Hämatologische Geräte Für Das Praxislabor--übersicht und Neue Entwicklungen
World Small Animal Veterinary Association World Congress Proceedings, 2010
Philadelphia, PA, USA

Es ist kaum mehr denkbar ohne Labordiagnostik zu praktizieren. Untersuchungen von Blut, Harn und andern Flüssigkeiten, sind erforderlich um Krankheiten zu diagnostizieren. Sie werden heute auch vielfach als Routineuntersuchung, präoperativ und zur Therapie-überwachung angewandt. Bis vor Kurzem wurden Untersuchungen meistens im Referenzlabor durchgeführt, doch werden immer mehr Kits und Geräte für das Praxislabor angeboten. All diese Methoden im Referenz- aber auch im Praxislabor, wurden zunächst für den Menschen entwickelt und mussten dann für unsere Kleintierarten adaptiert und validiert werden. Die Zahl der hämatologischen Labortests und Geräte ist zu gross um sie hier alle detailliert zu besprechen. Allein ein Dutzend Hämatologie-Geräte zur Bestimmung des Blutstatus stehen zur Verfügung, dazu kommen Kits und Geräte für Blutchemie, Harnanalyse, Infektionen, Koagluation, Thrombozytenfunktion, Bluttypisierung, etc. Deshalb werden hier Grundprinzipien bei der Selektion und Anwendung anhand von Beispielen gezeigt. Obschon der nachfolgende Text auf Englisch geschrieben ist, wird der Vortrag auf Deutsch gehalten.

Editor's Note: The rest of the text is duplicate of: "Point-of-Care Hematological Diagnostics and the Practice Laboratory"

A major advancement in veterinary medicine is the use of laboratory tests for diagnosing and managing animals' health and disease. Laboratory testing of blood, urine, and other bodily fluids has become standard for sick patients, and many of these tests are also used for regular health screenings, preoperative evaluations, and for monitoring animals' responses to therapy. Just a couple of decades ago, laboratory testing was rarely performed, and in-clinic testing was very limited. A few human hospital laboratories offered some form of extended testing of samples from animals, and soon many commercial, university, and governmental reference veterinary laboratories were established to screen samples from animals and cover multiple veterinary clinics in many different regions. Furthermore, laboratory testing opportunities have been specifically developed or adopted and validated for various animal species. Finally, many techniques initially used in reference laboratories are now available to small animal clinics by advances in technology and the development of smaller, less expensive, in-clinic instruments. Indeed, today's veterinary clinicians often have the choice of performing many tests in their own internal laboratory or sending their samples to several external veterinary reference laboratories. Both offer advantages and limitations in the speed, accuracy of test results, and support provided. Furthermore, there are often several instrument options to choose from, each offering different techniques and price ranges, making selection more difficult. Their performance similarities and differences and the potential for separate and combined usage to achieve optimal clinical results are illustrated here by examples from the author's experiences in hematological testing to reach optimal clinical evaluation.

The immediacy of test results is a major advantage of in-clinic testing, especially in emergency situations. Therefore, clinics with emergency and critical care services or that perform major surgical procedures requiring postoperative monitoring should be equipped with an in-house testing laboratory. Moreover, certain medical problems may not be recognized on initial presentation without laboratory test results, and the rapid availability of test results allows for immediate correction or treatment before the disease progresses. Hematology samples should be analyzed as quickly as possible to minimize artifacts and cellular alterations due to environmental, shipping, and storage conditions. Some potential cellular changes include the continuing segmentation of the neutrophils, swelling of red blood cells, and platelet clumping.

In-house testing also permits doctors to communicate with clients immediately and inform them of the patient's condition, prognosis, and therapeutic options. Clients certainly appreciate this, but clinicians also benefit because they can pursue additional diagnostic tests and initiate potential treatment options at the initial visit without the need for extensive follow-up communication. Historically, there was a major delay in receiving test results from reference laboratories, but many reference laboratories offer courier pickup services and will fax or e-mail test results by the next morning--which is early enough for many patients. Nevertheless, most veterinary clinics clearly can benefit from the combined use of internal and external laboratories.

