Clinical Management of the Cancer Patient
0195-5616/90 $0.00
+ .20
Taking a Biopsy
Ronald F. Carter, DVM, PhD,* and Victor Edwin Oswald Valli, DVM, PhDt
In this article we will review for the private practitioner the process of biopsy for cytologic and histologic study. We will emphasize a logical approach and critical elements of technique which together ensure ·a satisfactory interaction with pathologists. In accordance with the subject of this issue, we will emphasize biopsy of suspected malignancies.
THE ROLE OF THE BIOPSY IN THE DIAGNOSTIC APPROACH A biopsy is tissue obtained from a living animal by some method of surgical removal, such as excision, aspiration, imprint, scraping, or wash. Although sampling peripheral blood for cellular or biochemical studies constitutes a biopsy, we will not consider such tests here. Indications A biopsy is obtained to (1) diagnose disease, (2) monitor response to therapy, or (3) obtain living cells for studies such as tissue culture. For most practical purposes, a biopsy is obtained for cytologic or histologic study intended to provide a diagnosis for a clinical problem. Another reason related to diagnosis of disease is the selection of tissue for bacterial or viral culture. Repetitive biopsies of specific tissues under treatment are occasionally performed. Examples would be resections of recurring masses, repeated synovial taps for joint disease, and sequential bone marrow aspirations. More rarely, biopsies are obtained for the specific purpose of obtaining living cells for study. justification of Surgical Intervention. As with any intervention, it is implicit that potential benefit to the patient should exceed potential risk. *Postdoctoral Fellow, Department of Pathology, University of Toronto, Toronto, Ontario, Canada t Diplomate, American College of Veterinary Pathologists; Dean, University of Illinois College of Veterinary Medicine, Urbana, Illinois Veterinary Clinics of North America: Small Animal Practice-Val. 20, No. 4, July 1990
939
940
RONALD
F.
CARTER AND VICTOR EDWIN OSWALD VALL!
The decision to obtain a biopsy therefore depends in part upon an estimate of risk. The risks involved vary according to the severity of surgical intervention and anesthetic restraint required. For techniques such as fineneedle aspiration, risks may be very slight. In contrast, an exploratory laparotomy or thoracotomy of an aged and compromised patient may represent a significant risk of death. Biopsies obtained by laparoscopy or endoscopy may be reasonable alternatives to more invasive procedures despite the risk of diagnostic failure due to a smaller and possibly less representative specimen. While an estimate of risk may ultimately determine whether a biopsy should be taken from an animal, it is not the sole consideration. Estimate of risk alone is inadequate to determine the type of biopsy or technique to be used. Certain types of biopsy may result in more definitive and/or timely results. The least risky procedure is frequently not the most informative. Thus, the practitioner has to estimate the likelihood of obtaining the definitive answer versus the risk resulting from the necessary procedure. One should always attempt the most direct assay of the suspected lesion ppssible under the circumstances. A common approach is to investigate the nature of a suspicious lesion by cytology, which is rather noninvasive and provides a rapid diagnosis; if the findings are uncertain or indicate a serious lesion, follow-up excisional biopsy and confirmation by histologic interpretation is justified. Role of the Biopsy in the Diagnostic Plan. Biopsies should not be obtained unless there is a clear expectation of informative result. Although almost any tissue can be biopsied, biopsies provide specific information only about very localized areas of tissue; the surgical action required to obtain a biopsy may even preclude the simultaneous biopsy of a number of tissues while failing to provide information about those other tissues. Biopsies are unique in their ability to provide information about the living patient, because tissue is actually obtained for direct study. Biopsies are best used as the final test which confirms a diagnosis already suggested by history, clinical picture, panel tests, and/or ancillary diagnostic techniques. Biopsies performed as part of a survey based upon poorly formed clinical suspicions will often result in unnecessary injury with