Application Of Exfoliative Cytology In Obstetrics & Gynaecology
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APPLICATION OF EXFOLIATIVE CYTOLOGY IN OBSTETRICS & GYNAECOLOGY
DR. E.K. OKAGUA UPTH
INTRODUCTION
Exfoliative cytology is defined by the Morsby’s medical encyclopedia as the microscopic examination of dead cells for diagnosis. Epithelial cells of the body undergo a constant process of maturation/death/regeneration, and cells that die slough off or exfoliate. Four decades ago, Papanicolaou at the Cornell University Medical College laboratories initiated the examination of exfoliative material from the cervicovaginal area that would be valuable as a screening test for carcinoma of the cervix At the present time, cervical cytology is considered to be the only way to reduce cervical cancer incidence.
Griechische Wissenschaftler
George N. Papanicolaou was a Greek physician and anatomist in the United States (1883 - 1962).
SAMPLING TECHNIQUES
Natural exfoliation – urine, sputum Cervical PAP smear 1. 2. 3.
Ayres spatula Aylesbury device Cytobrush + extended tip device. One-day training workshop required.
Vaginal smear Endometrial aspiration cytology Smear of vulva lesions Fine needle aspiration cytology +/- imaging Peritoneal washings Buccal smear
USES OF THE PAP SMEAR
Detection of occult pathologic abnormalities of the uterine cervix in asymptomatic women Detection of recurrence of known pathologic abnormalities of the uterine cervix Evaluation of a suspected hormonal abnormality Monitoring of hormonal therapy
PATHOLOGIC ABNORMALITIES OF THE CERVIX ON PAP SMEAR
Pre-malignant and pre-invasive malignant conditions of the cervix
Infections such as:
Actinomyces israeli seen with IUCD users Lactobacillus species which is normally present in the secretory phase,
Gardenerella vaginalis a sexually transmitted coccobacillus, Chlamydia trachomatis a sexually transmitted bacterial pathogen that can be transmitted from the mothers cervix to the conjunctiva of her child, Candida albicans, Torulopsis glabrata, Trichomonas vaginalis classically presents as a profuse pungent malodorous vaginal discharge and puritis and is associated with the condition called strawberry cervix. Presentation my be subclinical or asymptomatic,
Enteroaemoba gingivalis, Enterobius vermicularis, Herpes simplex virus – The virus is not seen but there cytopathic effects are seen e.g. cell fusion, intranuclear inclusions, nuclear
INTERPRETATION OF RESULTS Result Action Taken Negative (Normal) Inflammation (infection/rxn) Mild Dysplasia
None. Repeat in one year. Treat then repeat Repeat Pap smear in 6 smear months.
Moderate Dysplasia
Colposcopy
Severe Dysplasia
Colposcopy with or without cone biopsy.
Class V - Cancer cells
Several types of surgical techniques are used depending upon
EFFECTIVENESS OF CERVICAL SCREENING % NUMBER
SCREENING SCHEDULE
REDUCTIO N IN RISK
OF SMEARS TAKEN
Every 10 years age 25-64
64%
Every 5 years age 20-64
83.8% 9
Every 5 years age 25-64
81.8% 8
Every 5 years age 35-64
69.6% 6
Every 3 years age 20-64
91.2% 15
Every 3 years age 25-64
89.8% 13
Every 3 years age 35-64
77.6% 10
Every year age 20-64
93.3% 45
5
PAP SMEAR PROCEDURE
Obtain informed consent Request form should include biodata, obs/ gynae history and details of prior abnormal smears Time test for second half of cycle Instruct patient to avoid intercourse/ douching 24 hours prior to test Sample is collected by the chosen spatula after exposing the cervix and including the squamou-columnar junction of the cervix
PAP SMEAR PROCEDURE (cont)
The material is evenly spread on a glass slide Smears are immediately fixed in alcohol solution Smears are thereafter placed in distilled water Stain for 2 - 3 minutes in Harris’ or Mayer’s Hematoxylin. This stains the nucleus. The cytoplasm is thereafter stained with OG6 or EA50. Smears which are considered abnormal by the bioscientist are re-screened and reported by a consultant cytopathologist All negative smears undergo an internal quality assurance check, called rapid
MODIFICATIONS OF PROCESSING METHODS FOR PAP SMEAR
Liquid based cytology - or thin layer
cytology, is a technique of preparing a monolayer of cells on a glass slide. The technique involves suspending the sample in a solution that is both mucolytic and haemolytic. The sample is concentrated either by centrifugation or by filtration, in the former method a monolayer of cells sediments onto the slide, in the filtration method a monolayer of cells is pressed onto the slide from the filter.
