Histopathological Pattern of Endometrial Hyperplasia in Peri- and Postmenopausal Women


Histopathological Pattern of Endometrial Hyperplasia in Peri- and Postmenopausal Women

*Fardousi F,1 Sultana SS,2 Kaizer N,3 Dewan RK,4 Jinnah MS,5 Jeba R,6 Hoque MN,7 Hussain M8

Abstract

Endometrial hyperplasia is one of the major gynaecological problem in peri- and post-menopausal women worldwide. It deserves special attention because of its relationship with endometrial carcinoma. The histopathological pattern of endometrial hyperplasia in peri- and post-menopausal women and their relationship with clinicopathological features are imperative to reach a diagnosis as well as early management. To find out the histopathological patterns of endometrial hyperplasia in peri- and post-menopausal women this descriptive cross-sectional study was carried out at the Department of Pathology, Dhaka Medical College, Dhaka during the period from January 2013 to December 2014. A total of seventy histopathologically diagnosed cases of endometrial hyperplasia were included in this study according to inclusion and exclusion criteria. Among the 70 cases, endometrial curettage biopsy specimens were 45 and hysterectomy specimens were 25. Routine Haematoxylin & Eosin staining was done on all cases. Out of all endometrial hyperplasia cases, 53 cases were simple endometrial hyperplasia without atypia, 8 cases were simple endometrial hyperplasia with atypia, 6 cases were complex endometrial hyperplasia without atypia and 3 cases were complex endometrial hyperplasia with atypia. Majority of the patients 35 (50%) were between 41-50 years of age. The study revealed that most common histopathological pattern of endometrial hyperplasia was simple endometrial hyperplasia without atypia, followed by simple endometrial hyperplasia with atypia. The importance of knowledge about the histological pattern of endometrium in abnormal uterine bleeding in different age group is useful in managing the cases with accuracy.

[Journal of Histopathology and Cytopathology, 2018 Jan; 2 (1):30-40]

 Key words: Endometrial hyperplasia, gynecological problem, peri- and postmenopausal women, histopathological pattern.

  1. *Dr. Farzana Fardousi, Lecturer, Department of Cytopathology, National Institute of Cancer Research and Hospital (NICRH), Mohakhali, Dhaka. farzanafardousi@yahoo.com
  2. SK Salowa Sultana, Assistant Professor, Department of Pathology, Ad Din Women’s Medical College.Dhaka
  3. Nahid Kaizer, Associate Professor (CC), Department of Pathology, Shahabuddin Medical College. Dhaka
  4. Professor Dr. Rezaul Karim Dewan, Head of the Department of Pathology, Dhaka Medical College.
  5. Mohammed Shahed Ali Jinnah, Associate Professor, Department of Pathology, Dhaka Medical College.
  6. Ruksana Jeba, Associate Professor Department of Pathology, Dhaka Medical College.
  7. Md. Nazmul Hoque, Ex Associate Professor, Department of Pathology, Dhaka Medical College
  8. Professor Dr. Maleeha Hussain , Ex Head of the Department of Pathology, Dhaka Medical College.

*For correspondence

Introduction

Endometrial hyperplasia is defined as an increased proliferation of the endometrial glands relative to the stroma, resulting in an increased gland-to-stroma ratio when compared with normal proliferative endometrium. Endometrial hyperplasia deserves special attention because of its relationship with endometrial carcinoma in peri and postmenopausal women.1 Worldwide, endometrial cancer is the most common invasive cancer of female genital tract.

In 50% cases of endometrial adenocarcinoma, endometrial hyperplasia particularly atypical hyperplasia is found as a premalignant lesion.2 Endometrial hyperplasia usually develops in the presence of continuous estrogen stimulation unopposed by progesterone. In the years before menopause, women may have numerous cycles without ovulation (anovulatory) during which there is continuous unopposed estrogen activity. Similarly, hormone replacement therapy consisting of estrogen without progesterone may lead to endometrial hyperplasia.3 The endometrium becomes atrophic after menopause as a result of ovarian failure. The postmenopausal endometrium which despite being atrophic, retain a weak proliferative pattern for many years probably as a response to continuous low level of estrogenic stimulation. These are at a higher risk of progression to endometrial hyperplasia and subsequently to endometrial malignancy.3 Many classifications of endometrial hyperplasia have been proposed over the years. The one that is currently preferred and which has been recognized by the World Health Organization (WHO) was originally proposed.4 It takes into account both the architectural and cytologic features, for reasons of dividing the hyperplasias into simple and complex based on architecture and subdividing each into typical and atypical on the basis of their cytological pattern.5 The type I endometrial cancers which is endometrioid variant is associated with unopposed estrogen exposure and is often preceded by atypical endometrial hyperplasia. However, type II endometrial cancers where a non-endometrioid histology (usually papillary, serous or clear cell) has an aggressive clinical course, is not preceded by any type of endometrial hyperplasia.6 According to WHO, endometrial hyperplasia is divided into four major categories: Simple hyperplasia without atypia, Simple hyperplasia with atypia, Complex hyperplasia without atypia and Complex hyperplasia with atypia. The confirmed diagnosis of endometrial hyperplasia can be made by histopathological examination.7 Endometrial hyperplasia is associated with prolonged estrogen stimulation of the endometrium, which can be due to anovulation, increased estrogen production from endogenous sources or exogenous estrogen. The factors associated with endometrial hyperplasia include obesity, menopause, polycystic ovarian diseases, functioning granulosa cell tumors of the ovary, excessive cortical function and prolonged administration of estrogenic substances.1 The primary presenting symptom of endometrial hyperplasia is abnormal uterine bleeding, which typically prompts an endometrial biopsy to rule out carcinoma.8 Approximately, 70% of women with abnormal uterine bleeding are diagnosed with benign findings and 15% are diagnosed with carcinoma. The remaining 15% receive a diagnosis of endometrial hyperplasia, which includes a broad range of lesions, from mild, reversible proliferations to the immediate precursors of carcinoma. Abnormal uterine bleeding is the chief complaints of endometrial hyperplasia and can be categorized into dysfunctional uterine bleeding or postmenopausal bleeding. Long reproductive life has risk for development of endometrial hyperplasia. Different studies have shown that, women who have achieved early menarche or late menopause have more risk for development of endometrial hyperplasia and endometrial carcinoma, as estrogen exposure especially unopposed by progesterone is a known risk factor for the development of endometrial carcinoma. Parity causes estrogen-hormonal environment throughout the fertile years of a woman, which may increase risk for the development of endometrial carcinoma.9 Obesity is a known risk factor for endometrial hyperplasia and this excess risk is associated with the endocrine and inflammatory effects of adipose tissue. Adipocytes express aromatase that converts ovarian androgens into estrogens, which induce endometrial proliferation.10 It was observed in different studies that, endometrial hyperplasia was found in diabetic and hypertensive patient, but whether this association is statistically significant or not or whether it carries a risk of endometrial carcinoma for diabetic and hypertensive women need to be ascertained with further studies in a larger series of population. It was observed in many studies that endometrial hyperplasia is associated with family history of endometrial cancer. Because a common genetic alteration found in a significant number of endometrial hyperplasias and related endometrial carcinomas i.e. inactivation of the PTEN tumour suppressor gene.11 The study was aimed to find out the histopathological patterns of endometrial hyperplasia in peri- and post-menopausal women.

Methods

Place and period of study

This is a descriptive cross-sectional study which was carried out at the Department of Pathology, Dhaka Medical College, Dhaka, during the period of January 2013 to December 2014.

Sample Size

For this study, 110 peri- and postmenopausal women with dysfunctional uterine bleeding or postmenopausal bleeding who underwent D&C or hysterectomy were screened. A total of seventy histopathologically diagnosed cases of endometrial hyperplasia who met the enrollment criteria (inclusion & exclusion criteria) were included in this study. Among 70 cases, endometrial curettage biopsy specimens were 45 and hysterectomy specimens were 25. Routine Haematoxylin & Eosin staining was done on all cases.

Collection of Sample

Ethical clearance was taken for this study from institutional ethical committee of Dhaka Medical College, Dhaka. Each patient was interviewed before collection of the specimen and relevant information was recorded in a prescribed clinical proforma. Detail history with particular attention to age, clinical features, age at menarche, parity, obesity, history of contraceptives, history of hormone replacement therapy, history of diabetes, history of hypertension, history of estrogen producing ovarian tumor, age at menopause were taken.

 Histopathological Examination

All specimens obtained either by endometrial curettage biopsy or hysterectomy were immersed in 10% formalin. The specimens were examined in the department of pathology, Dhaka Medical College with a particular emphasis on number, size, shape, color and consistency. This part was done by experienced pathologist in the department of Pathology of Dhaka Medical College. Tissue processing and staining: Routine tissue processing and Haematoxylin & Eosin staining were done at the Department of Pathology, Dhaka Medical College.

Microscopic Analysis

Following histopathological diagnoses were made according to WHO classification of endometrial hyperplasia: Simple endometrial hyperplasia without atypia, Simple endometrial hyperplasia with atypia, Complex endometrial hyperplasia without atypia, Complex endometrial hyperplasia with atypia.5

Statistical Analysis

Statistical analyses of the results were obtained by using window based computer software devised with Statistical Packages for Social Sciences (SPSS-16). Percentages were calculated to find out the proportion of the findings. The results were presented in Tables and Figures.

