Clinical Pathology in Nonclinical Toxicity Testing

Essential Services

Clinical pathology testing in nonclinical toxicity studies encompasses hematology, hemostasis, clinical chemistry, and urinalysis as the core elements. The scope of testing is evolving constantly to include new technologies as well as additional test parameters and novel biomarkers. Despite the rapid advances in laboratory instrumentation and biomarker development, the core set of clinical pathology tests for nonclinical safety studies typically is limited to the four standard test panels for nonclinical safety studies.

Hematology : Ethylenediaminetetraacetic acid (EDTA)–anticoagulated blood for determination of a complete blood count (CBC) is the most frequently submitted sample for hematological analysis in all species used in toxicity testing. The CBC provides rapid, inexpensive, and noninvasive assessment of the erythron (red blood cells [RBCs]), leukon (white blood cells [WBCs]), and thrombon (platelets) ( Table 10.2 ). A freshly made and stained blood smear from each animal is used to confirm cell counts and to evaluate blood cell morphology. These smears may be made by investigators at the time of phlebotomy or, provided that the blood samples are delivered to the clinical pathology laboratory within 1–2 h of collection, they can be made in the laboratory at the time of specimen processing.

Bone marrow cytologic smears, prepared at necropsy for each animal, are also submitted routinely for staining and potential future evaluation in toxicity studies. Although these smears are collected in most studies, they are used infrequently in test article evaluation. Their use is reserved for studies in which the CBC combined with bone marrow histopathology is insufficient to explain the cause(s) for unexpected nonregenerative decreases in erythroid mass, neutropenia, and/or thrombocytopenia. They may also be used less frequently to investigate unexplained test article–induced increases in various blood cell lines and certain types of morphologic changes in blood cells ( ; ).

Hemostasis : Citrated blood is the sample submitted most frequently for hemostasis evaluation. Citrated plasma is used for determining the prothrombin time (PT)—a rapid screen of the extrinsic and common pathways of coagulation; activated partial thromboplastin time (APTT)—a rapid screen of the intrinsic and common pathways of coagulation; and fibrinogen concentration—a measurement of the plasma protein precursor to fibrin ( Table 10.2 ).

Clinical Chemistry : A variety of fluids may be submitted for biochemical analysis: serum, plasma, urine, and less frequently cerebrospinal fluid, synovial fluid, organ lavage fluids, semen, body effusions, and cell culture supernatants. Of these fluids, serum (the supernatant remaining after blood has been allowed to clot) followed by plasma (the supernatant following centrifugation of anticoagulated blood) is analyzed most frequently in clinical pathology laboratories in the United States. Specific tests may be requested individually, or a panel of tests may be selected to screen multiple organ systems ( Table 10.2 ). These standardized panels are commonly used for safety assessment studies. A typical screening panel may include tests to evaluate renal function, liver injury, and electrolyte status as well as lipid, carbohydrate, and protein metabolism. The exact number of tests per panel may vary with the individual clinical pathology laboratory and institution as well as the sample size. If the amount of serum available for analysis is limited due to animal size, investigators may opt to modify the standard panel or select an organ-specific panel (such as a liver screen that includes total protein, albumin, and total bilirubin concentrations as well as the activities of the hepatobiliary enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and/or gamma-glutamyl transferase (GGT)).

Urinalysis : Aliquots of fresh urine are submitted for complete urinalysis. The full evaluation includes assessment of physical parameters of urine (e.g., volume, color, clarity, and specific gravity); reagent stick (semiquantitative) biochemistry determinations (pH, protein, glucose, bilirubin, ketones, blood, urobilinogen) or quantitation of specific analytes on automated instrumentation; and microscopic examination of the sediment to observe cells, crystals, casts, and infectious agents. Urine may be submitted and analyzed within 4 h of collection or may be refrigerated for up to 24 h prior to analysis. It is essential for appropriate data interpretation that investigators specify the method of urine collection (voided midstream catch, cage pan, catheterization, cystocentesis, or metabolism cage) when submitting urine for analysis since collection methods can influence proper interpretation of urinalysis results. For certain renal toxicity/physiology studies, urine may be analyzed quantitatively to determine the concentrations of albumin, protein, glucose, sodium, potassium, chloride, calcium, phosphate, and creatinine. True quantitative studies of urine proteins, creatinine, and electrolytes, often referred to as “renal physiology studies,” may require timed urine collection via metabolism cages, and values may be reported as urine analyte concentration and/or analyte concentration normalized to urine creatinine concentration.

