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The Technical Importance of Procedures in the Preparation of Histological Specimens from the Bovine Endometrium
THE TECHNICAL IMPORTANCE OF PROCEDURES IN THE PREPARATION OF HISTOLOGICAL SPECIMENS FROM THE BOVINE ENDOMETRIUM By P.
J.
HARTIGAN, AND
J.
J.
A.
F. T.
MURPHY,
W. R.
NUNN
GRIFFIN
School of Veterinary Medicine, University of Dublin
SUMMARY
The paper describes artifacts which developed as a result of some procedures which were employed in the sampling, storage and fixation of tissue specimens from the endometrium of the cow. A procedure is described which consistently produced histological sections free from artifacts. INTRODUCTION
Specimens for histological study of the endometrium in cattle may be obtained by biopsy methods or by section of the uterus from slaughtered animals. An advantage of the in vivo methods is that, even when contemporary microbiological samples are required, the biopsy specimens can be placed in a fixative solution almost immediately after cessation of blood flow through the excised tissue. When microbiological samples are required from the uteri of slaughtered cows there is often an interval of some hours between death and excision of the tissue specimen. This delay provides the opportunity for the onset of autolytic changes. The possibility that these changes might be mistaken for pathological lesions makes it imperative that the procedures employed in the collection and preparation of histological specimens from the uterus should not permit the development of artifacts in the tissue. Therefore, an investigation was made to determine the effects on endometrial specimens of the various procedures which can be employed in the sampling, storage and fixation of uterine tissue from slaughtered cows. MATERIALS AND METHODS
The observations are based on the study of endometrial samples obtained at slaughter from I4I cows. All the tissues were fixed in IO per cent formol saline; sections were cut at 6 microns and stained by haematoxylin and eosin. The influence of the following factors was assessed: (i) method of excision of tissue from the uterus; (ii) storage of the unfixed uterus at room temperature; (iii) storage of the unfixed uterus at -r6 °C.; (iv) fixation procedures. Excision of tissue from the uterus. Comparisons were made between specimens obtained by transverse incision through the entire uterine horn with scissors or
ENDOMETRIAL BIOPSY
165
a sharp blade and specimens obtained with the same instruments after exposure of the endometrium by a longitudinal incision through the dorsal wall of the uterine horn. Twenty uteri were employed in this experiment. Storage at room temperature. Uteri from 20 heifers and 6 cows were sampled repeatedly at various intervals up to 48 hours post mortem and the histological picture was compared with control specimens taken immediately after slaughter. Storage at minus 16°C. Tissue specimens were taken from 33 uteri which had been refrigerated at - 16°C for at least 24 hours prior to fixation. Control specimens were taken immediately after slaughter. Fixation procedures. The uteri from 24 cows were employed to compare three procedures. Immediately after removal of the genital tract from the carcase a transverse section approximately 1.0 cm long was removed from the base of each horn and placed at once in the fixative solution. Then the remainder of each horn was fixed by one of the following procedures: (a) incisions were made through the entire depth of the dorsal wall of the uterine horn at regular intervals throughout its length prior to immersion for 24 hours in a bath of fixative (Weber, Morgan & McNutt, 1948) ; or (b) fixative solution was infused into the uterine lumen prior to immersion for 24 hours in a bath of formol saline. A different method was employed for each horn and so it was possible to compare all three methods on each genital tract. To study the influence of parity and the stage of the oestrous cycle on the results obtained with the immersion method of Weber et al. (1948), the uteri from an additional sample of 12 nulliparous heifers and 26 pluriparous cows were examined. In this experiment control specimens were taken from the cervical end of each horn, the intercornual ligament was transected and one horn was fixed by the immersion method while the other was stored unfixed at room temperature. RESULTS
Tissue Excision The luminal epithelium was frequently lost from tissue specimens obtained after the dorsal wall of the uterine horn had been incised in a longitudinal direction to expose the endometrial surface. This did not occur when a crosssection of the entire uterine horn was removed. Excellent histological sections were obtained when the tissue specimen was obtained with a sharp blade (Fig. I & 2). Removal by scissors caused invagination of gland tubules, leading to a collection of hyperchromatic nuclei and cellular debris in the gland lumina (Fig. 3 & 4 )· Storage at room temperature Uteri stored at room temperature for up to four hours after slaughter yielded histological specimens free from artifacts. Minor autolytic changes became apparent in both the luminal and glandular epithelial cells after five hours and by 24 hours post mortem these degenerative changes had developed into serious artifacts (Fig. 5).
