3%), INFL (16/138; 11.6%), PSS (30/138; 21.7%), ID+INFL (63/138; 45.7%), ID+PSS (8/138; 5.8%), and MISC (4/138; 2.9%). Distribution in nonanemic dogs (67/205) was similar, except for a lower number of dogs with PSS.

Conclusions: Low RET-He values indicate IDE even in dogs with other CBC variables within the RIs.
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http://dx.doi.org/10.1111/vcp.12499DOI Listing
September 2017

Objective evaluation of analyzer performance based on a retrospective meta-analysis of instrument validation studies: point-of-care hematology analyzers.

Vet Clin Pathol 2017 Jun 3;46(2):248-261. Epub 2017 May 3.

Department of Clinical Sciences, Clinical Pathophysiology and Clinical Pathology, Faculty of Veterinary Medicine, Justus-Liebig-University Gießen, Gießen, Germany.

Background: Information on quality requirements and objective evaluation of performance of veterinary point-of-care analyzers (POCAs) is scarce.

Objectives: The study was aimed at assessing observed total errors (TE s) for veterinary hematology POCAs via meta-analysis and comparing TE to allowable total error (TE ) specifications based on experts' opinions.

Methods: The TE for POCAs (impedance and laser-based) was calculated based on data from instrument validation studies published between 2006 and 2013 as follows: TE = 2 × CV [%] + bias [%]. The CV was taken from published studies; the bias was estimated from the regression equation at 2 different concentration levels of measurands. To fulfill quality requirements, TE should be < TE . Measurands were considered as globally acceptable if > 60% of analyzers showed TE < TE .

Results: Six studies evaluating 11 analyzers and 5 studies evaluating 5 analyzers were included for canine and feline hematology variables, respectively. For the CBC, TE was < 15% for canine and < 13% for feline measurands, except for HGB and platelet counts. Measurands of the CBC, excluding differential WBC and platelet counts, and HGB concentration were considered globally acceptable. For most of the cell types in the WBC differential count, TE was > TE (data from 3 analyzers).

Conclusion: This meta-analysis is considered a pilot study. Experts' requirements (TE < TE ) were fulfilled for most measurands except HGB (due to instrument-related bias for the ADVIA 2120) and platelet counts. Available data on the WBC differential count suggest an analytic bias, so nonstatistical quality control is recommended.
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http://dx.doi.org/10.1111/vcp.12492DOI Listing
June 2017

Comparison of the Sysmex XT-2000iV with microscopic differential counts of canine bone marrow.

J Vet Diagn Invest 2017 Mar 8;29(2):148-153. Epub 2017 Feb 8.

Merck KGaA, Global Non-Clinical Safety, Global Early Development, Darmstadt, Germany (Pernecker, Johannes).

Canine bone marrow is frequently assessed in the advanced preclinical research environment. Automated analysis provides time savings and objectivity over the gold standard of microscopic (cytologic) evaluation. We compared the analysis of 90 canine bone marrow samples by the Sysmex XT-2000iV hematology analyzer (Sysmex Corp., Kobe, Japan) with cytologic evaluation. Gates for cell populations were created in the system's WBC/BASO channel. Variables "total nucleated red blood cells" (total_NRBC), "poly- and orthochromatic nucleated red blood cells" (poly_orth_NRBC), "total neutrophils" (total_NEUT), "mature neutrophils" (mature_NEUT), and myeloid-to-erythroid (M:E) ratio were compared with cytologic evaluation. Intra-assay repeatability and total error (TE) were calculated for both methods. Intra-assay repeatability was 0.95-2.48% for the XT-2000iV and 8.32-23.23% for cytology. Observed TE for the automated measurement was 5.16-46.8% and for cytology 22.70-76.74%. Spearman rank correlation was excellent for M:E ratio (0.91) and fair for the other populations (0.65-0.71). Absolute bias for M:E ratio was low (-0.114). A negative absolute bias of -7.71% for the XT-2000iV was found for poly_orth_NRBC, whereas the bias was positive for total_NEUT (7.10%) and mature_NEUT (14.67%). M:E ratio of canine bone marrow samples can be precisely determined using the Sysmex XT-2000iV WBC/BASO channel. Total_NRBC, poly_orth_NRBC, total_NEUT, and mature_NEUT can be estimated rapidly. With distinctly lower coefficient of variation and observed TE compared with cytology, automated measurement provides advantages in terms of standardization, and it is suited to the advanced preclinical research environment where large numbers of samples are investigated.
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http://dx.doi.org/10.1177/1040638717692011DOI Listing
March 2017

[Neutropenia in dogs: etiology and prognostic factors].

