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 3 RD
EDITION

Canine and Feline

Cytology
A COLOR ATL AS AND INTER PR ETATION GUIDE

RO S E E . R A S K I N, DVM, PhD, DAVCP

Professor Emerita of Veterinary Clinical Pathology
Department of Comparative Pathobiology
College of Veterinary Medicine
Purdue University
West Lafayette, Indiana;
Visiting Professor
Department of Physiological Sciences
College of Veterinary Medicine
Purdue University
West Lafayette, Indiana

DE N N Y J. M E Y E R , DVM, DACVIM, DACVP

Executive Director, Navigator Services
Senior Clinical Pathologist
Charles River Laboratories
Reno, Nevada
3251 Riverport Lane
St. Louis, Missouri 63043

CANINE AND FELINE CYTOLOGY: A COLOR ATLAS

AND INTERPRETATION GUIDE, THIRD EDITION ISBN: 978-1-4557-4083-3
Copyright © 2016 by Elsevier, Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechan-
ical, including photocopying, recording, or any information storage and retrieval system, without permission in
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sions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright
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than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden
our understanding, changes in research methods, professional practices, or medical treatment may become
necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and
using any information, methods, compounds, or experiments described herein. In using such information or
methods they should be mindful of their own safety and the safety of others, including parties for whom they
have a professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised to check the most
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Previous editions copyrighted 2010 and 2001.

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Vice President and Publisher: Loren Wilson

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Content Development Manager: Jolynn Gower
Content Development Specialist: Brandi Graham
Publishing Services Manager: Julie Eddy
Senior Project Manager: Celeste Clingan
Design Direction: Renee Duenow

Printed in Canada

Last digit is the print number: 9 8 7 6 5 4 3 2 1

 CONTRIBUTORS

Claire B. Andreasen, DVM, PhD, DACVP Ul Soo Choi, DVM, PhD

Professor and Associate Dean for Academic and Student Associate Professor
Affairs Department of Veterinary Clinical Pathology
College of Veterinary Medicine College of Veterinary Medicine
Iowa State University Chonbuk National University
Ames, Iowa Jeonjui, Republic of Korea
Oral Cavity, Gastrointestinal Tract, and Associated Structures Endocrine System

Tara P. Arndt, DVM, Cert LAM, Dip LAS (Path), DACVP Keith DeJong, DVM, DACVP
Staff Pathologist Veterinarian, Technical Services
Covance Laboratories, Inc. Boehringer Ingelheim Vetmedica, Inc.
Madison, Wisconsin St. Joseph, Missouri
Endocrine System Urinary Tract

Anne C. Avery, VMD, PhD Shannon J. Hostetter, DVM, PhD, DACVP

Associate Professor Assistant Professor, Veterinary Pathology
Department of Microbiology, Immunology, and Pathology Department of Veterinary Clinical Sciences
College of Veterinary Medicine & Biomedical Sciences College of Veterinary Medicine
Colorado State University Iowa State University
Fort Collins, Colorado Ames, Iowa
Advanced Diagnostic Techniques Oral Cavity, Gastrointestinal Tract, and Associated Structures

Paul R. Avery, VMD, PhD, DACVP Albert E. Jergens, DVM, PhD, DACVIM
Assistant Professor Professor and Associate Chair for Research and Graduate
Department of Microbiology, Immunology, and Pathology Studies
College of Veterinary Medicine & Biomedical Sciences Department of Veterinary Clinical Sciences
Colorado State University College of Veterinary Medicine
Fort Collins, Colorado Iowa State University
Advanced Diagnostic Techniques Ames, Iowa
Oral Cavity, Gastrointestinal Tract, and Associated Structures
Anne M. Barger, DVM, MS, DACVP
Clinical Professor, Pathobiology Davide De Lorenzi, PhD, DECVP, SCMPA
Clinical Professor, Veterinary Diagnostic Laboratory Specialist, Clinic and Pathology of Companion Animals
College of Veterinary Medicine Veterinary Hospital “I Portoni Rossi”
University of Illinois at Urbana-Champaign Bologna, Italy
Urbana, Illinois The Central Nervous System
Musculoskeletal System
Maria Teresa Mandara, DVM
Dori L. Borjesson, DVM, PhD, DACVP Neuropathology Laboratory
Professor Department of Biopathological Science and Hygiene of Animal
Department of Pathology, Microbiology & Immunology and Food Production
School of Veterinary Medicine School of Veterinary Medicine
University of California, Davis University of Perugia
Davis, California Perugia, Italy
Urinary Tract The Central Nervous System

Mary Jo Burkhard, DVM, PhD, DACVP Carlo Masserdotti, DVM, DECVCP

Associate Professor Consultant in Clinical Pathology
Department of Veterinary Biosciences San Marco Veterinary Laboratory
College of Veterinary Medicine Padua, Italy
The Ohio State University Reproductive System
Columbus, Ohio
Respiratory Tract

v
vi Contributors

Denny J. Meyer, DVM, DACVIM, DACVP Laia Solano-Gallego, DVM, PhD, DECVCP
Executive Director, Navigator Services Senior Researcher
Senior Clinical Pathologist Department of Animal Medicine and Surgery
Charles River Laboratories College of Veterinary Medicine
Reno, Nevada Autonomous University of Barcelona
The Acquisition and Management of Cytology Specimens Barcelona, Spain
The Liver Reproductive System
Microscopic Examination of the Urinary Sediment
Craig A. Thompson, DVM, DACVP
José A. Ramos-Vara, DVM, PhD, DECVP Clinical Assistant Professor of Clinical Pathology
Professor of Veterinary Pathology Department of Comparative Pathobiology
Department of Comparative Pathobiology School of Veterinary Medicine
Animal Disease Diagnostic Laboratory Purdue University
College of Veterinary Medicine West Lafayette, Indiana
Purdue University Body Cavity Fluids
West Lafayette, Indiana
Advanced Diagnostic Techniques Heather L. Wamsley, DVM, PhD, DACVP
Veterinary Clinical Pathologist
Rose E. Raskin, DVM, PhD, DACVP ANTECH Diagnostics
Professor Emerita of Veterinary Clinical Pathology Tampa, Florida
Department of Comparative Pathobiology Dry-Mount Fecal Cytology
College of Veterinary Medicine
Purdue University Amy L. Weeden, DVM
West Lafayette, Indiana Clinical Pathology Resident
General Categories of Cytologic Interpretation Department of Physiological Sciences
Skin and Subcutaneous Tissues College of Veterinary Medicine
Lymphoid System University of Florida
Appendix Gainesville, Florida
Eyes and Adnexa Dry-Mount Fecal Cytology

Alan H. Rebar, DVM, PhD, DACVP

Executive Director of Discovery Park
Senior Associate Vice President for Research
Professor of Veterinary Clinical Pathology
School of Veterinary Medicine
Purdue University
West Lafayette, Indiana
Body Cavity Fluids
From Rose:
I dedicate this third edition to the memory of John Van Vleet, DVM, PhD, DACVP, who as my
teacher, mentor, and close friend influenced my veterinary career path towards pathology. He
will be remembered and sorely missed.
To my parents, who always supported my career goals and provided a loving environment with
an abundance of household pets, I could not have succeeded without them!
To my brother Richard, other family members, and my lifetime friends, I am eternally grateful
for your love and steadfast support whenever I needed you.
Last but not least, to my daughter Hannah, who continues to win over her struggles, I am so
proud of you! You remain the love of my life.

From Denny:
To my dad, who instilled in me a work ethic and lifestyle founded on firm principles and prayer.
When thoughts of him float through my memory, a smile appears on my face . . . and to Mary,
his walking and prayer partner in life.
To my brother, Michael, who is . . . well . . . my favorite brother and to his family for their
c­ ontinued friendship.
My two greatest accomplishments in life are as father and husband. I have the undying love and
unwavering support of my amazing, beautiful family.
To son Christopher, daughter-in-law, Claudia, grandson Alexander, and granddaughter Lexi;
thank you for reaching across the miles to enrich my life and making me feel valued.
To daughter Jen, son-in-law, Ross, and granddaughter Bianca, my playmate, who grounds me to
this planet and keeps me young in heart and mind.
Saving the best for last, to my beautiful, vibrant, vivacious wife, Jae C, who has always been wise
beyond her years, although I did not realize it until recently! Now I get it! It is time to attack that
“bucket list” and continue having fun together. Thank you for your undying commitment to our
love affair.

From Both:
To all the veterinary students, residents, and practitioners who have touched our lives and made
us feel that what we do is worthwhile, we thank you.

People Touch Our Lives – Bits and Pieces

People important to you, people unimportant to you,
cross our life, touch it with love and carelessness and move on.
There are people that leave you, and you breathe a sigh of relief,
and you wonder why you ever came in contact with them.
There are people who leave, and you breathe a sigh of remorse,
and wonder why they had to go and leave such a gaping hole.
People move in and out of each other’s lives,
and each leaves a mark on the other.
You find you are made up of bits and pieces of all who have ever touched your life,
and you are more because of it, and would be less if they had not touched you.
Pray to always accept the bits and pieces in humility and wonder
and never question and never regret.
Adapted from a poem by Lois Cheney.
P R E FA C E
“Another source of fallacy is the vicious circle of illusions which
consists on the one hand of believing what we see and, on the other,
seeing what we believe.”
– Sir Clifford Allbutt, M.D. (1836-1925). He introduced the
diagnostic use of the microscope to the hospital ward.
The objective of the first and second editions of the Atlas was
to compile a practical guide to cytopathology that focused pri-
marily on the types of lesions that clinicians faced in routine
practice, yet be a user-friendly teaching tool for the soon-to-be
practitioner. We used tables, brief descriptions, and carefully se-
lected photomicrographs accumulated over decades of diagnos-
tic cytology with concise, informative figure legends to support
the microscopic examination of the cytology specimen. We at-
tempted to organize the presentations into logical and uniform
approaches, thereby facilitating readability, comprehension, and
learning. Based on the robust positive feedback we received, we
are pleased to surmise that we have generally achieved that ob-
jective.
Constructive suggestions indicated that the cytopathologist
desired additional lesions be covered, including those less com-
monly encountered, additional images of each disease, more
histopathology correlates, and a broader use of stains and im-
munocytochemistry for differential cytologic characterization.
The encouragement incentivized us to expand the photomicro-
graph portfolio, including more comparative histology, and the
attendant text and references. This was accomplished by adding
new authors, international subject matter experts, who injected
their pragmatic microscopic expertise into expanded chapters.
The enhanced portfolio of images has also been made possible
by the helpful assistance of other benevolent internationally
known cytopathologists, who generously contributed photomi-
crographs from their collections.
Specific changes to the third edition include substantial im-
provement of the quality of the images, providing closer rep-
resentation of the original microscopic hues. All chapters have
been updated according to current veterinary terminology,
classification schemes, and diagnostic testing availability. These
changes particularly affect the chapters on skin, hemolymphatic
viii
system, body cavity fluids, reproductive system, and advanced
diagnostic techniques. An exciting new section is the Appendix
covering microscope basics and telecytology, advanced staining
protocols, demonstrations of artifacts and polarizing substances,
handy nuclear chromatin chart, advanced cell preparation tech-
niques, lists of specialized diagnostic testing, and guides for
­cytology quality assurance.
Please note that image magnifications often change during
the publication process. As such, figures indicate structure sizes
either with internal bars or magnification noted relative to the
original objective lens used during image capture. The notations
for the objectives are: LP (low power) for 4x or 10x; IP (inter-
mediate power) for 20x or 40x; HP oil (high power oil) for 50x,
60x, or 100x oil immersion objectives.
It is our hope that by careful editing to ensure a clear and
concise narrative, seamless integration of new and updated in-
formation into the existing text, judicious selection of new and
enhanced photomicrographs, and the use of lists that highlight
criteria for differential diagnosis, we have produced a signifi-
cantly updated edition that will continue to find preferred res-
idence beside the microscope because of its utility. Students,
veterinary technologists, general practitioners, and veterinary
specialists will easily find what they need within well-referenced
chapters logically organized by body systems.
We present the third edition with considerable excitement
and hope that we have succeeded in transmitting to the user the
beauty of the expanded application of diagnostic cytology. We
share in the exhilaration of the microscopist when the unknown
cytologic specimen is translated into a cytologic diagnosis, a Eu-
reka moment, because they “believe in what they see,” with the
guidance of this Atlas.
Rose & Denny
 ACKNOWLED GMENTS

Teamwork = Cooperative effort by the members of a group or team to achieve

a common goal.
Achievement = Something accomplished successfully, especially by means of exertion,
skill, practice, or perseverance.
—American Heritage Dictionary, 4th edition

