HEMATOLOGY AND BIOCHEMICAL REFERENCE INTERVALS FOR FREE-RANGING PRONGHORN (ANTILOCAPRA AMERICANA) IN WEST TEXAS.

Pronghorn (Antilocapra americana) are considered a keystone species of North American grasslands and an important economic source for many landowners in Texas. Pronghorn restoration projects routinely capture and translocate individuals from surplus populations to restoration areas. The objective of this study was to generate normal hematological and biochemical reference intervals (RI) for free-ranging pronghorn populations in Texas as a health monitoring tool for pronghorn restoration efforts. Blood samples were collected by jugular venipuncture and divided among an EDTA tube, serum separator tube, and a single blood smear on site. Complete blood counts and biochemical profiles were completed at the Texas Veterinary Medical Diagnostic Laboratory. In total, 417 individuals (41 males, 376 females) were included in the analysis. RI were determined by robust methods (R Studio) and mixed models' analysis of variance (SPSS 28) to examine differences in blood parameters due to fever, sex, age (adult versus yearling [<1 yr of age]), cell abnormalities, and pathogen exposure reported by the testing laboratory. Sex, age, and pathogen exposure affected mean blood values, but did not warrant development of separate RI by class. Bluetongue virus was identified in 46.8% (195/417) of pronghorns and epizootic hemorrhagic disease in 89.4% (194/217) of pronghorns; 84.8% (184/217) of the pronghorns tested positive for both diseases. This information provides baseline hematology and biochemical parameters to assess the health of free-ranging pronghorn and guide wildlife managers in decision-making for future translocations and restoration objectives.

HEMATOLOGY AND BIOCHEMISTRY REFERENCE INTERVALS FOR FREE-LIVING ADULT LESSER KESTRELS (FALCO NAUMANNI) IN SOUTHERN ITALY.

The lesser kestrel (Falco naumanni) is a small falcon with a Euro-Central and Asian-Mediterranean range wintering in sub-Saharan Africa. In the second half of the 20th century, the European population experienced a steep decline and was classified as at risk; thus, its biological and ecological aspects have been widely investigated. Nonetheless, data on hematology and plasma chemistry are not yet available. Therefore, hematology and biochemistry parameters were investigated in a sampling population of clinically healthy lesser kestrels (21 female and 10 male adults) from an Italian rescue center during breeding season, estimating the 95% (2.5 - 97.5th percentile) reference intervals (RI) for standard tests based on either parametric or robust statistical methods. The effect of sex on the referenced parameters was also tested and showed no statically significant differences. The established 95% RI highlighted values comparable with those of other similar raptors such as American kestrel (Falco sparverius) and peregrine falcon (Falco peregrinus). As the first recorded hematology and serum chemistry RI, these clinical data could support conservation efforts and clarify the effects of various environmental and ecological factors on the clinical and health status of lesser kestrels, although they should be reinforced with further data from healthy wild animals.

HEMATOLOGY AND BIOCHEMISTRY REFERENCE VALUES IN NESTLING RED KITES (MILVUS MILVUS) IN SHORT-TERM HUMAN CARE IN ENGLAND.

Between the years 2022 and 2023, 62 red kite (Milvus milvus) nestlings were translocated from England to Spain to bolster declining populations in mainland Europe as part of a wider conservation initiative. Health examinations were undertaken by veterinarians ahead of translocation, including examination of hematology and biochemistry parameters from blood samples. This study aimed to establish reference values for these parameters in nestling red kites for use in future translocations or for other clinical purposes. All individuals included in the analysis were clinically healthy at the time of sampling. Biochemical reference intervals were comparable to published values for other Accipitridae, although differences in hematology were noted: PCV was generally lower; and WBC counts higher than (up to triple) those reported for related species of a similar age. It is hypothesized that these differences reflect species variations or the effects of the stress of recent capture on the immune system of the red kites. A Leucocytozoon species was identified on blood smears of six of the red kites. The reference intervals presented in this study are representative of free-living red kite nestlings in England that have recently been captured for conservation translocation purposes.

HEMATOLOGIC AND PLASMA BIOCHEMICAL REFERENCE VALUES FOR CAPTIVE LEOPARD SHARKS (TRIAKIS SEMIFASCIATA).