little diagnostic benefit. Relationship to Clinical Pathology and Autopsy. It is useful to review the relationship among clinical pathology (e. g., hematology and serum biochemistry), biopsy, and autopsy. Hematology constitutes a specific test for the hemopoietic system and also provides information about systemic disease and response. Serum biochemical panels are carefully constructed to provide surveys of systemic response as well as indications of specific organ function or injury. Biopsies provide premortem morphologic evidence of tissue function and disease. Autopsy provides a postmortem opportunity to selectively or exhaustively study all tissues for morphologic evidence of tissue function and disease. Autopsy will not provide information useful for the management of the patient in question, but it is essential for monitoring the accuracy of the diagnostic approach and the (lack of) effectiveness of clinical management. Summary. The decision to obtain a biopsy is reached after the history,
TAKING A BIOPSY
941
physical examination, and noninvasive tests suggest a specific list of diseases related to an identified tissue. Undertaking biopsy requires that the risk of surgical intervention is justified by the benefit of resulting information. The choice of biopsy technique and specimen preparation should maximize the diagnostic value of the biopsy while minimizing the risk. Cytopathology can be used as a safe screening technique to provide rapid diagnoses or indicate the need for excisional biopsy and histopathology. For many diseases, biopsy represents the decisive diagnostic approach for the living patient. Autopsy, on the other hand, provides the ultimate assessment of the adequacy of diagnosis and management.
COMPONENTS OF THE BIOPSY PROCEDURE The complete process of taking a biopsy involves many steps (Fig. 1). Throughout, there are two paramount considerations. There must be constant maintenance of correct patient and specimen identification, and the correct diagnostic interpretation must be returned to the practitioner. Failure in these aims compromises the entire procedure. Biopsies can be classified by the nature of tissue sampled (Table 1), which in turn reflects the diagnostic dilemma facing the practitioner. One may sample fluids by aspiration (e.g., ocular, pleurallpericardial, synovial, peritoneal, cerebrospinal, urine, or cystic fluids, and lung or prostatic washes). One may sample tissues by fine-needle aspiration, even wellorganized connective tissue. However, surgical excision of tissue is still the most common method of biopsy, applicable to almost all tissues except fluids. The method of biopsy determines the type of laboratory processing for routine evaluation (see Table 1). The most common division is into cytologic specimens (which are examined without prior sectioning) and histologic specimens (which involve fixation, paraffin embedment, and sectioning before evaluation). Fluids and fine-needle aspirations are processed for cytology; solid tissues obtained by excision can provide cytologic specimens by making imprints or scrapings on slides (often useful for quickly assessing the adequacy of the biopsy and obtaining a provisional diagnosis), but are ultimately examined as histologic sections to obtain the most information. Not covered in detail in this review are the other types of diagnostic tests that can be performed on biopsies. Outlines of appropriate procedures for immunohistochemistry, electron microscopy, and other tests are summarized in Table 2. Techniques
Important points for taking biopsies and preparing specimens for submission are summarized in Figures 2 and 3. Diagnostic Assessment. The initial step in the biopsy procedure consists of an evaluation of history, signalment, and physical findings, which leads to a problem list and associated diagnostic plans. After ancillary testing where necessary, the clinician forms a list of possible causes for the problems identified. Biopsy may be indicated as a logical development of the
:f t-:;1 PRACTITIONER
ASSESSMENT-.. PROBLEM LIST-.. RULE-INS
...
BIOPSY
/
EVALUATION AND MANAGEMENT
...
LABELLING, FORMS, HISTORY
t
•
TRANSPORT
TRANSPORT
t REPORTING
•
PREPARATION & FIXING
!
LABORATORY
...
INTERPRETATION ...
PROCESSING ...
staining sectioning embedment Figure l. Flow chart of biopsy procedure.
IDENTIFICATION
943
TAKING A BIOPSY
Table I. Types of Biopsies for Morphologic Study TISSUE TYPE
Fluid'-.......