Results

Out of 70 histopathologically diagnosed endometrial hyperplasia cases, 53 cases were simple endometrial hyperplasia without atypia, 8 cases were simple endometrial hyperplasia with atypia, 6 cases were complex endometrial hyperplasia without atypia and 3 cases were complex endometrial hyperplasia with atypia. Majority of the patients 35(50%) were between 41-50 years of age (Table I).

Table I: Distribution of the study patients by Age (n=70)

 

Age (Years) SEH without atypia

(n=53)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

Total

(n=70)

n %
35-40 12 1 4 1 18 25.7
41-50 30 5 0 0 35 50.0
51-60 11 2 1 2 16 22.9
61-70 0 0 0 0 0 0.0
>70 0 0 1 0 1 1.4
Total 53 8 6 3 70 100

 

(SEH without atypia- Simple endometrial hyperplasia without atypia, SEH with atypia- Simple endometrial hyperplasia with atypia, CEH without atypia – Complex endometrial hyperplasia without atypia and CEH with atypia- Complex endometrial hyperplasia with atypia)

Table II: Distribution of the study patients by menstrual history (n=70)

 

Menstrual history SEH without atypia

(n=53)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

Total

(n=70)

n %
Irregular menstrual bleeding 42 8 4 1 55 78.6
Post-menopausal bleeding 11 0 2 2 15 21.4

Majority of the patients (56 cases) achieved their menarche at 12-13 years of age in simple endometrial hyperplasia without atypia and no patient achieved early menarche (before 10 years) (Table III)

Table III: Distribution of the study patients by age of menarche (n=70)

 

Age of menarche (years) SEH without atypia

(n=53)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

Total

(n=70)

n %
12-13 47 3 4 2 56 80.0
14-15 6 5 2 1 14 20.0
Mean±SD 13.7±0.7 13.8±1.3 13.3±1.0 12.7±0.6 13.6 ±0.9
Range (Min-max) 12-15 12-15 12-15 12-13 12-15

 

Out of 70 cases, 45(57.14%) were multipara. Among 45 cases, 36(51.4%) cases had 3 children, 6(8.6%) cases had 4 children and 3(4.3%) cases had 5 children (Table V).

By Basal Metabolic Index, obesity (BMI more than 30) was found in 34(48.6%) cases, among them 27 cases in simple endometrial hyperplasia without atypia, 5 cases in simple endometrial hyperplasia with atypia and 2 cases in complex endometrial hyperplasia with atypia (Table VI).

 

Table IV: Distribution of the study patients by age of menopause (n=70)

 

Age of menopause SEH without atypia

(n=53)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

Total

(n=70)

n %
50 3 0 1 0 4 26.6
51 4 0 0 1 5 33.3
52 4 0 1 1 6 40.0
Mean±SD 51.09±0.8 0±0 51.0±0 51.5±0.7 51.1 ±0.8
Range (Min-max) 50-52 51-51 50-52 51-52 50-52

 

Table V: Distribution of the study patients by parity (n=70)

 

Para SEH without atypia

(n=53)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

Total

(n=70)

n %
2 18 4 2 1 25 35.7
3 29 4 2 1 36 51.4
4 4 0 1 1 6 8.6
5 2 0 1 0 3 4.3
Mean±SD 2.8±0.8 2.5±0.5 3.2±1.2 3.0±1.0 2.8±0.8
Range (Min-max) 2-5 2-3 2-5 2-4 2-5

 

 

Table VI: Distribution of the study patients by obesity by calculating Basal Metabolic Index (BMI) (n=70)

 

Obesity by BMI (kg/m2) SEH without atypia

(n=53)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

Total

(n=70)

n %
Present 27 5 0 2 34 48.6
Absent 26 3 6 1 36 51.4

 

Out of the total study cases, diabetes was found in 11 cases, hypertension was found in 24 cases, and history of associated estrogen producing tumor of ovary was found in 1 case (Table VII).

 

Table VII: Distribution of the study patients by diabetes, hypertension and history of associated estrogen producing tumour of ovary (n=70)

 

Diseases SEH without atypia

(n=53)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

Total

(n=70)

n %
Diabetes 4 5 1 1 11 15.7
Hypertension 20 2 1 1 24 34.3
History of associated estrogen producing tumour of ovary 0 0 0 1 1 1.4

 

History of oral contraceptive pill was found in 15(21.4%) cases and history of hormone replacement therapy was found in 12(17.15%) cases (Table VIII).

 

Table VIII: Distribution of the study patients by history of Oral contraceptive pill (OCP) and history of Hormone replacement therapy (HRT) (n=70)

 

History SEH without atypia

(n=53)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

Total

(n=70)

n %
H/O OCP 7 4 3 1 15 21.4
H/O HRT 10 0 0 2 12 17.15

 

 

 

 

 

 

Figure 1. Photomicrograph showing simple endometrial hyperplasia without atypia (H & E stain, x10)

 

 

 

 

 

Figure 2. Photomicrograph showing simple endometrial hyperplasia with atypia (H & E stain, x40)

 

 

 

 

 

Figure 3. Photomicrograph showing complex endometrial hyperplasia without atypia(H & E stain, x40)

 

 

 

 

 

 

Figure 4. Photomicrograph showing complex endometrial hyperplasia with atypia(H & E stain, x40)

Microscopic pictures of different types of endometrial hyperplasia are shown in figures 1, 2,3 and 4. Pie chart showing the commonest diagnosis in 70 patients was SEH without atypia (75.7%) followed by SEH with atypia (11.4%; Fig-5).

 

 

 

 

Figure 5. Pie chart showing distribution of the patients by diagnosis (n=70)

 

 

 

 

 

Figure 6.  Bar diagram showing distribution of the patients according to mean age with diagnosis (n=70)

Discussion

Endometrial hyperplasia has a significant place in gynecological morbidity in women of reproductive age (10% to 18%).12 It is associated with menstrual irregularities and anaemia in women and poses a high risk for malignant transformation into endometrial cancer.13 Worldwide endometrial cancer is the most common gynecological cancer in peri and postmenopausal women.14,15 The incidence of endometrial adenocarcinoma not only has remained high but in recent years has tended to significantly increase in many countries, including Bangladesh.12,16

 

The diagnosis and classification of endometrial hyperplasia can be made by histopathological examination. The present cross-sectional study was carried out with an aim to observe the histopathological pattern of endometrial hyperplasia in peri and post-menopausal women. In the present study, the commonest diagnosed lesion was simple endometrial hyperplasia without atypia which was 53(75.7%), followed by simple endometrial hyperplasia with atypia 8(11.4%), complex endometrial hyperplasia without atypia 6(8.6%) and then complex endometrial hyperplasia with atypia 3(4.3%).

In this study it was observed that, the mean age was 43.7±7.9 years in simple endometrial hyperplasia without atypia, 44.3±5.9 years in simple endometrial hyperplasia with atypia, 48.5±15.5 years in complex endometrial hyperplasia without atypia and 50.0±13.2 years in complex endometrial hyperplasia with atypia. In our study, the age ranged from 35 to 75 years with a mean age of 45 years. The highest number of cases 35(50%) were in the fourth decade. These findings are almost similar to the studies carried out.17,13 In their study, they also found maximum cases in the fourth decade. However, the present study differed from the study conducted by Trimble et al.2 who reported the mean age as 58 years and the age range between 25 to 89 years. Probably this discrepancy is due to small number of cases in our study. The present study also differed from the study conducted.18 They observed that, the incidence of simple and complex hyperplasia without atypia were highest in women aged 50 to 54 years, whereas the rate of atypical hyperplasia was highest in women aged 60 to 64 years. This variation may be due to high expectancy of life in developed countries.

We observed, 55 cases were in their reproductive age and on menstrual history, all of them had irregular menstrual bleeding. Among 55 cases, majority (42) were diagnosed as simple endometrial hyperplasia without atypical, followed by (8) as simple endometrial hyperplasia with atypia. In this study it was observed that, 15 cases achieved menopause and postmenopausal bleeding. Out of 15 cases, on histopathological examination, 11 were diagnosed as simple endometrial hyperplasia without atypia, 2 as complex endometrial hyperplasia without atypia and 2 as complex endometrial hyperplasia with atypia. This finding is in concordance with that found in the study of Farquhar et al.19 Thus, it can be concluded that, postmenopausal bleeding does not always indicate a risk for development of endometrial carcinoma as simple endometrial hyperplasia without atypia has only 1 to 3% risk for development of endometrial carcinoma.1

In the present study, postmenopausal patients were 15 in number. The mean age at which they achieved menopause was 51.09±0.8 years in simple endometrial hyperplasia without atypia, 51.0±0 years in complex endometrial hyperplasia without atypia and 51.5±0.7 years in complex endometrial hyperplasia with atypia. In this study it was observed that, most patients achieved menopause at the age of 52 years. This result is in concordance with the study done by Jetley et al.20 However, the number of menopausal patient is inadequate to come to any definite comment.

All the patients of this study were parous women. Parity causes estrogen-hormonal environment throughout the fertile years of a woman.9 In the present study it was found that, range of parity varied from 2 to 5 in all types of endometrial hyperplasia. This result is in concordance with the study done by Topcu et al.21,22

They also observed range of parity from 2 to 5 among the patients with endometrial hyperplasia. However, this finding has a pitfall of not having nullipara patient in the present study.