Leopard sharks (Triakis semifasciata) are temperate, Eastern Pacific elasmobranchs popular in public aquariums. Blood analysis is commonly used for assessing animal health, yet reference values have not been established for this species. This study analyzed T. semifasciata population data to characterize blood reference values for a collection of T. semifasciata housed at a public aquarium. Twenty-one captive leopard sharks were sampled. Blood was collected during annual health examinations from sedated animals. After collection, blood samples were anticoagulated with lithium heparin, and hematocrit and plasma biochemistry values were analyzed. The minimum-maximum ranges were hematocrit 11-31%, buffy coat 1-2%, glucose 4.94-9.38 mM/L, sodium 244-272 mM/L, potassium 3.7-5.5 mM/L, chloride 214-246 mM/L, aspartate aminotransferase 5-31 U/L, creatine kinase 36-1,136 U/L, calcium 3.65-3.95 mM/L, phosphorus 1.13-2.23 mM/L, total protein 21-38 g/L, and total CO2 12-18 mM/L. The values identified will contribute to a better understanding of captive leopard shark physiology and to improved veterinary care for captive leopard sharks. Further research can examine the validity of machines like the Vetscan VS2, which will expand the resources available to care professionals.

Diagnostic role of Sysmex hematology analyzer in the detection of malaria: A systematic review and meta-analysis.

BACKGROUND: Malaria control depends primarily on rapid and accurate diagnosis followed by successful treatment. Light microscopy is still used as a gold standard method for the diagnosis of malaria. The Sysmex hematology analyzer is a novel method for malaria detection. Therefore, the aim of this review was to investigate the diagnostic accuracy of the Sysmex hematology analyzer for malaria diagnosis. METHODS: Electronic databases like PubMed, PubMed Central, Science Direct databases, Google Scholar, and Scopus were used to find relevant articles from April to June 14, 2023. The studies' methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. Using Review Manager 5.4.1, the estimates of sensitivity and specificity, as well as their 95% confidence intervals, were shown in forest plots. Midas software in Stata 14.0 was utilized to calculate the summary estimates of sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio. Heterogeneity was assessed by using I2 statistics. In addition, publication bias was assessed using a funnel plot and Deeks' test. Sub-group and meta- regression analysis were also performed. RESULTS: A total of 15 studies were assessed for diagnostic accuracy. The sensitivity and specificity of Sysmex hematology analyzer for studies ranged from 46% to 100% and 81% to 100%, respectively. The summary estimate of sensitivity and specificity of Sysmex hematology analyzer were 95% (95% CI: 85%-99%) and 99% (95% CI: 97%-100%), respectively. It had excellent diagnostic accuracy. There were significant heterogeneity among the studies included in this meta-analysis. The summary estimate of sensitivity and specificity of Sysmex hematology analyzer using polymerase chain reaction as the gold standard was 97.6% (95% CI: 83.2, 99.7) and 99.4% (98.5, 99.8), respectively. CONCLUSION: In this review, Sysmex hematology analyzer had excellent diagnostic accuracy. Therefore, it could be used as an alternate diagnostic tool for malaria diagnosis in the hospital and health center. TRIAL REGISTRATION: Systematic review registration PROSPERO (2023: CRD42023427713). https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023427713.

Advances in Hematology Analyzers Technology.

The evolution of complete blood count (CBC) methodology from manual calculations to sophisticated high throughput hematology analyzers is the focus of this article. In recent years, hematology testing has greatly benefitted from the combination of various technologies with automated neural networks. In addition to an increasing complexity of the laboratory instrumentation, there is a demand on point of care CBC testing with its benefits and drawbacks. This article highlights exciting advancements of hematology testing from the past to the present and into the future.

Bayesian hierarchical modeling for bivariate multiscale spatial data with application to blood test monitoring.

Public health spatial data are often recorded at different spatial scales (or geographic regions/divisions) and over different correlated variables. Motivated by data from the Dartmouth Atlas Project, we consider jointly analyzing average annual percentages of diabetic Medicare enrollees who have taken the hemoglobin A1c and blood lipid tests, observed at the hospital service area (HSA) and county levels, respectively. Capitalizing on bivariate relationships between these two scales is not immediate as counties are not nested within HSAs. It is well known that one can improve predictions by leveraging correlations across both variables and scales. There are very few methods available that simultaneously model multivariate and multiscale correlations. We propose three new hierarchical Bayesian models for bivariate multiscale spatial data, extending spatial random effects, multivariate conditional autoregressive (MCAR), and ordered hierarchical models through a multiscale spatial approach. We simulated data from each of the three models and compared the corresponding predictions, and found the computationally intensive multiscale MCAR model is more robust to model misspecification. In an analysis of 2015 Texas Dartmouth Atlas Project data, we produced finer resolution predictions (partitioning of HSAs and counties) than univariate analyses, determined that the novel multiscale MCAR and OH models were preferable via out-of-sample metrics, and determined the HSA with the highest within-HSA variability of hemoglobin A1c blood testing. Additionally, we compare the univariate multiscale models to the bivariate multiscale models and see clear improvements in prediction over univariate analyses.