~
/ / Solid~ ~
SPECIMEN TYPE
PREPARATIOI\
Air-dried Film on Slide Direct/ Aspirate ~ Concentrated - - Wet-fixed Film on Slide (Optional) ....--"Air-dried Imprint on Slide~ Excisional or~ Wet-fixed Imprint on Slide~ Tru-Cut ~ (Optional) Biopsy Formalin Fixed, Paraffin~ Section
~
ROUTINE STAIN
Wright's Papanicolaou Wright's Papanicolaou Hematoxylin & Eosin
diagnostic plans, and the appropriate biopsy procedure should be selected through evaluation of the type oflesion, the risk associated with the biopsy, and the likelihood of obtaining a definitive answer. Biopsy. The biopsy should be representative of the lesion. Fine-needle aspiration should be done in a methodical spraying motion, with the lesion immobilized by one hand, to ensure representative sampling of the lesion. Aspiration should be stopped as soon as there is either sufficient material obtained or dilution of the intended sample by frank blood. When resection is performed, the biopsy may consist of part of the lesion or the entire lesion as part of the approach to therapy. Resection of a mass should be represented by a specimen which consists of the normal tissue surrounding the mass, the interface of the mass and the normal tissue, and the center of the mass. If an abscessed, necrotic, or cystic lesion is identified, the biopsy should consist of viable peripheral tissue rather than the disintegrating center. Surgical removal should disrupt the existing morphology of the tissue as little as possible. When fine-needle aspiration is complete, suction pressure should be released before the needle is withdrawn from the lesion, so that the aspirated material remains in the hub of the needle and is not explosively released into the syringe by inrushing air. The needle is then detached from the hub, air is drawn into the syringe, and the needle is replaced preparatory to expelling the sample onto microscope slides. For histology, excessive surgical manipulation, crushing, and congestion are common artifacts of surgical procedure which cause major difficulties in interpretation (Fig. 4). If imprints are made, care should be taken not to crush the tissue while making the imprints (a common error with Tru-Cut biopsies). Fixation. The material obtained by biopsy should be immediately fixed, that is, processed in a manner which ensures conservation of morphologic detail over time and throughout subsequent processing (see Tables 1 and 2). Unfixed or poorly fixed cells lose morphologic detail. They are also difficult to prepare for examination because they may undergo lysis, fail to adhere to slides, or fail to take up stains properly. Delayed or improper fixation of a specimen can render interpretation of subtle, or even otherwise obvious, lesions difficult. Cells with high metabolic rates, such as gut epithelial cells and hemolymphatic cells, will start changing in morphologic
944
RONALD
F.
CARTER AND VICTOR EDWIN OSWALD VALLI
Table 2. Outline of Specimens Required for Tests on Biopsies* DIAGNOSTIC TEST
POSSIBLE SPECIMENS
METHOD OF PREPAR"'TION
Cytology
Aspirated fluids or tissues, See Table l. Air-dried smears exudates, imprints, washes are routine. Assess (eg, bronchoalveolar lavage), requirement for scrapings if poor exfoliation concentration by initial quick stain
Histology
Any solid tissue, or any cells See Table l. Formalin fixative is routine; other fixatives which can be centrifuged to may be preferred for a pellet specific tissues, eg, B5 for bone marrow
Electron microscopy
Any solid tissue, or any cells Glutaraldehyde-Os04 fixation is routine; paraformaldehyde which can be centrifuged to a pellet may be best for immunoelectron microscopy. Formalin adequate for some purposes
Immunohistochemistry, eg, skin, autoimmune diseases
Any suspension of cells or solid tissue
Bacteriology, mycology, virology
Refrigerated transport and Any solid or fluid specimen holding until samples which can be collected cultured. Freezing aseptically. Solid tissue acceptable but room specimens should be large enough that cultures can be temperature to be avoided initiated from interior sites specifically
Cytochemistry & histochemistry, eg, storage diseases, leukemias
Cell suspensions or solid tissues
Submission of absolutely fresh specimens best. Transport in liquid nitrogen or specialized transport media (eg, Michel's media for skin samples) may be appropriate. Tests done on smears, frozen sections, or fixed tissue
Absolutely fresh tissues best. Other transport methods may be possible
*The first procedure should always be to contact the laboratory for exact requirements for a specific test.