Obesity is a known risk factor for endometrial cancer. This excess risk is associated with the endocrine and inflammatory effects of adipose tissue. Adipocytes express aromatase that converts ovarian androgens into estrogens, which induce endometrial proliferation.10 In the present study, it was observed that by Body Mass Index (BMI), obesity (BMI more than 30) was found in 34 cases (48.6%). Among them, majority (27) cases were simple endometrial hyperplasia without atypia. This result is in concordance with the study done by Epplein et al. 23,21,17 they also found 48.89% cases of endometrial hyperplasia in obese women.

In this study. it was observed that diabetes was found in 11 cases (15.7%). Similar study was done by Bera et al.17 They also found association of diabetes mellitus in 18 cases (15%) of endometrial hyperplasia.

In present study it was observed that, hypertension was found in 24 cases (34.3%). This result is in concordance with the study done by Bera et al.17 They also found 36 cases (35%) of endometrial hyperplasia in hypertensive patients. It is observed that, in the present study and other comparable studies, endometrial hyperplasia is found in diabetic and hypertensive patient, but whether this association is statistically significant or not or whether it carries a risk of endometrial carcinoma for diabetic and hypertensive women need to be ascertained with a large series of study population.

The history of associated estrogen producing tumor of ovary was found in 1 case and it was a case of complex endometrial hyperplasia with atypia. Though this number is very insignificant in our study, Gregory et al.24 have shown in their study that, women with ovarian tumor and polycystic ovarian syndrome (PCOS) have a higher risk of development of estrogen-induced endometrial hyperplasia and cancer.

We observed that history of OCP in 15 cases. Out of 15 cases, 7 cases were simple endometrial hyperplasia without atypia, 4 cases were simple endometrial hyperplasia with atypia, 3 cases were complex endometrial hyperplasia without atypia and 1 case was complex endometrial hyperplasia with atypia. Epplein et al.23 also the found association of OCP in 18 cases of endometrial hyperplasia in the women of reproductive age.

History of hormone replacement therapy (HRT) was found in 12 cases and majority (10) were simple endometrial hyperplasia without atypia. Epplein et al.23 have found similar association with HRT which is composed of estrogen only.

In the present study, no patient gave family history of endometrial hyperplasia or endometrial carcinoma. This may be due to unawareness of the study patients about their family history. Other studies also found no association with the family history of endometrial hyperplasia or endometrial carcinoma, though they were aware about their family history.17, 23

Limitation of the Study

Our study included few numbers of postmenopausal women. It was a cross-sectional study. To evaluate the risk factors for endometrial hyperplasia and the risk of endometrial hyperplasia to progress to endometrial carcinoma, ideally a cohort or follow-up study should be done.

 Conclusion

We found that most of the patients were in the fourth decade and all the patients had chief complaints of either irregular menstrual bleeding or postmenopausal bleeding. On USG, all of them had bulky uterus and all the patients were parous women. We found no significant association with obesity, diabetes mellitus, hypertension, oral contraceptive pill or estrogen producing ovarian tumour but significant association was found with HRT in postmenopausal women. None of the patient had family history of endometrial hyperplasia or endometrial carcinoma.

Recommendations

A large follow–up study is recommended for patients of endometrial hyperplasia selected for conservative treatment with progestogen and GnRH-agonists. Monitoring should be done by observing the Ki-67 expression in these patients. If the Ki-67 expression increases, they should be treated by surgical intervention.

References

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  2. Trimble CL, Kauderer J, Zaino R, Silverberg S, Lim PC, Burke JJ, Alberts D, Curtin J. Concurrent endometrial carcinoma in women with a biopsy diagnosis of atypical endometrial hyperplasia. Cancer, 2006; 106(4):812-9.
  3. Buckley CH. Fox H, Biopsy pathology of the endometrium. 2nd London Arnold, 2002 pp.209-240.
  4. Kurman RJ, Norris HJ. Evaluation of criteria for distinguishing atypical endometrial hyperplasia from well differentiated carcinoma. Cancer, 1982; 49(12):2547-59.
  5. Rosai J. Rosai and Ackerman’s surgical pathology e-book. Elsevier Health Sciences; 2011 Jun 20.
  6. Rakha E, Wong SC, Soomro I, Chaudry Z, Sharma A, Deen S, Chan S, Abu J, Nunns D, Williamson K, McGregor A. Clinical outcome of atypical endometrial hyperplasia diagnosed on an endometrial biopsy: institutional experience and review of literature. The American journal of surgical pathology, 2012; 36(11):1683-90.
  7. Pungal A, Balan R, Cotuţiu C. Hormone receptors and markers in endometrial hyperplasia. Immunohistochemical study. Revista medico-chirurgicala a Societatii de Medici si Naturalisti din Iasi, 2010; 114(1):180-4.
  8. Lacey JV, Chia VM. Endometrial hyperplasia and the risk of progression to carcinoma. Maturitas, 2009; 63(1):39-44.
  9. Bayo S, Bosch FX, de Sanjose S, Munoz N, Combita AL, Coursaget P, Diaz M, Dolo A, van den Brule AJ, Meijer CJ. Risk factors of invasive cervical cancer in Mali. International journal of epidemiology, 2002; 31(1):202-9.
  10. Schmandt RE, Iglesias DA, Lu KH. Understanding obesity and endometrial cancer risk: opportunities for prevention. American Journal of Obstetrics & Gynecology. 2011; 205(6):518-25.
  11. Fabjani G, Kucera E, Schuster E, Minai-Pour M, Czerwenka K, Sliutz G, Leodolter S, Reiner A, Zeillinger R. Genetic alterations in endometrial hyperplasia and cancer. Cancer letters, 2002; 175(2):205-11.
  12. Benyuk VA, Kurochka VV, Vynyarskyi YM, Goncharenko VM. Diagnostic algorithm endometrial pathology using hysteroscopy in reproductive age women. Women Health (ZdorovyaZhinky), 2009; 6(42):54-6.
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  14. Nicolaije KA, Ezendam NP, Vos MC, Boll D, Pijnenborg JM, Kruitwagen RF, Lybeert ML, van de Poll-Franse LV. Follow-up practice in endometrial cancer and the association with patient and hospital characteristics: a study from the population-based PROFILES registry. Gynecologic oncology, 2013; 129(2):324-31.
  15. Giordano G, Gnetti L, Merisio C, Melpignano M. Postmenopausal status, hypertension and obesity as risk factors for malignant transformation in endometrial polyps. Maturitas, 2007; 56(2):190-7.
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  17. Bera H, Mukhopadhyay S, Mondal T, Dewan K, Mondal A, Sinha SK. Clinicopathological study of endometrium in peri and postmenopausal women in a tertiary care hospital in Eastern India. OSR-JDMS, 2014; 13:16-23.
  18. Giuntoli RL, Zacur HA, Goff B, Garcia RL, Falk SJ. Classification and diagnosis of endometrial hyperplasia. Offical Reprint from UpToDate. 2014 Jun.
  19. Farquhar CM, Lethaby A, Sowter M, Verry J, Baranyai J. An evaluation of risk factors for endometrial hyperplasia in premenopausal women with abnormal menstrual bleeding. American Journal of Obstetrics & Gynecology, 1999; 181(3):525-9.
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  21. Topcu HO, Erkaya S, Guzel AI, Kokanali MK, Sarıkaya E, Muftuoglu KH, Doganay M. Risk factors for endometrial hyperplasia concomitant endometrial polyps in pre-and post-menopausal women. Asian Pac J Cancer Prev, 2014; 15(15):5423-5425.
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Imprint Cytology in the Diagnosis of Upper Gastrointestinal Lesions

Imprint Cytology in the Diagnosis of  Upper Gastrointestinal Lesions

*Keya  SA,1 Saha NK,2 Alam MJ,3 Ullah P,4 Shariar S,5 Hira AD,6 Ahmed NU,7 Islam N8

 Abstract

Endoscopic biopsy and cytology are well established techniques for the diagnosis of upper gastrointestinal tract malignances. Imprint cytology is a touch smear preparation which helps to diagnose malignant cell easily. This cross sectional study was done to see imprint cytological patterns of benign and malignant lesions of upper gastrointestinal tract,  to find out histopathological diagnoses and to compare imprint cytological diagnoses with histopathological diagnoses. Biopsy materials from 100 patients were obtained by endoscopist using video endoscope and were subjected to imprint smear technique followed by histopathological examination of the same materials. Cytological and histopathological diagnoses were compared using appropriate statistical methods. Among the 100 cases, 57(57%) were male & 43(43%) were female with a mean age of 56.7 years.  Out of 100 cases, gastric, esophageal and duodenal lesions were 63(63%), 32(32%) and 5(5%) respectively. Misinterpretation on cytological evaluation was seen in only 6 cases. The overall sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of imprint cytology were 98.46%, 91.42%, 95.52%, 96.97% and 96% respectively. The findings of the present study indicate that imprint cytological method is a reliable technique for rapid diagnosis of upper GIT lesions as it is simple, accurate and cost effective. Diagnosis can be provided within an hour of endoscopic procedure by the imprint smear technique.

[Journal of Histopathology and Cytopathology, 2018 Jan; 2 (1):23-29]

 Keywords:  Imprint  cytology,  Upper  Gastrointestinal Lesions

 

  1. *Dr. Shamim Ara Keya, Lecturer of Pathology, Shaheed Suhrawardy Medical College, Dhaka. keyaparash@gmail.com
  2. Professor Dr. Naba Kumar Saha, Professor and Head, Department of Pathology, Sylhet MAG Osmani Medical College, Sylhet.
  3. Md. Jahangir Alam, Associate Professor and Head, Department of Gastroenterology, Sylhet MAG Osmani Medical College, Sylhet.
  4. Parash Ullah, Medical Officer, Department of Medicine, Shaheed Suhrawardy Medical College Hospital, Dhaka.
  5. Sakib Shariar, Pathologist, Bangladesh Railway Hospital, Chittagong.
  6. Ananda Dyuti Hira, Pathologist, Khulna Medical College, Khulna.
  7. Nasir Uddin Ahmed, Pathologist, Faridpur Medical College, Faridpur.
  8. Nazmul Islam, Assistant Professor of Pathology, Army Medical College, Comilla.