Instrumentation

A detailed description of all instrumentation utilized in the clinical pathology laboratory is beyond the scope of this chapter. Excellent, comprehensive laboratory instrument reviews are provided by the College of American Pathologists ( ). The ASVCP publishes numerous instrument validation and methods comparison studies in the journal Veterinary Clinical Pathology ( ). Several reference textbooks review instrument methodology and the challenges of analyzing animal samples ( ; ; ). There are some important concepts that are common to laboratory instruments, and investigators should be aware of these when pursuing services from a clinical pathology laboratory. These guiding principles are covered briefly here.

The instrument for measurement of the CBC should be automated and accommodate high volumes of samples daily. Given the small sizes of laboratory mice and rats and the limited blood volumes that may be collected from these species, the hematology instrument should be capable of accurately analyzing a small EDTA blood sample (approximately 250 μL). Some newer automated hematology instruments that are under evaluation currently and not yet in widespread use have smaller sample volume requirements (approximately 125 μL) and may be very helpful for analysis of mouse blood samples. The instrument intended for animal hematologic analysis should be equipped with multispecies software to accommodate the laboratory animals that are being studied at that institution. The range of nonclinical test species most often includes mice, rats, dogs, rabbits, pigs, and nonhuman primates (NHPs); however, other species are also occasionally utilized such as guinea pigs, hamsters, gerbils, and/or cats. It is important to utilize appropriate instrumentation and optimize it accordingly in order to accurately analyze blood samples from a variety of laboratory animal species. An automated blood smear stainer also should be available for routine use. These instruments should provide an acceptable Romanowsky stain, such as modified Wright stain, Wright stain, or Wright–Giemsa stain, to highlight cytologic features of various cell types. This instrument also may be used to stain cytologic preparations of bone marrow and other body fluids, tissue aspirates, or impression smears. Manual staining of blood smears is a time-consuming and challenging task that may lead to much stain variation and so should be avoided.

Instruments dedicated to measurement of hemostasis assays are sometimes called “coagulometers.” These instruments vary markedly in size and complexity. In the modern clinical pathology laboratory, these instruments are usually semi- or fully automated, accommodate high volumes of samples daily, and employ physical method, optical end points (turbidometry or nephelometry), or a combination of these methods to detect conversion of fibrinogen to fibrin. Most hemostasis assays are simple clotting or chromogenic substrate assays. A limiting factor in hemostasis testing in animals is the volume of the citrated plasma sample required for completion of a panel of the more commonly used assays (PT, APTT, and fibrinogen concentration)—usually approximately 400 μL minimum. Manual assays (tilt tube method) that use visual inspection for clot detection are used rarely today.

In the clinical chemistry section of the laboratory, the main instrument for biochemical analysis should accommodate large numbers of samples daily. This instrument should be equipped with reagents and assays to measure the 20–30 more common analytes ( Table 10.2 ) of biochemical panels and should have open channels that can be programmed to accommodate unique assays (e.g., novel biomarkers) developed by the laboratory staff. Ideally, the sample volume requirement for the entire clinical chemistry panel should be small (≤250 μL serum or plasma, which equates to approximately 500 μL of whole blood) to accommodate the limited blood volume of rodents.

Urine specific gravity should be measured by refractometry as the urine reagent sticks have proven unsatisfactory for measurement of urine specific gravity in animals. The refractometer may be a hand-held device or might be incorporated into a large bench-top instrument that is used to evaluate the semiquantitative urine reagent stick chemistry determinations.

All clinical pathology laboratory instruments should have an appropriate maintenance log and a standard operating procedure (SOP). Competency of laboratory personnel to operate instruments should be reassessed according to institutional guidelines on a regular basis. Every clinical pathology laboratory should have a quality assurance program to ensure the proper operation of each instrument, the appropriate use of assay reagents, and the correct actions of the instrument operator to generate accurate and precise data. Readers are referred to summaries in textbooks ( ; ; ) and the numerous publications and guidelines produced by the Quality Assurance and Laboratory Standards Committee of the ASVCP ( ) for details on these important programs.