An Atlas that successfully covers the broad scope of cytopa-
thology could not be completed without the assistance of an
editorial staff, many of whom are transparent. Thanks to Heidi
Pohlman and Penny Rudolph for believing in us one more time
during the planning of the third edition. Noteworthy recog-
nition of folks at Elsevier is extended to Brandi Graham, who
exhibited remarkable patience as we missed timelines and ad-
ministered respectful, tenacious encouragement and respectful
prodding to keep the process in motion. Lastly, the worker bees
constituting the editing staff who, along with Celeste Clingan in
the final stages of the project, were technically terrific, consci-
entious, and attentive to detail. Collectively, they made us look
good and helped produce a quality product of which we are all
very pleased and proud.
We wish to express our sincere appreciation to the contrib-
uting authors of the third edition. They are represented both by
the seasoned and the newer, most promising purveyors of cy-
tology today. Their collective expertise has markedly extended
the range of information that is embedded in this new edition.
We could not have worked with a more energetic, enthusiastic
group of professionals; they spoiled us with their responsive-
ness. They altruistically added one more burden to their pri-
mary professional duties to share their cytologic expertise for
betterment of veterinary patient care. Thank you for successful-
ly partnering with us. We hope you share in our pride with the
final product, simply put, it is awesome!
Rose would like to acknowledge her co-editor, Denny, for
providing his remarkable skill of language massage and speedy
editorial assistance that complemented perfectly the deficits
she has.
Lastly, Denny takes the opportunity to acknowledge Rose.
She was clearly, again, the indefatigable driving force of the
third edition. Her passionate commitment to exhaustive com-
pleteness, accuracy, and detail translated into the differentiating
excellence of this benchmarking edition.
Rose & Denny

ix

The Acquisition and Management
of Cytology Specimens
The classification of events that depend on the accuracy of observation is limited by the
­ability of the observer to describe and of the interpreter to decipher.
For the microscopic examination of tissue, one important fac-
tor that affects the accuracy of observation is specimen man-
agement. The successful use of aspiration cytology depends on
several interrelated procedures: acquisition of a representative
specimen, proper application to a glass side, adequate staining,
and examination with a high-quality microscope. A deficiency
in one or more of these steps will adversely affect the yield of
diagnostic information. The objective of this chapter is to pro-
vide general recommendations for managing samples in order
to ensure accurate diagnosis.
GENERAL SAMPLING GUIDELINES
Before executing any sampling procedure, a cytology kit should
be prepared and dedicated for that purpose. An inexpensive
plastic tool caddie works well. Suggested contents are listed in
Box 1-1. Six or more slides are placed on a firm, flat surface such
as a surgical tray immediately before initiating the sampling pro-
cedure. The surface of the glass slide should be routinely wiped
with a paper towel, or at least on a shirtsleeve, to remove “invis-
ible” glass particles that interfere with the spreading procedure.
Table 1-1 lists biopsy techniques, example specimens, and
suggested cytologic preparation techniques. The collection of
specimens for cytologic evaluation from cutaneous and subcu-
taneous tissues and abdominal organs and masses in smaller
animals is generally accomplished with a 20- or 22-gauge, 1- to
1½-inch needle firmly attached to a 6- or 12-mL syringe. For
internal organs that are more difficult to reach, a 2½- to 3½-inch
spinal needle is used. The added length amplifies the area for cell
collection and enhances the diagnostic yield—cores of hepatic
tissue can be obtained with a longer needle. The stylet can be left
in place as the cavity is entered to avoid contamination during
the “searching” process of locating the tissue of sampling inter-
est. Coating the needle and syringe hub with sterile 4% disodium
ethylenediaminetetraacetic acid (EDTA) before aspiration biopsy
sampling of vascular tissues, notably the bone marrow, reduces
the risk of clot formation that will compromise the quality of the
cytologic specimen. For the relatively inexperienced, this may be
a practice to consider routinely when sampling any tissue. Clotted
specimens are a frequent cause of cytologic preps of poor quality.
The general steps for obtaining a cytologic specimen are
illustrated in Fig. 1-1A-E. Following appropriate cleansing and
CHAPTER 1
Denny J. Meyer
—Michael Podell, M.Sc., D.V.M.
disinfectant application, the tip of the needle is inserted into the
tissue of interest, the plunger retracted slightly (0.5 to 1 mL of
vacuum), the needle advanced and retracted in several different
directions, the plunger released, the needle withdrawn, and the
specimen placed on a glass slide or in an EDTA (purple-topped)
tube as appropriate. Commercial aspiration guns (Fig. 1-1B)
are available that can be loaded with various size syringes (Fig.
1-1B). The syringe plunger sits within the trigger, which allows
for easier and more stable retraction. If fluid is obtained from
a mass lesion, the site is completely drained, the needle with-
drawn, the fluid placed in an EDTA tube, and the procedure
repeated with a new needle directed at firm tissue. Both speci-
mens are examined microscopically. To enhance operator flex-
ibility, a butterfly needle can be used to attach the needle and
syringe. Positioning and redirection of the needle is easier and
accommodates patient movement (Fig. 1-1C).
Aspiration is not a prerequisite for obtaining a cytologic spec-
imen. A technique based on the principle of capillarity, referred
to as fine-needle capillary sampling, can be performed by placing
a needle into the lesion with or without a syringe attached (Mair
et al., 1989; Yue and Zheng, 1989). The technique has diagnos-
tic sensitivity similar to that of aspiration biopsy when used
to sample a variety of tissues. Its major advantage is to reduce
blood contamination from vascular tissues such as liver, spleen,
BOX 1-1 Contents of the Cytology Kit
Clippers
Cleansing and disinfectant wipes
Syringes: 6 to 12 mL, 20 mL if necessary
Needles: 1- and 1½-inch—20- to 22-gauge; 2½- or 3½-inch spinal needle with
stylet
Bone marrow aspiration needles and core biopsy materials
Scalpel blades: #10 and #11
Culture swabs and applicator sticks for slide preparation
Box of precleaned glass slides with frosted end
Tubes: EDTA (purple top) and serum (red top without separator)
Rigid, flat surface on which 6 to 10 slides can be spread out
Butterfly catheters 21- to 23-gauge and intravenous extension tubing
Pencil or solvent resistant slide-specific black marker 4% sterile EDTA
Hair dryer
1
2 Canine and Feline Cytology
TABLE 1-1 Biopsy Techniques, Associated Specimens, and Cytologic Preparation Techniques
BIOPSY TECHNIQUE SPECIMEN
A. Aspiration of Solid Tissue
1. Suction unknown mass
2. Nonsuction vascular tissue
B. Fluid Aspiration
1. Bloody fluid effusion (pericardial)
2. Non-bloody fluid effusions, synovial fluid, cerebrospinal fluid, urine
3. EDTA syringe bone marrow
C. Incisional Biopsy soft tissue, bone marrow core
D. Excisional Biopsy masses, lymph node, eye, testicle
E. Scraping firm tissue, conjunctiva
F. Swab vaginal, fecal, oral, ocular
G. Washes prostate, urinary bladder, respiratory, peritoneal lavage
kidney, and thyroid. Cells are displaced into the cylinder of the
needle by capillary action as the needle is incompletely retracted
and redirected into the tissue three to six times. Personal prefer-
ence is justified when deciding between aspiration and nonaspi-
ration sampling for collection of the specimen. Through trial
and error, the operator may determine that each has value for
sampling different tissues.
KEY POINT Acquisition of the cytology specimen is an art that can
be honed only by practice. Selecting an appropriate mode of sampling
enhances the probability of obtaining accurate diagnostic information.
KEY POINT Routinely dry-wipe the surface of the glass slide to re-
move “invisible” glass particles that cause spreading deficiencies. Never
reuse washed glass slides.
DIAGNOSTIC IMAGING-GUIDED SAMPLE
COLLECTION
Cytology sample collection can be performed under the guidance
of fluoroscopy, ultrasound, and computed tomography. Ultra-
sound guidance is the preferred method because of its widespread
availability and portability. In addition, ultrasound provides real-
time monitoring of precise needle placement. The technique
and indications are detailed elsewhere (Nyland et al., 2002a).
Ultrasound-guided fine-needle aspiration biopsy (FNAB) is indi-
cated for cytologic evaluation of nodules and masses detected on
ultrasound and to evaluate organomegaly when a diffuse cellular
infiltrate such as lymphoma and mast cell tumor is suspected.
Most sarcomas exfoliate sparsely or not at all. A surgical or
ultrasound-guided cutting needle biopsy is recommended if the
FNAB sample is not conclusive. Ultrasound-guided FNAB can
be performed in most patients without chemical restraint or local
anesthesia. If chemical restraint is needed, agents that promote
panting should be avoided because this will lead to excessive
movement and gas ingestion (Nyland et al., 2002a).
Biopsy Guidance
Ultrasound-guided FNAB can be performed by freehand tech-
nique or with the aid of a biopsy guide fastened to the trans-
ducer. Freehand technique consists of holding the transducer in
one hand and inserting the needle with the other at an oblique
PREPARATION TECHNIQUE
squash, suspension cytospin
squash, blood smear
buffy coat smear
direct, sediment, cytospin
particle squash
imprint, tissue roll
imprint
imprint, spread, squash
imprint, roll
sediment, cytospin
angle to the long axis of the transducer but still within the scan
plane (Fig. 1-1D). This technique requires more skill but allows
for greater flexibility. If the needle cannot be seen during the
procedure, slightly moving the transducer into the path of the
needle and gently agitating the needle or injecting microbubbles
in saline solution through the needle will usually allow the nee-
dle’s position to be determined. Better visualization of the needle
can be achieved by ensuring needle placement within the focal
zone of the transducer. The biopsy guide holds the needle firmly
and directs the needle along a predetermined course within the
scan plane of the ultrasound transducer (Fig. 1-1E). This may
be easier for the beginner because the lesion is more easily and
reliably sampled; however, the biopsy guide limits transducer
movement.
Equipment and Technique
Sterility is maintained during the procedure. Routine skin prepa-
ration should be performed before needle puncture through the
skin. The transducer can be sterilized with transducer-­compatible
disinfectant and sterilizing solutions (a list of which can be found
in the user manual of the ultrasound machine). Following the
diagnostic ultrasound evaluation of the site of interest, the cou-
pling gel is wiped off and alcohol or sterile water is used as the
coupling media during the FNAB procedure. The use of a cou-
pling gel is avoided because it can introduce potentially mislead-
ing artifact into the cytologic specimen (see Chapter 4).
The most commonly used needles are 20- to 23-gauge hypo-
dermic and spinal needles. These are inexpensive and long
enough to pass through the biopsy guide and still reach most
lesions. Larger-bore needles are easier to visualize and generally
increase the reliability of sample collection, but they increase
the risk of hemorrhage. A larger-bore needle is used when aspi-
rating viscous fluids. Once the needle is placed in the lesion, the
stylet is removed and the needle is moved up and down within
the lesion until a small amount of fluid is seen within the hub of
the needle (Fagelman and Chess, 1990). This method generally
produces a sample with less blood contamination. Alternatively,
a syringe can be attached to the needle for better handling—a
few milliliters of negative pressure can be applied while mov-
ing the needle up and down. The negative pressure should be
released before removing the needle from the lesion. When pos-
sible, two or three samples should be obtained from each biopsy
site; a new needle is used for each sample taken. A large lesion
may have a necrotic center; therefore, samples should also be
collected from the margins.
 CHAPTER 1 The Acquisition and Management of Cytology Specimens 3

C D

E
n FIGURE 1-1 A, Aspiration biopsy technique. The needle is inserted into the tissue and redirected three or four times using an aspiration or
nonaspiration technique. The same concept generally applies to the use of the technique for sampling sites within the thorax or abdomen. B, Aspi-
ration gun. The use of the aspiration gun facilitates better control and more deliberate retraction during the aspiration process. C, Butterfly needle.
Using a butterfly needle attached to the syringe will allow more flexibility with fractious patients when removing fluid. A three-way stopcock can be
placed between the butterfly tubing and syringe to facilitate the removal of large amounts of fluid. D, Ultrasound guidance. Free-hand technique for
ultrasound-guided fine-needle aspiration biopsy. E, Ultrasound guidance. Biopsy guide is attached to a linear transducer that holds a needle firmly
for ultrasound-guided fine-needle aspiration biopsy. (A from Meyer DJ: The management of cytology specimens, Compend Contin Educ Pract Vet
9:10-17, 1987. B, Courtesy of Delasco.)

Complications but is rare with 22-gauge or smaller aspiration needles.

Complications associated with ultrasound-guided FNAB are
uncommon and depend on the experience of the operator,
size of needle, and type of lesion aspirated (Léveillé et al.,
1993). Patients should be evaluated for bleeding disorders
before FNAB, especially when highly vascular tissues are
sampled. Occasional needle tract tumor implantation has
been reported in animals (Nyland et al., 2002b). In humans,
implantation is associated with the use of large-bore needles
Because pneumothorax can develop following FNAB of the
thoracic structures, the patient should frequently be observed
for 12 to 24 hours after the procedure. Hypertension and
paradoxic hypotension has been reported in dogs follow-
ing ultrasound-guided FNAB of pheochromocytoma of the
adrenal glands (Gilson et al., 1994). Therefore, FNAB of the
adrenal gland must be performed cautiously when pheochro-
mocytoma is suspected.
4 Canine and Feline Cytology

A B

C D
n FIGURE 1-2 Slide preparation. A, The application of only a small drop or a portion of the specimen on the glass slide near the frosted end is an
important initial step for making a quality cytologic preparation. Placing too much material on the slide results in a preparation that is too thick and/or
spreads too close to the slide edges for diagnostic purposes. B, The specimen is gently but firmly compressed between the two glass slides (B) and
in the same continuous motion (C) the top slide is glided along the surface of the slide with the material directed away from the frosted end, resulting
in a feather-shaped spread of the specimen (D) referred to as the “sweet spot.” C, The location of the “sweet spot” is illustrated by this appropriately
labeled and stained compression preparation of a lymph node specimen. D, A squash preparation can be made by gently placing the top slide parallel
to the bottom slide and gliding apart with even pressure. (B from Meyer DJ, Franks PT: Clinical cytology: Part I: Management of tissue specimens,
Mod Vet Pract 67:255-59, 1986. C from Meyer DJ: The management of cytology specimens, Compend Contin Educ Pract Vet 9:10-17, 1987.)