detail within minutes of cardiovascular compromise (Fig. 5). Fixation therefore should be initiated rapidly and should be complete before further tissue processing. Cytologic specimens are usually prepared by quickly making air-dried smears (see Fig. 2) or imprints (see Fig. 3). Imprints are best made by gently rolling a freshly cut face of tissue across the slide, after the tissue face has been gently blotted dry with paper gauze. Fine-needle aspirates can be prepared as smears (or films) and crushes (Fig. 6, A and B). Crush preparations, made by placing the aspirated material between a slide and a coverslip and drawing the two apart, often cause significant loss of
Text continued on page 955
immobilize tissue in one hand
.. ""'
cq:
II
~raying motion while aspirating
I ~
release vacuum before leaving tissue remove needle and reload syringe with air
,
propel tissue out of needle hub using air in syringe Essential elements of Technique: 1.) 22 gauge needle, 10 ml syringe 2.) Accurate sampling of lesion; no dilution by bleeding 3.) Sample retained in hub of needle, not lost in syringe 4.) Rapid preparation of slides directly from aspirate 5.) Smear technique used to spread slides spread tissue out behind leadir.g edge of slide 6.) Rapid fixation by air drying or wet fixation (spray is best). 7.) Transport at room temperature in protected package air dry and/or wet fix rapidly
..
Figure 2. Preparation of smears from fine-needle aspirates.
f
1:11
~
~
O'l
obtain biopsy tissue without comprising morphology slice into 2 to 3 mm thick sections in appropriate orientation
~
'-._____../
............
/ f6 = ,
~
[)
..,._ ( [ ) [ )
~
blot
'
,;;:::::"make imprints from sections by gently rolting slide over tissue face; air dry or wet fix rapidly
Essential elements of imprint technique: 1.) Gently cut fresh tissue face and blot off excess fluid 2.) Hold in one hand and gently roll slide over tissue face without creating a suction effect which rips cells 3.) Rapidly air-dry or wet fix (spray is best) 4.) Prepare only a few imprints from each tissue face
F
for~~
ORMALIN . lices into place ussue s arion and formalin laboratOry then tran'P? .for processmg
Essential elements of formalin fixation: 1.) Gently handled fresh tissue 2.) Abundant volume of fresh, buffered 3.7% formalin at room temperature in secure specimen bottle 3.) Distance formalin required to penetrate to desired lesion < 3 mm 4.) Fix at least 24 hrs before processing regardless of sample size 5.) Avoid freezing before embedding
Figure 3. Preparation of imprints and fixed histologic specimens.
~ ~
-l
Figure 4. Histologic section of laryngeal biopsy from a cat . Edge of tissue with crush artifact in fresh specimen . The irregular dark streaks are ruptured nuclei. 1-1 & E; bar = lO fJ.m.
~
..... oc
Figure 5. A, Histologic section of mediastinal mass from a cat with immunoblastic lymphoma. E arly autolysis is represented by cytoplasmic separation and contraction, and by irregular nuclear membranes and chromatin clearing. H & E ; bar = lO J.Lm. Illustration continued on opposite page
~
..... ~
Figure 5 (Continued). B, Histologic section of a mediastinal mass from a cat. More extensive autolysis is represented by ruptured nuclear membranes and fragmentation of chromatin. Cytoplasmic separation is again present. Stromal nuclei, in comparison, maintain a more normal appearance , except for vesiculation and peripheralized chromatin (arrow). The section is also too thick, obscuring cellular details. H & E ; bar = 10 fLm .
Illustration continued on following page
:ll
0
Figure 5 (Continued). C, Histologic section of liver from a cat. Autopsy specimen. Subcapsular tissue has undergone chromatin lysis and cytoplasmic degradation due to effects of bile leakage prior to tissue fixation. Edge effects may also be caused by desiccation. H & E ; bar = 100 J.Lm, or 0.1 mm.