166
BRITISH VETERINARY JOURNAL,
130, 2
Storage at minus 16°C When the uteri were stored at - 16°C for 24 hours or longer prior to fixation, severe artifacts developed. Some breakdown of the normal architecture of the endometrial stroma occurred and the tissues appeared to be oedematous but the outstanding changes were seen in the endometrial glands (Fig. 6). The periglandular connective tissue sheaths appeared to contract around a mass of pyknotic nuclei within each gland tubule, so that each gland tubule was indistinguishable from the "gland site mass lesion" described by Dawson (1963) as the significant lesion in endometritis in the cow. Fixation procedures The immersion method described by Weber et al. (1948) produced proper fixation only in the immediate vicinity of the incisions. The farther away from an incision and the longer the period of immersion, the more severe were the degenerative changes. In its most severe form the artifact was identical to that which developed in the unfixed tissue stored at room temperature for the same period. These autolytic changes could not be distinguished from those described by Weber et al. (1948) in heifers during the follicular phase of the oestrous cycle. We found that the incidence and severity of the artifacts were independent of the age or parity of the animal or of the phase of the oestrous cycle. Similar artifacts developed in cross-sections of the intact uterine horn when the original tissue sample was more than I cm in length. Again, there was inadequate penetration of fixation fluid and sections taken from the centre of the specimens showed autolytic changes. Infusion of fixation fluid into the uterine lumen prior to immersion of the entire uterus in a bath of fixative did not give consistently good results. DISCUSSION
The experiments demonstrated the importance of employing proper procedures in the sampling, storage and fixation of endometrial tissue specimens from slaughtered cows. Faulty procedures induced artifacts which could be mistaken for physiological or pathological changes. Excision of the tissue specimens with scissors caused invagination of the gland tubules. Histologically, the invagination was seen as a mass of hyperchromatic nuclei and cytoplasmic debris lying within a normal gland tubule. In specimens obtained at the appropriate stage of the oestrous cycle this artifact might have been thought to be due to the cyclic regressive changes which occur in the endometrial glands. Weber et al. (1948) described in detail the endometrial changes associated with metrorrhagia in the virgin heifer. They described destruction of the epithelial cells at the luminal surface and in the neck and middle regions of the endometrial glands. In controlled experiments we have shown that these degenerative changes develop in unfixed tissue or when there is inadequate penetration of fixation fluid into the tissues. We have not seen these changes in
ENDOMETRIAL BIOPSY
any of a small number of heifers sampled during the follicular phase of the oestrous cycle or in any of more than 1000 uteri from pluriparous cows from which histological sections have been prepared according to the procedure outlined below. When they stored uteri at 4°C for 18 hours prior to fixation, Peterson & Newsam (1964) observed changes similar to those described by Weber et at. ( 1948). It would appear that refrigeration at 4°C does not prevent the development of autolytic changes but is not responsible for additional tissue damage. However, refrigeration at - 16°C damages the endometrium, producing an artifact which resembles the 'gland site mass lesion' to which Dawson (1963) has attributed particular diagnostic significance . This artifact was induced in specimens obtained from virgin heifers, from non-pregnant primipara and pluripara, and from pregnant cows. The experiments showed that the following routine consistently yielded histological specimens free from artifacts. A transverse section, not exceeding one centimetre in length, was removed from the uterine horn with a sharp