Hematology Techniques and Concepts for Veterinary Technicians E-Book

Gregg L. Voigt

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 243

Veröffentlichungsjahr: 2011

Beschreibung

Now in full color, Hematology Techniques and Concepts for Veterinary Technicians, Second Edition is a thorough update to this introduction to the fundamental concepts of collecting, handling, and preparing hematology samples. Covering the basics of blood composition, cell morphology, and sample collection, handling, and preparation, the book is designed specifically for veterinary technicians and students to gain a full understanding of why each test is performed and ensure accurate test results. In addition to addressing advances in technology, equipment, and test techniques throughout, a new chapter covers automated testing, and a companion website provides review questions and images from the book for download at www.wiley.com/go/voigt.

Key concepts have also been added to each chapter to better promote learning, and terms are now defined throughout the text, with the definitions collected into a glossary.  User-friendly and well-illustrated with charts, reference values, algorithms and photomicrographs, Hematology Techniques and Concepts for Veterinary Technicians, Second Edition is a key reference for veterinary technicians and veterinary technology students.

Leseprobe

Contents

Preface

Acknowledgments

Chapter 1 Introduction to the Hematology Laboratory

Uses and Benefits of Hematology Results

Limitations of Laboratory Findings

Laboratory Safety

Chapter 2 Composition of Blood

PCV, Buffy Coat, and Plasma

Blood Cells

Chapter 3 Blood Volume and Effects of Blood Loss

Approximate Total Blood Volumes

Effects of Blood Collection and Blood Loss

Chapter 4 Blood Collection and Handling

Venipuncture Site

Blood Collection Equipment

Venipuncture Techniques

Anticoagulants

Potential Collection Errors

Chapter 5 Blood Smears and Staining

Blood Smear Preparation

Staining the Blood Smear

Chapter 6 Routine Hematology Laboratory Tests

Packed Cell Volume (PCV)/Hematocrit (Hct)

Erythrocyte Sedimentation Rate (ESR)

Hemoglobin Determination

Total Plasma Proteins

Plasma Fibrinogen

Microscopic Examination of the Blood Smear

Differential Leukocyte Count

Absolute Leukocyte Count

Total Cell Counts

Chapter 7 Automated Laboratory Methods and Instruments

Hematology and Biochemical Analyzer System Overview

Scientific Chemistry Methods

Advantages and Disadvantages of Automated Equipment

Chapter 8 Leukocyte Cell Types and Functions

Leukocyte Distribution and Responses

Neutrophils

Eosinophils

Basophils

Lymphocytes

Monocytes

Chapter 9 Introduction to the Immune System

Nonspecific Immunity

Specific (Acquired) Immunity

Testing Immune System Response

Chapter 10 Erythrocyte Form, Function, and Indices

Erythrocyte Morphology

Erythrocyte Function

Red Blood Cell Indices

Chapter 11 Erythrocyte Abnormalities

Variations in Cell Size

Variations in Cell Shape

Cytoplasmic Variations and Inclusions

Chapter 12 Anemias and Polycythemias

Anemia

Polycythemia

Chapter 13 Hemostasis and Coagulation

Hemostatic Mechanisms

Hemostatic Defects

Coagulation Tests

Chapter 14 Hematopoiesis and Bone Marrow Examination

Hematopoiesis

Indications for Bone Marrow Examination

Chapter 15 Collection and Handling of Cytology Samples

Sample Collection

Sample Evaluation

Vaginal Cytology

Glossary

Recommended Reading

Index

This edition first published 2011, © 2011 by John Wiley & Sons, Inc.

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Disclaimer

The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specific method, diagnosis, or treatment by practitioners for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom.

Library of Congress Cataloging-in-Publication Data

Available through the Library of Congress.

Preface

The major reason for the preparation of this second edition is to introduce the veterinary technology student to new technology, equipment, and test techniques that are becoming more available in the field of veterinary hematology. The intent of the new edition remains the same as in the previous edition. It is an introductory overview of veterinary hematology and a guide for the student to learn the fundamental concepts of collecting, handling, preparing, and testing blood and other samples in the hematology laboratory. It is not intended as a diagnostic aid, differential disease discussion, or complete atlas of blood cells.