Changes in Red Cell Morphology and Haematological Laboratory Parameters Associated With Alectinib.

BACKGROUND: Alectinib is a second-generation anaplastic lymphoma kinase (ALK) inhibitor indicated for ALK-mutated non-small-cell lung cancer. Recently, the association between alectinib and red cell morphological abnormalities has been reported in a few case series. This retrospective observational study aims to determine the frequency of occurrence of acanthocytosis in patients taking alectinib and to evaluate the red cell indices, biochemical markers of haemolysis and eosin-5-maleimide (EMA) binding assay results in patients receiving alectinib. METHODS: Patients who were on alectinib and had a complete blood count test performed in Queen Elizabeth Hospital Haematology Laboratory between 1 May 2021 and 31 August 2021 were included in the study. Haematological investigations that had been performed before and after the commencement of alectinib were reviewed. RESULTS: Fifty patients receiving alectinib were evaluated in this analysis. One hundred per cent of patients showed 3+ acanthocytes on the peripheral blood smears. Compared with the test results before starting alectinib, the post-alectinib blood tests showed a significantly lower haemoglobin concentration, red blood cell count and haematocrit; and a significantly higher mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and red cell distribution width. All the tested patients showed a marked reduction in EMA mean channel fluorescence compared with normal control. CONCLUSION: Our cohort revealed that alectinib caused significant acanthocytosis in all patients. Alectinib was also associated with changes in red cell indices and biochemical markers of haemolysis, compatible with a spherocytic and anisopoikilocytic morphology with haemolysis. Patients on alectinib had reduced EMA binding.

Admission blood tests predicting survival of SARS-CoV-2 infected patients: a practical implementation of graph convolution network in imbalance dataset.

BACKGROUND: Predicting an individual's risk of death from COVID-19 is essential for planning and optimising resources. However, since the real-world mortality rate is relatively low, particularly in places like Hong Kong, this makes building an accurate prediction model difficult due to the imbalanced nature of the dataset. This study introduces an innovative application of graph convolutional networks (GCNs) to predict COVID-19 patient survival using a highly imbalanced dataset. Unlike traditional models, GCNs leverage structural relationships within the data, enhancing predictive accuracy and robustness. By integrating demographic and laboratory data into a GCN framework, our approach addresses class imbalance and demonstrates significant improvements in prediction accuracy. METHODS: The cohort included all consecutive positive COVID-19 patients fulfilling study criteria admitted to 42 public hospitals in Hong Kong between January 23 and December 31, 2020 (n = 7,606). We proposed the population-based graph convolutional neural network (GCN) model which took blood test results, age and sex as inputs to predict the survival outcomes. Furthermore, we compared our proposed model to the Cox Proportional Hazard (CPH) model, conventional machine learning models, and oversampling machine learning models. Additionally, a subgroup analysis was performed on the test set in order to acquire a deeper understanding of the relationship between each patient node and its neighbours, revealing possible underlying causes of the inaccurate predictions. RESULTS: The GCN model was the top-performing model, with an AUC of 0.944, considerably outperforming all other models (p < 0.05), including the oversampled CPH model (0.708), linear regression (0.877), Linear Discriminant Analysis (0.860), K-nearest neighbours (0.834), Gaussian predictor (0.745) and support vector machine (0.847). With Kaplan-Meier estimates, the GCN model demonstrated good discriminability between low- and high-risk individuals (p < 0.0001). Based on subanalysis using the weighted-in score, although the GCN model was able to discriminate well between different predicted groups, the separation was inadequate between false negative (FN) and true negative (TN) groups. CONCLUSION: The GCN model considerably outperformed all other machine learning methods and baseline CPH models. Thus, when applied to this imbalanced COVID survival dataset, adopting a population graph representation may be an approach to achieving good prediction.

Reform of teaching and practice of the integrated teaching method BOPPPS-PBL in the course "clinical haematological test technique".