Durham Duplex * blade. It was placed in fixative solution at room temperature as soon as possible, and never later than four hours after death. Next day it was trimmed down to five millimetres in length and fixation was continued. Prior to embedding in paraffin wax, it was further trimmed so that it consisted of endometrium surrounded by a thin circle of myometrial tissue. It had been expected that a major problem with cross-sections of the uterine horn would be the production of artifacts due to 'bounce' as the microtome knife passed through the section from the myometrium to the endometrium. This was a problem, however, only in uteri from aged pluriparous cows in which the large blood vessels penetrating the endometrium from the stratum vasculare were very numerous and markedly hypertrophied. ACKNOWLEDGEMENTS
The authors are grateful to Chemical Services Ltd. for financial support and to Mr. T. Morley for assistance with the photography. REFERENCES
DAWSON, F. L. M. (1963). J. Reprod. Fert. 5· 397· PETERSON, J. E. & NEWSAM, 1. D. B. (1964). Br. vet. J. 120, 229. WEBER, A. F., MORGAN, B. B. & McNuTT, S. H. ( 1948). Am. J. Anat. 83, 309. L'itnportance des procedes de laboratoire pour la preparation de specitnens histologiques Ii partir de l'endoIDetre des bovins (Hartigan et al.) SoIDIDaire. Cet article decrit les artefacts qui sont presents a la suite de certains procedes de laboratoire utilises lors de la prise d'echantillon, du rangement et de la fixation de specimens
* Durham
Duplex Razor Co. Ltd., Petre Street, Sheffield 4, England.
168
BRITISH VETERI NARY JOURNAL,
130, 2
tissulaires provenant de l'endometre d e la vache. Un proced e es t d ecrit qui permet la preparation de coupes histologiques n e presentant pas d'artefacts. Die Wichtigkeit der Methodik bei der Anfertigung histologischer Pdtparate vOnt bovinen Endontetriunt (Hartigan et al.) ZusaDlDlenfassung. Beschreibung einiger Kunstprodukte, welche die Foige von Entnahmetechnik, Aufbewa hrung und Fixierung von Gewebe a us dem E ndometrium von Ktihen waren . Eine M ethod e wird beschrieben, di e stets kunstproduktfreie histologische Schnitte produzierte. La intportancia de los procedintientos entpleados en la preparacion de especintenes histologicos del endontetrio bovino (Hartigan et al.) ResUDlen. La ponencia describe artefactos que aparecieron como resultado de algunos procedimentos empleados en el muestreo, almacenaje y fijado de especimenes de tejido obtenidos del en dome trio de la vaca. Se d escribe un procedimiento que produjo consistentem ente secciones histologicas libres de artefactos.
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Fig. I. Sec tion of norm a l endom e trium ob ta in ed by th e techniqu e d escribed . It shows luminal e pithelium (L ), gland tubu les (G ) and a numb er of ca pillaries (C) in th e stro m a (S) . Includ ed for co mpariso n w ith Fig . 3- 6. (H & E) X 150.
•
Fig. 2. H ig h powe r view of a gla nd tubu le tn erosssection. Note th e la rge vesicul a r nucl ei. (H & E ) X 630.
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Fig. 3. Specimen excised with scissors. Many gland tubules contain invaginated masses of hyperchromati c nucl ei and cytoplasmic debris. (H & E ) X 150.
Fig. 4. High power view of gland tubule showing contrast between large vesicular nuclei of normal cells and d ense pyknotic nuclei of th e tissu e invaginated in th e gland lumen. M = mitosis. (H & E ) X 630.
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Fig. 5. Section showing th e a utolyti c changes in glandu la r epithelium in unfixed or inadequately fix ed tiss ue sp ecimens. M ost cells show cytoplasmic d ege neration. Some nucl ei show little change but ma ny are pykno ti c. (H & E ) X 400.
,
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Fig. 6. Section fr om uterus stored at _,6°C for 24 hours prior to fixati on. The tissues a re oedema to us, the stroma is d isrupted and th e periglandula r connective tissue sheaths ha ve co ntracted around a m ass of pyknotic epithelia l nucl ei. (H & E ) x ' 50.