MANAGING THE CYTOLOGIC SPECIMEN
Compression (Squash) Preparation
The compression (squash) technique is an important and adapt-
able procedure for the management of cytology specimens that
are semisolid, mucus-like, or pelleted by centrifugation. A small
amount of material is placed on a clean glass slide approxi-
mately ½ inch (1 cm) from the frosted end (Fig. 1-2A). A sec-
ond clean glass slide is placed over the specimen at right angles.
The specimen is gently but firmly compressed between the two
glass slides, and in the same continuous motion, the top slide
is pulled along the surface of the bottom slide, directing the
material away from the frosted end (Fig. 1-2B). The objective is
to redistribute the material, turning a multicellular mass into a
thin monolayer ideal for maximal flattening of individual cells
and even stain penetration. The compression preparation thus
optimizes the specimen for microscopic examination of cell
morphology. A properly prepared glass slide is characterized by
a feather-shaped (oblong) area, with a monolayer end referred
to as the sweet spot (Fig. 1-2C). An alternate method for the
squash preparation is placing the top slide parallel to the bottom
slide (Fig. 1-2D). A common mistake is the initial placement of
excess sample on the glass slide, resulting in a thick preparation
that is not possible to adequately examine microscopically.
KEY POINT Compression and spread of the specimen is a continuum;
there should be no momentary pause as the upper slide contacts the spec-
imen. Keep the flat surfaces of the two slides parallel. A common mistake
is to slightly angle the upper slide near the end of the gliding motion by
allowing a slight counterclockwise rotation of the wrist (clockwise if left
handed) to occur, causing cell lysis or uneven spread of the specimen. A
scraping sound of glass on glass can be heard when this occurs. Again,
wiping slides before the procedure will help ensure a uniform spread of
the cytologic specimen.
KEY POINT The term sweet spot refers to that area around the center
mass of a baseball bat, tennis racket, or golf club that is the most effective
part with which to make a successful hit. The same concept applies to the
location of the cytologic specimen if it is to make a successful diagnostic hit.
Cellular material too close to the ends or edges of the slide cannot be prop-
erly examined. When slides go through an automated stainer, their guiding
tracks can scrape off diagnostic material that is too close to the end of the
slide (Fig. 1-3). Material placed too far from the end the specimen may not
be exposed adequately to the stain. The ends and longitudinal edges of the
slide cannot be adequately examined because of the inability of the 40× dry
and 50× and 100× oil objectives to properly focus at those extremes.
 CHAPTER 1 The Acquisition and Management of Cytology Specimens
n FIGURE 1-3 Slide preparation. The clear area to the left represents
the guide track of the automated stainer that has partially scraped off
the only cytologic material present on the slide because it was located
too close to the slide’s end. (Wright; LP.)
KEY POINT If the compression preparation appears too thick, it prob-
ably is. Make another one. If the cytology specimen appears to be too
close to the end or edge of the slide, it probably is. Make another one. If
you have doubts regarding the quality of the preparation, make additional
preparations.
Management of Fluids
A fluid specimen should be immediately placed in an EDTA
tube to prevent clot formation. Fluid with a plasma-like consis-
tency can be handled in a fashion similar to the preparation of
a blood smear. A small drop of fluid is placed approximately ½
inch (1 cm) from the frosted end. The angled edge of a second
glass slide, with the acute angle facing the operator, is backed
into the specimen and drawn away from the frosted end as the
fluid begins to spread along its edge (Fig. 1-4A-C). The speed at
which the slide is moved depends on the viscosity of the sam-
ple—the thinner the specimen, the faster the slide should be
moved to distribute the specimen evenly and thinly. For a vis-
cous fluid specimen such as synovial fluid, the spreader slide is
moved with a slow and even movement.
All fluid initially applied to the slide must remain on the slide
(Fig. 1-4D&E). It is tempting to go off the end of the slide with
excess fluid, referred to as the “edge-of-the-cliff syndrome,” but
the result is the potential loss of diagnostic material, which is
thrown into the garbage with the spreader slide (Fig. 1-4D). The
“edge-of-the-cliff syndrome” poses a notable threat to pleural
and peritoneal fluids that contain clumps of neoplastic cells.
These cellular clumps often follow the spreader slide, finally
sticking to the surface when the fluid dissipates (Fig. 1-5A&B).
To avoid this cytologic disaster when excess fluid remains,
simply stop ½ inch from the end of the specimen slide, apply
the spreader slide to another clean glass slide, and repeat the
spreading procedure. When minimal excess fluid remains, the
fluid can be permitted to slowly flow back on itself for a short
distance. The thin part of the stained cytology slide preparation
can be used to estimate cell numbers and the relatively thick,
concentrated part (where the excess fluid is dried) can be eval-
uated for types of cells and/or infectious agents (Fig. 1-4E).
Although not an optimal preparation, this “poor man’s centri-
fuge” technique is useful in emergency settings for the initial,
rapid triage of a fluid specimen.
5
Sedimentation preparations can also be used to concentrate
cells in bloody, cloudy, or wash fluids. The sample can be cen-
trifuged in the same tube in which it was collected after direct
preparations have been made. After centrifugation, the major-
ity of supernatant is removed with a pipette and the cell pellet
resuspended in the remaining fluid. A smear and/or compres-
sion preparation can be made from the concentrated cell spec-
imen. It is important to remember that estimates of cell counts
cannot be made on concentrated samples, only from the direct
smear. Following these and other concentration techniques, cell
blocks can be made from the pelleted material to be evaluated
histopathologically and immunohistochemically. (See Appen-
dix for more information.)
The diagnostic yield of a predominantly bloody fluid speci-
men is enhanced with the buffy-coat concentration technique.
A microhematocrit tube is prepared as if to measure a hemato-
crit. The tube is broken just below the cellular concentrate (buffy
coat) and gently expelled onto two or three slides (Fig. 1-6A-C).
A direct smear or squash technique is used to spread the spec-
imen. The technique is valuable for hemorrhagic pericardial,
peritoneal, and pleural samples (Fig. 1-6A&B). It is also useful
for the examination of peripheral blood for neoplastic cells and
cell-associated infectious organisms.
Transudates and cerebrospinal fluid are low in protein and
cell numbers. The use of a cytocentrifuge (cytospin) is recom-
mended for the capture of all the cells (Fig. 1-7A&B). For cere-
brospinal fluid, a cytologic preparation should be made—ideally
within 30 to 60 minutes because the low specific gravity predis-
poses to cellular lysis. However, it appears that when inflamma-
tory and neoplastic cells and infectious agents are present, their
diagnostic cellular integrity is usually maintained for up to 12
hours with refrigeration.
KEY POINT For the management of fluid samples, routinely make di-
rect, centrifuged (or buffy coat), and cytospin (if possible) preparations and
assess each for the best diagnostic yield.
KEY POINT The refractometer-determined total solute (protein) con-
centration should be measured for all pleural and peritoneal fluids to
facilitate classification as transudate or modified transudate when that
information has diagnostic importance (Meyer and Harvey, 2004).
KEY POINT For low-protein fluids such as urinary sediments, cere-
brospinal fluid, and transudates, the cells can be washed off during the
staining process. The use of premade serum-coated slides facilitates the
adhesion of the cells, which can make a diagnostic world of difference.
Several drops of the excess serum not used for clinical chemistries are
applied to the entire surface of a glass slide, and the film of serum is
air-dried. Ten to 20 slide preparations are made. Once dry (not sticky to
the touch), the slides can be stacked together in an empty slide box and
placed in the freezer to prevent bacterial growth. Prior to use, several
slides are brought to room temperature. It is critical that no condensation
develops on the surface because it causes severe cell lysis.
HELPFUL HINT A hair dryer set on low heat or small personal fan
enhances even drying of fluid specimens. It can also be used to remove
condensation from the serum-covered slides taken from the freezer.
6 Canine and Feline Cytology

A B

C D

E
n FIGURE 1-4 Fluid material preparation. A, The procedure for making a cytologic preparation from a fluid specimen is illustrated. A small drop
of the specimen is placed approximately ½ inch (1 cm) from the frosted end of the slide. B, The spreader slide is slowly backed into the drop. C, Just
as the fluid begins to spread along its edge, the spreader slide is glided away from the frosted end. D, All of the original fluid drop should remain on
the slide, and the temptation to go off the end of the slide with excess fluid must be avoided. The lower slide illustrates a properly feathered fluid
specimen with the entire specimen remaining on the slide. The upper slide demonstrates the “edge-of-the-cliff syndrome” in which the excess fluid
was drawn off the slide’s end. E, Excess fluid that remains is allowed to partially flow back and is air-dried as illustrated by the small opaque dried
fluid triangle near the nonfrosted end of the slide. Alternatively, the edge of the spreader slide with the excess fluid adhering is transferred to another
clean slide and another smear made.
 CHAPTER 1 The Acquisition and Management of Cytology Specimens 7

A B
n FIGURE 1-5 Slide examination. A, Examination of the feathered edge of the lower slide pictured in Fig. 1-4D demonstrates clumps of cells
located along the point where the fluid feathers out, emphasizing the need to leave the excess fluid on the slide. The area to the right of the cell
clumps consisted of only erythrocytes. (Wright; IP.) B, A diagnosis of a neoplastic effusion (adenocarcinoma) was made by examining the cell clumps.
(Wright; HP oil.) The upper slide pictured in Fig. 1-4D of the same specimen contained only erythrocytes and a few mesothelial cells but no cell
clumps, precluding a cytologic diagnosis.

A B

C
n FIGURE 1-6 Slide examination. A, A microhematocrit tube is filled with bloody aspirated fluid, spun down, and broken just below the buffy
coat. The contents are gently expelled and spread onto one or more slides. B, This bloody aspirate was obtained by pericardiocentesis. A rare large,
atypical spindle-shaped cell suggestive of a sarcoma was observed among the many erythrocytes and a small number of reactive mesothelial cells.
(Wright; HP oil.) C, After making a smear from the buffy-coat preparation of the same bloody specimen, numerous spindle-shaped cells that show
malignant characteristics were observed, affording a cytologic diagnosis of a neoplastic effusion consistent with a sarcoma. (Wright; HP oil.)
8 Canine and Feline Cytology
A 1-8C&D). This technique works well on ulcerated cutaneous
B Small tissue samples such as those obtained with an endo-
n FIGURE 1-7 Slide examination. A, This is a direct smear of a pleu-
ral fluid specimen from a cat with a thoracic effusion. A small number
of small, medium, and large lymphocytes were observed. (Wright; HP
oil.) The triglyceride concentration of the fluid approximated the serum
value, making the diagnosis of a chylous effusion less likely. (Wright; HP
oil.) B, A cytospin preparation of the specimen easily demonstrates that
most of the cells are medium to large immature lymphocytes indicative
of malignant lymphoma. A normal small lymphocyte (long arrow) and a
neutrophil (short arrow) are useful size comparators. (Wright; HP oil.) (A
and B from Meyer DJ, Franks PT: Effusion: classification and cytologic
examination, Compend Contin Educ Pract Vet 9:123-28, 1987.)
Touch Imprint
Cells will often exfoliate from excised tissue when the cut sur-
face is touched to a glass slide. This type of cytologic prepa-
ration permits immediate evaluation of a biopsy, provides the
pathologist with a second means of evaluating the tissue, and
is a valuable instructional tool. The clinician’s interpretation
can be compared with the histopathologic findings. The cut
surface of the excised tissue is aggressively blotted on a paper
towel to remove blood and tissue fluid. The specimen is dry
enough to exfoliate cells without excessive blood contami-
nation when the paper towel is observed to stick to it. The
surface will have a dull, dry, tacky appearance. It is touched
firmly to the surface of a clean glass slide in several places
around the “sweet spot” (Fig. 1-8A&B). Alternatively, one can
hold the tissue in one hand and touch one or more times the
“up” side of a glass slide to the tissue using the other hand.
It is important to always be aware of the “up” surface of the
slide and the cut surface of the tissue. Imprinting the meso-
thelial or serosal surface will produce nondiagnostic results.
Properly prepared tissue momentarily sticks to the surface
of the glass. If excess blood or tissue fluid is noted, the tis-
sue is blotted again and a new touch imprint made. Imprint
areas that appear too thick can be finessed to a monolayer by
the gentle use of the compression technique. Touch imprints
should be made of each area of tissue specimen that appears
grossly different.
Tissues with a fibrous texture, such as fibromas, fibrosar-
comas, and cicatricial inflammation, may not exfoliate ade-
quately with this technique. The surface of these firm, often
pale-appearing, tissues needs to be roughened or scraped with
a scalpel and then touched to the surface of a glass slide. In
addition, the tissue on the edge of the scalpel can be used to
make touch imprints and/or compression preparations (Fig.
lesions when neoplasia or mycotic infection is suspected. The
surface is frequently contaminated with debris, bacteria, and
an attendant mixed inflammatory cell reaction composed of
neutrophils, macrophages, and fibroplasia that can obscure the
true etiology if a direct touch imprint is made. It is prudent to
aggressively debride the area by using moistened gauze and/
or by aggressive, deep scraping of the area with a scalpel. The
exfoliated material, including the tissue on the scalpel blade, is
used to make touch imprints and compression preparations. In
certain bullous skin diseases, touching a glass slide to a freshly
ruptured bulla can be used to identify acantholytic epithelial
cells along with nondegenerate neutrophils (Tzanck prepara-
tion), supporting a tentative diagnosis of an immune-mediated
skin disorder (see Fig. 3-8).
scopic biopsy instrument or cutting biopsy needle or bone mar-
row core biopsy needle can be rolled on a slide using another
glass slide or a 22- or 25-gauge needle (Fig. 1-8E). One may
perform a compression preparation if there is extra tissue not
needed for histologic examination.
Swabs are used to exfoliate cells from mucosal surfaces or
from viscous or fluid discharges. The applicator stick is rolled
generally once or twice over the surface, creating one or two
lines, respectively, while avoiding painting the slide (Fig. 1-8F).
An example of a stained slide with two rolled lines is shown in
Fig. 1-8G.
STAINING THE SPECIMEN
Romanowsky and new methylene blue stain are used predom-
inately in veterinary medicine to identify nucleated cells. Prior
to staining, slides should not be heat-fixed because this is likely
to damage cell morphology. Air-drying slides well is the pre-
ferred method of initial preparation. Rapid drying prevents cell
shrinkage. One exception is the procedure of wet fixation before
the slide dries for the Papanicolaou stain (see below).
Papanicolaou Stain
The Papanicolaou (Pap) stain is used routinely in the human
medical profession for cytologic specimens. The stain accentu-
ates nuclear detail and is valuable in detecting early morpho-
logic aberrations indicative of dysplasia and neoplasia. It is not
used commonly in veterinary medicine because of the multistep
staining procedure and its limitations in evaluating inflamma-
tory reactions. A rapid Papanicolaou staining procedure has
been described in veterinary medicine that may be advanta-
geous for enhancing the nuclear abnormalities of cancer cells
(Jorundsson et al., 1999).
 CHAPTER 1 The Acquisition and Management of Cytology Specimens 9