*For correspondence

Introduction

Any portion of the gastrointestinal tract may be affected by malignancy. The highest incidence of malignancy is in the esophagus, stomach and colorectal region. In fact esophagogastric and colorectal malignancies are amongst the commonest cancers in humans.1 Worldwide, gastric adenocarcinoma is the second most common cancer.2 Endoscopic biopsy and cytology are well established techniques for the diagnosis of upper gastrointestinal tract malignancies. Now a days, various cytologic techniques like brush cytology, crush preparation, touch smear or imprint cytology are commonly used along with routine endoscopic biopsy.3

Imprint cytology from endoscopic biopsy is highly sensitive and specific technique for the diagnosis of upper GI malignancy. Imprint cytology showed an overall accuracy of 97.6%, 100% and 100% for the diagnosis of malignancies of esophagus, stomach and duodenum respectively.4  But there is no systemic study on imprint cytology of gastrointestinal lesions in our country. Imprint cytology can act as an adjunct to histopathology as it increases the diagnostic efficacy and saves time but definitely it cannot replace histopathology as chances of false positives are high.5  Imprint cytology is a special variation of applied cytology that can be used for various purposes.6 Sometimes endoscopic biopsy fails to make a definitive histological diagnosis due to inadequate tissue from small lesion where tissues are distorted during histological processing; in that case imprint cytology may be useful in diagnosing upper gastrointestinal tract malignancy.7 In this study, imprint smears were taken before keeping the endoscopic specimens in fixative for histopathological examination. Endoscopic biopsy is an essential part of the evaluation of gastrointestinal pathology. In the diagnosis of upper GI lesions, histopathological examination is considered gold standard but is time-consuming  when compared to cytology. Most gastroenterologists and patients would like to get an immediate opinion regarding the adequacy of biopsy and nature of the lesion. Imprint cytology is a simple, rapid, reliable and cost effective method and it alone can serve as a useful tool for an immediate diagnosis within an hour after endoscopic procedure. Imprint cytology lets the surgeon to plan a therapeutic strategy approximately one week earlier.4,8  With this background, this study was designed to find out imprint cytological patterns of benign and malignant lesions of upper gastrointestinal tract, to find out histopathological diagnoses and to compare imprint cytological diagnoses with histopathological diagnoses.

 Methods

This study was a cross sectional study and carried out on 100 patients in the department of Pathology, Sylhet MAG Osmani Medical College, Sylhet in collaboration with the department of Gastroenterology, Sylhet MAG Osmani Medical College Hospital, Sylhet, Bangladesh from 1st January 2015 to 31st December 2015. During endoscopic examination by using video endoscope, biopsy was taken. Imprint smears were prepared from all biopsy samples. Imprint smear was done by placing tissue on the slides from the biopsy forcep with the help of a fine needle.  After placing tissue on the slides, thin smears were prepared by gently rotating the tissue with the needle. Then smears were fixed in 95% ethyl alcohol and stained with Papanicolaou stain.The same tissue after smear preparation was collected for histopathological examination. The tissue was examined thoroughly in day light  with particular emphasis on number, colour, consistency, presence or absence of hemorrhage and necrosis. Tissue was embedded as such and was placed in bottle containing 10% formalin with proper tagging and was kept for overnight fixation.  Routine tissue processing with paraffin impregnation was done and sections were stained with haematoxylin and eosin. The diagnosis of upper gastrointestinal tract malignancy was done according to the cytopathological and histopathological findings and grading was done by WHO classification. Then imprint smear diagnosis was compared with the histopathological diagnosis. The efficacy of imprint cytology was determined by sensitivity, specificity, positive predictive value, negative predictive value and accuracy.

 Results

Among the 100 patients, the age ranged from 21 to 100 years with a mean of 56.7 years. In this study, 57(57%) were male and 43(43%) were female with male to female ratio of 1.33:1. The highest number of patients  30(30%) was in the age group 61-70 years followed by 27(27%) in 41-50 years age group & 21(21%) in 51-60 years age group. In our study, 63% (63 cases) were located in the stomach, 32 % (32 cases) were in the esophagus, and 5% (5 cases) were in the duodenum. The most common presenting symptoms were pain and vomiting in case of gastric lesions and dysphagia in case of esophageal lesions.

 

Table I:  Imprint cytology and corresponding histopathological diagnosis in contingency (cross) table

 Cytological diagnosis

 

 

 

 

 

No

 

Histopathological diagnosis

T

 

o

 

t

 

a

 

 

l

Adenocarcinoma Squamous cell carcinoma Chronic non atrophic gastritis Suggestive of adenocarcinoma Chronic gastric ulcer Chronic duodenitis Dysplasia Suggestive of squamous cell carcinoma Chronic esophagitis Hyperplastic polyp Gastric adenoma
Adenocarcinoma 35 34 1 35
Squamous cell carcinoma 25 25 25
Chronic  gastritis 24 23 1 24
Suspicious of adenocarcinoma 5 4 1 5
Chronic gastric ulcer 3 3 3
Suspicious of squamous cell carcinoma 2 1 1 2
Chronic duodenitis 2 2 2
Normal findings 2 1 1 2
Chronic esophagitis 1 1 1
Dysplasia 1 1 1
Total 100 35 25 24 4 4 2 2 1 1 1 1 100

 

 

Ulcerative (41%) and ulceroproliferative lesions (41%) were the most common endoscopic findings.  Proliferative lesion was found in 17% of cases and benign polyp was noted in 1% of cases. In the present study, the most common lesion diagnosed by imprint cytology was adenocarcinoma (35%) followed by squamous cell carcinoma (25%) and chronic gastritis (24%) (Table-1). Histopathological evaluation revealed that the most common tumor was adenocarcinoma (35%) followed by squamous cell carcinoma (25%), chronic non atrophic gastritis (24%), suggestive of adenocarcinoma (4%), chronic gastric ulcer (4%), chronic duodenitis (2%),  dysplasia (2%) & suggestive of squamous cell carcinoma, chronic esophagitis, hyperplastic polyp, gastric adenoma  1 case each  (Table I).

Complete correlation between imprint cytological and histopathological diagnosis was obtained in 94(94%) cases (Table II) and 6 cases were misinterpreted on cytological examination. Out of six misinterpreted cases, two cases were diagnosed as normal findings on imprint smears which were subsequently diagnosed as adenocarcinoma in one case and chronic gastric ulcer in another case on histopathology. One case was diagnosed as adenocarcinoma on imprint smears which was subsequently diagnosed as chronic non atrophic gastritis on histopathology. Another one case was diagnosed cytologically as chronic gastritis was diagnosed histologically as hyperplastic polyp. Cytological diagnosis of suspicious of squamous cell carcinoma in one case was confirmed as moderate dysplasia by histopathology. Cytologically one case was interpreted as suspicious of adenocarcinoma which was diagnosed as gastric adenoma on histopathological examination.

 

Table II:  Correlation of histopathological and cytopathological diagnoses

 

Histopathological diagnosis No. of Cases Cytological correct diagnosis Percentage
Adenocarcinoma 35 34 97.05
Squamous cell carcinoma 25 25 100
Chronic nonatrophic gastritis 24 23 95.83
Suggestive of adenocarcinoma 4 4 100
Chronic gastric ulcer 4 3 75
Chronic duodenitis 2 2 100
Dysplasia 2 1 50
Suggestive of Squamous cell carcinoma 1 1 100
Chronic esophagitis 1 1 100
Hyperplastic polyp 1 0 0
Gastric adenoma 1 0 0
Total 100 94 94

Statistical evaluation of imprint cytology was done (Table III) and validity of imprint cytology like sensitivity, specificity, PPV, NPV and accuracy were found as 98.46%, 91.42%, 95.52%, 96.97% and 96% respectively (Table IV).

Table III:  Statistical evaluation of cytopathological diagnosis of 100 cases of  upper GIT lesions

 

Histopathological diagnosis                               Cytopathological Diagnosis
Disease positive (Malignant) Disease negative(Benign)
Positive(Malignant)

 

65

 

TP 64 FP 3
Negative(Benign)

 

35

 

FN 1 TN 32
Total 100 Total 65 Total 35

 

TP= True positive, TN= True negative, FP= False positive, FN= False negative.

 

Table IV:  Validity of cytopathological diagnosis of 100 cases of upper GIT lesions.

 

Sensitivity Specificity PPV NPV Accuracy
98.46% 91.42% 95.52% 96.97% 96%

 

 

 

 

 

 

Figure 1. Photomicrograph of imprint cytology of adenocarcinoma (Papanicolaou stain, x40).

 

 

 

 

 

Figure 2. Photomicrograph of imprint cytology of squamous cell carcinoma (Papanicolaou stain, x40).

 

 

 

 

 

 

Figure 3.  Photomicrograph imprint cytology of chronic gastritis (Papanicolaou’s stain, x40).

