Anne Geffré

Reference Value Advisor: a new freeware set of macroinstructions to calculate reference intervals with Microsoft Excel

International recommendations for determination of reference intervals have been recently updated, especially for small reference sample groups, and use of the robust method and Box-Cox transformation is now recommended. Unfortunately, these methods are not included in most software programs used for data analysis by clinical laboratories. We have created a set of macroinstructions, named Reference Value Advisor, for use in Microsoft Excel to calculate reference limits applying different methods. For any series of data, Reference Value Advisor calculates reference limits (with 90% confidence intervals [CI]) using a nonparametric method when n≥40 and by parametric and robust methods from native and Box-Cox transformed values; tests normality of distributions using the Anderson-Darling test and outliers using Tukey and Dixon-Reed tests; displays the distribution of values in dot plots and histograms and constructs Q-Q plots for visual inspection of normality; and provides minimal guidelines in the form of comments based on international recommendations. The critical steps in determination of reference intervals are correct selection of as many reference individuals as possible and analysis of specimens in controlled preanalytical and analytical conditions. Computing tools cannot compensate for flaws in selection and size of the reference sample group and handling and analysis of samples. However, if those steps are performed properly, Reference Value Advisor, available as freeware at http://www.biostat.envt.fr/spip/spip.php?article63, permits rapid assessment and comparison of results calculated using different methods, including currently unavailable methods. This allows for selection of the most appropriate method, especially as the program provides the CI of limits. It should be useful in veterinary clinical pathology when only small reference sample groups are available.

Estimation of reference intervals from small samples: an example using canine plasma creatinine.

BACKGROUND According to international recommendations, reference intervals should be determined from at least 120 reference individuals, which often are impossible to achieve in veterinary clinical pathology, especially for wild animals. When only a small number of reference subjects is available, the possible bias cannot be known and the normality of the distribution cannot be evaluated. A comparison of reference intervals estimated by different methods could be helpful. OBJECTIVE The purpose of this study was to compare reference limits determined from a large set of canine plasma creatinine reference values, and large subsets of this data, with estimates obtained from small samples selected randomly. METHODS Twenty sets each of 120 and 27 samples were randomly selected from a set of 1439 plasma creatinine results obtained from healthy dogs in another study. Reference intervals for the whole sample and for the large samples were determined by a nonparametric method. The estimated reference limits for the small samples were minimum and maximum, mean +/- 2 SD of native and Box-Cox-transformed values, 2.5th and 97.5th percentiles by a robust method on native and Box-Cox-transformed values, and estimates from diagrams of cumulative distribution functions. RESULTS The whole sample had a heavily skewed distribution, which approached Gaussian after Box-Cox transformation. The reference limits estimated from small samples were highly variable. The closest estimates to the 1439-result reference interval for 27-result subsamples were obtained by both parametric and robust methods after Box-Cox transformation but were grossly erroneous in some cases. CONCLUSION For small samples, it is recommended that all values be reported graphically in a dot plot or histogram and that estimates of the reference limits be compared using different methods.

A new approach for the determination of reference intervals from hospital-based data

BACKGROUND Reference limits are some of the most widely used tools in the medical decision process. Their determination is long, difficult, and expensive, mainly because of the need to select sufficient numbers of reference individuals according to well-defined criteria. Data from hospitalized patients are, in contrast, numerous and easily available. Even if all the information required for a direct reference interval computation is usually not available, these data contain information that can be exploited to derive at least rough reference intervals. METHODS In this article, we propose a method for the indirect estimation of reference intervals. It relies on a statistical method which has become a gold-standard in other sciences to separate components of mixtures. It relies on some distributional assumptions that can be checked graphically. For the determination of reference intervals, this new method is intended to separate the healthy and diseased distributions of the measured analyte. We assessed its performance by using simulated data drawn from known distributions and two previously published datasets (from human and veterinary clinical chemistry). RESULTS AND DISCUSSION The comparison of results obtained by the new method with the theoretical data of the simulation and determination of the reference interval for the datasets was good, thus supporting the application of this method for a rough estimation of reference intervals when the recommended procedure cannot be used.