A E

G
n FIGURE 1-8 A, Impression smear. The touch imprint technique is illustrated. The cut surface of the specimen is firmly blotted on a paper towel
(note wet spots, arrow) until tacky and then firmly touched multiple times to the surface of a clean glass slide. B, A well-prepared and well-stained
touch preparation is shown as an example. C, Tissue scraping. If the tissue does not adequately exfoliate, a scalpel blade is used to scrape or
roughen up the surface of the tissue. The tissue can be touched to a glass surface and/or the material on the edge of the blade, dragged along the
surface of a slide, air-dried, and stained or, if thick, a compression preparation made. D, A good scrape smear contains both thick and thin areas. E,
Tissue rolling. Small pieces of tissue that cannot be grasped with a forceps for imprinting can be gently rolled on a slide using a 25-gauge needle.
This will allow for exfoliation of a thin layer of cells. If the tissue is not friable, multiple slides can be made. F, The swab smear is made by gently
rolling over the slide in one or two lines. G, A good example of a stained vaginal smear is shown. (C from Meyer DJ: The management of cytology
specimens, Compend Contin Educ Pract Vet 9:10-17, 1987.)
10 Canine and Feline Cytology
A specimen to be examined. The excess stain is removed by tilting
B before it dries onto the slide, rinsing and fixing the slide with
C use. Wright or Giemsa stains are different types of MR stains
n FIGURE 1-9 New methylene blue. A, This unfiltered stain contains
chains of budding yeast as a contaminant. B, Contaminants may be
removed using a 0.45-μm filter attached to a syringe filled with stain.
Just before use, the stain is filtered as a simple drop that is directly
applied to an air-dried slide. C, The coverslip is applied, and excess stain
is removed by tilting the slide at right angles and blotting to an absor-
bent sheet.
New Methylene Blue Stain
New methylene blue (NMB) is a basic dye that stains nuclei,
most infectious agents, platelets, and mast cell granules. Eosin-
ophil granules do not stain nor do erythrocytes, which appear
microscopically as translucent circular areas. Because there is
no alcohol fixation, lipids associated with lipomas and adipose
tissue can be easily recognized. The cholesterol crystals asso-
ciated with follicular cysts are highlighted (see Fig. 2-23). The
staining solution consists of 0.5 g of NMB (Fisher Scientific,
Waltham, MA , USA ) dissolved in 100 mL of 0.9% saline. Full-
strength formalin (1 mL) is added as a preservative. The stock
solution is kept refrigerated. For clinical use, a small stoppered
bottle is replenished from the stock solution; the stain is passed
through filter paper first in order to remove all precipitate and
contaminants (Fig. 1-9A-E). A convenient filter alternative is
the use of a syringe filter (0.45 μm) (Fisher Scientific, Waltham,
MA, USA), as shown in Fig. 1-9B, which minimizes the waste
from filtering large amounts of stain. A small drop of stain is
applied directly to an air-dried cytology preparation. A dust-
free coverslip (wiped with a paper towel or shirtsleeve) is placed
on the drop of stain, which spreads by capillary movement.
Larger coverslips, 20 × 40 mm or 20 × 50 mm, allow more of the
and touching the glass slide onto a paper towel. The specimen
should be examined immediately because the water-based stain
will evaporate. An NMB-stained cytologic specimen is useful
for the detection of nucleated cells, bacteria (both gram-positive
and gram-negative bacteria stain dark blue), fungi (see Fig.
5-11C), and adipose tissue (see Fig. 3-44A). When applied to a
blood smear, leukocyte and platelet numbers can be estimated
and polychromatophils (as reticulocytes) recognized. This
makes it a valuable triage stain for blood and fluid specimens
examined on an emergency basis. When religiously filtered, it is
an ideal stain to detect hemoplasmosis because the erythrocyte
is essentially “invisible,” thus accentuating the surface silhou-
ette of the dark blue organism. Occasionally, the NMB-stained
slide can be successfully preserved by removing the coverslip
methanol, followed by a permanent Romanowsky stain, either
aqueous or methanolic.
HELPFUL HINT This is a valuable, cost-effective stain for examining
cytologic preparations, blood smears, and urine sediments in veterinary
practice. The added responsibility of filtering the stock stain periodically
is well worth the time invested.
Romanowsky Stains (Methanolic and Aqueous)
Romanowsky stains involve methanol fixation of an air-dried
slide. Methanolic Romanowsky (MR) stains are often utilized
in practice settings because they work rapidly and are easy to
that contain azures, which are basic dyes that attract acidic
nuclear proteins (DNA/RNA) and stain them blue or purple.
These stains also contain eosin, which is an acidic dye that
attracts alkaline cytoplasmic components such as hemoglobin
and major basic protein and stains them pink. This combination
of basic and acidic dyes is dissolved in methanol. These poly-
chromatic stains impart the basophilic and eosinophilic tinc-
torial properties observed on blood films. Wright stain (Wright
Stain Solution; Fisher Scientific, Waltham, MA, USA) is used
widely in human medical and veterinary laboratories because it
results in well-stained blood films. Other MR stains used alone
or in various combinations include Leishman and May-Grun-
wald-Giemsa. Some MR stains are intended to be used as a stat
stain. One example of a rapid Wright-Giemsa stain is Camco
Quik Stain II® (Fisher Scientific, Waltham, MA , USA).
Stains which are aqueous Romanowsky (AR) involve pop-
ular quick stains such as Diff-Quik® (Diff-Quik® Differential
Stain Set; Fisher Scientific, Waltham, MA, USA) and Quik-Dip
 CHAPTER 1 The Acquisition and Management of Cytology Specimens
10 m
A Mounting the coverslip before the specimen is dry
10 m
B demonstrated to vary considerably among suppliers and from
n FIGURE 1-10 Staining for distemper inclusion bodies. A, The
cytoplasmic viral inclusions within the neutrophil and erythrocyte stain
pale blue. (methanolic Romanowsky/Wright, HP oil.) B, Canine dis-
temper inclusion bodies stain dark purple, which markedly improves
its visibility among the neutrophils and erythrocytes shown. (aqueous
Romanowsky, HP oil.)
(Mercedes Medical). These polychromatic stains are commonly
used in veterinary practice because of their time-saving conve-
nience. Another advantage of AR over MR is the improved vis-
ibility of distemper viral inclusion bodies (Fig. 1-10A&B). For
certain specimens such as bone marrow samples, there may be
a tradeoff in staining quality. Mast cell granules, basophil gran-
ules, and cytotoxic lymphocyte granules do not stain reliably
with AR (Allison and Velguth, 2010). Primary granules and toxic
granulation may also stain weaker, according to a Wescor man-
ual comparing automated AR (7120/7121) and MR (7150/7151)
Aerospray instruments. With an AR stain, the granule contents
are washed away in the water-diluted stain solution (see Fig.
3-53E&F). In the MR stain, a precipitate initially forms primarily
from a concentrated anhydrous azure B dye solution that stabi-
lizes the granules prior to diluted staining. While both AR and
MR incorporate an initial methanol fixation, they differ in the
azure dye type and concentration, pH, and solvent medium for
the thiazine family of azures used in these methods. If a stain-
ing deficiency is suspected during the examination of a discrete
cell neoplasm, NMB or MR stain can be used to demonstrate the
presence of these granules.
11
BOX 1-2 Causes of Abnormal Staining
Excessive blue (erythrocytes appear blue green)
Prolonged contact time with the stain
Inadequate wash
Specimen too thick
Stain or diluent too alkaline—pH >7; check with pH paper
Exposure of specimen to formalin or its fumes (e.g., open formalin container)
Delayed fixation
Excessive pink
Prolonged washing
Insufficient contact time with the stain
Stain or diluent too acidic—pH >7; erythrocytes can appear orange or bright
red—formic acid can result from the oxidation of methanol with prolonged
exposure to air; fresh methanol is recommended
Inadequately stained nucleated cells and erythrocytes
Insufficient contact time with one or more of the staining solutions
Surface of a second glass slide covers the specimen on the first slide (can
occur when staining two slides back-to-back in Coplin jars)
Precipitate on the stained specimen
Inadequate washing of the slide at the end of the staining period
Inadequate filtration of the stain
Unclean slides
Poorly stained specimens can result from improper staining
times, weakened stain from overuse, and improperly managed
cytologic preparations. One should become familiar with one
kind of Romanowsky stain and not switch brands frequently.
The composition of dyes in polychromatic stains has been
batch to batch from the same supplier. Furthermore, prolonged
storage at room temperature (25°C; 77°F) can impair staining
intensity because of the formation of degradation products in
methanol. It is most convenient to purchase stains in liquid
form. Box 1-2 lists the factors that can cause poorly stained
specimens with Romanowsky stains.
Staining times vary depending on the thickness of the spec-
imen and the freshness of the stain. The frequency with which
the solutions are changed or refreshed depends on the number
of slides processed. The appearance of dull-blue–appearing
nuclei that lack sharp chromatin detail is one indication of a
weak solution. Solutions should be changed completely when-
ever infectious agents or cellular elements inappropriately
appear on specimens. The staining times for AR stain solutions
need to be increased depending on the thickness of the cyto-
logic preparation and the freshness of the stain. A pleural effu-
sion with low cellularity may be stained adequately with three
to five dips in each solution. A thick preparation from a lymph
node or bone marrow specimen may require 60 to 120 seconds
in each solution to stain optimally (Fig. 1-11A&B). Box 1-3 lists
staining time guidelines.
At the end of the staining process, the slide is washed with
cold running water for 20 seconds to remove stain precipitate
and allowed to dry in a nearly vertical position (also see KEY
POINT regarding the use of a hair dryer or fan). Any stain film
on the back of the slide can be removed with an alcohol-moist-
ened gauze sponge. The stained specimen is examined micro-
scopically using the 10× or 20× objective for staining quality and
uniformity. If acceptable, a coverslip is placed on the specimen
12 Canine and Feline Cytology
A
B rates of these areas and firmer tissue are planned. The area of
n FIGURE 1-11 Staining technique. A, This aspirate from an
enlarged lymph node was stained with approximately five dips in the
fixative and each of the staining solutions. Cell outlines can be seen,
but the detailed cytomorphology cannot be adequately examined. (Diff-
Quik®; HP oil.) B, The same slide was replaced into the fixative and the
staining solutions for approximately 60 seconds in each station while
it was slowly moved up and down. A cytologic diagnosis of malignant
lymphoma now can be made. A small lymphocyte near center is a help-
ful size comparator. (Diff-Quik®; HP oil.)
BOX 1-3 Suggested Procedure for Staining
Cytologic Specimens Using Aqueous
Romanowsky Solutions*
Fixative: 60 to 120 seconds
Solution 1: 30 to 60 seconds
Solution 2: 5 to 60 seconds†
Rinse under cold tap water: 15 seconds
Examine staining adequacy using low power; eosinophilia or basophilia can
be enhanced by returning to Solution 1 or Solution 2, respectively, followed
by a rinse.
Air-dry and examine
*Suggested times are based on fresh stains; the stains weaken
with time and use, and longer times will be required. Consistently
understained specimens are an indication for replenishing with fresh
stain.
†The shortest times are suggested for hypocellular fluids that are
low in protein such as transudates, cerebrospinal fluid, and urine
sediments.
Modified from Henry MJ, Burton LG, Stanley MW, et al: Application of a
modified Diff-Quik® stain to fine needle aspiration smears: rapid staining
with improved cytologic detail, Acta Cytol 31:954-955, 1987.
if the 40× objective is to be used. A temporary mount is made
by placing a drop of immersion oil on the specimen followed by
a coverslip. A permanent mount is made with a commercially
available coverslip mounting glue (e.g., Eukitt®; Sigma-Aldrich).
KEY POINT A coverslip is always required for sharp focus when the
40× objective is used to examine hematologic and cytologic specimens. A
second drop of oil is placed on the coverslip when using the oil objective.
KEY POINT Two staining stations are routinely recommended. One
is used for “clean” specimens such as blood films, effusions, and lymph
node aspirates, and the other is used for “dirty” specimens such as skin
scrapings, fecal and intestinal cytology, and suspected abscesses.
SITE-SPECIFIC CONSIDERATIONS
Cutaneous Nodule and the Lymph Node
The cutaneous nodule and the enlarged lymph node are readily
accessible tissues for exfoliative cytology. A minimum of two
lymph nodes should be sampled if there is generalized lymph-
adenopathy. The center of an enlarged lymph node should be
avoided to minimize the risk of obtaining necrotic debris and
nondiagnostic cytologic material. The tissue is palpated for
consistency, and the margins are defined. Softer areas sugges-
tive of fluid or necrotic tissue are identified, and separate aspi-
interest is clipped and scrubbed before aspiration. The tissue
is immobilized firmly between the thumb and forefinger. The
needle is inserted into the tissue, an aspiration or nonaspira-
tion technique is used, and the needle is advanced into (but
not through) the tissue of interest. The needle is redirected
several times (Fig. 1-1A). The plunger of the syringe is gently
returned to the start position, and the needle is withdrawn.
Maintaining vacuum while removing the needle from the tis-
sue causes splattering of the material in the syringe barrel and
enhances the potential of blood contamination from a cutane-
ous vessel. When fluid is encountered, it should be completely
removed and handled as a fluid specimen. A separate sampling
procedure is executed for the firmer tissue with a new needle
and syringe combination.
KEY POINT The exfoliation of cells occurs as a consequence of the
needle’s passage through the tissue. Thus, repeated movement of the
needle through the tissue is the critical component of obtaining diagnostic
material from nonfluid tissues.
KEY POINT Not all solid tissues can be adequately sampled with ex-
foliation cytology. If diagnostic cells are not obtained with FNAB after
triaging the stained specimen, consider an excisional or incisional biopsy.
Liver, Spleen, and Kidney
The use of exfoliative cytology for the investigation of organo-
megaly of the liver, spleen, and kidney is the most rewarding
indication. The cellular or cell-associated causation of the
enlarged organ often exfoliates from these tissues. Ultraso-
nographic examination of these organs has increased the use
 CHAPTER 1 The Acquisition and Management of Cytology Specimens
n FIGURE 1-12 Liver biopsy. Fine-needle aspiration biopsy of the
liver can be accomplished with the dog in right lateral recumbency. In
the picture, the head is to the reader’s left. The needle is inserted in
a craniodorsal direction at the triangle formed by the left lateral edge
of the xiphoid process and the union of the last rib with the sternum.
Once the needle touches the surface of the liver, the hub of the nee-
dle moves in concert with the movement of the diaphragm but in the
opposite direction.
of FNAB for the examination of focal lesions. The diagnostic
efficiency of cytology is reduced in support of this indication.
There is a greater possibility that the cell type may not exfoliate
or the lesion will be missed and the surrounding tissue exam-
ined, resulting in an erroneous impression and misdiagnosis.
In addition, greater expertise is required for the examination of
FNAB specimens from these organs because nodular hyperplas-
tic lesions of the spleen and canine liver become more prevalent
in the geriatric patient (see Chapters 4&9).
KEY POINT The nonaspiration sampling technique reduces blood con-
tamination from vascular organs such as the liver and spleen.
KEY POINT Remember that the liver is a moving target due to its in-
timate association with the movement of the diaphragm. Consequently,
a craniodorsal positioning of the needle reduces the risk of laceration
(Fig. 1-12).
KEY POINT Two actions should be taken if a bloody sample is ob-
tained from the liver or spleen. First, place the sample immediately into
an EDTA tube. A direct smear (similar to a peripheral blood film) and a
buffy-coat preparation should be triaged for diagnostic material such
as malignant mesenchymal cells (hemangiosarcoma). Do not attempt to
“coat” an entire glass slide with the bloody specimen in hopes of a diag-
nostic specimen. The result will be a dismal diagnostic failure. Second, if
no fluid-filled lesion is present upon ultrasound examination, repeat FNAB
with a clean needle and use a nonaspiration technique.
Nose and Lung
Evaluation of the nasal cavity is often compromised by the
occult nature of the underlying pathology. Radiography always
should precede an attempt to obtain a specimen for cytology or
histopathology: It can define the area of the nasal cavity that is
predominantly involved and thus suggest the side of the cavity
13
to be sampled. In addition, manipulation within the nasal cav-
ity often results in hemorrhage, which obscures radiographic
detail. After radiography, the oropharynx is examined visually
and by digital palpation. The dorsal area of the soft palate is
examined with a dental mirror and by palpation. If no abnor-
mal tissue is identified for aspiration and/or excisional biopsy,
the recesses of the nasal cavity are sampled by a washing or aspi-
ration technique. Examination of the nasal cavity with an oto-
scope can allow visualization of abnormal tissue and can assist
in procuring a tissue specimen.
Superficial lesions, such as eosinophilic or fungal rhinitis,
can occasionally be identified by examination of nasal mucus
or superficial mucosal scrapings. Most of the time, superficial
swab-obtained specimens are nondiagnostic or yield only non-
specific inflammation and bacteria. More aggressive cytologic
specimens from the nasal cavity can be obtained by flush or
aspiration techniques. A soft, rubber urinary catheter is flex-
ible enough for the retrograde flushing procedure. The saline
flush is collected and squash preps are made from mucoid globs
and bits of tissue. The remaining saline is centrifuged in a coni-
cal-tip tube and preps (squash and/or direct smears) made from
the pellet. A rigid, large-bore polyurethane urinary catheter or
the plastic needle guard from a Sovereign® intravenous catheter
is effective for obtaining a nasal specimen (Fig. 1-13A&B). The
depth of the nasal cavity is approximated, and a corresponding
length of catheter is cut at an angle. The catheter is attached to
a syringe and firmly advanced into the nasal cavity until mod-
erate resistance is encountered. Aspiration is applied while the
catheter is manipulated within the nasal cavity (Fig. 1-13B).
For this procedure, aspiration and manipulation of the catheter
can be more aggressive because of the mucosal and cartilagi-
nous nature of diseased tissue. Another deviation is the main-
tenance of negative pressure when withdrawing the catheter in
an attempt to exteriorize bits of tissues. Fluid and bits of tissue
can be used for cytologic preparations. Larger pieces of tissue
fragments and clotted blood can be placed in 10% formalin for
histopathologic examination to maximize the diagnostic yield.
FNAB of the lung parenchyma is rewarding when the inter-
stitial infiltrative disease is diffuse or large focal lesions are iden-
tified radiographically. Unless the cellular infiltrate is notable by
imaging procedures, the diagnostic yield cannot be expected to
be fruitful. Ultrasonographic guidance of the needle is a more
accurate way of ensuring that the desired lesion is sampled.
For small or ill-defined lesions, guessing the location of needle
placement from the radiograph is problematic. Consideration
should be given to the possibility of tumor seeding of pulmo-
nary adenocarcinoma before attempting cytologic aspiration
(Vignoli et al., 2007; Warren-Smith et al., 2011).
Successful use of the transtracheal wash and bronchoalveo-
lar lavage for assessing pulmonary changes depends on the dis-
ease process involving the mucosa and/or the alveolar lumen,
sampling of the diseased region, and adequate collection of the
saline lavage. There must be a relatively aggressive attempt to
recover the wash or lavage that includes angling the patient’s
head downward to facilitate a diagnostic yield. Mucosal brush-
ings/scrapings can enhance the cytologic yield of mucosal
lesions (Clercx et al., 1996).
KEY POINT The lung is a dynamic organ prone to laceration by the
needle. Momentary apnea can be achieved occasionally by touching or
gently blowing on the patient’s nose.
14 Canine and Feline Cytology