Automated devices
ADVANTAGES OF LIQUID BASED CYTOLOGY
Clean background
Excellent fixation
Well defined nuclear detail
Instant preservation with no air drying artefact
Removal of blood and mucus
Increased productivity
Standardisation of collection and laboratory techniques
Reduction in unsatisfactory as well as low-grade abnormality rates
Ability to use sample for other techniques such as HPV typing, immunocytochemistry or DNA probes.
Reduction in costs of women unnecessarily referred for colposcopic assessment and treatment.
DISADVANTAGES OF LIQUID BASED CYTOLOGY
Training needed for smear takers
Lengthy laboratory preparation time
Training required to operate equipment
Training required to interpret the smears
Morphology may be different from conventional preparations
Cost and storage of reagents
Loss of existing screener skills in interpreting conventional smears
Loss of ability for future staff to develop skills in
AUTOMATED DEVICES FOR INTERPRETING CERVICAL SMEARS GUIDELINES OF THE INTERNATIONAL ACADEMY OF CYTOLOGY task force, 1997
100% specificity and sensitivity The professional in charge of a clinical laboratory should continue to bear medical responsibility for diagnostic decisions made by machines. Automation should not lead to a reduction in standards nor expose the patient to any increased risk. Supervisory personnel should continue a visual quality control on a percentage of slides diagnosed as normal, and recognition should be made that human expert opinion represents the gold standard. The setting of performance criteria should remain the responsibility of the regulatory authorities, and not the manufacturers, and the professional cytology community should participate in the development of procedures that
VAGINAL SMEARS & HORMONAL ABNORMALITY Examination of vaginal smears for hormonal assessment can be done with the Papanicolaou stains; rapid Schorr method; other rapid suparvital stains; Use of phase microscopy in addition reveals bacterial flora. Cells demonstrated include:
Superficial squamous cells are shed from a fully
mature squamous epithelium that has developed its full thickness under the influence of oestrogen and will be most numerous at the middle of the menstrual cycle. The cells stain pink with the Papanicolaou stain, are angular outline, 40-60 micrometres in diameter, and contain a shrunken hyperchromatic pyknotic nucleus. Avitaminosis A & C can mimic changes similar to oestrogenic effect.
Intermediate cells are shed from the surface of a semi-
mature epithelia that shows a diminished response to oestrogen or the effects of progesterone and are commonly seen at the latter stages of the menstrual cycle, they are 30-60 micrometres in diameter, contain glycogen and stain blue/green with the Papanicolaou stain and contain a clearly defined round or oval vesicular nucleus 8 micrometres in diameter. Doderleins bacilli destroys these cells preventing maturation to superficial cells.
Parabasal and basal cells are seen in the absence of either oestrogen or progesterone, and are found in pre pubertal smears, post-menopausal smears or post partum smears. Also seen in anorexia nervosa. The cells are small, 15 -20 micrometres in diameter,
ENDOCRINOPATHIES ON VAGINAL SMEARS PRIMARY AMENORRHEA
Gonadal Dysgenesis: In Turner’s syndrome, the smear is atrophic (cf
atypical dysgenesis), and in 80% of cases, sex chromatin (SC) body is -ve (cf mosacism). Pituitary Infantilism: The smear is atrophic/intermediately proliferative. SC +ve. Ovarian Eunuchoidism: The smear is atrophic. SC +ve. Testicular Femininization Synd.: The smear is proliferative, but SC is -ve. Simple Congenital Absence of Uterus: The smear shows a normal cyclic pattern. SC is +ve. Congenital Adrenal Hyperplasia: SC is +ve. The smear is atrophic or intermediate proliferative.
PRECOCIOUS PUBERTY SYNDROME
Constitutional or Idiopathic: The smear is proliferative and may show cycling.
Lesions of CNS: The degree of proliferation varies. Granulosa & Theca Cell Ovarian Tumors: The smear is highly proliferative.
2o AMENORREA of HYPOTHALAMIC ORIGIN
Psychogenic: Smears vary. The degree of estrogen effect is a guide to the
ENDOCRINOPATHIES ON VAGINAL
SMEARS
SMEARS Syndromes of Oligomenorrhea and Hirsutism
Stein-Leventhal syndrome: The proliferation of the smear varies from intermediate to good. Large parabasal and intermediate cells of the navicular type may be present. Adrenogenital syndrome: The smear may vary from atrophic to small intermediate cell proliferation. Cushing’s syndrome: Generally, cell proliferation is intermediate. Masculinizing tumors of the ovary: Generally, the smear is atrophic. Genetic: The smear is proliferative and may be cyclic.