Although changes have been made throughout the text, the basic format of the first edition has been retained for the benefit of those currently using the book in the classroom. The major change is the addition of a new chapter, Automated Laboratory Methods and Instruments, by Dr. S. L. Swist. Since publication of the first edition, automated analyzers specifically designed for the veterinary professio have become more available, affordable, accurate, and easier to operate and are more commonly seen in veterinary clinics. This new chapter describes the various types of automatic analyzers, the basics behind their operating systems, and compares the benefits and drawbacks of their use. Dr. Swist has also added her expertise to other areas of the text.

Other changes made to the new edition include a “Key Concept” area at the beginning of each chapter to inform the reader of the major concepts being presented. Many color photographs and photomicrographs have been added to help new students recognize blood cells, techniques, and equipment of the hematology laboratory. Whenever a new term is used in the text, it is accompanied by a definition or recognizable synonym. In addition, a glossary of terms has been added at the end of the chapters with a brief description or definition of terms.

Although the metric system is the standard throughout the medical field, many students accustomed to the American system have difficulty picturing metric units. In many places in the text where metric units are used that relate to animal size and weight, or blood volumes, the approximate US customary units are also given.

Since the original publication of this book, student use of the Internet has increased markedly and an accompanying website has been prepared by the publishers with further information, photographs, and study questions from the authors.

About the Review Questions

Review questions are provided at the end of each chapter and on the website. These questions are provided to stimulate the student to review the material in the chapter, and in any lecture or laboratory notes, in order to better understand the topic being presented by personal study or discussion groups with fellow students. Since the chapters themselves (with associated subheadings, figures, and diagrams) are the answers, a formalized list of answers is not provided. As the title of the book states, the concepts and techniques of hematology and how blood samples are tested are more important than the individual’s ability to pick a word off a list of possible answers.

Acknowledgments

The authors would like to thank the personnel from the following veterinary clinics for their help in the preparation of this text:

The Stock Doc (Riverton, WY), Amy Stockton, D.V.M., Miranda Townsend, D.V.M., Lisa Deutsch, R.V.T., and assistants, Jo Ann O’Neal, Randy Townsend, Amanda Miller, and Angela Mellville.

Neel Veterinary Hospital (Oklahoma City, OK), Tina Neel, D.V.M., Karen Anderson, D.V.M., Misty Fox, C.V.T., and Kat Yeatman.

Westfield Animal Hospital (Westfield, NJ), Ronald Swist, D.V.M. and Margaret Swist

The staff at the Wyoming State Veterinary Laboratory (Laramie, WY), Mariah Hall, Katherine Bardsley, Victoria Creager, Kara Robbins, Hank Edwards, Tracy Dunn, and Jessica Prescott

Chapter 1

Introduction to the Hematology Laboratory

Key Concepts

In the hematology laboratory, manual and automated test techniques are used in the study of blood and other body cells and fluids.

Blood tests obtained in the laboratory are used by the clinician for screening the health status of an animal or in making a diagnosis of a disease condition.

Quality control of laboratory procedures is essential to ensure that test results reflect the true status of the patient.

Hematology is the study of blood and the tissues that form, store, or circulate blood cells. Examination of the blood is a very common and useful procedure for several reasons. Blood bathes all the other cells of the body carrying nutrients, oxygen, and waste products, and is exposed to almost all metabolic processes of these cells, often reflecting any alteration from normal function. Blood is essential in water and electrolyte balance, temperature control, and the functioning of the immune system, which is the defense mechanism of the body. Obtaining, studying, and testing a blood sample is a relatively easy way of gathering information on many parts of the body.

Even though the term “hematology” literally means “the study of blood,” many of the techniques of collection, sample preparation, and cell identifications learned by the technician can be applied to other regions of the body, such as joint fluid, cerebrospinal fluid, thoracic and abdominal fluids, aspirants from abnormal growths or swellings, and cells collected from mucous membranes (e.g., the oral cavity, trachea, or vagina).