"'......
0195-5616/90 $0.00
+ .20
Taking a Biopsy
Ronald F. Carter, DVM, PhD,* and Victor Edwin Oswald Valli, DVM, PhDt
In this article we will review for the private practitioner the process of biopsy for cytologic and histologic study. We will emphasize a logical approach and critical elements of technique which together ensure ·a satisfactory interaction with pathologists. In accordance with the subject of this issue, we will emphasize biopsy of suspected malignancies.
THE ROLE OF THE BIOPSY IN THE DIAGNOSTIC APPROACH A biopsy is tissue obtained from a living animal by some method of surgical removal, such as excision, aspiration, imprint, scraping, or wash. Although sampling peripheral blood for cellular or biochemical studies constitutes a biopsy, we will not consider such tests here. Indications A biopsy is obtained to (1) diagnose disease, (2) monitor response to therapy, or (3) obtain living cells for studies such as tissue culture. For most practical purposes, a biopsy is obtained for cytologic or histologic study intended to provide a diagnosis for a clinical problem. Another reason related to diagnosis of disease is the selection of tissue for bacterial or viral culture. Repetitive biopsies of specific tissues under treatment are occasionally performed. Examples would be resections of recurring masses, repeated synovial taps for joint disease, and sequential bone marrow aspirations. More rarely, biopsies are obtained for the specific purpose of obtaining living cells for study. justification of Surgical Intervention. As with any intervention, it is implicit that potential benefit to the patient should exceed potential risk. *Postdoctoral Fellow, Department of Pathology, University of Toronto, Toronto, Ontario, Canada t Diplomate, American College of Veterinary Pathologists; Dean, University of Illinois College of Veterinary Medicine, Urbana, Illinois Veterinary Clinics of North America: Small Animal Practice-Val. 20, No. 4, July 1990
939
940
RONALD
F.
CARTER AND VICTOR EDWIN OSWALD VALL!
The decision to obtain a biopsy therefore depends in part upon an estimate of risk. The risks involved vary according to the severity of surgical intervention and anesthetic restraint required. For techniques such as fineneedle aspiration, risks may be very slight. In contrast, an exploratory laparotomy or thoracotomy of an aged and compromised patient may represent a significant risk of death. Biopsies obtained by laparoscopy or endoscopy may be reasonable alternatives to more invasive procedures despite the risk of diagnostic failure due to a smaller and possibly less representative specimen. While an estimate of risk may ultimately determine whether a biopsy should be taken from an animal, it is not the sole consideration. Estimate of risk alone is inadequate to determine the type of biopsy or technique to be used. Certain types of biopsy may result in more definitive and/or timely results. The least risky procedure is frequently not the most informative. Thus, the practitioner has to estimate the likelihood of obtaining the definitive answer versus the risk resulting from the necessary procedure. One should always attempt the most direct assay of the suspected lesion ppssible under the circumstances. A common approach is to investigate the nature of a suspicious lesion by cytology, which is rather noninvasive and provides a rapid diagnosis; if the findings are uncertain or indicate a serious lesion, follow-up excisional biopsy and confirmation by histologic interpretation is justified. Role of the Biopsy in the Diagnostic Plan. Biopsies should not be obtained unless there is a clear expectation of informative result. Although almost any tissue can be biopsied, biopsies provide specific information only about very localized areas of tissue; the surgical action required to obtain a biopsy may even preclude the simultaneous biopsy of a number of tissues while failing to provide information about those other tissues. Biopsies are unique in their ability to provide information about the living patient, because tissue is actually obtained for direct study. Biopsies are best used as the final test which confirms a diagnosis already suggested by history, clinical picture, panel tests, and/or ancillary diagnostic techniques. Biopsies performed as part of a survey based upon poorly formed clinical suspicions will often result in unnecessary injury with little diagnostic