 

 

 

 

 

Figure 4. Photomicrograph of imprint cytology of gastric ulcer (Papanicolaou stain, x40).

Discussion

The primary role of gastrointestinal tract cytology is the detection of cancer. Gastrointestinal cytology is still in its early stages of development and few studies have described the role of brush cytology and imprint cytology in interpretation of upper GI biopsy.

The age of study patients ranged from 21 years to 100 years with a mean of 56.7 years.  Of the 100 patients, 57(57%) were male and 43(43%) were female with male to female ratio of 1.33:1.  This observation is similar to other studies who also observed that the frequency of gastrointestinal lesions was more in male.4,9  Highest number (30%) of gastrointestinal lesions were found in the age group 61 to 70 years followed by 27(27%) in 41 to 50 years age group and 21(21%) in the age group of 51 to 60 years which have consistence with the study of Vijayanarasimha D et al9  who observed highest number in fifth to seventh decades of life with a mean age of 55 years.  In the present study, 63% lesions were in the stomach, 32% were in the esophagus and 5% were in the duodenum which are also close to the findings of the  study  done by Vijayanarasimha D et al9  who observed that out of 110 cases, 52(47.3%) were gastric, 45(40.9%) were esophageal and 13(11.8%) were duodenal lesions. The clinical presentations of study patients were upper abdominal or epigastric pain, dysphagia, vomiting, anorexia, distended abdomen and sometimes abdominal mass.  Abdominal or epigastric pain was the commonest symptom in case of gastric and duodenal lesions and dysphagia in case of esophageal lesions. This clinical observation is similar to the clinical findings of the study done by Vijayanarasimha D et al.9 In our study, endoscopic findings of upper GIT lesion were ulcerative (41%), ulcero-proliferative (41%), proliferative (17%) and polypoid (1%).  In this study, among 100 cases of upper GIT lesions, 65(65%) were malignant and 35(35%) were benign. Diagnostic accuracy of imprint cytology of esophageal, gastric and duodenal lesions were 96.88%, 95.24% and 100% respectively which corresponds to or even better than some of the other studies.4,5,9,10,11 Histopathological evaluation of biopsies from 100 cases revealed that the most common lesion  was adenocarcinoma (35%) followed by squamous cell carcinoma(25%), chronic  non atrophic gastritis (24%), suggestive of adenocarcinoma(4%), chronic gastric ulcer (4%), chronic duodenitis (2%) and dysplasia (2%). Chronic esophagitis, hyperplastic polyp, gastric adenoma constituted  one case each.

Complete correlation between imprint cytological and histopathological diagnosis was obtained in 94(94%) cases and 6 cases were misinterpreted on cytological examination. One false negative case found in this study that was observed in gastric lesion. This false negative result may be due to subepithelial location of the lesion which was missed on imprint smear.  False negative diagnosis in imprint cytology was also observed in subepithelial lesions studied by Asha M et al.10 On endoscopic examination, ulceroproliferative lesion was seen in two cases in the gastric antrum. In one case, imprint cytology smears showed anaplastic epithelial cells and a diagnosis of adenocarcinoma was made. Histopathological examination showed chronic non atrophic gastritis and did not reveal any evidence of malignancy. Other case was interpreted as suspicious of adenocarcinoma by imprint smear which was subsequently diagnosed as gastric adenoma with dysplastic change by histopathology. On endoscopic examination, an ulceroproliferative lesion was seen at the lower end of esophagus which was interpreted as suspicious of squamous cell carcinoma on imprint smear but histopathological examination revealed moderate dysplasia. In the present study, three false positive diagnoses were made by imprint cytology. False positive diagnosis was also reported by other researchers and they concluded that the false positive diagnosis was due to regenerative atypia.9,11

The highest correlation (100%) was observed between imprint cytology and histopathology in the diagnosis of squamous cell carcinoma, suggestive of adenocarcinoma, suggestive of squamous cell carcinoma, chronic duodenitis and chronic esophagitis.

The overall sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of imprint cytology in the diagnosis of upper gastrointestinal lesions were 98.46%, 91.42%, 95.52%, 96.97% and 96% respectively which are similar to other studies.4,9

 Conclusion

The findings of the present study indicate that imprint cytology is a reliable technique for rapid diagnosis of upper GIT lesions as it is simple, accurate and cost effective. Diagnosis can be provided within an hour of endoscopic procedure by the imprint smear technique. So, gastroenterologist or surgeon can take a therapeutic decision approximately one week earlier.

References

  1. Conrad R, Castelino-Prabhu S, Cobb C, Raza A. Role of cytopathology in the diagnosis and management of gastrointestinal tract cancers. Journal of Gastrointestinal Oncology 2012; 3: 285-98.
  2. Zhang XF, Huang CM, Lu HS et al. Surgical treatment and prognosis of gastric cancer in 2613 patients. World Journal of Gastroenterology 2004; 10: 3405-08.
  3. Batra M, Handa U, Mohan H, Sachdev A. A Comparison of cytohistologic techniques in diagnosis of gastroesophageal malignancy. Acta Cytologica 2008; 52: 77-82.
  4. Sanjeevreddy M, Kittur SK, Jakareddy RB, Patil SY, Yelikar BR. Role of imprint cytology in the diagnosis of upper gastrointestinal tract lesions. Indian Journal of Public Health Research and Development 2013; 4: 190-95.
  5. Sharma P, Misra V, Singh PV, Misra SP, Gupta SC. A correlative study of histology and imprint cytology in the diagnosis of gastrointestinal tract malignancies. Indian Journal of Pathology & Microbiology 1997;40:139-146.
  6. Muller HA. Imprint cytology, advantages and possibilities. Entrez Pub Med 1976; 94: 7-10. [Online]
  7. Lal N, Bhasin DK, Malik AK, Gupta NM, Singh K, Mehta SK. Optimal number of biopsy specimens in the diagnosis of ca esophagus. Gut 1992; 33: 724-26.
  8. Cubukçu A, Gönüllü NN, Kaçar SO, Alponat A, Paksoy N. Imprint cytology in the endoscopic diagnosis of gastrointestinal malignancies. Hepatogastroenterology 2002; 49: 198-200.
  9. Vijayanarasimha D, Mahadevappa A, Manjunath GV, Sunila R. Imprint cytology: A diagnostic aid in interpretation of upper gastrointestinal endoscopic biopsies. Journal of Digestive Endoscopy 2014; 5: 144-148.
  10. Asha M, Divya V, Manjunath GV, Sunila R. Application of imprint cytology in interpretation of esophageal biopsies. Journal of Evolution of Medical and Dental Sciences 2013; 2: 4350-7.
  11. Mysorekar VV, Dandekar CP, Satyaprakash BS, Sarkar A. Role of imprint cytology in the diagnosis of gastrointestinal tract malignancies. Indian Journal of Pathology & Microbiology 2003; 46: 37- 43.

 

Computed Tomogram Guided Fine-Needle Aspiration Cytology of Lung and Mediastinal Masses: A Study of 166 Cases

Computed Tomogram Guided Fine-Needle Aspiration Cytology of Lung and Mediastinal Masses: A Study of 166 Cases

*Alam MA,1 Islam MR,2 Haque MR, Nath SK4

 Abstract
Computed tomogram guided fine needle aspiration cytology (FNAC) is an important and useful investigation to differentiate between benign and malignant lesions of lung and mediastinum. To evaluate the lung and mediastinal masses and to analyze and compare the results with cytological findings, 166 patients were retrospectively studied who underwent CT guided FNAC over a period of January 2015 to December 2016. The study was carried out in patients who presented with respiratory symptoms with a localized lung and mediastinal masses which were confirmed by radiologically was sent for FNAC. 155 cases of lung masses and 11 cases of mediastinal mass were included in this study. Patients’ age ranged from 15 to 95 year and the male to female ratio was 4:1. Radiologically, out of 166 cases, 140 cases were diagnosed as malignant, 8 cases as benign and 18 cases as inflammatory lesions. Cytologically, 146 cases were diagnosed as malignant, 20 cases were benign inflammatory lesion. Most common lung malignancy was squamous cell carcinoma (72 cases) followed by adenocarcinoma (32 cases), small cell carcinoma (10 cases), large cell carcinoma (8 cases), 18 cases of lung metastasis were seen. Compared to biopsy, CT guided FNAC shortens the diagnostic interval and helps in differentiating lung malignancy into different cytopathological types which aids in proper management of the malignant lesion. Out of 11 mediastinal masses 6 cases were malignant lymphoma, 3 cases specific inflammatory lesions (tuberculosis) and 2 cases was non-specific inflammatory lesions.

[Journal of Histopathology and Cytopathology, 2018 Jan; 2 (1):19-22]

Keywords: Computed tomogram, Cytology, Guided FNAC, Lung mass, Mediastinal mass.

 

  1. *Dr. Md. Ashraful Alam, Associate Professor, Department of Pathology, Rangpur Medical College. drashraful09@gmail.com
  2. Md. Rezaul Islam, Senior Consultant, Radiology & Imazing, Sadar Hospital, Nilphamari.
  3. Md. Rashedul Haque, Associate Professor, Department of Biochemistry, Rangpur Medical College.
  4. Professor Swapan Kumar Nath, Department of Radiotherapy, Rangpur Medical College.