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J.
HARTIGAN, AND
J.
J.
A.
F. T.
MURPHY,
W. R.
NUNN
GRIFFIN
School of Veterinary Medicine, University of Dublin
SUMMARY
The paper describes artifacts which developed as a result of some procedures which were employed in the sampling, storage and fixation of tissue specimens from the endometrium of the cow. A procedure is described which consistently produced histological sections free from artifacts. INTRODUCTION
Specimens for histological study of the endometrium in cattle may be obtained by biopsy methods or by section of the uterus from slaughtered animals. An advantage of the in vivo methods is that, even when contemporary microbiological samples are required, the biopsy specimens can be placed in a fixative solution almost immediately after cessation of blood flow through the excised tissue. When microbiological samples are required from the uteri of slaughtered cows there is often an interval of some hours between death and excision of the tissue specimen. This delay provides the opportunity for the onset of autolytic changes. The possibility that these changes might be mistaken for pathological lesions makes it imperative that the procedures employed in the collection and preparation of histological specimens from the uterus should not permit the development of artifacts in the tissue. Therefore, an investigation was made to determine the effects on endometrial specimens of the various procedures which can be employed in the sampling, storage and fixation of uterine tissue from slaughtered cows. MATERIALS AND METHODS
The observations are based on the study of endometrial samples obtained at slaughter from I4I cows. All the tissues were fixed in IO per cent formol saline; sections were cut at 6 microns and stained by haematoxylin and eosin. The influence of the following factors was assessed: (i) method of excision of tissue from the uterus; (ii) storage of the unfixed uterus at room temperature; (iii) storage of the unfixed uterus at -r6 °C.; (iv) fixation procedures. Excision of tissue from the uterus. Comparisons were made between specimens obtained by transverse incision through the entire uterine horn with scissors or
ENDOMETRIAL BIOPSY
165
a sharp blade and specimens obtained with the same instruments after exposure of the endometrium by a longitudinal incision through the dorsal wall of the uterine horn. Twenty uteri were employed in this experiment. Storage at room temperature. Uteri from 20 heifers and 6 cows were sampled repeatedly at various intervals up to 48 hours post mortem and the histological picture was compared with control specimens taken immediately after slaughter. Storage at minus 16°C. Tissue specimens were taken from 33 uteri which had been refrigerated at - 16°C for at least 24 hours prior to fixation. Control specimens were taken immediately after slaughter. Fixation procedures. The uteri from 24 cows were employed to compare three procedures. Immediately after removal of the genital tract from the carcase a transverse section approximately 1.0 cm long was removed from the base of each horn and placed at once in the fixative solution. Then the remainder of each horn was fixed by one of the following procedures: (a) incisions were made through the entire depth of the dorsal wall of the uterine horn at regular intervals throughout its length prior to immersion for 24 hours in a bath of fixative (Weber, Morgan & McNutt, 1948) ; or (b) fixative solution was infused into the uterine lumen prior to immersion for 24 hours in a bath of formol saline. A different method was employed for each horn and so it was possible to compare all three methods on each genital tract. To study the influence of parity and the stage of the oestrous cycle on the results obtained with the immersion method of Weber et al. (1948), the uteri from an additional sample of 12 nulliparous heifers and 26 pluriparous cows were examined. In this experiment control specimens were taken from the cervical end of each horn, the intercornual ligament was transected and one horn was fixed by the immersion method while the other was stored unfixed at room temperature. RESULTS
Tissue Excision The luminal epithelium was frequently lost from tissue specimens obtained after the dorsal wall of the uterine horn had been incised in a longitudinal direction to expose the endometrial surface. This did not occur when a crosssection of the entire uterine horn was removed. Excellent histological sections were obtained when the tissue specimen was obtained with a sharp blade (Fig. I & 2). Removal by scissors caused invagination of gland tubules, leading to a collection of hyperchromatic nuclei and cellular debris in the gland lumina (Fig. 3 & 4 )· Storage at room temperature Uteri stored at room temperature for up to four hours after slaughter yielded histological specimens free from artifacts. Minor autolytic changes became apparent in both the luminal and glandular epithelial cells after five hours and by 24 hours post mortem these degenerative changes had developed into serious artifacts (Fig. 5).