In the past, the scope of in-clinic testing was extremely limited; however, these original tests, such as the minimal database, remain indispensable in any clinic. They include packed cell volume (PCV) by microcentrifugation, total protein and urinary specific gravity with a refractometer, few biochemical parameters of urine and blood with dipsticks and a glucometer, and microscopic examination of blood, urine sediment, other fluids, fecal specimens, and tissue aspirates. These test results belong in a typical patient's minimum database.

It is peculiar that veterinary clinicians continue to rely mostly on the PCV, while human physicians use blood hemoglobin values to assess patients for anemia and polycythemia. Veterinary practitioners could also determine hemoglobin values with a drop of blood in a disposable cuvette to monitor anemias. The microhematocrit (PCV) requires more time for the centrifugation step but allows clinicians also to assess the buffy coat, plasma color (detecting lipemia, intravascular hemolysis, or icterus), and total plasma protein and thereby offers advantages, if no other testing is performed. However, a simple validated hemoglobin tool accurately assesses the degree of free hemoglobin in animals even in the presence of lipemia, intravascular hemolysis and those receiving Oxyglobin (Biopure) therapy.

Clinicians often under-use their microscopes despite the important information gained from a blood smear. These evaluations are fairly simple, can be rapidly accomplished, and are still indispensable. Similarly, practitioners should perform urinalyses that include a dipstick, specific gravity, and microscopic evaluation of sediment for cells and crystals. Beside these essential basic laboratory techniques, many other tools have become standard in small animal practices.

The progressive small animal clinician uses many more in-house and reference laboratory tests to practice high-quality veterinary medicine. Among the extended in-clinic testing are simple kits, such as SNAP tests for infectious diseases (including FeLV, heartworm antigen tests, tests for antibodies against parvovirus, FIV, and tick-borne pathogens), and serum progesterone test kits to assist with appropriately timed breeding.

With respect to in-clinic hematology tests, a simple activated clotting time (ACT) test and now also an activated PTT and PT can be performed in clinical practice to identify any coagulopathy. The buccal mucosal bleeding time is useful to assess primary hemostatic disorders, such as thrombopathias and von Willebrand's disease, after thrombocytopenia has been excluded. The canine d-dimer kit (Agen Biomedical) promised to be an easy test to screen for fibrin degradation products--for instance, those associated with overwhelming internal bleeding, thrombosis, and disseminated intravascular coagulation--but it is unfortunately no longer commercially available. Moreover, thromboelastography is being used with increased frequency in critical care settings to identify hypo- and particularly hypercoagulable states and monitor treatment responses. The canine erythrocyte antigen (DEA) and feline AB typing cards and cartridges, as well as a recently introduced tube crossmatch test, ensure canine and feline blood compatibilities for transfusions.

However, advanced diagnostic tests generally require some form of a measuring instrument, including a hematology analyzer, coagulation instrument, or chemistry or blood gas analyzer. Although such equipment can be expensive and is mostly intended for reference laboratory use, several have been developed specifically for affordable in-house application, and are, consequently, referred to as in-clinic or point-of-care units. While clinical pathology laboratories used to perform most CBCs, practitioners can now use automated, in-house hematology instruments. Automated cell counters offer several advantages over the manual differential, including improved accuracy of results, reduced technician time, and more immediate results. There are several methods available, each with strengths and limitations unique to its technology.

The first one available was quantitative buffy coat (QBC) analysis, which used sedimentation in a larger microhematocrit tube to provide white blood cell (WBC) and platelet counts. The QBC analyzers are easy to use, very economical, and efficient at screening blood samples, but they do not provide many hematological parameters. The QBC technology was followed by more sophisticated in-clinic cell counters. The technology, breadth of measured parameters, usefulness, and accuracy of in-house instruments vary greatly. The pros and cons of each instrument should be weighed before deciding which hematology analyzer is most appropriate for a given clinic. While some have come close to provide accurately many red cell parameters and WBC differentials, none has replaced the microscopic evaluation of a blood smear.

