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Getting Started with Clinical Pathology

Amy L. MacNeill

Colorado State University, Fort Collins, CO, USA

KEY TERMS

• Clinical pathology
• Laboratory
• Equipment
• Supplies
• Maintenance
• Safety

LEARNING OBJECTIVES

1. Become familiar with the equipment used to perform clinical pathology testing.
2. Understand when to use different types of blood collection tubes.
3. Know the sample types needed for clinical pathology tests.
4. Be able to process and store samples for clinical pathology tests.
5. Follow basic laboratory safety procedures.

Case example 1

A cytology sample slide was placed onto a microscope. Cells were visible using the 10× objective lens but could not be focused on using the 40× objective lens. What troubleshooting can you do to resolve this issue?

Case example 2

A urine sample needs to be centrifuged for 5?minutes at a speed of 450×g to collect a concentrated urine sediment for microscopic analysis without damaging the cells in the urine. Your centrifuge settings are reported as rpm, not g. How do you determine what rpm setting is needed to process the urine sample?

Case example 3

The total protein concentration of a patient's serum needs to be measured. You place a small amount of patient serum onto a refractometer and close the lid. When you look through the instrument, you cannot see a clear line that determines the protein concentration. What do you do?

Case example 4

A feline blood sample was collected into a blood tube containing EDTA, but the amount of blood in the tube was below the volume indicator on the side of the tube. The veterinary technician loaded the appropriate amount of the sample into the automated hematology analyzer. Results indicated that the cat had low erythrocyte and platelet counts. The technician recorded that the tube was underfilled before the results were reported. Why is it critical that the technician recorded the fact that the sample was underfilled?

Case example 5

A glass slide with dried blood on it was dropped on the floor and shattered. The animal caretaker saw the mess and began to pick up the pieces of glass with her bare hands. What should you do?

Introduction

Clinical pathology evaluates disease in animals using laboratory data collected during analysis of blood, urine, body fluids, and tissue aspirates. Laboratory data sets collected in sick animals typically include hematology data, serum or plasma chemistry concentrations, urinalysis results, and cytology interpretations. This chapter introduces the equipment used to collect accurate laboratory data.

Standard Equipment

1. Microscope.

   A well-maintained and properly aligned microscope is an important tool for analysis of blood smears, fecal samples, and urine sediment samples. This is an expensive tool that requires proper training to maximize the full potential use of the instrument.

   • Types of microscopes.

     There are several types of microscopes available (e.g., upright binocular light microscopes, inverted binocular fluorescent microscopes, and dissection monocular light microscopes). The most commonly used microscope in a veterinary clinic is an upright binocular light microscope. This type of microscope has a light source located below the sample stage, an objective lens located above the sample stage, and two eyepiece lenses that the user looks through simultaneously to visualize the sample. Figure 1.1 is a diagram of a typical upright binocular light microscope.

     Figure 1.1 Microscope diagram. Components of an upright binocular light microscope are indicated. Common microscope components include two eyepieces, two draw tubes, a body tube, an arm, objective lenses, a revolving nosepiece, the microscope stage, stage clips, a mechanical stage control, a course adjustment knob, a fine adjustment knob, a condenser, an iris diaphragm, a lamp, and a rheostat

     (Source: David Ahn/Getty Images).

   • Components of an upright binocular light microscope.
     • Eyepieces typically hold lenses that magnify the sample by 10-fold (10×).
     • Draw tubes may or may not be present, depending on the brand of microscope. Draw tubes allow for minimal adjustment of sample focus to accommodate differences in visual acuity between each eye.
     • The body tube is the hollow section of the microscope between the eyepieces and the objective lenses.
     • The arm of the microscope supports the body tube and connects the eyepieces to the base of the microscope.
     • Objective lenses magnify the sample. The strength of magnification and whether or not the lens requires immersion oil to focus on the sample are indicated on the side of the objective lens. For most clinical pathology tests performed in veterinary clinics, at least three objective lenses are recommended: a low-power lens, a dry high-power lens, and a high-power lens that requires immersion oil for fine focusing.
       • Objective lenses are attached to a revolving nosepiece so that the user can easily switch between objective lenses while looking at a sample. Properly aligned microscopes require very minimal movement of the fine adjustment knob to sharpen the focus between the different objective lenses.
     • The microscope stage holds the sample under the objective lens and over the light source. A stage may have stage clips that secure the sample onto the stage if the microscope has a mechanical stage control.
     • A mechanical stage control allows the user to move the sample around the stage via a joystick.
     • A course adjustment knob allows the user to focus the sample using scanning and low-power lenses. This knob should not be adjusted when using high-power lenses.
     • The fine adjustment knob is used to make subtle changes that bring the sample into fine, crisp focus when using high-power lenses.
     • A condenser is located between the bottom of the stage and the light source so that light scatter can be minimized.
       • There is a condenser height adjustment knob in front of the coarse adjustment knob that changes the location of the condenser relative to the stage.
       • Condenser adjustment knobs are also located just below the condenser and are used to center the light source on the sample and align the light with the field of view through the eyepieces.
     • An iris diaphragm is found over the light source of a microscope.
     • The lamp of the microscope is the light source. Most microscopes have a rheostat located on the base that can be used to adjust the brightness of the light source.
     • The microscope base should be placed on a clean, flat surface for proper use of the instrument.
   • Magnification (TECHNICIAN TIP 1-1).
     • 2×, 4×, and 5× dry lenses are considered scanning lenses.
     • 10× and 20× dry lenses are examples of low-power lenses.
     • 40× and 60× lenses are high-power lenses that usually are dry lenses. A cover slip should be used with these objectives to prevent contaminating the lens with the sample.
     • 50× and 100× lenses are high-power lenses that usually require immersion oil to allow the user to focus on the sample.
     • Most microscopes in veterinary practices have 10× and 40× dry lenses and a 100× oil lens.

       TECHNICIAN TIP 1-1 TOTAL MAGNIFICATION

       
       

       The total magnification of a sample equals the product of the objective lens magnification and the magnification of the eyepiece lens. For example, if you are looking at a sample at "high power" (40× objective lens) and you have a 10× eyepiece lens, the total magnification of the sample is (40?×?10) = 400×.

   • Specimen evaluation.

     Different sample types are prepared for microscopic examination in different ways. Whole blood typically is smeared onto a glass slide, dried, and stained before examination. Urine sediment, on the other hand, is examined as a wet mount after the sample has been centrifuged, concentrated, resuspended in a smaller volume of urine, dropped onto a slide, and then covered with a cover slip. Feces can be examined as a direct smear after being stained or as a wet mount after fecal flotation has been performed. These processes are described in detail in other chapters of this book. The focus of this section is to describe how to examine dry, stained smears, and wet mounts using a microscope. Additional tips for adjusting a microscope are listed in TECHNICIAN TIP 1-2.

     • The microscope needs to be on a flat surface near access to an electrical power outlet to plug in the light source.
     • Microscope lenses should be properly installed and clean.
     • Typically, the iris is kept opened when examining a sample for clinical pathology tests but may be closed slightly to reduce the amount of light reaching the sample or to minimize light scatter, if needed.
     • Samples must be thin enough to allow light to shine through the specimen.
     • A scanning objective lens or 10×...