Tierarztl Prax Ausg K Kleintiere Heimtiere 2016 Oct 16;44(5):307-315. Epub 2016 Aug 16.

Andrea Manuela Cook, Berrenrather Straße 1a, 50169 Kerpen, E-Mail:

Objective: The aim of this retrospective study was to evaluate frequency, prognostic factors, and differences for various etiologies of neutropenia in dogs.

Material And Methods: A total of 391 dogs with neutrophil counts < 2.78 x 10/l (January 2008 to December 2012) were included and, depending on the etiology of neutropenia, assigned to seven diagnostic groups: nonbacterial infectious disease, increased demand due to marked inflammation, drug-associated, bone-marrow diseases, immune-mediated, physiologic, miscellaneous. Absolute neutrophil counts, evidence of neutrophil toxicity or left shift, case history, rectal temperature, hospitalization, and survival were compared among groups.

Results: Increased demand due to marked inflammation (90/391, 23%) and nonbacterial infectious disease (70/391, 18%) were the most common causes for neutropenia, followed by drug-associated neutropenia (43/391, 11%) and bone-marrow disease (32/391, 8%). Immune-mediated and physiologic neutropenia (both 16/391, 4%) were uncommon. Almost one third (124/391, 32%) of dogs were assigned to the miscellaneous group. Absolute neutrophil counts were significantly higher (p < 0.01) in dogs of the physiologic and miscellaneous groups than in the other groups. Dogs with immune-mediated neutropenia or nonbacterial infectious disease displayed significantly lower absolute neutrophil counts than dogs with neutropenia due to an increased demand (p < 0.001) and were most commonly referred with a history of fever (11/16, 69%) or gastrointestinal signs (52/70, 74%), respectively. Neutrophil toxicity and left shift were most commonly associated with an increased demand due to marked inflammation (60/90 and 25/90, 67% and 28%, respectively) and the mortality rate was highest in this group (32/90, 36%).

Conclusion: Neutrophil toxicity and left shift are associated with an increased demand due to marked inflammation and may indicate a poor prognosis. The lower the absolute neutrophil count, the greater the probability of an immune-mediated neutropenia.

Clinical Relevance: Neutropenia should be assessed in context with case history, clinical examination, and neutrophil morphology.
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http://dx.doi.org/10.15654/TPK-160142DOI Listing
October 2016

Optimized gating and reference ranges of reticulated platelets in dogs for the Sysmex XT-2000iV.

BMC Vet Res 2016 Jul 22;12(1):148. Epub 2016 Jul 22.

Department of Veterinary Clinical Sciences, Clinical Pathophysiology and Clinical Pathology, Justus-Liebig University Giessen, Frankfurter Str. 126, 35392, Giessen, Germany.

Background: Canine reticulated platelets (r-PLTs) i.e., juvenile PLTs reflecting thrombopoiesis can be measured automatically with the hematology analyzer Sysmex XT-2000iV using manual gating options. However, the impact of interferences on r-PLT measurements performed with the gates published previously (Pankraz et al., Vet Clin Path 38:30-38, 2009; Gelain et al., High fluorescent platelets fraction in macrothrombocytopenic Norfolk terrier, 2010) is largely unknown. The aim was to compare different published gates for measurement of r-PLTs with the Sysmex XT-2000iV with an own, optimized gate ("Oellers-gate") and to establish reference intervals (RIs) in > 120 dogs. Data of 362 measurements of diseased and healthy dogs were analyzed retrospectively. Several gates were applied and RIs for r-PLTs and platelet indices were established for pet dogs and a group of 153 healthy Beagles kept under defined housing conditions. Intra-assay precision (CV) was also assessed.