Impedance counters are based on the Coulter principle and separate cell populations by size. They are economical and provide the fastest results but are unable to provide a complete differential count or reticulocyte data. Because cells are separated by size, platelet counts can be very inaccurate and not all types of white blood cells can be differentiated. Laser flow cytometry is the method employed in most reference laboratories. As cells pass through a laser beam, the pattern of light scattered by the individual cells is analyzed. This pattern is characterized according to cell size, nucleus and cytoplasmic contents. Laser flow cytometers offer a differential WBC count, reticulocyte count, and thereby the most complete assessment of blood cells. Recently about 10 impedance or laser-based hematology analyzers have become available for in-clinic use, and some can analyze nearly the same range of hematologic parameters as reference laboratory instruments.

Reference laboratories generally rely on large hematology instruments, such as the Advia, Cell-Dyn, and Sysmex. These laboratories offer economical though not immediate hematology results to veterinarians. They employ trained technicians, who can manually better examine blood smears from diseased animals, and have well established regular quality control programs to ensure accuracy. Board-certified clinical pathologists are available to review slides and blood test results and often consult on challenging cases.

Similarly, not only reference laboratory chemistry analyzers can offer dry or fluid phase analyses of specific chemistry screen panels for common clinical circumstances, as well as single parameters for monitoring. In general, however, external laboratory instruments offer more test parameters than internal ones. In addition, many emergency clinics consider electrolyte and blood gas analyses critical and use in-clinic instruments to measure these parameters.

Accuracy of test results: It is generally expected, when receiving test results from either in-clinic instruments or reference laboratories, that they are accurate and meaningful when interpreted in light of the patient's clinical signs. However, pre-analytical, analytical, and post-analytical mistakes can produce erroneous and misleading test results. To minimize these errors, individuals involved in laboratory testing need to be trained in standard operating procedures (SOP) and quality assurance.

Pre-analytical errors can result from improper collection techniques, inadequate sample sizes, patient anxiety, and recent food intake. Atraumatic and rapid blood collection in the correct tube ensures appropriate anticoagulation for blood cell and plasma analyses. Failure to use proper collection techniques can cause platelet activation, thrombus formation, and red blood cell (RBC) lysis, all of which affect cell counts and plasma parameters. To avoid collection errors, practitioners should keep these guidelines in mind:

 Whether samples are tested in-house or at a reference laboratory, each one needs to be clearly marked with identification number, name of the owner and animal, date, and time of collection. Any unlabeled samples should be discarded.

 The jugular veins are the preferred site of collection in small animals, but other peripheral veins (cephalic and saphenus) may also be useful. With bleeding patients holding of the vein for several minutes is imperative.

 Vacutainer systems are preferred over syringe methods whenever possible, except in tiny animals or when accessing small vessels (vein may collapse). Needle gauge size is of lesser importance.

 Collection from catheters should be avoided (dilution and heparin effects) or performed only with an appropriate volume catheter and flushing of the catheter.

 Proper anticoagulant selection and the ratio of blood to anticoagulant are important considerations. Excessive EDTA concentration causes cell shrinking, morphologic artifacts of blood cell, and dilution and thus can lead to a lower PCV and an inability to examine blood cells properly. Hence, EDTA blood tubes need to be filled at least half full for a CBC.

The 9:1 ratio of blood to citrate as an anticoagulant when the tube is properly filled by the predetermined vacuum of a tube is critical to obtain interpretable coagulation results, because a specific amount of calcium is added to the assay to counter the citrate; in other words, over-citrated samples (too little blood) may have reduced coagulation activity and under-citrated samples (too much blood) may appear hypercoagulable. Moreover, the ratio may be affected by severe anemia and polycythemia, and adjustments (adding more citrate or less, respectively) may be needed for the sample to reach the appropriate plasma to anticoagulant ratio. Heparinized blood samples are rarely useful in hematology (except in plasma ammonia and a few hormone measurements), and the slightest contamination from a heparinized catheter can invalidate the coagulation and chemistry test results. Following collection, samples may have to be specifically handled and processed. Blood smears from EDTA anticoagulated blood should be freshly prepared upon collection for subsequent staining and microscopic examination and stored at room temperature, regardless of internal or external laboratory analysis.