benefit. Relationship to Clinical Pathology and Autopsy. It is useful to review the relationship among clinical pathology (e. g., hematology and serum biochemistry), biopsy, and autopsy. Hematology constitutes a specific test for the hemopoietic system and also provides information about systemic disease and response. Serum biochemical panels are carefully constructed to provide surveys of systemic response as well as indications of specific organ function or injury. Biopsies provide premortem morphologic evidence of tissue function and disease. Autopsy provides a postmortem opportunity to selectively or exhaustively study all tissues for morphologic evidence of tissue function and disease. Autopsy will not provide information useful for the management of the patient in question, but it is essential for monitoring the accuracy of the diagnostic approach and the (lack of) effectiveness of clinical management. Summary. The decision to obtain a biopsy is reached after the history,
TAKING A BIOPSY
941
physical examination, and noninvasive tests suggest a specific list of diseases related to an identified tissue. Undertaking biopsy requires that the risk of surgical intervention is justified by the benefit of resulting information. The choice of biopsy technique and specimen preparation should maximize the diagnostic value of the biopsy while minimizing the risk. Cytopathology can be used as a safe screening technique to provide rapid diagnoses or indicate the need for excisional biopsy and histopathology. For many diseases, biopsy represents the decisive diagnostic approach for the living patient. Autopsy, on the other hand, provides the ultimate assessment of the adequacy of diagnosis and management.
COMPONENTS OF THE BIOPSY PROCEDURE The complete process of taking a biopsy involves many steps (Fig. 1). Throughout, there are two paramount considerations. There must be constant maintenance of correct patient and specimen identification, and the correct diagnostic interpretation must be returned to the practitioner. Failure in these aims compromises the entire procedure. Biopsies can be classified by the nature of tissue sampled (Table 1), which in turn reflects the diagnostic dilemma facing the practitioner. One may sample fluids by aspiration (e.g., ocular, pleurallpericardial, synovial, peritoneal, cerebrospinal, urine, or cystic fluids, and lung or prostatic washes). One may sample tissues by fine-needle aspiration, even wellorganized connective tissue. However, surgical excision of tissue is still the most common method of biopsy, applicable to almost all tissues except fluids. The method of biopsy determines the type of laboratory processing for routine evaluation (see Table 1). The most common division is into cytologic specimens (which are examined without prior sectioning) and histologic specimens (which involve fixation, paraffin embedment, and sectioning before evaluation). Fluids and fine-needle aspirations are processed for cytology; solid tissues obtained by excision can provide cytologic specimens by making imprints or scrapings on slides (often useful for quickly assessing the adequacy of the biopsy and obtaining a provisional diagnosis), but are ultimately examined as histologic sections to obtain the most information. Not covered in detail in this review are the other types of diagnostic tests that can be performed on biopsies. Outlines of appropriate procedures for immunohistochemistry, electron microscopy, and other tests are summarized in Table 2. Techniques
Important points for taking biopsies and preparing specimens for submission are summarized in Figures 2 and 3. Diagnostic Assessment. The initial step in the biopsy procedure consists of an evaluation of history, signalment, and physical findings, which leads to a problem list and associated diagnostic plans. After ancillary testing where necessary, the clinician forms a list of possible causes for the problems identified. Biopsy may be indicated as a logical development of the
:f t-:;1 PRACTITIONER
ASSESSMENT-.. PROBLEM LIST-.. RULE-INS
...
BIOPSY
/
EVALUATION AND MANAGEMENT
...
LABELLING, FORMS, HISTORY
t
•
TRANSPORT
TRANSPORT
t REPORTING
•
PREPARATION & FIXING
!
LABORATORY
...
INTERPRETATION ...
PROCESSING ...
staining sectioning embedment Figure l. Flow chart of biopsy procedure.
IDENTIFICATION
943
TAKING A BIOPSY
Table I. Types of Biopsies for Morphologic Study TISSUE TYPE
Fluid'-.......