Laboratory Staffing

Clinical pathology laboratories may vary in size and number of employees, depending on the management objectives for individual institutions. In some organizations, the clinical pathology laboratory focuses on services in hematology, hemostasis, clinical chemistry, and urinalysis in laboratory animals only. In other institutions, the clinical pathology laboratory may be involved in active biomarker research, microbiological testing, development and validation of emerging molecular pathology techniques, and/or processing samples for test article absorption and metabolism determinations. Many of the Good Laboratory Practice (GLP)–compliant clinical pathology laboratories in biopharmaceutical research, contract research organizations, and the chemical industry are restricted to analysis of animal samples. Infrequently, institutions will have both human (where the GLP-equivalent standard is Clinical Laboratory Improvement Amendments [CLIA] compliance) and animal samples analyzed within the same clinical pathology laboratory. The common denominators for these laboratories should be high-quality analysis of samples in a timely manner.

The job titles for laboratory personnel vary widely among companies and organizations. Position titles such as research associate, research specialist, technologist, and technician often lack universally accepted definitions for education, training, skill level, and duties. These titles may confuse investigators and can be defined differently at various institutions. Since high-quality sample analysis depends on appropriate education and training of laboratory employees, it is desirable for clinical pathology laboratory employees to have earned certification in clinical pathology testing by a professional society of pathology such as the American Society for Clinical Pathology (ASCP). Individuals credentialed by the ASCP as Medical Laboratory Technician or Medical Laboratory Scientist [formerly known as Clinical Laboratory Scientist or Medical Technologist] are highly valued clinical pathology professionals. Many of these individuals, while originally educated in hospital- or university-based clinical pathology training programs focused on human sample testing, have completed additional years of training in a veterinary clinical pathology laboratory to familiarize themselves with the myriad species differences in hematology, cytology, clinical chemistry, hemostasis, and urinalysis. Clinical pathology laboratory personnel should be fluent in operation of the latest automated instruments for hematology, hemostasis, clinical chemistry, and urinalysis as well as have skills in manual techniques and procedures such as creation of blood and bone marrow smears, production of buffy coat preparations, analysis of body fluids, and performance of manual “quick” stains on cytologic specimens. Lastly, it is desirable that a veterinary clinical pathology laboratory have a board-certified veterinary clinical pathologist as the supervisor or scientific liaison to advise on challenging issues such as cellular identification in hematology or cytology, unique assay and instrument validation procedures, study designs, and data interpretation as well as to provide continuing education opportunities for laboratory employees ( ; ; ). Commonly, clinical pathologists in animal facilities are veterinary pathologists with certification as a diplomate of the American College of Veterinary Pathologists (ACVP) or European College of Veterinary Clinical Pathology (ECVCP). The practice in some nonclinical facilities of having clinical pathology data evaluated solely by the toxicologist, the study director, or by the anatomic pathologist who is performing the histopathologic evaluation may be suitable in cases where few or obvious clinical pathology alterations are found, but unusual or challenging clinical pathology changes generally should be analyzed or reviewed by a veterinary clinical pathologist.

Types of Samples and Identification

All specimens delivered to the clinical pathology laboratory should be properly labeled. An ideal specimen label contains the following information: study number, unique animal identifier, date the sample was collected, and species; in some institutions, a unique computer tracking bar code may be included to speed the analysis (by reducing the need for direct entry of the unique animal identifiers for all subjects of large studies). Upon receipt in the clinical pathology laboratory, all specimens from a study should be reviewed by laboratory personnel and matched to the study protocol. The number of samples is reviewed, and any missing samples should be noted immediately and recorded in the written and/or computer record for the study.

Quality of Samples—Volume, Hemolysis, Lipemia, and Icterus

Ideally, blood samples submitted to the clinical pathology laboratory for hematologic, hemostasis, and clinical chemistry analysis are obtained from rats, dogs, and NHPs fasted overnight. Fasting improves specimen quality by reducing variability in glucose, triglyceride, and some lipid concentrations compared to those analyte values obtained from nonfasted animals. Fasting also reduces the occurrence of lipemia, which may interfere with some test methods in hematology, hemostasis, and clinical chemistry. However, fasting of mice for longer than 4 h in preparation for clinical pathology sampling typically is not recommended since fasted mice also may decrease water consumption and become dehydrated, which can impact several clinical pathology parameters. Urine for urinalysis may be obtained from fasted or nonfasted animals.