BACKGROUND: In order to meet the demand for laboratory talents in the clinical laboratory industry and address the current curriculum characteristics and shortcomings of the teaching mode of "Clinical Hematology Laboratory Technology", we investigated the effectiveness of the bridge-in, objective, pre-assessment, participatory learning, post-assessment, and summary model combined with problem-based learning (BOPPPS-PBL) in undergraduate teaching of this course. METHOD: Seventy students majoring in Medical Laboratory Technology from the Army Medical University in the past 5 years have been selected and divided into two groups with the same teaching content and time. The control group (2015 and 2016 grades) used traditional teaching methods, while the experimental group (2017, 2018 and 2019 grades) used the BOPPPS-PBL model. After class, diverse evaluation methods were used to analyze the formative and summative exam scores of the two groups of students. RESULTS: After the reform, students performed significantly better in exams than before. In addition, the new teaching methods have had a positive impact, with students demonstrating high motivation for self-directed learning and problem-solving abilities. CONCLUSION: Compared to traditional teaching methods. The BOPPPS-PBL integrated case study education model is a relatively effective teaching method to improve students' problem-solving ability and comprehensive practical ability.

Predictive value of abnormal blood tests for detecting cancer in primary care patients with nonspecific abdominal symptoms: A population-based cohort study of 477,870 patients in England.

BACKGROUND: Identifying patients presenting with nonspecific abdominal symptoms who have underlying cancer is a challenge. Common blood tests are widely used to investigate these symptoms in primary care, but their predictive value for detecting cancer in this context is unknown. We quantify the predictive value of 19 abnormal blood test results for detecting underlying cancer in patients presenting with 2 nonspecific abdominal symptoms. METHODS AND FINDINGS: Using data from the UK Clinical Practice Research Datalink (CPRD) linked to the National Cancer Registry, Hospital Episode Statistics and Index of Multiple Deprivation, we conducted a population-based cohort study of patients aged ≥30 presenting to English general practice with abdominal pain or bloating between January 2007 and October 2016. Positive and negative predictive values (PPV and NPV), sensitivity, and specificity for cancer diagnosis (overall and by cancer site) were calculated for 19 abnormal blood test results co-occurring in primary care within 3 months of abdominal pain or bloating presentations. A total of 9,427/425,549 (2.2%) patients with abdominal pain and 1,148/52,321 (2.2%) with abdominal bloating were diagnosed with cancer within 12 months post-presentation. For both symptoms, in both males and females aged ≥60, the PPV for cancer exceeded the 3% risk threshold used by the UK National Institute for Health and Care Excellence for recommending urgent specialist cancer referral. Concurrent blood tests were performed in two thirds of all patients (64% with abdominal pain and 70% with bloating). In patients aged 30 to 59, several blood abnormalities updated a patient's cancer risk to above the 3% threshold: For example, in females aged 50 to 59 with abdominal bloating, pre-blood test cancer risk of 1.6% increased to: 10% with raised ferritin, 9% with low albumin, 8% with raised platelets, 6% with raised inflammatory markers, and 4% with anaemia. Compared to risk assessment solely based on presenting symptom, age and sex, for every 1,000 patients with abdominal bloating, assessment incorporating information from blood test results would result in 63 additional urgent suspected cancer referrals and would identify 3 extra cancer patients through this route (a 16% relative increase in cancer diagnosis yield). Study limitations include reliance on completeness of coding of symptoms in primary care records and possible variation in PPVs if extrapolated to healthcare settings with higher or lower rates of blood test use. CONCLUSIONS: In patients consulting with nonspecific abdominal symptoms, the assessment of cancer risk based on symptoms, age and sex alone can be substantially enhanced by considering additional information from common blood test results. Male and female patients aged ≥60 presenting to primary care with abdominal pain or bloating warrant consideration for urgent cancer referral or investigation. Further cancer assessment should also be considered in patients aged 30 to 59 with concurrent blood test abnormalities. This approach can detect additional patients with underlying cancer through expedited referral routes and can guide decisions on specialist referrals and investigation strategies for different cancer sites.

Hematology and serum biochemistry of free-ranging mantled howler monkeys (Alouatta palliata) at La Pacifica, Costa Rica.

BACKGROUND: Hematologic and blood biochemical values are key tools for assessing primate health. A long-term behavioral study of howler monkeys at a single site (La Pacífica, Guanacaste, Costa Rica), afforded the opportunity to develop baseline values for a large group of animals, evaluating differences between adult males and females and comparing to a report in the same population two decades later. METHODS: In 1998, 64 free-ranging mantled howler monkeys were anesthetized and sampled for hematologic and biochemical analysis. RESULTS: Blood analysis is reported for 29 adult females, 9 juvenile females, 19 adult males and 3 juvenile males. Four adults were excluded due to external injury or disease. There were few significant differences between adult females, juvenile females, and adult males. CONCLUSIONS: Baseline blood parameters are useful for determining normal values for howler monkey populations. The values for total protein, blood urea nitrogen, glucose, liver enzymes and potassium differed from a later study in 2019 may indicate changes that are influencing howler monkey health.