 

* For correspondence

 Introduction

A Computed tomography (CT) guided fine needle aspiration cytology (FNAC) is a well known modality for characterization of mediastinal masses. CT guided FNAC of lung lesions is a well established technique for the cytologic diagnosis of peripheral malignant lung lesions, with a reported diagnostic accuracy rate more than 93% and a sensitivity rate less than 95%.1,2 It has been used to differentiate mediastinal masses into benign, malignant and inflammatory types. Furthermore, its use has been extended in differentiating lung malignancy into different cytopathological types which aids in proper management of the malignant lesions. CT guided FNAC is widely recognized technique in indeterminate mass. It is a simple diagnostic method of relatively low cost, with negligible mortality and limited morbidity.3 The accuracy of CT guided FNAC for discriminating benign from malignant lesion has been recorded to vary from 64% to 97%.4 Several post procedural complications have been reported for CT guided FNAC such as pulmonary hemoptysis and pneumothorax. The risk for developing pneumothorax has been observed to be 22% – 45% due to high sensitivity of CT in detecting pneumothorax.5 Relative contraindications to image guided FNAC are severe chronic obstructive airway disease, bleeding diathesis, contra lateral pneumonectomy and pulmonary arterial hypertension.6 The purpose of our study is to evaluate the accuracy of CT and CT guided FNAC in differentiating and recording the pathological spectrum of the mediastinal and lung masses.

 Methods

This is a retrospective study conducted in a private medical college hospital at Rangpur and two private laboratories in Rangpur city from Janary 2015 to December 2016.The study was carried out in 166 patients who presented with lung and mediastinal mass attended to different physicians and Rangpur Medical College Hospital and were sent for Fine needle aspiration cytology. Relevant clinical history and investigations were obtained from the patient to narrow down the differential diagnosis and to decide if patient was eligible for FNAC, such as history of bleeding diathesis, thrombocytopenia, dyspnea, uncontrolled cough, other feature of chronic obstructive airway diseases (COPD), pulmonary arterial hypertension etc. CT guided FNAC was performed in patients with peripheral lung and mediastinal mass or masses which were only approachable by spinal needle. Patient inclusion criteria included: cooperative patient who was able to hold breath for a short while, no bleeding tendency, patient who was to undergo chemo or radio-therapy and lesions not approachable by USG. Informed and written consent was taken from the patient explaining the risk and benefits of the procedure. Axial section of the area of interest was taken after a scanogram. A feasible approach was judged and the patient positioned accordingly with radiopaque marker placed at the site of puncture. Then under all aseptic precaution aspiration done by 21-22 G spinal needle and 10 cc disposable syringe and smear was prepared in glass slide for fixation in 95% alcohol. Routine Papaniculau stain were done in all cases.

 Results

The data were collected from January 2015 to December 2016. Our study included 166 patients, out of which 155 with lung and 11 with mediastinal mass were subjected to CT guided FNAC. Their ages ranged from 15 to 95 years with mean age of 65 years (Table I). The male to female ratio was 4:1. Out of 155 lung  malignant cases squamous cell carcinoma (Fig 1, 72 cases) was the commonest followed by adenocarcinoma ( fig 2, 32 cases), 10 cases of small cell carcinoma, 8 cases of large cell carcinoma were seen. Out of 18 cases of metastatic tumors, 10 cases were from gastrointestinal tract, 2 cases from testis and 6 cases from thyroid follicular carcinoma (Table IV). Out of 15 inflammatory cases 7 cases was specific inflammatory (tuberculosis) 8 cases was non- specific inflammatory lesion was observed. (Table IV). Out of 11 mediastinal masses 6 cases were malignant lymphoma,3 cases ware specific inflammatory lesions(tuberculosis) and 2 cases were non-specific inflammation was observed (Table V).

Table I: Age distribution (n=166)

Age Groups
(Years)
Male Feamle Total %
15-25 6 3 9 5.42%
26-35 5 3 8 4.81%
36-45 13 4 17 10.42%
46-55 24 13 37 22.28%
56-65 30 12 42 25.30%
66-75 34 3 37 22.28%
>75 15 1 16 9.6%

 

Table II: Sites of the lesions (n=166)

Sites No %
Pulmonary 155 93.37
Mediastinal 11 6.62

Table III: Lung lesions by site and sex

 

Sex Site
Right Lung Left lung Total
Male 102(76.69%) 31(18.67%) 133(80.12%)
Female 20(60.60% 13(39.39%) 33(19.87%)
Total 122(73.49%) 44(26.50%) 166

 

Table IV: CT guided FNAC diagnosis of intrathoracic and mediastinal masses (n=166)

 

Cytological Findings: No(%)
Squamous cell carcinoma 72(43%)
Adenocarcinoma 32(19.27%)
Small cell carcinoma 10(6.02%)
Large cell carcinoma 8(4.81%)
Metastatic carcinoma 18(10.84%
Malignant Lymhoma 6(3.61%)
Specific Inflammatory lesions(TB) 10(6.02%)
Non specific inflammatory lesion 10(6.02%)

 

Table V: CT guided FNAC diagnosis of mediastinal masses (n=11)

 

Cytological Findings: No (%)
Malignant lymphoma 6(54.54%)
Specific Inflammatory lesion(TB) 3 (27.27%)
Non-specific inflammatory lesion 2(18.18%)

TB=Tuberculosis

 

 

 

 

 

 

Figure 1. Photomicrograph of sqamous cell carcinoma of lung (Cytopathology)

 

 

 

 

 

Figure 2. Photomicrograph of adeno carcinoma of lung (Cytopathology)

 Discussion

CT guided transthoracic FNAC is a safe and accurate means of diagnosing benign and malignant intrathoracic lesions. In this study, Out of 166 patients 5.42% were in the age group from 15-25 years and 25.30%were in the age group of 56-65 years which is not similar with the study of Sarker RN et. al 7 who found patients of intra-thoracic mass 36% in the age group ranging from 46-55 years and 21% in the age group of 56-65 years, these two groups were predominant in terms of age. There were 133  male (80.12%) and 33 female (19.67%). In the study of Sarker RN et. al7 out of 100 cases there were 77 men (77%) and 23 (23%) were women. This correlates with the well-known fact that intrathoracic mass occurs most commonly in older age group and in males than in females. Female cases are less because malignant pulmonary lesions are less in females in our population. Male: Female ratio was 4:1 in our study. That is similar to the study done by Ahmed et al.8 The locations of the pulmonary lesions were in right lung 122 (73.49%), and 44 (26.50%) in left lung. In the study of Ahamad et al8 lesion in right lung was 98 (60.49%), in left lung 64 (39.41). In the final diagnosis, squamous cell carcinoma was the commonest malignant tumour followed by adenocarcinoma and metastatatic carcinoma. These findings are similar to the findings of the study done by Mostafa et al9 although his study was not guided by CT and the number of cases was less. Our experience is similar to the study of Singh et al10 where fatal complications like tension pnemothorax, air embolism, endo bronchial haemmorhage etc were absent. The complication rate depends on the distance of the lesion from pleura and lesion size. The more the amount of the lung tissue traversed by the needle the more was the complication rate and smaller the lesion the more was the complication rate. In this study fine needle of 21- 22G was used where the chance of complication seems to be minimum which correlates well with the study of Zavala et al.11  Saha A et al12 in their series have reported cases of mediastinal masses, 3 (5.6%) cases was NHL and (1cases) was Hodgkin’s lymphoma.In our study 6 cases (54.54%) was malignant lymphoma. This discrimination may be due to total number of cases.

Conclusion

This study concludes that CT guided lung and mediastinium needle aspiration cytology by spinal needle is a highly effective procedure in the diagnosis and sub- classification of mass lesions. It is a relatively simple, cost effective procedure with good patient compliance and low morbidity. The use of CT-guided FNAC of intrathoracic mass lesions reduces the diagnostic interval and cost. It also avoids unnecessary thoracotomy for diagnostic purposes. As the facilities continue to improve; it is likely to have a greater role in the initial evaluation of intrathoracic and mediastinal mass in the near future.

 References

  1. Wallace MJ, Krishnamurthy S, Broemeling LD, Gupta S,Ahrar K, Morello FA Jr, et al. CT-guided percutaneous fine needle aspiration biopsy of small (<1 cm) pulmonary lesions. Radiology 2002; 225:823-8.
  2. Laurent F, Latrabe V, Vergier B, Mountadon M, MernejouxJM, Dubrez J. CT-guided transthoracic needle biopsy of pulmonary nodules smaller than 20 mm: results with an automated 20-gauge coaxial cutting needle. Clin Radiol 2000; 55:281-7.
  3. Santambrogio L, Nosotti M, Bellaviti N et al. CT Guided Fine Needle Aspiration Cytology of Solitary Pulmonary Nodules. Chest1997; 112:423-5.
  4. Mohammad GM. CT guided fine needle aspiration cytology in diagnosis of thoracic lesions. JIMA 2001; 99(10):1-5.
  5. Herman PG, Hessel SJ. The diagnostic accuracy and complications of closed lung biopsies. Radiology 1977; 125:11-4.
  6. Hensell DM: Interventional techniques. In Armstrong P,Wilson AG, Dee P, et al (eds): Imaging Of Diseases Of TheChest. 2nd ed . St. louis, Mosby, 1995, p. 894-912.
  7. Sarker RN, Rabbi AF, Hossain A, Quddus MA, Chowdhury N, Sarker T. Computed tomography guided transthoracic fine needle aspiration cytology in the diagnosis of Sonographically non-approachable intrathoracic masses-A study of 100 cases.J Dhaka Med Coll 2011; 20(1):25-31.
  8. Ahmed S, Ahamad M S U. Computed tomography guided fine needle aspiration cytology of lung lesions: A study of162 cases. JCMCTA 2009; 20 (1):50-2.
  9. Mostafa MG. Computed tomographic guided fine needle aspiration cytology in the diagnosis of thoracic lesions. J Indian Med Assoc 2001; 99(10): 550-3.
  10. Singh JP, Garg L, Setia V. Computed tomography (CT) guided transthoracic needle aspiration cytology in difficult thoracic mass lesions – not approachable by USG. IJRI, 2004; 14(4):395-400.
  11. Zavala DC, Bedell GN. Transthoraciclung biopsy with a cutting needle. Am Rev Respir Dis 1972; 106: 186-93.
  12. Saha A, Kumar K, Choudhuri M K. Computed tomography – guided fi ne needle aspiration cytology of thoracic mass lesions: A study of 57 cases. J cytol 2009; 26 (2):55-9.