Body fluid specimens (anticoagulated blood, serum, plasma, urine) should be evaluated for adequacy of sample volume. Any sample with inadequate volume to complete the tests listed in the study protocol should be recorded.

Anticoagulated blood samples (EDTA, citrate, and heparin) should be gently rocked back and forth and evaluated for clotting. A wooden applicator stick should be inserted and gently stirred in the anticoagulated samples to check for microclots. Any anticoagulated blood sample with gross or microclots should be rejected and not analyzed since clotting will alter some parameters by consuming proteins, causing release of some cellular constituents, and removing cells from the sample. Clotted samples also may potentially disable a hematology analyzer if large cellular clumps occlude the fluid mechanics of the instrument.

Samples of whole blood that are processed to serum and anticoagulated blood samples processed to plasma should be evaluated for the presence of hemolysis, lipemia, and icterus. If present, the magnitude of hemolysis, lipemia, or icterus should be assessed objectively by visually matching to a color chart or by biochemical determination using the chemistry analyzer. Any sample quality observations should be recorded and reported along with the study analyte data to the clinical pathologist to assist in data interpretation. Laboratory personnel should contact the study director to discuss any clotted samples or samples with an inadequate volume for analysis. Collection of another blood sample may be possible for certain prestudy or interim time points from animals with larger blood volumes (e.g., dogs, NHPs, and pigs). Body fluid samples from mice or rats are seldom redrawn as they are usually obtained at the time of necropsy.

If it is impossible to obtain adequate sample volume to complete all the tests listed in the study protocol, the study director should be asked to provide a list of prioritized tests that can be completed using the sample submitted to the laboratory. It is most expedient if the prioritized list of testing is established and communicated in advance in the study protocol and/or a facility SOP to avoid delays if this situation arises as an urgent need for a critical study time point.

Erythrogram

The erythrogram is comprised of several erythroid measurements including RBC count, Hgb, Hct, and reticulocyte count as well as several erythrocyte indices (e.g., MCV, MCH, MCHC, red cell distribution width (RDW), corpuscular hemoglobin concentration mean (CHCM), and/or hemoglobin distribution width (HDW). The RBC count, Hgb, and Hct are often collectively referred to as “red cell mass” parameters. Erythrocyte indices are a collection of instrument-based calculations generated from direct measurements of various characteristics (i.e., size, density) of RBC populations. Although erythrocyte indices can sometimes be used to provide additional supportive or mechanistic information, they most often are limited in their interpretive value and largely reflect expected changes associated with RBC turnover (regeneration and kinetics). MCV is a measure of RBC size, while RDW relates to the degree of size variability overall in the RBC population; changes to these parameters can be used to help characterize erythropoietic activity of the bone marrow (see Interpretation and Reporting of Clinical Pathology Results in Non-clinical Toxicity Testing Vol 2, Chap 14). MCH, MCHC, CHCM, and HDW are indices related to Hgb content and the overall degree of variability of Hgb content with the RBC population. Unlike most of the erythrocyte indices including MCHC, CHCM is a direct measure of Hgb content within RBCs which can be used in conjunction with other markers to help characterize certain hematologic abnormalities.

Enumeration of reticulocytes is an important aspect of an erythrogram as reticulocytes are anucleate but immature RBCs. Reticulocyte counts are an indicator of bone marrow regeneration and overall erythropoietic activity. Reticulocytes correspond to polychromatophilic erythrocytes on blood smear evaluation, and assessment should be made based on absolute rather than relative reticulocyte counts. Nucleated RBCs should also be enumerated by counting the number per 100 WBCs, which is typically done by the automated hematology analyzer and incorporated into cell count results.

Leukogram

The leukogram is comprised of the total leukocyte (WBC) count and differential leukocyte (relative and absolute) counts, which include neutrophils, lymphocytes, monocytes, eosinophils, basophils, and large unstained (or unclassified) cells (LUC, which are large peroxidase-negative cells that cannot be further categorized). Interpretation of leukocyte responses should be made based on absolute rather than relative percentage counts, even if the laboratory includes both absolute and relative percent differential counts in the report. Changes in the number of individual leukocyte types in blood reflect the balance of cell production and release to the blood, margination (adhesion to endothelial cells [ECs]), migration to tissues, and destruction/consumption. Microscopic evaluation of blood smears can reveal important morphologic alterations to leukocytes that can further aid in the characterization of pathologic processes, particularly those involving inflammation.