Patient safety in actioning and communicating blood test results in primary care: a UK wide audit using the Primary care Academic CollaboraTive (PACT).

BACKGROUND: Errors associated with failures in filing, actioning and communicating blood test results can lead to delayed and missed diagnoses and patient harm. This study aimed to audit how blood tests in primary care are filed, actioned and communicated in primary care, to identify areas for patient safety improvements. METHODS: UK primary care clinicians were recruited through the Primary Care Academic CollaboraTive (PACT). PACT members audited 50 recent sets of blood tests from their practice and retrospectively extracted data on blood test result coding, actioning and communication. PACT members received a practice report, showing their own results, benchmarked against other participating practices. RESULTS: PACT members from 57 general practices across all four UK nations collected data on 2572 patients who had blood tests in April 2021. In 89.9% (n=2311) they agreed with the initial clinician's actioning of blood tests; 10.1% disagreed, either partially (7.1%) or fully (3.0%).In 44% of patients (n=1132) an action (eg, 'make an appointment') was specified by the filing clinician. This action was carried out in 89.7% (n=1015/1132) of cases; in 6.8% (n=77) the action was not carried out, in 3.5% (n=40) it was unclear. In the 117 cases where the test result had not been actioned 38% (n=45) were felt to be at low risk of harm, 1.7% (n=2) were at high risk of harm, 0.85% (n=1) came to harm.Overall, in 47% (n=1210) of patients there was no evidence in the electronic health records that results had been communicated. Out of 1176 patients with one or more abnormal results there was no evidence of test communication in 30.6% (n=360). There were large variations between practices in rates of actioning and communicating tests. CONCLUSION: This research demonstrates variation in the way blood test results are actioned and communicated, with important patient safety implications.

Trends in primary care blood tests prior to lung and colorectal cancer diagnosis-A retrospective cohort study using linked Australian data.

INTRODUCTION: Abnormal results in common blood tests may occur several months before lung cancer (LC) and colorectal cancer (CRC) diagnosis. Identifying early blood markers of cancer and distinct blood test signatures could support earlier diagnosis in general practice. METHODS: Using linked Australian primary care and hospital cancer registry data, we conducted a cohort study of 855 LC and 399 CRC patients diagnosed between 2001 and 2021. Requests and results from general practice blood tests (six acute phase reactants [APR] and six red blood cell indices [RBCI]) were examined in the 2 years before cancer diagnosis. Poisson regression models were used to estimate monthly incidence rates and examine pre-diagnostic trends in blood test use and abnormal results prior to cancer diagnosis, comparing patterns in LC and CRC patients. RESULTS: General practice blood test requests increase from 7 months before CRC and 6 months before LC diagnosis. Abnormalities in many APR and RBCI tests increase several months before cancer diagnosis, often occur prior to or in the absence of anaemia (in 51% of CRC and 81% of LC patients with abnormalities), and are different in LC and CRC patients. CONCLUSIONS: This study demonstrates an increase in diagnostic activity in Australian general practice several months before LC and CRC diagnosis, indicating potential opportunities for earlier diagnosis. It identifies blood test abnormalities and distinct signatures that are early markers of LC and CRC. If combined with other pre-diagnostic information, these blood tests have potential to support GPs in prioritising patients for cancer investigation of different sites to expedite diagnosis.

Defining blood hematology reference values in female pig-tailed macaques (Macaca nemestrina) using the Isolation Forest algorithm.

BACKGROUND: Pig-tailed macaques (PTMs) are commonly used as preclinical models to assess antiretroviral drugs for HIV prevention research. Drug toxicities and disease pathologies are often preceded by changes in blood hematology. To better assess the safety profile of pharmaceuticals, we defined normal ranges of hematological values in PTMs using an Isolation Forest (iForest) algorithm. METHODS: Eighteen female PTMs were evaluated. Blood was collected 1-24 times per animal for a total of 159 samples. Complete blood counts were performed, and iForest was used to analyze the hematology data to detect outliers. RESULTS: Median, IQR, and ranges were calculated for 13 hematology parameters. From all samples, 22 outliers were detected. These outliers were excluded from the reference index. CONCLUSIONS: Using iForest, we defined a normal range for hematology parameters in female PTMs. This reference index can be a valuable tool for future studies evaluating drug toxicities in PTMs.