Canine reference intervals for the Sysmex XT-2000iV hematology analyzer

BACKGROUND The laser-based Sysmex XT-2000iV hematology analyzer is increasingly used in veterinary clinical pathology laboratories, and instrument-specific reference intervals for dogs are not available. OBJECTIVE The purpose of this study was to establish canine hematologic reference intervals according to International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute guidelines using the Sysmex XT-2000iV hematology analyzer. METHODS Blood samples from 132 healthy purebred dogs from France, selected to represent the most prevalent canine breeds in France, were analyzed. Blood smears were scored for platelet (PLT) aggregates. Reference intervals were established using the nonparametric method. PLT and RBC counts obtained by impedance and optical methods were compared. Effects of sex and age on reference intervals were determined. RESULTS The correlation between impedance (I) and optical (O) measurements of RBC and PLT counts was excellent (Pearson r=.99 and .98, respectively); however, there were significant differences between the 2 methods (Students paired t-test, P<.0001). Differences between sexes were not significant except for HCT, PLT-I, and PLT-O. WBC, lymphocyte, and neutrophil counts decreased significantly with age (ANOVA, P<.05). Median eosinophil counts were higher in Brittany Spaniels (1.87 × 10(9) /L), Rottweilers (1.41 × 10(9) /L), and German Shepherd dogs (1.38 × 10(9) /L) than in the overall population (0.9 × 10(9) /L). PLT aggregates were responsible for lower PLT counts by the impedance, but not the optical, method. CONCLUSION Reference intervals for hematologic analytes and indices were determined under controlled preanalytical and analytical conditions for a well-characterized population of dogs according to international recommendations.

Comparison of a new device for blood sampling in cats with a vacuum tube collection system—Plasma biochemistry, haematology and practical usage assessment

Using paediatric devices to collect venous blood from a cephalic vein in cats offers numerous practical advantages over traditional jugular venepuncture and vacuum closed systems: minimal restraint is required; there is minimal risk of serious injury to the cat; the discomfort associated with venepuncture is reduced by the use of small diameter (25 gauge) needles; very small volumes (200 μl) of blood are extracted; and the risk of vein collapse or haematoma is low. The aim of this study was to compare the haematological and plasma chemistry results obtained from six healthy cats using the two sampling techniques. Five plasma biochemical analytes were measured and a complete haematological examination was performed on each specimen. No clinically relevant difference between the two blood sampling techniques was observed for any variable, indicating that paediatric devices provide a useful alternative to vacuum tubes for venous blood collection in the cat.

Comparison of Platelet Clumping and Complete Blood Count Results with Sysmex XT-2000iV in Feline Blood Sampled on EDTA or EDTA plus CTAD (Citrate, Theophylline, Adenosine and Dipyridamole)

False thrombocytopenia may result from platelet aggregation, especially in feline ethylenediamine tetra-acetic acid (EDTA) blood specimens. Citrate, theophylline, adenosine and dipyridamole (CTAD) was added to 46 feline EDTA specimens to test its anti-aggregation action. Platelet aggregation was estimated from blood films and a complete blood count was performed with a Sysmex XT-2000iV analyser. Platelet aggregation score was >2 in 11/46 EDTA tubes and only in one EDTA + CTAD specimen. The platelet count was higher in all CTAD-supplemented tubes except one, medians measured by cytometry being 225.5 times 109/1 and 249.0 times 109/1 in EDTA and EDTA + CTAD, respectively (P = 0.007). Adding CTAD had statistically and analytically significant but moderate effects on other blood variables, the most intense variations being observed for reticulocytes (about 3% higher in EDTA specimens) and reticulocyte indexes. Addition of CTAD to EDTA when sampling feline blood is a useful option to reduce platelet clumping.