Thrombogram

The thrombogram is composed of tests that evaluate platelets or circulating platelet mass. The key components of the thrombogram are a count of platelet number and various calculated platelet indices; these parameters are core components of hemostasis testing in all nonclinical species. The numbers of platelets in circulation vary considerably by species and reflect a balance of production, destruction, consumption, and redistribution to tissues such as the spleen ( ). While the platelet count is the most frequently reported parameter in the thrombogram, other parameters may be measured by the analyzer, but not always reported, depending on the study protocol.

More recently developed measurements of platelets are available from some types of automated cell counting instruments. These new techniques include the following: thrombocrit, large platelets, mean platelet volume (MPV), and platelet distribution width (PDW). The thrombocrit (or plateletcrit) is the percentage of blood volume occupied by platelets and is an assessment of circulating platelet mass. MPV is the average volume of all the particles in a blood sample that are counted as individual platelets. PDW is a measurement of platelet anisocytosis (size differences) calculated from the distribution of individual platelet volumes. These biomarkers may provide additional data regarding platelet numbers and bone marrow response to treatment in some specialized studies, especially those in which serial determinations occur in a longitudinal fashion ( ; ).

Additional biomarkers used to assess platelets qualitatively are available from certain automated cell counters and can be measured during routine CBCs. Mean platelet component and platelet component distribution width are two biomarkers that change in association with platelet activation and have proven helpful in some safety assessment studies in rodents, dogs, and NHPs.

Hemostasis Screening

Historically, screening of the hemostatic system in safety assessment studies has been focused on the identification of potential bleeding liabilities. For most studies, platelet counts, APTT, PT, and less frequently fibrinogen concentration are included in protocols as markers of hemostasis in rats, dogs, and NHPs. Results of these tests are correlated with clinical observations and macroscopic and microscopic tissue examination to assess hemostatic liabilities. An appropriate battery of other, more specialized hemostasis tests (e.g., fibrinogen/fibrin degradation products [FDPs], specific factor assays, measures of fibrinolysis, antithrombin activity, thrombin generation tests, thromboelastography and other viscoelastic assays, and overall hemostasis potential) can provide significant additional information for safety assessment of test agents ( ; ; ). It is important to select the relevant tests in accordance with the characteristics of the test agent or toxicological events ( ).

Samples for Hemostasis Screening

Platelet counts and platelet biomarkers may be obtained from EDTA-anticoagulated blood when the CBC is measured on certain automated hematology analyzers. Most coagulation tests, such as the APTT, PT, and fibrinogen concentration, require platelet-poor plasma obtained from blood collected in citrate. However, many platelet function tests, such as adhesion and aggregation studies, require platelet-rich plasma obtained from citrated blood and therefore are not routine endpoints in toxicity studies.

Blood sample quality is essential for successful hemostasis testing. Blood samples should be collected from high-flow, large-caliber vessels such as the jugular vein, abdominal aorta, and/or vena cava in rats. Blood obtained by techniques that cause tissue trauma and platelet activation during sampling, such as periorbital sinus collection in rats, is unsatisfactory for hemostasis evaluations. Due to blood volume limitations, mice are rarely used for hemostasis assessment in safety assessment studies. In dogs, the jugular vein and less frequently the cephalic and/or saphenous veins are used for blood collection for hemostasis testing. In NHPs, blood is usually collected from the femoral vein. It is important to fill the blood collection tube properly and maintain a 9:1 ratio of blood:citrate anticoagulant. Vacuum blood tubes with citrate anticoagulant that are underfilled (too little blood) may result in artifactually prolonged APTT and PT. Vacuum blood tubes with citrate anticoagulant that are overfilled with blood (too little citrate) may result in clotted samples and potentially yield falsely shortened APTT and PT. The citrate-anticoagulated blood sample for hemostasis testing should be held at ambient temperature for delivery to the clinical pathology laboratory within 1 h and must be free of clots or hemolysis resulting from improper collection to be useful for coagulation testing ( ; ).