Joint
Lameness and swollen joints are common indications for the
examination of the synovial fluid. A review of the skeletal anat-
omy for the joint of interest is prudent before beginning the
sampling procedure. In general, an appropriate interosseous
location is determined by digital palpation, with the affected
joint in a slightly flexed position. The site should be clipped and
prepared using sterile techniques to avoid contamination of the
sample with bacteria, especially if the synovial fluid is also going
to be submitted for culture, because this will ensure its accurate
assessment for presence of bacteria. A 22- to 25-gauge needle
attached to a 3-mL syringe is used. Normal synovial fluid is vis-
cous, and even inflamed synovial fluid may retain this quality.
A Consequently, gentle aspiration must be linked to patience as
the thick fluid slowly rises up the smaller needle. Quantity is less
important than quality of the specimen. One drop is adequate
for a slide preparation and two or three drops in a sterile tube or
applied to a culturette for potential culture will suffice. If non-
localizing polyarticular disease is suspected, two or more joints,
including at least one carpal joint, should be routinely sampled.

Vertebral Body Lesions

B ing a diagnostic specimen using an imaging-guided spinal needle.
n FIGURE 1-13 Nasal biopsy. A, This schematic representation
demonstrates a method of altering an intravenous catheter for use in
obtaining nasal cytologic specimens by aspiration. One end of the outer
plastic shield is cut at an angle and the needle is cut close to the plas-
tic hub. The outer plastic shield is wedged firmly over the hub. B, A
sagittal schematic representation of a dog’s head, demonstrating two
possible techniques for obtaining a cytologic specimen from the nose.
The altered intravenous catheter or a relatively rigid large-bore urinary
catheter is aggressively inserted via the external nares and aspiration
applied when resistance is encountered. Alternatively, a flexible rubber
urinary catheter can be inserted above the soft palate and the nasal
cavity flushed retrograde. The fluid and solid material are collected in a
container. (A and B from Meyer DJ: The management of cytology spec-
imens, Compend Contin Educ Pract Vet 9:10-17, 1987.)
Vertebral body pathology may be an incidental finding on radio-
graphs or be suspected based on neurologic abnormalities such
as ataxia, inability to rise in either end, or neck pain. Obtaining
a cytologic specimen is challenging in such cases because of the
difficulty of locating the site by palpation and the proximity to the
spinal cord. Experienced radiologists may be successful in obtain-
SUBMITTING CYTOLOGY SPECIMENS
TO A REFERENCE LABORATORY
The busy practitioner often finds it more convenient to submit
cytology specimens to a commercial veterinary laboratory for
examination. Many of these facilities have personnel specifically
trained to make buffy-coat and cytospin preparations of fluid
specimens and experienced microscopists to examine cytologic
specimens. Their expertise is effective only if the specimen is
submitted properly.
Fluid specimens should be placed immediately in EDTA
tubes to prevent clot formation. If the fluid will be in transit

A B
n FIGURE 1-14 Formalin effects. A, This lymph node specimen was inadvertently exposed to formalin fumes. Most of the elements present
cannot be recognized as lymphocytes, precluding a cytologic interpretation. Formalin fumes alter the cytomorphology and staining characteristics of
nucleated cells; this should be considered as a reason for a nondiagnostic specimen. (Wright; HP oil.) A cytologic diagnosis of lymphoid hyperplasia
was made from a second aspirate (not shown). B, This sample was also exposed to formalin. Notice how the erythrocyte morphology lacks clarity
and has a greenish tint. (Romanowsky; HP oil.)
 CHAPTER 1 The Acquisition and Management of Cytology Specimens
longer than 24 hours, a direct slide preparation should be made
to accompany the tube. Red-topped and purple-topped blood
collection tubes should not be considered sterile; contaminant
bacterial growth can occur in the specimen submitted for cul-
ture. Only use containers supplied by the laboratory dedicated
for bacterial and fungal culture. (Contact the laboratory.) See
the Appendix for additional information about submitting
specimens for specialized diagnostic testing.
As previously indicated, touch imprints can be helpful
adjuncts to the histologic examination of formalin-fixed tissues.
Formalin vapors can alter the staining characteristics of touch
imprints drastically (Fig. 1-14A&B). When touch imprints
are sent along with formalin-fixed tissues to the laboratory,
REFERENCES
Allison RW, Velguth KE: Appearance of granulated cells in blood films stained
by automated aqueous versus methanolic Romanowsky methods, Vet Clin
Pathol 39:99–104, 2010.
Clercx C, Wallon J, Gilbert S, et al: Imprint and brush cytology in the diagnosis
of canine intranasal tumours, J Sm Anim Pract 37:423–437, 1996.
Fagelman D, Chess Q: Nonaspiration fine-needle cytology of the liver: a new
technique for obtaining diagnostic samples, Am J Roentgenol 155:1217–1219,
1990.
Gilson SD, Withrow SJ, Wheeler SL, et al: Pheochromocytoma in 50 dogs,
J Vet Intern Med 8:228–232, 1994.
Henry MJ, Burton LG, Stanley MW, et al: Application of a modified Diff-Quik
stain to fine needle aspiration smears: rapid staining with improved cytologic
detail, Acta Cytol 31:954–955, 1987.
Jorundsson E, Lumsden JH, Jacobs RM: Rapid staining techniques in cytopa-
thology: a review and comparison of modified protocols for hematoxylin and
eosin, Papanicolaou and Romanowsky stains, Vet Clin Pathol 28:100–108,
1999.
Léveillé R, Partington BP, Biller DS, et al: Complications after
­ultrasound-guided biopsy of abdominal structures in dogs and cats: 246
cases (1984-1991), J Am Vet Med Assoc 203(3):413–415, 1993.
Mair S, Dunbar F, Becker PJ, et al: Fine needle cytology—Is aspiration suction
necessary? A study of 100 masses in various sites, Acta Cytol 33:809–813,
1989.
Meyer DJ: The management of cytology specimens, Compend Contin Educ
Pract Vet 9:10–17, 1987.
15
they must be placed in separate packages and never submitted
together to avoid formalin effects to cytologic materials. Break-
age is a common problem when glass slides are mailed in card-
board containers. Rigid plastic or Styrofoam containers offer
reliable protection. If there is a lack of familiarity with a particu-
lar sample submission procedure, the laboratory should always
be contacted for advice before collection.
KEY POINT Formalin fumes are pervasive and rapidly penetrating.
They alter the staining and morphology of hematology and cytology speci-
mens. Keep open formalin containers away from these specimens even if
opened only momentarily.
Meyer DJ, Franks PT: Clinical cytology: Part I: Management of tissue specimens,
Mod Vet Pract 67:255–259, 1986.
Meyer DJ, Harvey JW: Evaluation of fluids: effusions, synovial fluid, cerebro-
spinal fluid. In Meyer DJ, Harvey JW (eds): Veterinary laboratory medicine:
interpretation and diagnosis, Philadelphia, 2004, Saunders, pp 245–259.
Nyland TG, Mattoon JS, Herrgesell EJ, et al: Ultrasound-guided biopsy. In
Nyland TG, Mattoon JS (eds): Small animal diagnostic ultrasound, ed 2,
Philadelphia, 2002a, Saunders, pp 30–48.
Nyland TG, Wallack ST, Wisner ER: Needle-tract implantation following
US-guided fine-needle aspiration biopsy of transitional cell carcinoma of
the bladder, urethra and prostate, Vet Radiol Ultrasound 43(1):50–53, 2002b.
Podell M: Epilepsy and seizure classification: a lesson from Leonardo, J Vet
Intern Med 13:3–4, 1999.
Vignoli M, Rossi F, Chierici C, et al: Needle track implantation after fine
needle aspiration biopsy (FNAB) of transitional cell carcinoma of the
urinary bladder and adenocarcinoma of the lung, Schweiz Arch Tierheilk
149(7):314–318, 2007.
Warren-Smith CMR, Roe K, de la Puerta B, et al: Pulmonary adenocarcinoma
seeding along a fine needle aspiration tract in a dog, Vet Rec 169:181–182,
2011.
Yue X, Zheng S: Cytologic diagnosis by transthoracic fine needle sampling
without aspiration, Acta Cytol 33:806–808, 1989.
 2 CHAPTER
General Categories of
Cytologic Interpretation
Rose E. Raskin
One use of cytology is to classify lesions so as to assist with
the diagnosis, prognosis, and management of a case. Cytologic
interpretations are generally classified into one of five cytodiag-
nostic groups (Box 2-1). A sixth category can be used for non-
diagnostic or artifact interpretations. Nondiagnostic samples
usually result from insufficient cellular material or excessive
blood contamination.
KEY POINT Interpretation of cytologic material may include more than
one category, such as inflammation along with a response to tissue injury
or neoplasia with inflammation.
NORMAL TISSUE
Normal tissue is generally composed primarily of mature cell
types, although some organs such as integumentary normally
contain immature basal epithelium. Normal cells display
uniformity in cellular, nuclear, and nucleolar size and shape.
Cytoplasmic volume is usually high relative to the nucleus
(Figs. 2-1 and 2-2).
HYPERPLASTIC TISSUE
Hyperplasia is a non-neoplastic enlargement of tissue that can
occur in response to hormonal disturbances or tissue injury.
Hyperplastic tissue has a tendency to enlarge symmetrically
in comparison to neoplasia. Cytologically, hyperplastic cells
may appear similar to normal tissue but have a higher nucle-
ar-to-cytoplasmic ratio than normal mature cells. Examples of
hyperplastic responses include nodular proliferations within
the parenchyma of the prostate (Fig. 2-3), liver (see Fig. 9-20A),
and pancreas (Fig. 2-4).
BOX 2-1 General Categories of
Cytodiagnostic Interpretation
Normal or hyperplastic tissue
Cystic mass
Inflammation or cellular infiltrate
Response to tissue injury
Neoplasia
Nondiagnostic sample
16
CYSTIC MASS
Cystic lesions contain liquid or semisolid material. The low-
protein liquid usually contains a small number of cells. These
benign lesions may result from proliferation of lining cells or
tissue injury. Examples include seroma (Fig. 2-5), salivary
mucocele, apocrine sweat gland cyst, epidermal/follicular cyst
(see Fig. 3-2), and cysts associated with noncutaneous glands
such as the mammary gland or prostate (Fig. 2-6).
INFLAMMATION OR CELLULAR INFILTRATE
Inflammatory conditions are classified cytologically by the pre-
dominance of the cell type involved. Recognition of the inflam-
matory cell type often suggests an etiologic condition.
Purulent or suppurative lesions contain greater than 85%
neutrophils; they are then classified by the presence or absence
of nuclear degeneration of the neutrophil. Nondegenerate neu-
trophils are morphologically normal with mature condensed
chromatin and well-segmented purple lobes. These neutro-
phils predominate in relatively nontoxic environments such
as immune-mediated diseases (Fig. 2-7A), neoplastic lesions
(Fig. 2-7B), and sterile conditions caused by irritants such as
urine and bile (Fig. 2-7C). Degenerate neutrophils display early
signs of oncotic necrosis with observable cellular and nuclear
swelling with decreased nuclear stain intensity. In cytology,
this early recognizable change is termed karyolysis (Fig. 2-8).
Karyolysis is attributed to decreased mitochondria function
to produce ATP that maintains transmembrane ion pumps
resulting in an influx of sodium, calcium, and water (hydropic
degeneration) as well as to damaged cell membranes that result
in release of endonucleases that degrade RNA and DNA. This
often indicates rapid cell death in a toxic or injurious environ-
ment (Perman et al., 1979). Degenerate neutrophils predom-
inate in bacterial infections, particularly gram-negative types
that produce endotoxins. Cytologically, under conditions of
neutrophil degeneration, small infectious agents must be found
intracellularly to confidently report it as septic neutrophilic
inflammation (Fig. 2-9).
Karyolysis as a histologic term describes cell death in the
form of a ghost nucleus or nuclear remnants that precede even-
tual nuclear loss or dissolution. In contrast to acute cell injury
involving cellular and nuclear swelling, cell death may occur
more slowly through a shrinkage phenomenon (apoptosis).
While apoptosis often occurs in isolated cells during normal
 CHAPTER 2 General Categories of Cytologic Interpretation
n FIGURE 2-1 Normal skeletal muscle. Tissue aspirate. Dog. Numer-
ous threadlike myofibrils compose each cell with small, condensed, and
oval nucleus. Cross-striations, characteristic of skeletal muscle, are visible
against the dark blue cytoplasm. (Modified Wright; HP oil.)
n FIGURE 2-2 Normal salivary gland. Tissue aspirate. Dog. The
gland has uniform features of nuclear size, nuclear-to-cytoplasmic ratio,
and cytoplasmic content. (Wright-Giemsa; HP oil.)
n FIGURE 2-3 Canine prostatic hyperplasia. Tissue aspirate. Dog.
The presenting clinical sign in this case involves blood dripping from the
prepuce. Cytologically, the nuclear size is uniform; however, the nucle-
ar-to-cytoplasmic ratio is increased as indicated by the close proximity
of nuclei to each other. (Wright-Giemsa; HP oil.)
17
n FIGURE 2-4 Nodular hyperplasia of the pancreas. Tissue aspi-
rate. Dog. Ultrasound examination revealed a hypoechoic mass in the
area of the pancreas. Cytologically, hyperplastic parenchymal organs
commonly display binucleation (arrows). (Wright-Giemsa; HP oil.)
n FIGURE 2-5 Seroma. Tissue aspirate. Dog. Blood-tinged fluid is
removed from a swelling on the neck. There is low cellularity (3800/μL)
and low protein content (2.5 g/dL). Cytologically, the direct smear
contains a mixed cell population with large mononuclear cells having
fine cytoplasmic granularity along with low numbers of erythrocytes.
(Wright-Giemsa; HP oil.)
n FIGURE 2-6 Prostatic cyst. Histopathology. Dog. Cuboidal epithe-
lial cells line large cystic spaces that represent dilated ducts. (H&E; LP.)
18 Canine and Feline Cytology