Veterinary technicians should expect to encounter a wide range of laboratory facilities and instrumentation in various veterinary clinics. This can vary from a separate room fully equipped with the latest automated analyzers to a small area on one counter with a microscope, slides, and stains in smaller clinics (Figs. 1.1, 1.2, and 1.3). Even if the clinic sends out all of its hematology tests to a commercial lab, or to the local human hospital, the technician is still usually responsible for correctly collecting, preparing, and mailing or delivering the sample as well as reporting and recording the results.

Hematology is one of several specialties in the field of clinical pathology, a field that encompasses any manual or automated laboratory procedure used on the animal to aid in diagnosing a clinical condition. Clinical chemistry, parasitology, and urinalysis are additional classical aspects of clinical pathology. Other diagnostic tools used by the veterinarian that qualify as clinical pathology under this definition are microbiology, diagnostic imaging (radiology and ultrasound), and the microscopic interpretation of biopsies (tissue samples).

Figure 1.1 Some clinics may have limited laboratory equipment and may routinely send samples to hospital or commercial laboratories.

Figure 1.2 Technicians will find clinics with an area dedicated to laboratory work with some of the latest automated equipment.

Figure 1.3 Larger veterinary hospitals often have a complete laboratory room that may be staffed by one or more full-time laboratory technicians.

Uses and Benefits of Hematology Results

The most common use of the hematology laboratory is to screen the general health of an animal and to assess its overall ability to transport oxygen and defend against infectious agents. Hematology results provided by the technician are also used by the veterinarian as tools that, when combined with the history, physical examination, and other laboratory findings, help to form a diagnosis. Although occasionally a blood test will yield a definitive diagnosis (e.g., blood parasites), most laboratory results should be viewed as large or small pieces of a diagnostic puzzle that must be assembled.

Hematology results may also indicate a course of treatment for the animal. An example of this is the use of hematology to differentiate between anemia caused by internal hemorrhage and that caused by bone marrow depression. Another example is finding specific infectious organisms within blood cells. Laboratory findings may also suggest other beneficial tests, such as a bone marrow biopsy.

Serial sampling, which is the collection and testing of a series of blood samples over a period of time (hours, days, or even weeks), can demonstrate the severity of a disease process and the ability of the animal to respond. By combining these results with clinical evaluation, the veterinarian will be better able to understand the disease process and make a prognosis for the patient.

It has been said that a clinician who relies entirely on laboratory results to make a diagnosis is probably inexperienced and a clinician who claims not to need a laboratory is uninformed.

Limitations of Laboratory Findings

The validity and usefulness of both manual and automated laboratory results can be influenced by many factors that should be understood by the technician, so they may be eliminated or minimized as much as possible. Technical errors associated with individual tests are discussed in later chapters, but those of general laboratory procedures are addressed in this section.

The first potential problems arise in the collection and handling of the sample. The blood sample, or any other tissue sample, should not be subjected to traumatic physical forces, such as being forced rapidly through a small needle or violently shaken, or to extreme temperatures of heating or freezing. Care must be taken to avoid contaminating the sample with foreign material such as dirt, infectious agents, chemicals, or even water. Test results are always more meaningful with fresh samples, but when this is not possible samples should be refrigerated since blood, like other organic material, begins to degenerate after removal from the body. A more extensive list of sample handling errors is found in Chapter 4.

Quality control, or more accurately “quality assurance,” should be considered a laboratory commitment, rather than a laboratory problem. Whether the test procedures are performed manually, carried out by automated equipment, or even sent to a local hospital or a commercial or state laboratory, it is essential to know that the results reflect the patient’s status and not a difference in machines, technicians, or techniques.