Syndromes of Amenorrhea and Galactorrhea
Chiari-Frommel syndrome. Typically, the smear is markedly atrophic. Pituitary tumors and CNS lesions (Forbes-Albright syndrome): The smear varies. Del Castillo’s syndrome (nonpuerperal, dysfunctional): Usually, small intermediate cells predominate. Pseudocyesis: Proliferation often with progestational characteristics.
Menopausal Syndrome
Great individual variation. Early in the climacteric, the smear may show high proliferation, but no cycling. In some cases, atrophic changes may occur abruptly, whereas in others, varying degrees of proliferation may be evident for years, with a gradual shift in the maturation index to the left.
FINE NEEDLE ASPIRATION CYTOLOGY
This is frequently used to provide a rapid diagnosis with or without the use of imaging from ascitic fluid, ovarian tumours, lymph nodes, vulva cysts etc.
Advantages of FNA over open surgical biopsy Diagnosis with a simple, cheaper, outpatient procedure
Avoids biopsy in some cases and may allow treatment of cancers at a planned surgery
Surgical team and theatre time not required
Avoids frozen section
Reduces patient uncertainty and anxiety
Low complication rate. Disadvantages of FNA over open surgical biopsy Errors in diagnosis may lead to over treatment or delay in making the correct diagnosis
Occasional complications such as bleeding or pneumothorax
Requires skilled personnel to take the sample
ASPIRATION NEEDLE & SYRINGE
OTHER APPLICATIONS OF CYTOLOGY IN OBSTETRICS/GYNECOLOGY
Cytology of serous fluid – Most exudates are metastatic and without good clinical information it is not possible to ascertain the primary site by cytology. Cytology of peritoneal washings important for staging ovarian & endometrial malignancies Cytology of nipple discharge for diagnosis of breast malignancy Exfoliative vaginal cytology with cervical mucus studies is a dependable non-invasive method for assessment of ovulation. Endometrial Cytology is however required for conclusion. Vulva cytology for vulva dystrophies,
DR. E.K. OKAGUA UPTH
INTRODUCTION
Exfoliative cytology is defined by the Morsby’s medical encyclopedia as the microscopic examination of dead cells for diagnosis. Epithelial cells of the body undergo a constant process of maturation/death/regeneration, and cells that die slough off or exfoliate. Four decades ago, Papanicolaou at the Cornell University Medical College laboratories initiated the examination of exfoliative material from the cervicovaginal area that would be valuable as a screening test for carcinoma of the cervix At the present time, cervical cytology is considered to be the only way to reduce cervical cancer incidence.
Griechische Wissenschaftler
George N. Papanicolaou was a Greek physician and anatomist in the United States (1883 - 1962).
SAMPLING TECHNIQUES
Natural exfoliation – urine, sputum Cervical PAP smear 1. 2. 3.
Ayres spatula Aylesbury device Cytobrush + extended tip device. One-day training workshop required.
Vaginal smear Endometrial aspiration cytology Smear of vulva lesions Fine needle aspiration cytology +/- imaging Peritoneal washings Buccal smear
USES OF THE PAP SMEAR
Detection of occult pathologic abnormalities of the uterine cervix in asymptomatic women Detection of recurrence of known pathologic abnormalities of the uterine cervix Evaluation of a suspected hormonal abnormality Monitoring of hormonal therapy
PATHOLOGIC ABNORMALITIES OF THE CERVIX ON PAP SMEAR
Pre-malignant and pre-invasive malignant conditions of the cervix
Infections such as:
Actinomyces israeli seen with IUCD users Lactobacillus species which is normally present in the secretory phase,
Gardenerella vaginalis a sexually transmitted coccobacillus, Chlamydia trachomatis a sexually transmitted bacterial pathogen that can be transmitted from the mothers cervix to the conjunctiva of her child, Candida albicans, Torulopsis glabrata, Trichomonas vaginalis classically presents as a profuse pungent malodorous vaginal discharge and puritis and is associated with the condition called strawberry cervix. Presentation my be subclinical or asymptomatic,
Enteroaemoba gingivalis, Enterobius vermicularis, Herpes simplex virus – The virus is not seen but there cytopathic effects are seen e.g. cell fusion, intranuclear inclusions, nuclear
INTERPRETATION OF RESULTS Result Action Taken Negative (Normal) Inflammation (infection/rxn) Mild Dysplasia
None. Repeat in one year. Treat then repeat Repeat Pap smear in 6 smear months.
