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General Laboratory Medicine

Raquel M. Walton

IDEXX Laboratories, Inc., Langhorne, PA, USA

Acronyms and abbreviations that appear in this chapter include: Hb, hemoglobin; MCH, mean cell Hb; MCHC, mean cell Hb concentration; MCV, mean cell volume; PCV, packed cell volume; POC, point of care; POCT, point-of-care testing; RBC, red blood cells; TP, total protein; TPRef, refractometer total protein; TS, total solids.

1.1 Introduction to Laboratory Medicine

Laboratory medicine, more commonly referred to as clinical pathology (or bioanalytical pathology), is a distinct specialty that overlaps other medicine specialties such as internal medicine and oncology in the area of diagnostics. In contrast to internists, clinical pathologists practice a systems-based rather than problem-based approach when interpreting hematological and biochemical results. However, in addition to recognizing disease-associated changes, two other phenomena contribute to test interpretation: how test results are generated and how "normal" is defined. Artifacts due to sample preparation, sample condition or disease processes need to be identified and distinguished from true disease-associated changes. Similarly, test interpretation is always performed in context - the context of health. The accuracy and sensitivity of tests and the use of appropriately established reference intervals are essential to the ability to diagnose disease.

This chapter will provide selected information on hematological and biochemical test methodologies and validation, and will discuss the basic knowledge needed for generating and/or using reference intervals. The remainder of the book will address test interpretation using a systems-based approach.

1.2 Preanalytical Factors

Preanalytical factors that may affect test results should be minimized in order to ensure result accuracy [1]. Specimens should be collected according to standard practices and transported to the laboratory in a timely manner under conditions appropriate for the type of specimen and its stability. The minimum information on a specimen label for laboratory evaluation should include the full name of the patient (animal and owner), the patient signalment, and the specimen type (e.g., whole blood, serum or plasma). Especially for hematological evaluation, it is important that the patient's signalment be correct as analyzer settings vary with respect to species.

Anticoagulated specimens for hematology that have visible macroclots in the tube will produce variably erroneous results. Because the degree of inaccuracy cannot be predicted, clotted specimens are unsuitable for analyzis and these specimens should not be analyzed or submitted for analysis.

Blood films and cytology smears should not be refrigerated and should be protected from condensation and freezing during transport to the laboratory to avoid condensation artifact (Figure 1.1). Failure to fully dry blood films or cytology preparations before placing them into slide holders can also result in moisture artefact.

Figure 1.1 Condensation artifact caused by exposure of unfixed slides to moisture. The nucleated cells are lyzed and many hemoglobin crystals are present, formed from erythrocytes.

1.3 Basic Hematological Techniques

1.3.1 Packed Cell Volume and Plasma Evaluation: Disease and Artifacts

Measurement of the percentage of red blood cells in whole blood can provide more information than simply the packed cell volume (PCV). In addition to the packed erythrocytes at the bottom of a microhematocrit tube, there is the white buffy coat layer and a plasma layer. The size of the buffy coat is related to the white blood cell (WBC) (and platelet) count; a thick buffy coat would indicate a high leukocyte (and/or platelet) count, whereas a scant buffy coat suggests leukopenia. The character of the plasma can also yield valuable information pertaining to a disease process, as well as contributing to spurious results. The plasma can appear hemolyzed, icteric or lipemic (Figure 1.2).

Hemolysis in samples from horses usually indicates an in vivo phenomenon due to toxins or immune-mediated disease (see Chapter 4). However, hemolysis can also occur during blood collection if excessive force or too small needle gauge is used in phlebotomy. Whether in vivo or in vitro, hemolysis produces a color change that can make refractometer readings difficult or interfere with spectrophotometric tests.

Icterus indicates hyperbilirubinemia that usually exceeds 1.5?mg/dL (see Chapter 5). However, in herbivorous animals yellow-colored plasma is not a reliable indicator of hyperbilirubinemia due to the presence of diet-associated carotene pigments, which impart a yellow color to plasma. Icterus has not been demonstrated to interfere with refractometer readings [2]. Depending upon the chemistry analyzer, icterus can cause interference with some serum chemistry tests.

Figure 1.2 Evaluation of plasma. From left to right: normal plasma color and consistency; lipemic and slightly hemolyzed plasma; hemolyzed plasma; icteric plasma.

Lipemia is visible to the eye as increased turbidity in plasma or serum at triglyceride concentrations >300?mg/dL. Whether physiological (postprandial) or pathological (see Chapter 9), lipemia can cause spuriously high refractometer readings and will interfere with many chemistry tests.

1.3.2 Protein Measurement by Refractometer

Protein can be rapidly and accurately measured by hand-held refractometers. Because refractometers measure protein via a total solids-based technique, the total dissolved solids in the sample affect light refraction. In addition to protein, total solids include electrolytes, glucose, urea, and lipids. The term "total solids" has caused much confusion in the reporting of refractometric protein results. Total protein (TP) and total solids (TS) are not synonymous. Currently, the vast majority of refractometers incorporate a conversion factor in their design so that the scales report TP and not TS. Contributing to the confusion is the fact that at least one refractometer is named the "TS meter" (AO Corporation) when it is in fact calibrated to report TP. While the altered refraction of plasma is mostly due to protein content, increases in lipid, glucose or urea content interfere with refractometric protein measurements. However, marked increases in urea or glucose (273?and 649?mg/dL, respectively) are needed to increase protein measurement by 0.4-0.5?g/dL. Increases in plasma cholesterol of 39?mg/dL are shown to increase the refractometer TP (TPRef) by 0.14?g/dL [2].

Another potential cause of erroneous refractometer readings is the addition of EDTA from K3EDTA anticoagulant tubes. At the standard concentration of EDTA (5?µmol/mL), K3EDTA by itself has minimal effect on the plasma's refraction (=0.1?g/dL increase). This is not true for peritoneal fluid, however, where overestimation of TPRef by 0.7 +/- 0.1?g/dL was reported in one study (see Chapter 18). At higher concentrations of EDTA (10 and 20?µmol/mL), EDTA can increase TPRef by 0.9-1.0?g/dL. Underfilling of EDTA tubes has the effect of increasing the EDTA concentration and will cause spurious increases in the TPRef [3]. Some commercial tubes with K3EDTA anticoagulant may also contain additives to prevent crystallization of the EDTA. Tubes that contain the additive may increase TPRef readings by up to 0.9?g/dL, even when properly filled. In general, polypropylene (plastic) tubes are more likely to include additives to prevent evaporation than glass tubes [4]. While sodium heparin anticoagulant has no effect on TPRef, heparin has deleterious effects on cellular morphology and is not recommended for samples that will be evaluated cytologically.

1.4 Point-of-Care Testing

Point-of-care testing (POCT) is defined as testing done at or near the patient with the expectation that results will be available quickly to facilitate immediate diagnosis and/or clinical intervention [5]. Whilst POCT provides quick, relatively inexpensive results with small volumes of blood, it also comes with its own set of risks. The major sources of error associated with POCT were categorized in one study as most often due to operator incompetence, nonadherence to test procedures, and the use of uncontrolled reagents and testing equipment [6]. Instrument calibrations and quality control measures may be omitted due to ignorance or the need for fast results. And, in veterinary medicine, analyzers may be used with species for which the instrument has not been validated. It should also be noted that diagnostic instruments for veterinary use are not subject to government regulations as they are for human use, which means that devices may not have been independently evaluated or tested [7]. Finally, poorly maintained instruments that are carried from one area to another may be a source of nosocomial infection or may transmit...