 

Evaluation of Space Occupying Lesion of Liver by Fine Needle Aspiration Cytology and Cell Block Examination

Evaluation of Space Occupying Lesion of Liver by Fine Needle Aspiration Cytology and Cell Block Examination

*Sultana SS,1Dewan RK,2 Ferdousi F,3 Sarker R,4 Jinnah SA,5 Jeba R,6 Haque N,7 Hussain M8

 To differentiate between benign from  malignant  tumor and hepatocellular carcinoma from metastatic carcinoma in hepatic space occupying lesion on the basis of cytology and cell block examination this study was done. This was a descriptive cross sectional study comprising of 48 cases, carried out at the department of pathology, Dhaka Medical College during the period of July 2013 to June 2015. Results of all patients were collected and tabulated. Statistical analysis was performed  on tabulated data. Out of 48 cases, the cytological diagnosis  revealed the highest number of cases of hepatocellular carcinoma 22(45.8%), followed by metastatic carcinoma 13(27.1%), abscess 6(12.5%), hepatocellular dysplasia 3(6.3%) and negative for malignant cell 4(8.3%). Of the total 42 cases of space occupying lesion evaluated by cytology, the diagnoses were similar in cell block, the another six cases contains necrotic debris and cytologically proved as abscess. This measure of agreement is statistically significant with substantial agreement between cell block and cytology status in evaluation of space occupying lesion in liver. Fine needle aspiration cytology in case of space occupying lesion of liver can be relied upon to differentiate between benign and malignant lesion and also primary from secondary lesion. Simultaneous preparation of cell block give no hazard to the patient but provide maximum benefit.

[Journal of Histopathology and Cytopathology, 2018 Jan; 2 (1):11-18]

 Key words: Liver, Lesions, cytology, Cell block

  1. *Dr. Sk Salowa Sultana, Assistant Professor, Department of Pathology, Ad-Din Women’s Medical College, Dhaka. salowasultana257@gmail.com
  2. Professor Dr. Rezaul Karim Dewan, Professor & Head, Department of Pathology, Dhaka Medical College, Dhaka
  3. Farjana Ferdousi Lecturer, Department of Cytopathology, National Institute of Cancer Research & Hospital.
  4. Rabindranath Sarker, Associate Professor, Department of Radiology and Imaging, Dhaka Medical College. Dhaka
  5. Shahed Ali Jinnah, Associate Professor, Department of Pathology, Dhaka Medical College, Dhaka
  6. Ruksana Jeba, Associate Professor, Department of Pathology, Dhaka Medical College, Dhaka
  7. Najmul Haque, Former Associate Professor, Department of Pathology, Dhaka Medical College, Dhaka
  8. Professor Dr. Maleeha Hussain, Former Professor & Head, Department of Pathology, Dhaka Medical College, Dhaka.

 

*For correspondence

 Introduction

Liver diseases are common health problem throughout the world. Liver diseases are broadly categorized as diffuse and focal lesion. The differential diagnosis of focal lesions are primary liver tumors (benign and malignant), metastatic deposits, congenital and acquired cysts and abscess.1 Appropriate clinical management depends on accurate diagnosis but evaluation of the lesion is a common clinical problem.2 Imaging techniques and serological markers are useful in narrowing the differential diagnosis. Fine needle aspiration cytology (FNAC) mainly indicated in the diagnosis of malignant focal lesions both primary and secondary. FNAC also performed to rule out neoplasm from inflammatory lesion when radiologically inconclusive.3-6

Hepatocellular carcinoma (HCC) is responsible for a large proportion of cancer death worldwide. Also there are demographic variation in the incidence of HCC.7 GLOBOCAN global analysis published moderately high incidence (11-20 per 100,000) in Southeast Asia and also shows 82% of liver cancer cases occurring in developing countries. HCC is preceded by cirrhosis of the liver in most cases. The majority of them are due to viral hepatitis. Indeed, worldwide 50-80% of HCC is due to HBV and (10-25)% of cases are due to HCV infection respectively.8 Dual infection with HBV and  HCV is not uncommon in southeast Asia.9 Other causes include alcoholic liver disease, nonalcoholic steatohepatitis, intake of aflatoxin contaminated foods, diabetes and obesity.10

Liver is the most common site of distant metastasis as it filters most of the blood from the body.11 Metastasis commonly arise from tumor of colon, pancreas, breast and lung. Accurate diagnosis of the metastatic lesions is essential in determining the stage of tumor and also for therapeutic and prognostic purposes.  The  treatment vary  from  palliative care to partial hepatectomy, specially in those which are potentially chemosensitive or hormonally manipulable. Correlation of clinical, laboratory and radiologic findings is necessary. Radiologically multiple nodules of various sizes distributed randomly suggest metastases5.

The present study was done to evaluate the space occupying lesion of liver by fine needle aspiration cytology accompanying with cell block examination. There are some pitfalls in cytology associated with aspiration of necrotic material and presence of  regenerative atypia in hepatocytes. Some of these pitfall can be minimized by using cell block.  In cell block, architectural pattern,  thickening of cell plate and traversing blood vessels with their lining endothelial cells can be seen. Simultaneous preparation of cellblock  from the residual material after smear preparation can help to evaluate the difficult cases.12

 Methods

This is a descriptive cross sectional study which was carried out at the department of pathology, Dhaka Medical College, during the period of July, 2013 to June, 2015. The study was done on fine needle aspiration material of liver SOL that were received from Dhaka Medical College and Hospital, and Bangabandhu Sheikh Mujib Medical University (BSMMU).

Patients with radiologically diagnosed SOL in liver and suspected as a case of hepatic neoplasm were included in this study. Patient with bleeding disorders, prolonged prothrombin time,  Patient with liver abscess, cyst, haemangioma and already diagnosed cases were excluded from this study.

Relevant clinical informations were recorded. Patients having suspected hepatic neoplasm with good coagulation profile underwent ultrasound guided FNAC. According to standard protocol FNAC was done by an expert pathologist or radiologist and USG  guidance was provided by an expert radiologist.

Several cytologic smears were prepared and fixed immediately in 95% alcohol. The smears were left in alcohol at least for 30 minutes at room temperature before staining. The residual material remaining after completion of cytological smears  were fixed in 10% formalin and later processed to prepare paraffin embedded blocks .

 Smears prepared were stained by papanicolaou stain. Cell blocks were prepared by fixed sediment and bacterial agar method and stained by Hematoxyllin and eosin. Cytopathological examination of the stained slides of hepatic SOL were carried out under light microscope on the same day or next day. Satisfactory smears contained adequate number of representative cells from the target sites. Stained cell block sections were examined to compare  the cytological diagnosis.

Results

Table I shows age of the study patients, half of the patients belonged to age 51-70 years. The mean age was found 53.0±15.0 years with range from 18 to 90 years.

It was observed that three fourth (36, 75.0%) patients were male and 12(25.0%) patients were female. Male female ratio was 3:1.

Table II shows cytological diagnosis of the study patients. It was observed that almost half of the patients (45.8%) were found HCC followed by 13(27.1%) were metastasic Ca, 6(12.5%) were abscess and 3(6.3%) were hepatocellular dysplasia .

Table III shows presenting complaints of the study patients. Total 29 patients present with abdominal pain only. Nine patients presented with abdominal lump and  pain.

Table IV shows 22 patients were cytologicaliy  diagnosed as HCC. Among them 11(50.0%) were HBsAg positive and  2(9.1%)  were Anti HCV positive. No case was dual positive. In case of, 42 patients the tumor size was >3 cm, among them 2(66.7%) patients cytologically diagnosed as hepatocellular dysplasia, 22(100.0%)  HCC, 13(100.0%) metastatic carcinoma and 5(83.3%)  abscess. Total 28 patients presented with multiple SOL. Among them 10 (45.5%)  were HCC and 11(84.6%)  were metastatic carcinoma.

The association between cell block and cytology status in evaluation of space occupying lesion of liver is given in table VI. Of the total 42 cases of space occupying lesion evaluated by cytology, the diagnosis of 3(75.0%) negative for malignant cell, 2(66.7%) hepatocellular dysplasia, 20(90.9%) HCC and 8(61.5%) metastatic tumour were also similar by cell block. The results of the two methods (cell block and cytology status) analysis found Kappa value = 0.680 with p<0.05. This measure of agreement is statistically significant with substantial agreement between cell block and cytology status in evaluation of space occupying lesion in liver. One of the three cytologically diagnosed hepatocellular dysplasia one was finally proved as HCC after cell block examination.