Results: In 30/362 samples, interferences consistent with small erythrocytes/reticulocytes were seen in the previously published gates but not in the "Oellers-gate". Good correlation was found between the different gates (rs: 0.88-1.00). RIs for the "Pankraz-gate", the "Gelain-gate", and the "Oellers-gate" were 0.0-1.2, 0.2-3.7 and 0.2-3.9 % respectively. CVs were ranging between 22 and 41 %.

Conclusions: Optimization of previously published gates minimized interferences of small erythrocytes with r-PLT measurements.
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http://dx.doi.org/10.1186/s12917-016-0779-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4957853PMC
July 2016

Quality requirements for veterinary hematology analyzers in small animals-a survey about veterinary experts' requirements and objective evaluation of analyzer performance based on a meta-analysis of method validation studies: bench top hematology analyzer.

Vet Clin Pathol 2016 Sep 18;45(3):466-76. Epub 2016 Jul 18.

Department of Clinical Pathophysiology and Clinical Pathology, Faculty of Veterinary Medicine, Justus-Liebig-University, Gießen, Germany.

Background: Scarce information exists about quality requirements and objective evaluation of performance of large veterinary bench top hematology analyzers.

Objective: The study was aimed at comparing the observed total error (TEobs ) derived from meta-analysis of published method validation data to the total allowable error (TEa ) for veterinary hematology variables in small animals based on experts' opinions. Ideally, TEobs should be < TEa .

Methods: An online survey was sent to veterinary experts in clinical pathology and small animal internal medicine for providing the maximal allowable deviation from a given result for each variable. Percent of TEa = (allowable median deviation/clinical threshold) * 100%. Second, TEobs for 3 laser-based bench top hematology analyzers (ADVIA 2120; Sysmex XT2000iV, and CellDyn 3500) was calculated based on method validation studies published between 2005 and 2013 (n = 4). The percent TEobs = 2 * CV (%) + bias (%). The CV was derived from published studies except for the ADVIA 2120 (internal data), and bias was estimated from the regression equation.

Results: A total of 41 veterinary experts (19 diplomates, 8 residents, 10 postgraduate students, 4 anonymous specialists) responded. The proposed range of TEa was wide, but generally ≤ 20%. The TEobs was < TEa for all variables and analyzers except for canine and feline HGB (high bias, low CV) and platelet counts (high bias, high CV).

Conclusions: Overall, veterinary bench top analyzers fulfilled experts' requirements except for HGB due to method-related bias, and platelet counts due to known preanalytic/analytic issues.
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http://dx.doi.org/10.1111/vcp.12383DOI Listing
September 2016

Morphology and staining behavior of neutrophilic and eosinophilic granulocytes of the common marmoset (Callithrix jacchus).

Exp Toxicol Pathol 2016 Jun 7;68(6):335-43. Epub 2016 May 7.

Pathology Unit, German Primate Center, Leibniz-Institute for Primate Research, Göttingen, Germany.

Common marmosets (Callithrix jacchus) are frequently used as translational animal models for human diseases. However, a comparative study of cytological and histochemical detection methods as well as morphometric and ultrastructural characterization of neutrophils and eosinophils in this species is lacking. Blood samples of house dust mite sensitized and allergen challenged as well as lipopolysaccharide (LPS) challenged marmosets were analyzed with different cytological and histological staining methods. Furthermore, cell size and number of nuclear segments were compared between neutrophils and eosinophils. Electron microscopy was performed to characterize the ultrastructure of granulocytes. Of all applied cytological stains, three allowed differentiation of eosinophils and neutrophils and, thus, reliable quantification in blood smears: May-Grünwald-Giemsa stain, Congo Red and Naphthol AS-D Chloroacetate-Esterase. For histology, Hematoxylin-Eosin (H&E) could not demonstrate clear differences, whereas Sirius Red, Congo Red, and Naphthol AS-D Chloroacetate Esterase showed capable results for identification of eosinophils or neutrophils in lung tissue. Morphometry revealed that marmoset neutrophils have more nuclear segments and are slightly larger than eosinophils. Ultrastructurally, eosinophils presented with large homogeneous electron-dense granules without crystalloid cores, while neutrophils were characterized by heterogeneous granules of different size and density. Additionally, sombrero-like vesicles were detected in tissue eosinophils of atopic marmosets, indicative for hypersensitivity-related piecemeal degranulation. In conclusion, we provide a detailed overview of marmoset eosinophils and neutrophils, important for phenotypic characterization of marmoset models for human airway diseases.
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http://dx.doi.org/10.1016/j.etp.2016.05.002DOI Listing
June 2016

Impaired extracellular matrix structure resulting from malnutrition in ovariectomized mature rats.