166
BRITISH VETERINARY JOURNAL,
130, 2
Storage at minus 16°C When the uteri were stored at - 16°C for 24 hours or longer prior to fixation, severe artifacts developed. Some breakdown of the normal architecture of the endometrial stroma occurred and the tissues appeared to be oedematous but the outstanding changes were seen in the endometrial glands (Fig. 6). The periglandular connective tissue sheaths appeared to contract around a mass of pyknotic nuclei within each gland tubule, so that each gland tubule was indistinguishable from the "gland site mass lesion" described by Dawson (1963) as the significant lesion in endometritis in the cow. Fixation procedures The immersion method described by Weber et al. (1948) produced proper fixation only in the immediate vicinity of the incisions. The farther away from an incision and the longer the period of immersion, the more severe were the degenerative changes. In its most severe form the artifact was identical to that which developed in the unfixed tissue stored at room temperature for the same period. These autolytic changes could not be distinguished from those described by Weber et al. (1948) in heifers during the follicular phase of the oestrous cycle. We found that the incidence and severity of the artifacts were independent of the age or parity of the animal or of the phase of the oestrous cycle. Similar artifacts developed in cross-sections of the intact uterine horn when the original tissue sample was more than I cm in length. Again, there was inadequate penetration of fixation fluid and sections taken from the centre of the specimens showed autolytic changes. Infusion of fixation fluid into the uterine lumen prior to immersion of the entire uterus in a bath of fixative did not give consistently good results. DISCUSSION
The experiments demonstrated the importance of employing proper procedures in the sampling, storage and fixation of endometrial tissue specimens from slaughtered cows. Faulty procedures induced artifacts which could be mistaken for physiological or pathological changes. Excision of the tissue specimens with scissors caused invagination of the gland tubules. Histologically, the invagination was seen as a mass of hyperchromatic nuclei and cytoplasmic debris lying within a normal gland tubule. In specimens obtained at the appropriate stage of the oestrous cycle this artifact might have been thought to be due to the cyclic regressive changes which occur in the endometrial glands. Weber et al. (1948) described in detail the endometrial changes associated with metrorrhagia in the virgin heifer. They described destruction of the epithelial cells at the luminal surface and in the neck and middle regions of the endometrial glands. In controlled experiments we have shown that these degenerative changes develop in unfixed tissue or when there is inadequate penetration of fixation fluid into the tissues. We have not seen these changes in
ENDOMETRIAL BIOPSY
any of a small number of heifers sampled during the follicular phase of the oestrous cycle or in any of more than 1000 uteri from pluriparous cows from which histological sections have been prepared according to the procedure outlined below. When they stored uteri at 4°C for 18 hours prior to fixation, Peterson & Newsam (1964) observed changes similar to those described by Weber et at. ( 1948). It would appear that refrigeration at 4°C does not prevent the development of autolytic changes but is not responsible for additional tissue damage. However, refrigeration at - 16°C damages the endometrium, producing an artifact which resembles the 'gland site mass lesion' to which Dawson (1963) has attributed particular diagnostic significance . This artifact was induced in specimens obtained from virgin heifers, from non-pregnant primipara and pluripara, and from pregnant cows. The experiments showed that the following routine consistently yielded histological specimens free from artifacts. A transverse section, not exceeding one centimetre in length, was removed from the uterine horn with a sharp Durham Duplex * blade. It was placed in fixative solution at room temperature as soon as possible, and never later than four