Test results often vary with different test conditions, and the “normal” animal may routinely test higher or lower than published “normal” ranges. Commercial and state laboratories usually provide their established reference values for each test being performed on each species with the laboratory report. Clinical laboratories should establish reference values for any manual or automated test procedure performed in the clinic. For reference values to be statistically valid, a large number of tests must be performed, which may be difficult in a small clinical setting. By keeping a record of all tests run (and data on the patients), plus checking medical records for other similar test results, clinics can establish a database to compare with published values. At a minimum, tests on several known normal or control samples should be run for each procedure by each technician who will be performing the tests. These control samples can also be sent to the outside laboratory being routinely used and the results compared with the in-house results. Some slight variation in results should be expected, but they should fall within the established range for that procedure. Paired samples, half sent to an outside source and half run in-house for comparison, can also be quite instructive. Running such tests will ensure that current test procedures and results are reliable, or the tests can be used to establish reference readings for a new technique, technician, or a piece of equipment. The frequency of checking in-house tests will vary with the type and a number of tests performed and often varies from daily to weekly to monthly but should never be overlooked.

Another area of quality control that must not be overlooked is the variation in skill levels, accuracy, and care of the technician, especially in manual procedures. While the decision about which laboratory test to perform in the clinic or hospital and which to send out depends a great deal on the cost-effectiveness and timely availability of outside services, the number of tests being performed should also be considered. Routinely, if a test is not being done at least a few times per week, it does not provide the laboratory personnel with adequate opportunities to become skilled at, and comfortable with, the procedure or the equipment.

The veterinary technician is often responsible for collecting, preparing, and examining the sample and reporting the results. All counts or measurements should be done on a “blind” basis, that is, with no comparisons to normals or expectations of changes that could lead to bias in reported results. Since the technician may be the only person to actually see the sample, it is imperative that all observations, whether normal or abnormal, be recorded. Often the “comments” section of a lab report is as informative as the recorded results.

Laboratory Safety

Although most diseases of domestic animals routinely handled by the technician are not communicable to humans (zoonotic), the ones that tend to be quite severe, possibly fatal. All biological samples, whether blood or other body tissues and fluids, should be handled as if potentially infectious. Routine hand washing and disinfection of glassware and working areas is essential. There should be no drinking, eating, use of tobacco products, or other hand-to-mouth activities, nor any storage of food or beverages in the laboratory area. A laboratory coat should be worn at all times, long hair confined (especially around Bunsen burners and centrifuges!), and sandals, open-toed shoes, or canvas shoes should be avoided. Laboratory gloves should be worn whenever potentially infectious samples are being handled (Fig. 1.4). All disposable pointed or cutting instruments (e.g., needles and scalpel blades) should be placed in an appropriate “sharps” container, such as an empty gallon jug or commercial container, prior to disposal. Laboratory safety policies should not only be posted in the laboratory area but also be read and followed.

Figure 1.4 A laboratory smock should be worn at all times, and protective gloves, eyewear, and confining of long hair are important when working around potentially infectious or hazardous conditions.

Review Questions

1. What is the major function of the hematology laboratory?

2. Compare the benefits and limitations of the hematology laboratory.

3. What is meant by the term “quality control” in relation to reporting test results?

4. Why is laboratory safety important to the hematology technician?

Chapter 2

Composition of Blood

Key Concepts

Blood is a tissue composed of a fluid called plasma and seven cellular elements called erythrocytes, neutrophils, eosinophils, basophils, lymphocytes,monocytes, and platelets.

The cellular elements of the blood can be identified by their individual characteristics and the staining qualities of their cellular contents on a prepared glass slide.

Blood is a type of connective tissue, and collecting a blood sample is essentially taking a tissue biopsy. Blood is composed of cells surrounded by a noncellular substance, just like other connective tissues, such as fibrous tissue, bone, or cartilage. The major difference is, of course, that the extracellular substance in blood is liquid, called plasma. This characteristic, plus the fact that much of this “tissue” is located near the surface of the animal, makes collecting a sample of blood comparatively easier than sampling deeper, more solid organs and tissues.

PCV, Buffy Coat, and Plasma

If drawn blood is kept from clotting by the addition of an anticoagulant (see Chapter 4), we can separate the blood into its various components. If allowed to set undisturbed (or placed in a centrifuge to speed up the rate of separation of the components), three distinct layers will appear: the heavy red cells on the bottom, the lighter white cells in the middle, and the plasma on the top (Fig. 2.1).