10 m
A B

C
n FIGURE 2-7 Nondegenerate neutrophils. Dog. A, Synovial fluid from a Doberman Pinscher with an immune-mediated response to trimetho-
prim-sulfadiazine. There are eight neutrophils and five large mononuclear cells in a windrowing formation. (Wright-Giemsa; HP oil.) B, Nonseptic
inflammation of synovial fluid with predominately well-segmented neutrophils appears secondary to adjacent neoplasia of the bone. (Wright-Giemsa;
HP oil.) C, Abdominal fluid following bile duct rupture with intact neutrophils, one of which has phagocytized green-grey mucus. (Modified Wright;
HP oil).

n FIGURE 2-8 Degenerate neutrophils, karyorrhexis. Tissue aspi-

rate. Dog. Mild to moderate karyolysis of neutrophils is evident by the
decreased nuclear stain intensity and swollen nuclear lobes. Pyknosis
of multiple nuclear segments appears as dark, dense, round struc-
tures, termed karyorrhexis (arrows), in this case of bacterial dermatitis.
(Wright-Giemsa; HP oil.)
n FIGURE 2-9 Bacterial sepsis. Tissue aspirate. Dog. Markedly
karyolytic neutrophils are present with intracellular coccoid bacteria.
Karyolysis is so severe that the cells are barely recognizable as neu-
trophils. A fragmenting erythrocyte is helpful for size comparison to
demonstrate neutrophil swelling. (Modified Wright; HP oil.)
 CHAPTER 2 General Categories of Cytologic Interpretation
physiologic cell aging (Fig. 2-10A), it may be found alongside
pathologic cell death characterized by widespread nuclear
destruction and necrosis.
Increased nuclear staining (hyperchromia) with coalescence
of the nucleus into a single or two dark basophilic round seg-
ments characterizes pyknosis (Fig. 2-10B). If pyknosis is related
to a slow, progressive change within a relatively nontoxic envi-
ronment, an intact cell membrane may be present around the
shrunken, more eosinophilic cell as occurs with normal cell
aging. An end stage of nuclear breakdown termed karyorrhexis
or karyorhexis (Mastrorilli et al., 2013) may be seen as the result
of pyknosis of hypersegmented nuclei (Fig. 2-8) or fragmen-
tation of chromatin of an individual dying cell (Fig. 2-11) as
seen on both cytology and histology. Histiocytic or macrophagic
lesions contain a predominance of macrophages, suggesting
chronic inflammation (Fig. 2-12). Foamy, often vacuolated,
and phagocytic cells characterize this type of inflammation.
A
n FIGURE 2-10A Pyknosis. Blood. Dog. Two-day-old blood displays
cell aging and early pyknosis with rounded dense nuclear condensa-
tion and increased cytoplasmic eosinophilia. This change in the color of
the cytoplasm is attributed to consolidation of cellular components or
loss of ribosomal RNA which is responsible for cytoplasmic basophilia.
(Modified Wright; HP oil.)
B
n FIGURE 2-10B Pyknosis. Chylous effusion. Dog. Chronic inflam-
mation of this fluid produces neutrophils with nuclei that have con-
densed into a large, often single, dark, round structure (arrow) related to
the slow progression of cellular change in this nonseptic environment.
The pyknotic cell (arrow) in this case also contains a second, smaller
round nuclear fragment. (Wright; HP oil.)
19
In contrast, granulomatous lesions consisting of activated mac-
rophages that morphologically resemble epithelial cells form in
response to foreign material or persistent intracellular infec-
tious agents and have a secretory rather than phagocytic activ-
ity. These cells are therefore termed epithelioid macrophages and
recognized by their abundant basophilic cytoplasm and large
polygonal shape (Fig. 2-13A). Epithelioid macrophages under
the influence of cytokines and other inflammatory mediators
undergo macrophage fusion to form giant multinucleated forms
(Fig. 2-13B). Granulomas are often associated with foreign body
reactions and mycobacterial infections and may be recognized
cytologically by the presence of epithelioid macrophages and/or
multinucleate cells.
Mixed cell inflammatory lesions contain a mixture of neu-
trophils and macrophages (Fig. 2-14) that also may include
increased numbers of lymphocytes or plasma cells. This type
of inflammation is often associated with foreign body reac-
tions, fungal infections, mycobacterial infections, pannicu-
litis, lick granulomas, and other chronic tissue injuries. The
term pyogranulomatous should be reserved for a population
of neutrophils and epithelioid macrophages with or without
multinucleate giant cells (Fig. 2-13A).
n FIGURE 2-11 Karyorrhexis. Bone marrow aspirate. Dog. Frag-
mentation of the nucleus in this leukemic patient. (Modified Wright;
HP oil.)
n FIGURE 2-12 Macrophagic inflammation. Tissue imprint. Dog.
Nodular lung disease with numerous large mononuclear cells having
abundant foamy gray cytoplasm that also contains multiple colorless
vacuoles. (Wright-Giemsa; HP oil.)
20 Canine and Feline Cytology
A
n FIGURE 2-13A Pyogranulomatous inflammation. Tissue aspi-
rate. Dog. Long-standing bacterial infection created a mixture of degen-
erate neutrophils, epithelioid macrophages (arrows), binucleated giant
cell, lymphocytes, and a vacuolated phagocytic macrophage. Note the
presence of two cells displaying karyorrhexis. A plump fibroblast is seen
in the upper left. (Modified Wright; HP oil.) (From Raskin RE: Tail mass
in a dog, NAVC Clinician’s Brief Nov:13-15, 2006.)
B with allergic or immune reactions, early viral infections, and
n FIGURE 2-13B Multinucleate giant cell. Tissue aspirate. Cat.
Skin lesion with pyogranulomatous inflammation, including many giant
cells related to the presence of fungal hyphae (not shown). Pictured is
a cell with seven distinct nuclei and abundant blue-gray granular cyto-
plasm. (Wright-Giemsa; HP oil.)
n FIGURE 2-14 Mixed cell inflammation. Chylous effusion. Dog.
Chronic chylous effusion contains a variety of cell types, including non-
degenerate neutrophils, vacuolated macrophages, small to medium
lymphocytes, and two mature plasma cells. (Wright; HP oil.)
n FIGURE 2-15 Eosinophilic inflammation. Transtracheal wash.
Cat. Clinical presentation of a chronic cough in this cat with suspected
pulmonary allergy. Fluid contains 95% eosinophils. Pictured are several
eosinophils that stain pale pink to blue-green and adhere to pink mucous
material that prevents full stain penetration. (Wright-Giemsa; HP oil.)
Eosinophilic lesions contain greater than 10% eosinophils in
addition to other inflammatory cell types (Fig. 2-15). They are
seen with or without mast cell involvement. It is not uncommon
to see rust or brown granules in the cytoplasm of eosinophils on
cytology in contrast to the pink red cell color. This inflamma-
tory response is associated with eosinophilic granuloma, hyper-
sensitivity or allergic conditions, parasitic migrations, fungal
infections, mast cell tumors, and other neoplastic conditions
that induce eosinophilopoiesis. This combination of eosino-
philic inflammatory conditions has been referred to as “worms,
wheezes, and weird diseases.”
Lymphocytic or plasmacytic infiltration is often associated
chronic inflammation. The lymphoid population is hetero-
geneous, with small or intermediate-sized lymphocytes and
plasma cells mixed with other inflammatory cells (Fig. 2-14). In
contrast, a monomorphic population of lymphoid cells without
other inflammatory cells present suggests lymphoid neoplasia.
RESPONSE TO TISSUE INJURY
Cytologic samples often contain evidence of tissue injury in
addition to cyst formation, inflammation, or neoplasia. These
changes include hemorrhage, proteinaceous debris, cholesterol
or calcium crystals, necrosis, and fibrosis.
Hemorrhage that is pathologic can be distinguished from
blood contamination encountered during the cytologic collec-
tion: Blood contamination is associated with the presence of
numerous erythrocytes and platelets, whereas acute hemorrhage
is associated with engulfment of erythrocytes by macrophages
termed erythrophagocytosis (Fig. 2-16A). Care must be taken to
evaluate direct smears first prior to reporting the findings of pro-
cessed materials. For example, the simple act of centrifugation
to create a sediment smear from body fluids can activate mac-
rophages to engulf nearby erythrocytes, which is not observed
in the direct smear (Fig. 2-16B). Chronic hemorrhage is asso-
ciated with active macrophages containing degraded blood pig-
ment within their cytoplasm—for example, blue-green to black
hemosiderin granules (Figs. 2-17 and 2-18) or yellow rhomboid
hematoidin crystals (Fig. 2-18). Hemosiderin represents an
 CHAPTER 2 General Categories of Cytologic Interpretation 21

A
n FIGURE 2-16A Erythrophagocytosis. Cerebrospinal fluid. Cat.
Many erythrocytes are in the background of this direct smear along
with one large macrophage that has engulfed numerous intact red cells.
The cat had a confirmed infection (titer 1:1600) with feline coronavirus
(feline infectious peritonitis). Erythrophagocytosis in this case supports
the presence of acute hemorrhage. (Wright; HP oil.)
n FIGURE 2-17 Chronic hemorrhage with hemosiderin. Tissue aspi-
rate. Dog. Several foamy macrophages are present in this follicular cyst
lesion. The macrophage directly below the cholesterol crystal contains
blue-green granular material the cytoplasm consistent with hemosiderin,
a breakdown product of erythrocytes. On the left edge is a macrophage
with large black granules suggestive of hemosiderin. (Wright; HP oil.)