~
/ / Solid~ ~
SPECIMEN TYPE
PREPARATIOI\
Air-dried Film on Slide Direct/ Aspirate ~ Concentrated - - Wet-fixed Film on Slide (Optional) ....--"Air-dried Imprint on Slide~ Excisional or~ Wet-fixed Imprint on Slide~ Tru-Cut ~ (Optional) Biopsy Formalin Fixed, Paraffin~ Section
~
ROUTINE STAIN
Wright's Papanicolaou Wright's Papanicolaou Hematoxylin & Eosin
diagnostic plans, and the appropriate biopsy procedure should be selected through evaluation of the type oflesion, the risk associated with the biopsy, and the likelihood of obtaining a definitive answer. Biopsy. The biopsy should be representative of the lesion. Fine-needle aspiration should be done in a methodical spraying motion, with the lesion immobilized by one hand, to ensure representative sampling of the lesion. Aspiration should be stopped as soon as there is either sufficient material obtained or dilution of the intended sample by frank blood. When resection is performed, the biopsy may consist of part of the lesion or the entire lesion as part of the approach to therapy. Resection of a mass should be represented by a specimen which consists of the normal tissue surrounding the mass, the interface of the mass and the normal tissue, and the center of the mass. If an abscessed, necrotic, or cystic lesion is identified, the biopsy should consist of viable peripheral tissue rather than the disintegrating center. Surgical removal should disrupt the existing morphology of the tissue as little as possible. When fine-needle aspiration is complete, suction pressure should be released before the needle is withdrawn from the lesion, so that the aspirated material remains in the hub of the needle and is not explosively released into the syringe by inrushing air. The needle is then detached from the hub, air is drawn into the syringe, and the needle is replaced preparatory to expelling the sample onto microscope slides. For histology, excessive surgical manipulation, crushing, and congestion are common artifacts of surgical procedure which cause major difficulties in interpretation (Fig. 4). If imprints are made, care should be taken not to crush the tissue while making the imprints (a common error with Tru-Cut biopsies). Fixation. The material obtained by biopsy should be immediately fixed, that is, processed in a manner which ensures conservation of morphologic detail over time and throughout subsequent processing (see Tables 1 and 2). Unfixed or poorly fixed cells lose morphologic detail. They are also difficult to prepare for examination because they may undergo lysis, fail to adhere to slides, or fail to take up stains properly. Delayed or improper fixation of a specimen can render interpretation of subtle, or even otherwise obvious, lesions difficult. Cells with high metabolic rates, such as gut epithelial cells and hemolymphatic cells, will start changing in morphologic
944
RONALD
F.
CARTER AND VICTOR EDWIN OSWALD VALLI
Table 2. Outline of Specimens Required for Tests on Biopsies* DIAGNOSTIC TEST
POSSIBLE SPECIMENS
METHOD OF PREPAR"'TION
Cytology
Aspirated fluids or tissues, See Table l. Air-dried smears exudates, imprints, washes are routine. Assess (eg, bronchoalveolar lavage), requirement for scrapings if poor exfoliation concentration by initial quick stain
Histology
Any solid tissue, or any cells See Table l. Formalin fixative is routine; other fixatives which can be centrifuged to may be preferred for a pellet specific tissues, eg, B5 for bone marrow
Electron microscopy
Any solid tissue, or any cells Glutaraldehyde-Os04 fixation is routine; paraformaldehyde which can be centrifuged to a pellet may be best for immunoelectron microscopy. Formalin adequate for some purposes
Immunohistochemistry, eg, skin, autoimmune diseases
Any suspension of cells or solid tissue
Bacteriology, mycology, virology
Refrigerated transport and Any solid or fluid specimen holding until samples which can be collected cultured. Freezing aseptically. Solid tissue acceptable but room specimens should be large enough that cultures can be temperature to be avoided initiated from interior sites specifically
Cytochemistry & histochemistry, eg, storage diseases, leukemias
Cell suspensions or solid tissues
Submission of absolutely fresh specimens best. Transport in liquid nitrogen or specialized transport media (eg, Michel's media for skin samples) may be appropriate. Tests done on smears, frozen sections, or fixed tissue
Absolutely fresh tissues best. Other transport methods may be possible
*The first procedure should always be to contact the laboratory for exact requirements for a specific test.