The bottom one-third to one-half of the tube will be dark red and will contain the heaviest (most dense) structures, the erythrocytes (red blood cells). The measurement of the percentage of these cells in the blood is called the packed cell volume (PCV) or hematocrit (Hct) and is commonly one of the first blood evaluations performed (see Chapter 6).

Directly on the top of the red blood cells will be a relatively thin, white to tan layer called the buffy coat. This layer contains the leukocytes (white blood cells) and the platelets. The thickness of this layer can be a basis for a rough estimate of the relative numbers of these cells in the sample. The occasional appearance of a light pink to red color in the buffy coat indicates the entrapment of some lighter density erythrocytes in this layer.

At the top of the tube will be the fluid portion of the blood sample, called plasma since the blood has been separated without clotting. If the blood had been allowed to clot, several proteins in the fluid would have been used to form the clot and the fluid would then be called serum (Fig. 2.2). The plasma color will vary from clear to straw to yellow-orange (due to species variation, diet, and physiologic or pathologic conditions) but should be transparent. The technician should always observe the visual characteristics of the plasma, especially any cloudiness, abnormal coloration, or layering, and write descriptive comments on the laboratory report. (Remember, the technician may be the only one to see the sample and should pass on the information!)

Figure 2.1 The components of unclotted blood.

Figure 2.2 Physical difference between plasma and serum. (a) Mixed fluid and cells in whole blood. (b) With anticoagulant added, cells separate from plasma. (c) Without an anticoagulant, cells and clotting factors separate from serum.

Blood Cells

In order to see and evaluate individual blood cells, a drop of blood must be spread on a slide and stained. This is one of the most common and important hematology procedures. Techniques for making and staining the blood smear, as well as suggested methods of viewing the slide under the microscope to ensure optimum examination of the cells, are given in Chapters 5 and 6.

There are seven “formed elements” (cells or cell fragments) found in the blood:

Erythrocytes (red blood cells or RBCs) (1) Leukocytes (white blood cells or WBCs) Granulocytes Neutrophils (2) Eosinophils (acidophils) (3) Basophils (4) Agranulocytes Lymphocytes (5) Monocytes (6) Thrombocytes (platelets) (7)

A diagram of the basic differences in appearance of each of these cells is shown in Fig. 2.3. A brief description of the appearance and function of each is then presented to allow the reader to become familiar with and begin to identify these cells. More detailed descriptions are given in later chapters.

Figure 2.3 The seven “formed elements” (cells and cell fragments) of the blood.

Erythrocytes

The most common cell encountered in the blood is the erythrocyte; overall, there are approximately 1,000 erythrocytes for every leukocyte. On a slide, the erythrocyte of most mammals is a round, homogeneous, nonnucleated cell that stains pink to salmon to red ( Fig. 2.4). In the dog, the center of the cell is thinner than the edges (biconcave) and more lightly stained, but this shape is less pronounced in the other common species. The erythrocyte in the members of the camel family (camels, llamas, etc.) is routinely oval, and in birds, reptiles, amphibians, and fish, it is oval and nucleated (see Chapter 10).

Figure 2.4 Normal canine erythrocytes. The central pale areas are due to the biconcave nature of the cells. In most other species of domestic animals, erythrocytes are flatter.

The major function of the erythrocyte is to transport oxygen from the lungs to be released in the cells and tissues throughout the body. The carbon dioxide that is generated by the cells is then carried back to the lungs and exchanged for oxygen.

Leukocytes

Scattered among the erythrocytes on the slide preparation are nucleated cells of varying sizes, some of which contain granules that stain various colors. These are the leukocytes, of which there are five types. The neutrophil, eosinophil, and basophil routinely have granules present in the cytoplasm (cellular fluid) and are categorized as “granulocytes,” while the lymphocyte and monocyte are “agranulocytes.” Although the leukocytes have somewhat different individual functions, which are discussed in Chapter 8; in general, their activities are related to recognizing and responding to any substance foreign to the body, especially potential disease-causing agents, such as bacteria, viruses, and fungi.