Histochem Cell Biol 2015 Nov 26;144(5):491-507. Epub 2015 Jul 26.

Laboratory of Experimental Trauma Surgery, Justus-Liebig University, Giessen, Germany.

Bone loss is a symptom related to disease and age, which reflects on bone cells and ECM. Discrepant regulation affects cell proliferation and ECM localization. Rat model of osteoporosis (OVX) was investigated against control rats (Sham) at young and old ages. Biophysical, histological and molecular techniques were implemented to examine the underlying cellular and extracellular matrix changes and to assess the mechanisms contributing to bone loss in the context of aging and the widely used osteoporotic models in rats. Bone loss exhibited a compromised function of bone cells and infiltration of adipocytes into bone marrow. However, the expression of genes regulating collagen catabolic process and adipogenesis was chronologically shifted in diseased bone in comparison with aged bone. The data showed the involvement of Wnt signaling inhibition in adipogenesis and bone loss due to over-expression of SOST in both diseased and aged bone. Further, in the OVX animals, an integrin-mediated ERK activation indicated the role of MAPK in osteoblastogenesis and adipogenesis. The increased PTH levels due to calcium and estrogen deficiency activated osteoblastogenesis. Thusly, RANKL-mediated osteoclastogenesis was initiated. Interestingly, the data show the role of MEPE regulating osteoclast-mediated resorption at late stages in osteoporotic bone. The interplay between ECM and bone cells change tissue microstructure and properties. The involvement of Wnt and MAPK pathways in activating cell proliferation has intriguing similarities to oncogenesis and myeloma. The study indicates the importance of targeting both pathways simultaneously to remedy metabolic bone diseases and age-related bone loss.
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http://dx.doi.org/10.1007/s00418-015-1356-9DOI Listing
November 2015

Characterization of bone turnover and energy metabolism in a rat model of primary and secondary osteoporosis.

Exp Toxicol Pathol 2015 Apr 12;67(4):287-96. Epub 2015 Mar 12.

Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig University Giessen, Frankfurterstraße 126, 35392 Giessen, Germany.

Background: An experimental rat model served for evaluation of bone- and energy metabolism in early and late stages of osteoporosis. For the early stage, we hypothesized that bilateral ovariectomy (OVX)+multi-deficiency diet (OVXD; depletion of vitamin D, calcium, vitamin K, phosphorus) would induce increased bone turnover while the late stage would be characterized by enhanced bone catabolism. Obesity, insulin resistance and hyperleptinemia would be seen during the whole course of disease. Healthy female Sprague Dawley rats (n=41) aged 10 weeks were randomly assigned to sham and treatment groups and sacrificed at 3, 12, and 14 months after the study began.

Results: In the early phase, OVXD was associated with an increase in body weight, but not, however, in later stages. There was a decrease in bone mineral density and relative bone volume (BV/TV) as assessed by Dual Energy X-ray Absorptiometry and micro computed tomography that was most severe in the later stages of disease, indicating bone catabolism. Osteocalcin limiting bone formation was increased initially, whereas later stages (14 months) were characterized by elevated osteopontin, suggesting bone remodeling. Severe hyperparathyroidism was present during all stages of disease. Only the early phases of disease were characterized by hyperinsulinemia and increased adrenocorticotrophic stimulating hormone, whereas in the late stage hypoleptinemia rather than hyperleptinemia was seen.

Conclusion: Markers of bone and energy metabolism reflected both an increased bone turn over and ongoing bone remodeling associated with initial hyperinsulinemia. Osteopontin and osteocalcin can be used to differentiate early and late stages of osteoporosis.
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http://dx.doi.org/10.1016/j.etp.2015.01.004DOI Listing
April 2015

Rate of manual leukocyte differentials in dog, cat and horse blood samples using ADVIA 120 cytograms.

BMC Vet Res 2014 Jun 5;10:125. Epub 2014 Jun 5.