B
n FIGURE 2-16B Erythrophagocytosis post-centrifugation arti- n FIGURE 2-18 Chronic hemorrhage with hematoidin and hemo-
siderin. Pericardial fluid. Dog. Vacuolated macrophages with bright
fact. Pleural fluid. Dog. Sedimentation of the fluid induced macro-
yellow rhomboid crystals (hematoidin) of variable size appear in this
phage engulfment of erythrocytes. Blood contamination, not acute
hemorrhagic fluid related to hemoglobin breakdown in an anaerobic
hemorrhage, is present in this case; this was supported by frequent
environment. Several macrophages also contain black granular material
platelets and the absence of erythrophagocytosis in the direct smear.
consistent with hemosiderin. (Wright-Giemsa; HP oil.)
(Modified Wright; HP oil.)

excess aggregation of ferritin molecules or micelles. This form of

iron storage becomes visible by light microscopy and stains blue
with the Prussian blue reaction. Hematoidin crystals do not con-
tain iron and are often formed during anaerobic breakdown of
hemoglobin such as may occur within tissues or cavities. Hema-
tomas often contain phagocytized erythrocytes if the lesion is
acute or hemosiderin-laden macrophages if the lesion is chronic.
Proteinaceous debris may be seen within the background of
the preparation. Mucus stains lightly basophilic and appears
amorphous (Fig. 2-19). Lymphoglandular bodies (Fig. 2-20)
are cytoplasmic fragments from fragile cells, usually lympho-
cytes, which are discrete, round, lightly basophilic structures
(Flanders et al., 1993). Nuclear streaming refers to linear pink
to purple strands of nuclear remnants (Fig. 2-21) produced by
excessive tissue handling during cytologic preparation or with n FIGURE 2-19 Mucus. Salivary mucocele. Dog. The background
necrotic material when sampled. Clear to light-pink amorphous contains pale pink-blue amorphous material representative of mucus.
strands representing collagen (Fig. 2-22A) may be admixed with Numerous activated macrophages or mucinophages compose the pre-
spindle cells and endothelium within a fibrovascular stroma. dominant population. (Wright; HP oil.)
22 Canine and Feline Cytology

n FIGURE 2-20 Lymphoglandular bodies. Tissue aspirate. Dog.
The background of this lymph node preparation contains numerous
small, blue-gray cytoplasmic fragments called lymphoglandular bodies
that are related to the rupture of the fragile neoplastic lymphocytes. A
large vacuolated macrophage has phagocytized cellular debris appear-
ing as large blue-black particles. (Wright; HP oil.)
n FIGURE 2-21 Nuclear streaming. Tissue aspirate. Purple strands
of nuclear material are formed from ruptured cells either as an artifact
of slide preparation or from fragile cells that are frequently neoplas-
tic. (Wright-Giemsa; HP oil.) (Courtesy of Denny Meyer, University of
Florida.)

A B 50 m

10 m
C
n FIGURE 2-22 A, Collagenous fibers. Tissue aspirate. Dog. Clear to light pink strands of intact fibrous connective tissue may resemble fungal
hyphae. Collagenous fibers will have poorly defined margins and a variable diameter, unlike hyphae, which have uniform width and distinct borders.
(Wright-Giemsa, HP oil.) B, Collagenolysis. Tissue aspirate. Dog. Haphazard bands of collagen appear bright pink and hyalinized owing to the break-
down of the fibers through release of collagenase by degranulating eosinophils. This type of connective tissue damage occurs commonly in canine
mast cell tumors. Interspersed among tumor cells are eosinophils and their granules. (Wright; IP.) C, Amyloid. Tissue aspirate. Dog. Amorphous
magenta material surrounds a hepatocyte from a Shar Pei with familial amyloidosis. (Modified Wright, HP oil.)
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temper. I was proud and sullen, and—ungrateful.”
“Not always that.”
“I think I hated almost everybody. I did not want to be governed or
counseled. And Stephen was so—rigid and prompt. He treated me
like a little boy—”
“Oh, hush!” I interrupted.
“Some of it is true. He admits it. And when that awful affair
happened I expected he would disown me. He is so proud, then he
never did anything bad in all his life. So I felt that I had no mercy to
expect from him.”
“But you were mistaken,” I said eagerly.
“I couldn’t have gone there and in that way but for you. Perhaps he
has told you—” and his eyes questioned mine.
“No,” I answered, glad that we had not discussed it.
“I went to him. I believe it was the first manly step of my life. But,
oh, I felt so forlorn and miserable—I can’t tell you! If he had been
cold and cross I believe I should have gone and thrown myself in the
river.”
“He was not.”
“Oh, Rose, it was like the story of the prodigal son. ‘Fell upon his
neck and kissed him.’ I remember his kissing me the day father was
buried, and I do not believe any one ever did since till then. It melted
all my soul. Somehow I think he is wonderfully changed. His
goodness is so tender.”
“And you love him?”
“Love isn’t any word. I absolutely adore him! I did not think it was
in me, or in him. And all through the weeks that followed, for I was
very ill and miserable, he was so good. I never talked to any one
before, except you, somehow I could not. But he found his way to my
heart and said he would help me, that we would both try together, for
he had many faults to correct, that God had given us the tie of
brotherhood for a high and holy purpose, that we were to help and
strengthen each other; as if, Rose—as if I could do anything for him!”
“Yes, you can,” I replied. “You can keep him tender and cordial and
brotherly.”
“So he said. We did not come to this all at once, and Mrs.
Whitcomb’s cheerfulness helped. I had to try hard to be patient. I
was so used to flying out at everything. You see, at uncle’s they all
knew that I had a bad temper, they expected me to explode or sulk
on the slightest provocation, and only laughed or tormented me. If I
had been taught to control myself, it would never have been so
dreadful.”
“It is good to have the lesson learned now.”
“I never can forget it, never! I am not an angel yet, Rose, cherubim
or seraphim, I suppose Miss Fanny would say;” and he smiled oddly,
“but I am trying. I do not disdain the helps as I used to. I do not feel
that patience and self-control are exclusively girlish virtues.”
“No,” I returned, “we girls will not rob you of them.”
“You are generous. But then you always were. I am beginning to
learn that the grand corner-stones for the human soul are truth and
love, the truth that leads us to be fair and just to others, and the love
to our neighbor.”
“Here we are,” I said. “Do you want to come in?”
He followed me and we did our errand.
“I could not understand last summer why you loved to do these
things;” he began when we were homeward-bound.
“You considered it an evidence of a depraved taste?”
He smiled rather sadly.
“I supposed people consulted their own pleasure first. Doing any
rather distasteful deed and hunting around until you found a bright
side to it was like so much Sanscrit to me.”
“He came not to please—Himself;” I said solemnly.
 “I understand a little now. Yet when He had redeemed the world
there must have been a great joy in His own mind, as well as in
heaven.”
“We cannot do anything like that,” I said. “But as He loved us, so
we are to love the brethren, the whole world.”
“To be willing to do for them. To seek not our own pleasure
altogether. It is very hard, Rose, and sometimes I get discouraged.
Then Stephen tells me of his failures. It doesn’t go on continually. It
is a little doing all the time, work and healing, and he says it will have
to be so in this world.”
“Yes,” I answered. “We cannot hinder nor change. God sets the
work before us, and though the pleasant fields are all about us, we
have no right to choose our own paths. He knows best in what ways
He wants us to walk.”
“I talked to your father yesterday. I did not think I could talk to
anybody but you and Stephen. I was sorry for all the pain and
anxiety I had caused him—and—it was almost like having a father of
one’s own. I don’t wonder that you all have such sweet pleasant
natures.”
We met Lily and Tim taking a walk, their hands full of grasses and
wild flowers, so we turned them about and all went home together.
The visit proved a very delightful one. We went to the Cascade
one day, taking a lunch with us, and on another day the Churchills
sent their family carriage over and we had a royal time, crowding it
full, and taking turns in driving.
We all noticed the great change in Louis. Not that he was perfect
or saintly. In fact I think he was more of a boy, when it came to that,
than the summer before. He still had a dangerous tendency to
quickness of temper, sometimes he would flush deeply when
annoyed, but he always spoke afterward in a low, even tone of voice,
as if he had gained the mastery within. His feelings were more
healthy-toned, he had a heartsomeness that was genuine. You never
mistrusted it as you did Stuart’s.
 We ended the festivities with a croquet and tea-party on Saturday
afternoon, asking in a half dozen young people who all enjoyed
themselves amazingly. To the surprise of everybody, right in the
midst of the gayety who should drop down upon us but Stephen
Duncan.
“I was homesick to see you all,” he began, with a comically
lugubrious face.
“If you think you are going to be purely ornamental you are much
mistaken;” declared Fanny. “Here is a mallet and here is a place.”
“If you will excuse me—”
“But I will not. No running away to the study to talk with papa, or to
play with Edith. If you will come uninvited to a party you must take
the consequences.”
“Can I not soften your heart, if like the old man I should ‘sit on the
stile and continue to smile?’”
“Not any smiles. I am obdurate.”
He pretended to be much aggrieved, but in reality he was very
gay. I had never seen him so amusing and entertaining.
“I don’t see how you get acquainted with such loads of nice
people;” said Allie West. “And you always have such good times
here.”
The good times came without any trying. There are numberless
gates called Beautiful all along life, at which you give such as you
have, and find it more precious than silver or gold.
It was a lovely moonlight night, so after supper we walked part of
the way with the merry crowd. It did not seem to me that I had ever
been so happy in my life. I could not tell why but I felt as if I must
have wings somewhere that were lifting me off the ground at every
step.
We rambled around under the trees and by the way side. Louis
came back to my vicinity and we fell into a rather grave talk about
the future.
 “I never thought I should want to stay here so much,” he said. “I
was glad enough to get away last summer. I cannot forgive myself
for being such a boor! Now I shall want to come again and again.”
“Well why not?” I returned.
“I am afraid you will become tired of me.”
“Try us and see. We are not easily wearied.”
“You are all so generous with yourselves.”
I smiled a little. “Why not give of your best?”
“True.” Then there was a silence. We reached the gate presently.
“Do not go in just yet;” he pleaded, so we remained in the silvery
light that was flooding the whole earth. Moonlight always stirs the
tender and thoughtful side of one’s soul.
“I am glad that to-morrow will be Sunday. I can just think how I
shall enjoy going to church and hearing your father preach.”
This from him who had despised religion and sneered at sermons.
It did startle me.
“And to have Stephen here.”
“I am rejoiced that you feel so kindly toward one another,” I replied.
“You are getting to be brothers indeed.”
“And then will come weeks and weeks of study,” he went on in a
musing tone. “I like it. Books seem to me—well, better than some
people. Only—if you could all come down in the winter. Stephen and
Mrs. Whitcomb were planning for it, but there! it was a secret and I
have betrayed it.”
“I can keep secrets;” and I smiled up into his remorseful face.
“Yes; I have proved that. Rose”—after a pause—“I have half a
mind to tell you another, to ask some—advice; at least, I would like
to know how it appears to you.”
“Will it be of any real avail?” I asked, noting the perplexed lines on
his countenance. “I am not as wise as you think. Because I just
happened to stumble into one matter without making a mess of it—”
 “This is only an idea. I cannot ask Stephen. I think it would please
him and he might judge wrongfully.”
“If I can help you;” I replied encouragingly.
“It is about the future. It may never come to anything to be sure,
and perhaps I never can be good enough. Stuart will go into
business. He does not love study and he needs an active life. He
wanted Stephen to put him in a store this Autumn. But I—”
I knew then what he meant. Somehow I could not help laying my
hand on his arm with a touch of confidence.
“Whether I ever could so govern my temper and my impatient
desires;” bowing his head humbly. “But if I had some guard about
me, if I felt that I must try continually—would it be wrong to think of
it?”
“Surely not;” I returned warmly. “Nor to do it if God gives the
strength and the grace.”
“I like to think of that grand, earnest Saint Paul, with his ‘thorn in
the flesh.’ Perhaps it was some giant temper or desire. I fancy it
must have been, for you know how he persecuted the Christians
unto death. And though God would not take it away, there was the
promise of His grace being sufficient.”
“As it is, always.”
“There are some years to live before I decide positively. But if they
were spent in a worthy manner, and I mean them to be, with God’s
help.”
“Oh, you could, surely. And papa would be your best friend;” I
rejoined eagerly.
“Keep my secret—I have your promise,” he said in a hurried
manner, for a step sounded on the walk.
“It is sacred to me until you wish to take others into your
confidence.”
Stephen spoke and we turned, walking slowly up to the house.
Louis sat down on the step beside papa. I stood undecided whether
to go in or not, when Stephen took my arm and drew me around the
corner of the porch. There was a long grape arbor whose gloom was
made a pleasant twilight by the silver sifted through the openings
between the leaves, and we took a turn up and down.
“I want to tell you,” he began almost abruptly, and his voice had a
hard, strained sound, “that I heard—the last of what you said. I could
not help it. And I know your secret.”
I was a trifle annoyed, but I controlled myself.
“Oh,” I said, “then you will be tender and helpful and do all in your
power to strengthen Louis. He feels so humble. I would hardly have
thought it of him. And there are so few young men who have any
desire to take such a life upon them. With his means and his talents
he can do so much good.”
He stopped suddenly. “Rose, what are you talking about?” he
asked. “Did not Louis—”
“He confessed to me his desire—no, it was hardly that, as he is
afraid he can never be good enough for a clergyman. But you will
assist him—you do not disapprove of it?”
“Louis! Ah, I understand. It would be the delight of my heart. But I
thought—I knew he liked you so much. Oh, my little darling!”
He turned and gathered me in his arms. My heart beat and my
cheeks were in a blaze as the whole story came to me, dazing me
with its strange, sweet suddenness. I believe I cried and then I
laughed hysterically, but somehow the cool, steady voice quieted me
and made me feel the truth and earnestness of what he was saying,
so presently I grew still with a great awe.
“You will come,” he was saying. “We both need you. We want just
this steady, cheerful, loving influence. I think I have a tendency to be
impatient when people cannot see my ways, perhaps requiring a
little too much, and your sweetness will temper this. Then we can
both help him.”
Could I? How strange that any one should care for me alone. Not
for mamma, or Fanny, but to want me!
 “Mr. Duncan,” I began as we were going back to the porch—“have
you forgotten that my hair is—red?”
“Well, what of that?” in a gay tone.
“I do not believe you—like it.”
“You foolish little girl, set your heart at rest. Do you remember
when I came upon you suddenly last summer? You were standing on
the porch in a tiny glint of sunshine, and looked like some of the old
pictures! Why, I believe it was your hair that I fell in love with first of
all.”
“I am glad it was, for I am not half as good as you imagine I am.”
“Children,” mamma said, standing on the porch step. “Do you
realize how late it is?”
I felt that she knew all, perhaps had known it long before, indeed.
But I was glad that the knowledge had come to me so suddenly, and
not any sooner. Even now I was half afraid of it. Her kiss and tender
clasp re-assured me.
“Mother!” Stephen Duncan said with reverent sweetness.
 CHAPTER XVII.