Moderate Dysplasia
Colposcopy
Severe Dysplasia
Colposcopy with or without cone biopsy.
Class V - Cancer cells
Several types of surgical techniques are used depending upon
EFFECTIVENESS OF CERVICAL SCREENING % NUMBER
SCREENING SCHEDULE
REDUCTIO N IN RISK
OF SMEARS TAKEN
Every 10 years age 25-64
64%
Every 5 years age 20-64
83.8% 9
Every 5 years age 25-64
81.8% 8
Every 5 years age 35-64
69.6% 6
Every 3 years age 20-64
91.2% 15
Every 3 years age 25-64
89.8% 13
Every 3 years age 35-64
77.6% 10
Every year age 20-64
93.3% 45
5
PAP SMEAR PROCEDURE
Obtain informed consent Request form should include biodata, obs/ gynae history and details of prior abnormal smears Time test for second half of cycle Instruct patient to avoid intercourse/ douching 24 hours prior to test Sample is collected by the chosen spatula after exposing the cervix and including the squamou-columnar junction of the cervix
PAP SMEAR PROCEDURE (cont)
The material is evenly spread on a glass slide Smears are immediately fixed in alcohol solution Smears are thereafter placed in distilled water Stain for 2 - 3 minutes in Harris’ or Mayer’s Hematoxylin. This stains the nucleus. The cytoplasm is thereafter stained with OG6 or EA50. Smears which are considered abnormal by the bioscientist are re-screened and reported by a consultant cytopathologist All negative smears undergo an internal quality assurance check, called rapid
MODIFICATIONS OF PROCESSING METHODS FOR PAP SMEAR
Liquid based cytology - or thin layer
cytology, is a technique of preparing a monolayer of cells on a glass slide. The technique involves suspending the sample in a solution that is both mucolytic and haemolytic. The sample is concentrated either by centrifugation or by filtration, in the former method a monolayer of cells sediments onto the slide, in the filtration method a monolayer of cells is pressed onto the slide from the filter.
Automated devices
ADVANTAGES OF LIQUID BASED CYTOLOGY
Clean background
Excellent fixation
Well defined nuclear detail
Instant preservation with no air drying artefact
Removal of blood and mucus
Increased productivity
Standardisation of collection and laboratory techniques
Reduction in unsatisfactory as well as low-grade abnormality rates
Ability to use sample for other techniques such as HPV typing, immunocytochemistry or DNA probes.
Reduction in costs of women unnecessarily referred for colposcopic assessment and treatment.
DISADVANTAGES OF LIQUID BASED CYTOLOGY
Training needed for smear takers
Lengthy laboratory preparation time
Training required to operate equipment
Training required to interpret the smears
Morphology may be different from conventional preparations
Cost and storage of reagents
Loss of existing screener skills in interpreting conventional smears
Loss of ability for future staff to develop skills in
AUTOMATED DEVICES FOR INTERPRETING CERVICAL SMEARS GUIDELINES OF THE INTERNATIONAL ACADEMY OF CYTOLOGY task force, 1997
100% specificity and sensitivity The professional in charge of a clinical laboratory should continue to bear medical responsibility for diagnostic decisions made by machines. Automation should not lead to a reduction in standards nor expose the patient to any increased risk. Supervisory personnel should continue a visual quality control on a percentage of slides diagnosed as normal, and recognition should be made that human expert opinion represents the gold standard. The setting of performance criteria should remain the responsibility of the regulatory authorities, and not the manufacturers, and the professional cytology community should participate in the development of procedures that
VAGINAL SMEARS & HORMONAL ABNORMALITY Examination of vaginal smears for hormonal assessment can be done with the Papanicolaou stains; rapid Schorr method; other rapid suparvital stains; Use of phase microscopy in addition reveals bacterial flora. Cells demonstrated include:
Superficial squamous cells are shed from a fully
mature squamous epithelium that has developed its full thickness under the influence of oestrogen and will be most numerous at the middle of the menstrual cycle. The cells stain pink with the Papanicolaou stain, are angular outline, 40-60 micrometres in diameter, and contain a shrunken hyperchromatic pyknotic nucleus. Avitaminosis A & C can mimic changes similar to oestrogenic effect.