detail within minutes of cardiovascular compromise (Fig. 5). Fixation therefore should be initiated rapidly and should be complete before further tissue processing. Cytologic specimens are usually prepared by quickly making air-dried smears (see Fig. 2) or imprints (see Fig. 3). Imprints are best made by gently rolling a freshly cut face of tissue across the slide, after the tissue face has been gently blotted dry with paper gauze. Fine-needle aspirates can be prepared as smears (or films) and crushes (Fig. 6, A and B). Crush preparations, made by placing the aspirated material between a slide and a coverslip and drawing the two apart, often cause significant loss of
Text continued on page 955
immobilize tissue in one hand
.. ""'
cq:
II
~raying motion while aspirating
I ~
release vacuum before leaving tissue remove needle and reload syringe with air
,
propel tissue out of needle hub using air in syringe Essential elements of Technique: 1.) 22 gauge needle, 10 ml syringe 2.) Accurate sampling of lesion; no dilution by bleeding 3.) Sample retained in hub of needle, not lost in syringe 4.) Rapid preparation of slides directly from aspirate 5.) Smear technique used to spread slides spread tissue out behind leadir.g edge of slide 6.) Rapid fixation by air drying or wet fixation (spray is best). 7.) Transport at room temperature in protected package air dry and/or wet fix rapidly
..
Figure 2. Preparation of smears from fine-needle aspirates.
f
1:11
~
~
O'l
obtain biopsy tissue without comprising morphology slice into 2 to 3 mm thick sections in appropriate orientation
~
'-._____../
............
/ f6 = ,
~
[)
..,._ ( [ ) [ )
~
blot
'
,;;:::::"make imprints from sections by gently rolting slide over tissue face; air dry or wet fix rapidly
Essential elements of imprint technique: 1.) Gently cut fresh tissue face and blot off excess fluid 2.) Hold in one hand and gently roll slide over tissue face without creating a suction effect which rips cells 3.) Rapidly air-dry or wet fix (spray is best) 4.) Prepare only a few imprints from each tissue face
F
for~~
ORMALIN . lices into place ussue s arion and formalin laboratOry then tran'P? .for processmg
Essential elements of formalin fixation: 1.) Gently handled fresh tissue 2.) Abundant volume of fresh, buffered 3.7% formalin at room temperature in secure specimen bottle 3.) Distance formalin required to penetrate to desired lesion < 3 mm 4.) Fix at least 24 hrs before processing regardless of sample size 5.) Avoid freezing before embedding
Figure 3. Preparation of imprints and fixed histologic specimens.
~ ~
-l
Figure 4. Histologic section of laryngeal biopsy from a cat . Edge of tissue with crush artifact in fresh specimen . The irregular dark streaks are ruptured nuclei. 1-1 & E; bar = lO fJ.m.
~
..... oc
Figure 5. A, Histologic section of mediastinal mass from a cat with immunoblastic lymphoma. E arly autolysis is represented by cytoplasmic separation and contraction, and by irregular nuclear membranes and chromatin clearing. H & E ; bar = lO J.Lm. Illustration continued on opposite page
~
..... ~
Figure 5 (Continued). B, Histologic section of a mediastinal mass from a cat. More extensive autolysis is represented by ruptured nuclear membranes and fragmentation of chromatin. Cytoplasmic separation is again present. Stromal nuclei, in comparison, maintain a more normal appearance , except for vesiculation and peripheralized chromatin (arrow). The section is also too thick, obscuring cellular details. H & E ; bar = 10 fLm .
Illustration continued on following page
:ll
0
Figure 5 (Continued). C, Histologic section of liver from a cat. Autopsy specimen. Subcapsular tissue has undergone chromatin lysis and cytoplasmic degradation due to effects of bile leakage prior to tissue fixation. Edge effects may also be caused by desiccation. H & E ; bar = 100 J.Lm, or 0.1 mm.
"'......