Effects of intravenous, low-dose ketamine-diazepam sedation on the results of hematologic, plasma biochemical, and coagulation analyses in cats

OBJECTIVE To evaluate the effects of an IV, low-dose ketamine-diazepam combination used for short-duration chemical restraint on the results of clinicopathologic testing in cats and to assess its practicality and tolerance. DESIGN Prospective case series. ANIMALS 42 client-owned cats of various breeds, ages, and health status. PROCEDURES Blood samples were obtained just prior to and just after IV injection of ketamine chlorhydrate (10 mg) and diazepam (0.5 mg). A CBC, plasma biochemistry panel, and coagulation profile were performed on each sample (ie, before and after chemical restraint). Practicality of the procedure was assessed, and cats were monitored for immediate and delayed effects. RESULTS Significant changes were observed for most of the analytes tested. However, the magnitude of the observed changes was notably low and likely not of clinical relevance. The chemical-restraint procedure appeared effective, safe, and well tolerated. CONCLUSIONS AND CLINICAL RELEVANCE The IV, low-dose ketamine-diazepam combination used for short-duration chemical restraint in the present study may be suitable to assist physical restraint for blood sampling for assessment of hematologic, serum biochemical, and coagulation parameters in cats.

Canine Reference Intervals for Coagulation Markers Using the STA Satellite® and the STA-R Evolution® Analyzers

The aim of the current study was to determine canine reference intervals for prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, and antithrombin (AT) according to international recommendations. The STA Satellite® coefficients of variation of within-laboratory imprecision were 3.9%, 1.3%, 6.9%, and 5.1% for PT, APTT, fibrinogen, and AT, respectively. At 4°C, citrated specimens were stable up to 8 hr for whole blood and 36 hr for plasma, except for APTT, which increased slightly (<1 sec). Nonparametric reference intervals determined in citrated plasma from 139 healthy fasting purebred dogs were 6.9–8.8 sec, 13.1–17.2 sec, 1.24–4.30 g/l, and 104–188% for PT, APTT, fibrinogen, and AT, respectively. Based on Passing–Bablok comparison between STA Satellite and STA-R Evolution® using 60 frozen specimens from a canine plasma bank, the corresponding reference intervals were transferred to the STA-R Evolution: 7.1–9.2 sec, 12.9–17.3 sec, 1.20–4.43 g/l, and 94–159% for PT, APTT, fibrinogen, and AT, respectively.

Changes in hematology measurements in healthy and diseased dog blood stored at room temperature for 24 and 48 hours using the XT‐2000iV analyzer

BACKGROUND Changes in canine hematology measurements may occur when analyses are delayed due to shipment of specimens to a laboratory. OBJECTIVE The purpose of this study was to report changes in hematologic variables in healthy and diseased canine blood measured with a Sysmex XT-2000iV during storage at room temperature for 24 and 48 hours. METHODS EDTA-K3 blood samples from 42 healthy and diseased dogs were measured on a Sysmex XT-2000iV analyzer within one hour of sampling, and after storage for 24 and 48 hours at room temperature in the dark. RESULTS Storage caused little or no change in RBC count, HGB concentration and MCH, while there was a moderate increase in HCT, MCV and reticulocytes count, and a moderate decrease in MCHC. Decreased platelet counts by impedance (PLT-I) and optical (PLT-O) measurements were associated with increased mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet distribution width (PDW), including a right shift in the platelet histogram and a dispersion of the platelet dot plot on the scattergram. The total and differential WBC count remained stable except for decreased monocyte counts. In the scatterplots, monocytes shifted into the lymphocyte population after 24 hours, and neutrophil population shifted to the right appearing in the eosinophil gate at 48 hours of storage. The disease status had only a small effect on storage-induced changes, and observed changes had no consequences for clinical decisions. CONCLUSIONS Blood storage at room temperature was accompanied by moderate variations in some hematologic variables, awareness of which helps in avoiding misinterpretations.

Validation of the Medonic CA620/530 Vet 20-μ1 Microcapillary Sampler System for Hematology Testing of Feline Blood

The aim of the current study was to compare feline hematologic variables in blood collected in microcapillary tubes (20 μl) and conventional blood tubes with the Medonic CA620/530 Vet in-house hematologic analyzer. A comparison of results obtained in 60 cats presented at the clinics of the veterinary school showed that the correlations between the 2 methods were 0.97 for white blood cell, 0.95 for red blood cell, and 0.93 for platelet counts; 0.92 for hemoglobin concentration; and 0.99 for mean corpuscular volume. No clinically relevant differences between the 2 blood sampling techniques were observed for any variable, which suggests that both techniques are interchangeable in cats. Moreover, microcapillary tubes would allow easier repeated sampling in the same cat and would likely be useful in other small species.