hours after death. Next day it was trimmed down to five millimetres in length and fixation was continued. Prior to embedding in paraffin wax, it was further trimmed so that it consisted of endometrium surrounded by a thin circle of myometrial tissue. It had been expected that a major problem with cross-sections of the uterine horn would be the production of artifacts due to 'bounce' as the microtome knife passed through the section from the myometrium to the endometrium. This was a problem, however, only in uteri from aged pluriparous cows in which the large blood vessels penetrating the endometrium from the stratum vasculare were very numerous and markedly hypertrophied. ACKNOWLEDGEMENTS
The authors are grateful to Chemical Services Ltd. for financial support and to Mr. T. Morley for assistance with the photography. REFERENCES
DAWSON, F. L. M. (1963). J. Reprod. Fert. 5· 397· PETERSON, J. E. & NEWSAM, 1. D. B. (1964). Br. vet. J. 120, 229. WEBER, A. F., MORGAN, B. B. & McNuTT, S. H. ( 1948). Am. J. Anat. 83, 309. L'itnportance des procedes de laboratoire pour la preparation de specitnens histologiques Ii partir de l'endoIDetre des bovins (Hartigan et al.) SoIDIDaire. Cet article decrit les artefacts qui sont presents a la suite de certains procedes de laboratoire utilises lors de la prise d'echantillon, du rangement et de la fixation de specimens
* Durham
Duplex Razor Co. Ltd., Petre Street, Sheffield 4, England.
168
BRITISH VETERI NARY JOURNAL,
130, 2
tissulaires provenant de l'endometre d e la vache. Un proced e es t d ecrit qui permet la preparation de coupes histologiques n e presentant pas d'artefacts. Die Wichtigkeit der Methodik bei der Anfertigung histologischer Pdtparate vOnt bovinen Endontetriunt (Hartigan et al.) ZusaDlDlenfassung. Beschreibung einiger Kunstprodukte, welche die Foige von Entnahmetechnik, Aufbewa hrung und Fixierung von Gewebe a us dem E ndometrium von Ktihen waren . Eine M ethod e wird beschrieben, di e stets kunstproduktfreie histologische Schnitte produzierte. La intportancia de los procedintientos entpleados en la preparacion de especintenes histologicos del endontetrio bovino (Hartigan et al.) ResUDlen. La ponencia describe artefactos que aparecieron como resultado de algunos procedimentos empleados en el muestreo, almacenaje y fijado de especimenes de tejido obtenidos del en dome trio de la vaca. Se d escribe un procedimiento que produjo consistentem ente secciones histologicas libres de artefactos.
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--.J
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:;,;,
...~
~
OJ
'"
Jq
~
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~
Fig. I. Sec tion of norm a l endom e trium ob ta in ed by th e techniqu e d escribed . It shows luminal e pithelium (L ), gland tubu les (G ) and a numb er of ca pillaries (C) in th e stro m a (S) . Includ ed for co mpariso n w ith Fig . 3- 6. (H & E) X 150.
•
Fig. 2. H ig h powe r view of a gla nd tubu le tn erosssection. Note th e la rge vesicul a r nucl ei. (H & E ) X 630.
•
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IV
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Fig. 3. Specimen excised with scissors. Many gland tubules contain invaginated masses of hyperchromati c nucl ei and cytoplasmic debris. (H & E ) X 150.
Fig. 4. High power view of gland tubule showing contrast between large vesicular nuclei of normal cells and d ense pyknotic nuclei of th e tissu e invaginated in th e gland lumen. M = mitosis. (H & E ) X 630.
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Fig. 5. Section showing th e a utolyti c changes in glandu la r epithelium in unfixed or inadequately fix ed tiss ue sp ecimens. M ost cells show cytoplasmic d ege neration. Some nucl ei show little change but ma ny are pykno ti c. (H & E ) X 400.
,
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Fig. 6. Section fr om uterus stored at _,6°C for 24 hours prior to fixati on. The tissues a re oedema to us, the stroma is d isrupted and th e periglandula r connective tissue sheaths ha ve co ntracted around a m ass of pyknotic epithelia l nucl ei. (H & E ) x ' 50.
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