Blood in red top tubes should be kept at room temperature for 15 to 30 minutes to allow clotting before serum separation by centrifugation for serum chemistry and other tests. While citrated samples are generally chilled, centrifuged, and then frozen and shipped quickly for reference laboratory evaluation, in-house testing does not require this often-flawed handling of samples. However, in-clinic instruments that analyzes prothrombin time and activated partial thromboplastin time--using citrated whole blood used within an hour from blood collection--offer a simple means to obtain coagulation screening test results and eliminates the need to process and ship frozen citrated samples. There is also a point-of care instrument (PFA-100) that can assess platelet function and von Willebrand's activity in whole citrated blood. However, this instrument is fairly expensive, its use is limited to humans and dogs, and any degree of anemia affects the test results.

Analytical errors are those referring to the actual false testing and reporting of results. They are introduced by instrument malfunctions, reagent problems, or operator mistakes. To avoid these types of errors, all laboratory personnel should follow standard operating procedures for each test method and use only standardized and validated tests. Where available and appropriate, they should also use manufacturer controls or in-clinic or laboratory controls to check instruments and reagents, running one healthy control sample and one abnormal sample to ensure that they fall in the expected range. This can be readily achieved with blood chemistry parameters. Unfortunately, it is difficult to obtain good controls for blood cell counting because these cells age rapidly and sample values change. It is practically impossible to get controls that cover the entire range from low to high values. Therefore, actual blood samples are often substituted with controls from humans, or are replaced by artificial particles for calibration and quality control. Validated techniques, careful performance of tests, regular quality control, and appropriate control samples can greatly improve the accuracy of test values from in-house and reference laboratories. Generally, these guidelines are more closely followed in reference laboratories, but practitioners in private clinics can also train dedicated laboratory personnel to maintain similarly high standards for testing.

Nevertheless, test results can only be as good as the instrument and samples used. Considerable variation exists among laboratory tools and techniques, and although reference laboratories might be considered superior, this may not be true. For some laboratory values, such as the PCV, reference laboratories use precisely the same method as private practitioners, while for other values they employ different techniques. With respect to canine and feline blood typing, most reference laboratories use typing cards or cartridges while the reference laboratories have switched just a gel test method; the same principle is also used for crossmatching and the Coombs' test. Blood hemoglobin measurements are generally obtained with the blood cell count analyzers.

With in-house screenings, samples are usually analyzed while fresh, which is particularly important for blood cell counting and coagulation studies that are affected by testing delays. Hence, in-clinic and reference laboratories may produce different values for the same sample if they analyze the sample at different times. This has been clearly shown with MCV and blood cell counting on the same instrument over time. Furthermore, each instrument is calibrated differently; therefore, there may be a bias in test results, which is considered a systemic error. This emphasizes the need for laboratories to recalibrate, compare, and establish internal reference ranges for all instruments, despite the huge labor and monetary costs. Finally, quality control with accuracy and precision determinations should be repeated on a regular basis, and results should be tabulated as instruments may drift away from their original range.

Post-analytical errors relate to mistakes in reporting and misinterpretation of test results. Practitioners may ascribe test results to the wrong patient or wrongly transcribe results. The reference range posted with the result may be incorrect for the species or the animal's age. Reference ranges may not be available for particular instruments, especially if a technique has been validated only in people or one species and extrapolations are inappropriate. Furthermore, some results are important if they are anywhere outside the reference range, while others are meaningful only if they deviate markedly. And finally, clinicians should keep in mind that, by definition, the reference range applies only to 95% of a population.

Hence, it is important to consider the patient's clinical manifestations, sample condition, and methods used when interpreting results. Specialized laboratory personnel and clinical pathologists may detect abnormalities more readily, may be better suited for troubleshooting, and can likely provide additional expertise to ensure accuracy and in-depth interpretation of test results. Just like in human medicine, repeat testing is advisable to confirm an unusual, unexpected, or important result, as is a complete clinical evaluation of the patient before making treatment decisions.

In summary, many in-clinic and reference laboratory techniques have become available for diagnostic screening and monitoring of patients. Multiple instruments are available for the assessment of various hematological parameters, yet few unbiased comparative field studies are available to select the one most appropriate for a particular clinic setting. Practitioners should embrace both in-house and reference laboratory testing to reach definitive diagnoses, monitor disease in animals, and ensure patient health.

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Philadelphia, PA, USA

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