The most common of the leukocytes in most animals, and the second most common in the rest, is the neutrophil. Even with slight differences in appearance between species, it is usually the most easily recognized leukocyte. The neutrophil is somewhat larger than an erythrocyte and is characterized by its densely stained, clumped-appearing nucleus, which is elongate and usually deeply indented or constricted (Fig. 2.5a). This clumping often makes the nucleus appear to have two to five separate lobes or segments, which leads to the common names of “segmenter” or “polymorphonuclear” (PMN), meaning many shapes to the nucleus. When stained, the cytoplasm is clear to pale blue or gray and contains scattered to numerous neutrophilic (gray to light pink) granules. In some species, the granules are quite obvious, but in many species, they appear as a fine dust or are too small to see clearly with the light microscope, and so the cytoplasm will appear clear. If the nucleus is more ribbon-shaped, with parallel sides, it is called a band or stab neutrophil, and if more than five segments are seen, it is referred to as hypersegmented. Differentiating between these developmental stages is an important diagnostic aid and is discussed in Chapter 8.

Figure 2.5 (a) Horse neutrophil, (b) cow eosinophil, (c) cat basophil, (d) cat lymphocyte, (e) sheep monocyte, and (f) horse platelets. The indistinct cell borders and cytoplasmic contents are typical of cells viewed on most microscopes under very high magnification. (Amount of magnification and final printed cell sizes do not reflect normal relative sizes.)

The eosinophil (or acidophil) is the same size or slightly larger than the neutrophil and has a nucleus that may be band-shaped or constricted into a bilobed or trilobed appearance (Fig. 2.5b). The cytoplasm is usually a light blue but may be difficult to see due to the presence of distinctive granules. The size, shape, number, and staining qualities of these granules vary greatly between species and are so characteristic that the eosinophil can often be used to determine the species from which the sample was obtained. These cytoplasmic granules are called eosinophilic because they chemically attract eosin, the red dye used in staining. In general, they appear pink to orange to salmon colored, and occasionally bright red, in routine laboratory stains. They often refract (bend and scatter) light as it passes through the granules, which makes focusing directly on them difficult, and they may look like bright dots. Since overall staining qualities will vary with different techniques and species, a useful comparison is that the color of the granules should be similar to, or slightly lighter staining than, the surrounding erythrocytes.

The basophil is rarely encountered in normal blood smear preparations. Intermediate in size between the neutrophil and eosinophil, it has an elongate to slightly indented nucleus that has less densely staining chromatin. The nuclear outline is often obscured by basophilic granules in the cytoplasm that stain from light or dark purple to almost black (Fig. 2.5c). The number of granules may vary from few to densely packed and may be very small and light staining (as in the cat) or larger and very dark (as in the ruminant).

The lymphocyte is the most common leukocyte seen in many ruminants and rodents. It is unique among the leukocytes in that when it is stimulated it has the ability to change its size and shape into large, medium, and small forms, and it can form more oval cells called plasma cells. The mature lymphocyte is the smallest of the leukocytes, often barely larger than the erythrocyte, and has a round nucleus surrounded by scanty, blue-staining cytoplasm, which is often barely detectable (Fig. 2.5d). The nuclear chromatin is coarse and clumped, and a small indentation may be present at the nuclear periphery. Much larger lymphocytes are also seen, especially in cattle, with more abundant cytoplasm and a larger nucleus that may become more oblong or rectangular. These cells are easily distorted by surrounding cells. When the cytoplasm stains a more intense blue, the term “immunocyte” is often applied. Some lymphocytes will form plasma cells (plasmacytes), which are oval and have a round, eccentrically placed nucleus in which the clumped chromatin often has a cartwheel appearance. These cells are discussed in more detail in Chapters 8 and 9.

The monocyte is the largest of the leukocytes and is similar in appearance in all common species. It is characterized by a pleomorphic, or ameboid, nucleus that can assume various shapes from elongate to rounded and is often in kidney bean, horseshoe, butterfly, or Hshaped (Fig. 2.5e