Intermediate cells are shed from the surface of a semi-
mature epithelia that shows a diminished response to oestrogen or the effects of progesterone and are commonly seen at the latter stages of the menstrual cycle, they are 30-60 micrometres in diameter, contain glycogen and stain blue/green with the Papanicolaou stain and contain a clearly defined round or oval vesicular nucleus 8 micrometres in diameter. Doderleins bacilli destroys these cells preventing maturation to superficial cells.
Parabasal and basal cells are seen in the absence of either oestrogen or progesterone, and are found in pre pubertal smears, post-menopausal smears or post partum smears. Also seen in anorexia nervosa. The cells are small, 15 -20 micrometres in diameter,
ENDOCRINOPATHIES ON VAGINAL SMEARS PRIMARY AMENORRHEA
Gonadal Dysgenesis: In Turner’s syndrome, the smear is atrophic (cf
atypical dysgenesis), and in 80% of cases, sex chromatin (SC) body is -ve (cf mosacism). Pituitary Infantilism: The smear is atrophic/intermediately proliferative. SC +ve. Ovarian Eunuchoidism: The smear is atrophic. SC +ve. Testicular Femininization Synd.: The smear is proliferative, but SC is -ve. Simple Congenital Absence of Uterus: The smear shows a normal cyclic pattern. SC is +ve. Congenital Adrenal Hyperplasia: SC is +ve. The smear is atrophic or intermediate proliferative.
PRECOCIOUS PUBERTY SYNDROME
Constitutional or Idiopathic: The smear is proliferative and may show cycling.
Lesions of CNS: The degree of proliferation varies. Granulosa & Theca Cell Ovarian Tumors: The smear is highly proliferative.
2o AMENORREA of HYPOTHALAMIC ORIGIN
Psychogenic: Smears vary. The degree of estrogen effect is a guide to the
ENDOCRINOPATHIES ON VAGINAL
SMEARS
SMEARS Syndromes of Oligomenorrhea and Hirsutism
Stein-Leventhal syndrome: The proliferation of the smear varies from intermediate to good. Large parabasal and intermediate cells of the navicular type may be present. Adrenogenital syndrome: The smear may vary from atrophic to small intermediate cell proliferation. Cushing’s syndrome: Generally, cell proliferation is intermediate. Masculinizing tumors of the ovary: Generally, the smear is atrophic. Genetic: The smear is proliferative and may be cyclic.
Syndromes of Amenorrhea and Galactorrhea
Chiari-Frommel syndrome. Typically, the smear is markedly atrophic. Pituitary tumors and CNS lesions (Forbes-Albright syndrome): The smear varies. Del Castillo’s syndrome (nonpuerperal, dysfunctional): Usually, small intermediate cells predominate. Pseudocyesis: Proliferation often with progestational characteristics.
Menopausal Syndrome
Great individual variation. Early in the climacteric, the smear may show high proliferation, but no cycling. In some cases, atrophic changes may occur abruptly, whereas in others, varying degrees of proliferation may be evident for years, with a gradual shift in the maturation index to the left.
FINE NEEDLE ASPIRATION CYTOLOGY
This is frequently used to provide a rapid diagnosis with or without the use of imaging from ascitic fluid, ovarian tumours, lymph nodes, vulva cysts etc.
Advantages of FNA over open surgical biopsy Diagnosis with a simple, cheaper, outpatient procedure
Avoids biopsy in some cases and may allow treatment of cancers at a planned surgery
Surgical team and theatre time not required
Avoids frozen section
Reduces patient uncertainty and anxiety
Low complication rate. Disadvantages of FNA over open surgical biopsy Errors in diagnosis may lead to over treatment or delay in making the correct diagnosis
Occasional complications such as bleeding or pneumothorax
Requires skilled personnel to take the sample
ASPIRATION NEEDLE & SYRINGE
OTHER APPLICATIONS OF CYTOLOGY IN OBSTETRICS/GYNECOLOGY
Cytology of serous fluid – Most exudates are metastatic and without good clinical information it is not possible to ascertain the primary site by cytology. Cytology of peritoneal washings important for staging ovarian & endometrial malignancies Cytology of nipple discharge for diagnosis of breast malignancy Exfoliative vaginal cytology with cervical mucus studies is a dependable non-invasive method for assessment of ovulation. Endometrial Cytology is however required for conclusion. Vulva cytology for vulva dystrophies,
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