Donor Blood Tests do Not Predict Pancreas Graft Survival After Simultaneous Pancreas Kidney Transplantation; a National Cohort Study.

Simultaneous pancreas-kidney (SPK) transplantation improves quality of life and limits progression of diabetic complications. There is reluctance to accept pancreata from donors with abnormal blood tests, due to concern of inferior outcomes. We investigated whether donor amylase and liver blood tests (markers of visceral ischaemic injury) predict pancreas graft outcome using the UK Transplant Registry (2016-2021). 857 SPK recipients were included (619 following brainstem death, 238 following circulatory death). Peak donor amylase ranged from 8 to 3300 U/L (median = 70), and this had no impact on pancreas graft survival when adjusting for multiple confounders (aHR = 0.944, 95% CI = 0.754-1.81). Peak alanine transaminases also did not influence pancreas graft survival in multivariable models (aHR = 0.967, 95% CI = 0.848-1.102). Restricted cubic splines were used to assess associations between donor blood tests and pancreas graft survival without assuming linear relationships; these confirmed neither amylase, nor transaminases, significantly impact pancreas transplant outcome. This is the largest, most statistically robust study evaluating donor blood tests and transplant outcome. Provided other factors are acceptable, pancreata from donors with mild or moderately raised amylase and transaminases can be accepted with confidence. The use of pancreas grafts from such donors is therefore a safe, immediate, and simple approach to expand the donor pool to reach increasing demands.

Indirect Determination of Hematological Reference Intervals in Healthy Moroccan Adults Using Outpatient Data.

BACKGROUND: Reference intervals are essential for the interpretation of clinical laboratory tests and patient management. This study aims to determine age and gender reference intervals of complete blood count (CBC) in the Moroccan population by using the indirect approach. METHODS: The study used data of ostensibly healthy adults collected retrospectively using the laboratory information system (LIS) of the Laboratory for Research and Medical Analysis of the Fraternal Royal Gendarmerie in Rabat (Morocco), between January 2018 and February 2020. The study included 5,898 men and 10,172 women ranging in age from 18 to 90 years. The lower and upper reference limits of CBC parameters were calculated using the nonparametric technique, as suggested by the Clinical and Laboratory Standards Institute (CLSI). RESULTS: All hematological parameters showed no clinically significant gender-related differences, except small differences in the values of hemoglobin (HB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). There were also no clinically significant agerelated differences for median values of all hematology analytes in both genders, except for platelet count (PLT) that continued to decline with increasing age in men and women, and Red blood cell count (RBC), Hematocrit (HCT), and hemoglobin (HB) that tended to increase with age but decrease in older age groups in men while they tended to increase with age in women. CONCLUSIONS: The indirect method can be used to establish reference intervals for CBC, with appropriate selection criteria and statistical tools. Our findings differed from the reference ranges provided in the textbook and also in other countries' reports.

Baseline haematological parameters in three common Australian frog species.

Amphibians are experiencing declines globally, with emerging infectious diseases as one of the main causes. Haematological parameters present a useful method for determining the health status of animals and the effects of particular diseases, but the interpretation of differential cell counts relies on knowing the normal ranges for the species and factors that can affect these counts. However, there is very little data on either normal haematological parameters or guides for blood cell types for free-ranging frog species across the world. This study aims to 1) create a visual guide for three different Australian frog species: Litoria paraewingi, Limnodynastes dumerilii, and Crinia signifera, 2) determine the proportions of erythrocytes to leukocytes and 3) differential leukocytes within blood smears from these three species and 4) assess the association between parasites and differential counts. We collected blood samples from free-ranging frogs and analysed blood smears. We also looked for ectoparasites and tested for the fungal disease chytridiomycosis. Overall, we found that the differentials of erythrocytes to leukocytes were not affected by species, but the proportions of different leukocytes did vary across species. For example, while lymphocytes were the most common type of leukocyte across the three species, eosinophils were relatively common in Limnodynastes dumerilii but rarely present in the other two species. We noted chytridiomycosis infection as well as ectoparasites present in some individuals but found no effect of parasites on blood parameters. Our results add baseline haematological parameters for three Australian frog species and provide an example of how different frog species can vary in their differential blood cell counts. More information is needed on frog haematological data before these parameters can be used to determine the health status of wild or captive frogs.