WISHED there could be no such thing as breakfast the

next morning, but there was, and I had to go through
with it, feeling that I was no longer I, that Rosalind
Endicott was some dream-girl of the past. Stephen was
very good and did not notice me much, and Fan
appeared wonderfully pre-occupied. Mamma helped me over the
trying places, and papa just said with his tender morning kiss,—“And
this little girl, too.”
When I was all dressed for church I opened a little drawer to get
my gloves. There lay the box containing Stephen’s gift. I had never
worn it, but it seemed to me as if I ought to put it on now. He liked
me and the misunderstandings were at an end. I had accepted a
share of his burthens, his crosses, whatever they might be, so I
clasped it around my neck. It was so beautiful. I did not envy the
queen her diadem.
We walked to church together. Louis glanced back now and then. I
believe he began to suspect.
It was quite different from the Sunday when I had gone to church
with that strange sense of Fan’s new love. I felt quiet and restful, yet
it was such a great thing to have another’s heart in one’s keeping, to
take in a new life beside the old.
They both left us on Monday. Stephen was to come up soon
again. In the meanwhile, letters.
“I have one of yours to begin with,” he whispered.
It was a silent day for us. No one appeared to care about talking,
yet we were not gloomy. Indeed, I think mother, Fan and I
understood as we never had before, how much we loved one
another.
 I went on wearing my cross. In the first letter there came a pearl
ring for me. Fan had a handsome diamond but she seldom wore it
except when she was going to the Churchills. I slipped mine on my
finger with a slight presentiment that I should turn the pearl inside if
any one looked at me.
Richard Fairlie and Jennie came home bright and happy as birds.
They took possession of the great house, altered a little, re-arranged
to their liking and had Mrs. Ryder in their midst. There was no grand
party, but some pleasant tea-drinkings and hosts of calls. No one
could afford to slight Mrs. Fairlie, and people began to realize what a
noble girl Jennie Ryder had always been.
I am almost ashamed to confess how much talking it took to settle
our affairs. Stephen wanted to be married in the Spring. That was
too soon, mamma and I thought. But there were so many good
reasons.
Miss Churchill heard of it presently and came over to have a
consultation with mamma.
“It will have to be sometime,” she said. “It will make a little
confusion, a break, and no end of strangeness in adapting
yourselves to the new order. But here are Nellie and Daisy right
behind.”
“I don’t want to lose all my girls in this fashion,” said mamma.
Miss Churchill smiled and then admitted that she had a plan to
propose.
They wanted Fanny. The murder was out then.
“Kenton and I have discussed the matter a good while. Winthrop
will have the farm when we are done with it—he is the only nephew.
Kenton has been sorry for some years that we did not take him when
his father died. He is very fond of country life, and surely there are
enough to toil and moil in the cities. Then, although Lucy was
improved by her summer trip, we can understand that it is not
permanent. She wears out slowly. I should like her to have a happy
year or two with Fanny, and I should like the marriage well out of the
way of any sad memories.”
 “You are very thoughtful,” returned mamma.
“And it will hardly be like parting with Fanny, for you—as you can
see her every day. One thing and another has brought us so near
together. Kenton and I are growing old and the presence of these
young people will keep us from getting too queer and whimsical.”
It was settled some time in January.
“We shall have to do the best we can,” said mamma. “The
wardrobes must be simple. It is our station that they go out of, and
we never have been ashamed of our poverty.”
“What does a few clothes signify,” commented papa. “If the young
men are not satisfied we will give them a double portion of dry-goods
and keep our girls.”
Fan laughed over the idea.
So it was arranged that she and Fanny should go to New York. I
did not desire to accompany them, and I was sure they could choose
as well for me as if I hunted the whole town over. Besides, I wanted
the nice quiet time with papa, since I was the one who would have to
go away.
“Isn’t it funny!” said Fan. “I feel like the heroine of some hundred
year old novel, going up to town to buy wedding clothes, instead of a
girl of the period of puffs, paniers, chignons, Grecian bends, and all
that! Why Rose, think of it! We have never had a silk dress in all our
lives, except that once we had one ruffled with an old one of
mamma’s; and we have been very tolerably happy.”
“Yes, just as happy as one need be. All that could be crowded into
our small lives.”
“I dare say we should be absolute curiosities to some people.
Everybody now-a-days has a silk walking-suit, and some handsome
thread lace, and I don’t believe there are any poor people but just us.
But then we have had the love and comfort and enjoyment and no
time to worry about our rich neighbors. It has been a life full of
pleasantness and peace.”
 That was true enough. There were many, many things beside
raiment, if one could only get at the real completeness and harmony,
the secret of soul life.
Jennie Fairlie would help us sew. With their good servant she
declared she had nothing to do. Miss Churchill sent us both an
elegant poplin suit, or at least the materials. It was a simple
wardrobe to be sure. One pretty light silk dress, one dark silk with a
walking-jacket. We made morning robes and some inexpensive
house garments. Then it would be summer so soon, and there was
nothing equal to fresh, cool white. We were not used to crying for the
moon, we had found early in life that it was quite a useless
proceeding.
Altogether we kept our secrets pretty well, and when the truth
leaked out at last, everybody was so surprised that they could only
exclaim. Aunt Letty Perkins was brave enough to come and see if it
was really so.
“Well, I am beat!” she declared. “And doing well, too! I always said
there never was anyone like Mis’ Endicott for luck. Girls often do
hang on so where there is a lot, and you’ve enough left. Fanny is the
flower of the family to be sure, but she is making a big step to get in
with the Churchills. Ain’t afraid she’ll be puffed up with pride and
vanity, are you?”
“I think I can trust her,” replied mamma with a funny smile in the
corners of her mouth.
I remember the morning as one recalls a half dream, the misty
impression between sleeping and waking. The peculiar confusion
pervading the house, the strange mislaying of handkerchiefs and
gloves, the voices that were so full of tears and gay little laughs, the
half sentences, the clasp of hands as one went in or out of a room,
the long, loving glances as if each would fain garner all the past into
one sweet remembrance. Winthrop and Stephen, one rather grave
but very tender to mamma and the little ones, the other full of life and
vivacity, the happiest of the happy.
Fan had one little say though her eyes were bright with tears.
 “I hope I can be as good and sweet in my life as mamma has been
in hers. And I will not ask any higher happiness.”
We walked up the church aisle. The children stood around, back of
them Louis, Nelly and mamma, and then a host of eager parish
faces. Does any one take it all in then, the solemn questions, the still
more solemn promises?
Mr. Churchill gave us both away. Papa’s voice had a little falter in
it, and I dared not look up. “For better, for worse,” “till death do us
part,” rang clearly in heart and brain. The forever of human love,
when it is love and no base counterfeit.
A little kissing, a few tears, some tremulous whispers and sad, sad
good-byes. We whose farthest journey had been the brief sojourn at
Martha’s Vineyard, took up the great pilgrimage of a new life.

I cannot tell you anything about it, or Stephen. It was a happy

confusion of strange places and watchful care, bits of affection
shining out of the tiniest rift. Honeymoons, I suppose, are much
alike, but it is right for each to think his and hers the best and most
delightful.
One afternoon the carriage set us down in so quiet a street that I
could hardly believe it was New York. And when I entered the house,
my new house, I doubted more than ever, for everybody was there.
One kissed me until I thought the breath of life was surely gone, then
another took me up. I have a dim suspicion that my sleeves were
worn threadbare, and if my hair had not been all fast in my head, I
am afraid the difference would have been discoverable.
“Why you are rounder and rosier than ever!” declared Fan,
inspecting me.
She was elegant as a princess, and had her light silk dress
trimmed with applique lace.
 It seemed as if I never could get done looking at mamma, and
papa hovered around me as if I was indeed an unusual sight.
Somehow I managed to get up-stairs to my own pretty room, to
wash my face, what there was left of it, and straighten my gown. And
there was Beauty, my lovely half-grown kitten that some one had
brought from the old home.
I heard Stuart’s voice outside the door and called him in.
“Stuart,” I said with much dignity, “this is Miss Beauty Endicott, a
nice, orderly, well brought-up kitten, and mine. I want you to respect
her and treat her with the courtesy of a gentleman.”
“Oh, fudge!” he returned. “What are you doing with a kitten when
you are married? I thought it was only old maids who were death on
cats.”
“It is boys who are death on cats,” I replied severely. “And then—I
never did expect to be married. I always supposed—”
“Oh, you couldn’t have been an old maid! your nose never can be
sharp, and your chin has that great dimple in it, and you are such a
funny little dumpling altogether! If you say much I’ll put you in my
pocket and carry you off. No doubt Stephen would feel immensely
relieved, but what could the cat do?”
“You are an incorrigible boy!”
“But we will have jolly times for all that,” and he whistled to Tim,
who put her head within the door.
“Fan,” I exclaimed with remorseful tenderness as I was going
down stairs with her arm over my shoulder; “I have Mrs. Whitcomb.
But you know you half gave her to Stephen. And as you are not to
keep house—”
“I will lend her to you a little while longer.”
We had such a merry, enjoyable supper, such a lovely long
evening, and were brimfull of happiness.
But the next morning papa gathered up his flock, “what there was
left of them,” he said with a certain comical grimace.
 “I don’t know as you need lament,” answered Stephen. “I think the
sons are coming in pretty rapidly.”
“And if there should be seven! Mother what would we do with them
all?”
Mamma smiled a little as Stephen went around and kissed her.
“Remember that I am the first one; I will never be crowded out of
my place.”
“No,” she answered softly.
They all went away that noon, and left us to begin our home life.
We had talked it over, what we were to do for the boys, what for
ourselves, and what for the world outside. For the true life is not
bounded with a narrow—thou and I. The world takes us in, and over
and above all, God takes us in. His vineyard, His day, and first and
always His everlasting love.
 Young Folks’ Heroes of the Rebellion.
By Rev. P. C. HEADLEY.

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Admiral Farragut is rich in brave deeds and heroic example.
THE MINER BOY AND HIS MONITOR. The Career and
Achievements of John Ericsson, Engineer.
One of the most thrilling incidents of the war was the sudden appearance of the
Little Monitor in Hampton Roads to beat back the Merrimac. The life of the inventor
is crowded with his wonderful inventions, and the story of his boyhood in the coal
mines of Sweden is particularly interesting.
OLD STARS. The Life and Military Career of Major-Gen. Ormsby
McKnight Mitchel.
 “Old Stars” was the pet name given the brave general by his soldiers, who
remembered his career as an astronomer before he became a soldier. His story is
full of stirring events and heroic deeds.

☞Sold by all booksellers, or sent by mail, postpaid, on receipt of

price.

LEE AND SHEPARD, Publishers, Boston.

 YOUNG FOLKS’ HEROES OF HISTORY.
By GEORGE MAKEPEACE TOWLE.
Handsomely Illustrated. Price per vol., $1.25. Sets in neat boxes.

VASCO DA GAMA:
HIS VOYAGES AND ADVENTURES.
“Da Gama’s history is full of striking adventures, thrilling incidents, and perilous
situations; and Mr. Towle, while not sacrificing historical accuracy, has so skilfully
used his materials, that we have a charmingly romantic tale.”—Rural New-Yorker.

PIZARRO:
HIS ADVENTURES AND CONQUESTS.
“No hero of romance possesses greater power to charm the youthful reader than
the conqueror of Peru. Not even King Arthur, or Thaddeus of Warsaw, has the
power to captivate the imagination of the growing boy. Mr. Towle has handled his
subject in a glowing but truthful manner; and we venture the assertion, that, were
our children led to read such books as this, the taste for unwholesome, exciting,
wrong-teaching boys’ books—dime novels in books’ clothing—would be greatly
diminished, to the great gain of mental force and moral purpose in the rising
generation.”—Chicago Alliance.

MAGELLAN;
OR, THE FIRST VOYAGE ROUND THE WORLD.
“What more of romantic and spirited adventures any bright boy could want than
is to be found in this series of historical biography, it is difficult to imagine. This
volume is written in a most sprightly manner; and the life of its hero, Fernan
Magellan, with its rapid stride from the softness of a petted youth to the sturdy
courage and persevering fortitude of manhood, makes a tale of marvellous
fascination.”—Christian Union.

MARCO POLO:
HIS TRAVELS AND ADVENTURES.
“The story of the adventurous Venetian, who six hundred years ago penetrated
into India and Cathay and Thibet and Abyssinia, is pleasantly and clearly told; and
nothing better can be put into the hands of the school boy or girl than this series of
the records of noted travellers. The heroism displayed by these men was certainly
as great as that ever shown by conquering warrior; and it was exercised in a far
nobler cause,—the cause of knowledge and discovery, which has made the
nineteenth century what it is.”—Graphic.

RALEGH:
HIS EXPLOITS AND VOYAGES.
“This belongs to the ‘Young Folks’ Heroes of History’ series, and deals with a
greater and more interesting man than any of its predecessors. With all the black
spots on his fame, there are few more brilliant and striking figures in English
history than the soldier, sailor, courtier, author, and explorer, Sir Walter Ralegh.
Even at this distance of time, more than two hundred and fifty years after his head
fell on the scaffold, we cannot read his story without emotion. It is graphically
written, and is pleasant reading, not only for young folks, but for old folks with
young hearts.”—Woman’s Journal.

DRAKE:
THE SEA-LION OF DEVON.
Drake was the foremost sea-captain of his age, the first English admiral to send
a ship completely round the world, the hero of the magnificent victory which the
English won over the Invincible Armada. His career was stirring, bold, and
adventurous, from early youth to old age.

Sold by all Booksellers, and sent by mail, postpaid, on receipt of

price.

LEE & SHEPARD, Publishers BOSTON.

 Transcriber’s Notes

pg 64 Changed: She smiled in her irresistable fashion,

to: She smiled in her irresistible fashion,
pg 128 Changed: papa would say,” was my rejoiner
to: papa would say,” was my rejoinder
pg 150 Changed: And she enjoys everything so thorougly.
to: And she enjoys everything so thoroughly.
pg 168 Changed: our engagements and geting everything
to: our engagements and getting everything
pg 183 Changed: Here were sandwitches dripping with jelly
to: Here were sandwiches dripping with jelly
pg 185 Changed: great double lucious blossoms
to: great double luscious blossoms
pg 208 Changed: with a certain funny lugubriouness
to: with a certain funny lugubriousness
pg 238 Changed: Dosn’t she take care of sick people
to: Doesn’t she take care of sick people
pg 264 Changed: nothing but complaint and discouragment
to: nothing but complaint and discouragement
pg 264 Changed: went to neigboring towns
to: went to neighboring towns
pg 274 Changed: I struck out blindy
to: I struck out blindly
pg 284 Changed: I have gussed
to: I have guessed
pg 285 Changed: It is not—Winthop Ogden.
to: It is not—Winthrop Ogden.
pg 287 Changed: bound by a promise of secresy
to: bound by a promise of secrecy
pg 300 Changed: no expensive trosseau
to: no expensive trousseau
pg 317 Changed: spoken of the probabilty
to: spoken of the probability
pg 337 Changed: It was the begining
to: It was the beginning