Department of Veterinary Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig University Giessen, Frankfurterstr, 126, Giessen 35392, Germany.

Background: Modern automated haematology instruments are capable of performing leukocyte differentials faster, cheaper and with a higher precision than the traditional 100-cell manual differential count. Thus, in human laboratories, criteria are defined for performing a manual review of the blood smear resulting in a marked reduction of manual differential counts. While common in human laboratories, this approach to reducing the number of manual differentials in veterinary laboratories is still not commonly performed. Thus, our aim was to determine the rate and causes of manual leukocyte differentials in a university clinical pathology laboratory using the automated laser-based haematology analyser ADVIA 120. Overall, 14,953 complete blood cell counts from dogs, cats and horses were reviewed. Manual leukocyte differentials were requested if abnormal ADVIA peroxidase and baso cytograms were detected (i.e. suspicion of left shift or atypical lymphocytes/blasts, inappropriate separation of cell populations).

Results: In 21% of canine, 32% of feline and 20% of equine samples, a manual differential was requested. Indistinct separation of the cell population was present in 10% to 15% of the cases. Depending on the species, atypical lymphocytes were suspected in 2% to 12%, left shift in 13% to 25% and suspicion of blasts was present in less than 0.4% of the cases.

Conclusions: The obtained results are comparable to those published for human medicine and the rate of manual differentiation could be markedly reduced in veterinary laboratories if microscopic examination was used as a validation procedure rather than as a reflexive substitute for automated differentiation.
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http://dx.doi.org/10.1186/1746-6148-10-125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053280PMC
June 2014

Bone matrix, cellularity, and structural changes in a rat model with high-turnover osteoporosis induced by combined ovariectomy and a multiple-deficient diet.

Am J Pathol 2014 Mar 31;184(3):765-77. Epub 2013 Dec 31.

Laboratory of Experimental Trauma Surgery, Justus-Liebig University, Giessen, Germany; Department of Trauma Surgery, University Hospital of Giessen-Marburg, Giessen, Germany. Electronic address:

In estrogen-deficient, postmenopausal women, vitamin D and calcium deficiency increase osteoporotic fracture risk. Therefore, a new rat model of combined ovariectomy and multiple-deficient diet was established to mimic human postmenopausal osteoporotic conditions under nutrient deficiency. Sprague-Dawley rats were untreated (control), laparatomized (sham), or ovariectomized and received a deficient diet (OVX-Diet). Multiple analyses involving structure (micro-computed tomography and biomechanics), cellularity (osteoblasts and osteoclasts), bone matrix (mRNA expression and IHC), and mineralization were investigated for a detailed characterization of osteoporosis. The study involved long-term observation up to 14 months (M14) after laparotomy or after OVX-Diet, with intermediate time points at M3 and M12. OVX-Diet rats showed enhanced osteoblastogenesis and osteoclastogenesis. Bone matrix markers (biglycan, COL1A1, tenascin C, and fibronectin) and low-density lipoprotein-5 (bone mass marker) were down-regulated at M12 in OVX-Diet rats. However, up-regulation of matrix markers and existence of unmineralized osteoid were seen at M3 and M14. Osteoclast markers (matrix metallopeptidase 9 and cathepsin K) were up-regulated at M14. Micro-computed tomography and biomechanics confirmed bone fragility of OVX-Diet rats, and quantitative RT-PCR revealed a higher turnover rate in the humerus than in lumbar vertebrae, suggesting enhanced bone formation and resorption in OVX-Diet rats. Such bone remodeling caused disturbed bone mineralization and severe bone loss, as reported in patients with high-turnover, postmenopausal osteoporosis. Therefore, this rat model may serve as a suitable tool to evaluate osteoporotic drugs and new biomaterials or fracture implants.
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http://dx.doi.org/10.1016/j.ajpath.2013.11.011DOI Listing
March 2014

Effects of multi-deficiencies-diet on bone parameters of peripheral bone in ovariectomized mature rat.

PLoS One 2013 16;8(8):e71665. Epub 2013 Aug 16.

Laboratory of Experimental Trauma Surgery, Justus-Liebig University, Giessen, Germany.

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071665PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3745426PMC
April 2014