 Table I: Distribution of the study patients by age (n=48)

 

Age (years) Number of patients Percentage
≤30 4 8.3
31-50 17 35.4
51-70 24 50.0
>70 3 6.3
Mean±SD 53.0 ±15.0
Range (min-max) 18 -90

 

Table II: Distribution of the study patients by cytology (n=48)

 

Cytology Number of patients %
Negative 4 8.3
HD 3 6.3
HCC 22 45.8
Abscess 6 12.5
MetastasicCa 13 27.1
        Adenocarcinoma 9 18.8
        Sq. CC 1 2.1
        RCC 1 2.1
        GIST 1 2.1
        Small cell Ca 1 2.1

 

Table I11: Distribution of the study patient with different  cytological diagnosis according to  clinical feature (n=48)

 

Clinical feature Cytological diagnosis
Negative

(n=4)

HD

(n=3)

HCC

(n=22)

Metastatic Ca

(n=13)

Abscess

(n=6)

 

n % N % n % n % n %
Abd lump only 0 0.0 0 0.0 0 0.0 3 23.1 0 0.0
Abd pain only 3 75.0 1 33.3 15 68.2 5 38.5 5 83.3
Lump + pain 0 0.0 1 33.3 7 31.8 1 7.7 0 0.0
Pain + ascitis 0 0.0 0 0.0 0 0.0 1 7.7 0 0.0

 

Table IV: Distribution of cytologically diagnosed cases  with viral marker (n=48)

 

Viral marker Cytologcal diagnosis
Negative

(n=4)

HD

(n=3)

HCC

(n=22)

MetastasicCa

(n=13)

Abscess

(n=6)

n % n % N % N % n %
HBsAg
Positive 1 25.0 0 0.0 11 50.0 0 0.0 0 0.0
Negative 3 75.0 3 100.0 11 50.0 13 100.0 6 100.0
Anti HCV
Positive 0 0.0 0 0.0 2 9.1 0 0.0 0 0.0
Negative 4 100.0 3 100.0 20 90.9 13 100.0 6 100.0

 

Table V: Association between cytology status with USG finding (n=48)

 

USG finding Cytologycal diagnosis P value
Negative

(n=4)

HD

(n=3)

HCC

(n=22)

Metastatic Ca

(n=13)

Abscess

(n=6)

 

 

n % N % n % N % n %
Size
        ≤3 cm 4 100.0 1 33.3 0 0.0 0 0.0 1 16.7 0.001s
        >3 cm 0 0.0 2 66.7 22 100.0 13 100.0 5 83.3
SOL
        Single 1 25.0 1 33.3 12 54.5 2 15.4 4 66.7 0.122ns
        Multiple 3 75.0 2 66.7 10 45.5 11 84.6 2 33.3
Diagnosis
        Primary 0 0.0 0 0.0 13 59.1 0 0.0 0 0.0
        Secondary 3 75.0 2 66.7 6 27.3 12 92.3 0 0.0 0.001s
        Not diagnosed 1 25.0 1 33.3 3 13.6 1 7.7 6 100.0

 

Table VI: Association between cytological diagnosis with cell block (n=42)

 

Cell block Cytological diagnosis
Negative

(n=4)

Hepatocellular dysplasia

(n=3)

HCC

(n=22)

Metastatic Tumour

(n=13)

Inconclusive
n % N % n % n %
Negative for malignah 3 75.0 0 0.0 0 0.0 0 0.0 0
Hepatocellular dysplasia 0 0.0 2 66.7 0 0.0 0 0.0 0
HCC 0 0.0 1 33.3 20 90.9 0 0.0 0
Metastatic tumour 0 0.0 0 0.0 0 0.0 8 61.5 0
Inconclusive 1 25.0 0 0.0 2 9.1 5 38.5 0

 

 

 

 

 

Figure 1. Sex distribution of the study patients

 

 

 

 

 

 

Figure 2. Photomicrograph showing hepatocellular carcinoma (Cytology, Pap stain x40)

 

 

 

 

 

 

Figure 3. Photomicrograph showing hepatocellular carcinoma (Cell Block, H&E stain x20)

 

 

 

 

 

 

 

Figure 4. Photomicrograph showing hepatocellular carcinoma (Cell Block, H&E stain x10)

 

 

 

 

 

Figure 5. Photomicrograph showing adenocarcinoma (Cell Block, H&E stain x40)

 

 

 

 

 

 

Figure 6. Photomicrograph showing adenocarcinoma (Cytology, H&E stain x20)

 Discussion

Liver diseases particularly neoplasia  form focal lesion and are often asymptomatic. Even relevant biochemical tests may not show significant changes.13 Diagnosis and management of space occupying lesions in liver is a great challenge. The present study was carried out to evaluate the space occupying lesion of liver by USG guided FNAC.

The mean age of this study cases with hepatic SOL was forth to fifth decades. In Bangladesh Rahman et al (2014)14 showed similar observations. Similar findings were also observed by Nasit et al (2013)3 in India and Nazir et al (2010)4 in Pakistan. In this study, the incidence was more in male than female. According to World Factbook 2014, the finding is similar. In Asia socioeconomic condition and lifestyle favour exposure to hepatitis more in male than female. Risk factors for HBV and HCV infection such as transfusion related spread, sharing  of needle and syringes which is common for drug abuser, unprotected sex etc are more prevalent in male.18

Common clinical features were abdominal pain, lump and other constitutional symptom. Most of the patients with HCC presented with only abdominal pain. In metastatic group abdominal pain was frequently accompanied by lump in abdomen. Most of the patients with abscess presented with the complain of abdominal pain.  Similar observations regarding the clinical presentations were observed in the study done by Nasit et al (2013)3 and Hossain et al (2010).15

Viral marker was significantly positive in patients with hepatic malignancy. Half of the patients with HCC were HBsAg positive and 9% were anti-HCV positive in this study. Rahman et al (2014)13 and Rahman et al (2010)15 also observed similar findings in Bangladesh.

In present study, most of the cytological diagnosis cases of metastatic carcinoma  had multiple SOL in USG. Similar findings regarding distribution of cases according to cytological diagnosis was observed by Mohammed et al (2013)18 and Najir et al (2010). Hepatocellular carcinoma were more than metastatic neoplasm. Also metastatic cases include mostly adenocarcinoma. Similar findings regarding distribution of cases according to cytological diagnosis was observed by Mohammed et al (2013)15 and Najir et al (2010).4 To evaluate the accuracy of USG guided FNAC in hepatic SOL, the cytological diagnosis was compared with cell block.  Dysplastic nodules are precursor lesions of HCC and need careful evaluation. In such difficult cases cell block can help in examining architectural pattern as well as ancillary studies. Sometimes cirrhosis, progressing to HCC may have SOL. Cytology of cirrhosis may reveal pleomorphism, multinucleation, stippled cytoplasm and mimic HCC. Cell block in these cases show  hepatocytes and bile duct epithelial cells in monolayered sheets.  Other pitfall of FNAC related to  the diagnosis of well differentiated HCC and poorly differentiated HCC to be distinguished. Architectural pattern, thickening of cell plate, lining and traversing endothelial cell can determine and differentiate in such cases.18 Thus, cell block in addition to smears improve the diagnostic performance and decrease the non diagnostic result.

Conclusion

Treatment modalities are rapidly developing worldwide. Long term survival requires detection of small tumors. The patients with chronic liver diseases and other known primary need regular and proper evaluation. FNAC is a safe, minimum invasive procedure and multiple sample can be obtained with the small diameter needle. FNA cytology in case of SOL of liver can be  relied upon to differentiate between benign and malignant lesion and also from primary from secondary. However the indeterminate or  inconclusive report is a pitfall , which needs to be minimized. The result can be improved considering with availablity  of cell block examination. To get maximum benefit combined approach of FNAC and cell block   can be applied.

References

  1. Leiman G, Liver and Spleen. In: Ovell SR, Stennet GF, Whitaker D, editors. Fine needle aspiration cytology. 3rd New Delhi: Churchil Livingstone; 2003. pp 293-316.
  2. Swamy M. Arathi CA and Kodardaswamy CR. Value of ultrasonography-guided fine needle aspiration cytology in the investigative sequence of hepatic lesions with an emphasis on hepatocellular carcinoma. J. Cytol, 2011, 28(4): 178-184.
  3. Nasit J, Patel V, Parikh B, Shah M, Davara K. Fine-needle aspiration cytology & biopsy in hepatic masses: A minimally invasive diagnostic approach. Clin cancer investig J 2013; 2: 132-42.
  4. Nazir R, Sharif M, Iqbal M and Amin M. Diagnostic Accuracy of Fine Needle Aspiration Cytology in Hepatic Tumors. J of the college of Physicians & surgeons Pakistan 2010; 20(6): 373-376.
  5. Conrad R, Castelino-Prabhu S, Cobb C, Raza A. Cytopathologic diagnosis of liver Mass lesions. J of Gastrointestinal Oncology, 2013; 4(1): 53-61.
  6. Asghar F &Riaz S. Diagnostic Accuracy of percutaneous cytodiagnosis of Hepatic Masses by Ultrasound guided Fine Needle Aspiration Cytology. 2010, ANNALS,16:184-188.
  7. Ng J. Wuu J. Hepatitis B related and Hepatitis C related Hepatocellular Carcinoma in the United States; Similarities and Differences Hepat Mon, 2012, 7635.
  8. Venook AP, Papandreou C, Furuse J, Guevara L.L.D. The incidence and epitemiology of Hepatocellular carcinoma: A Global and Regional Perspective. The Oncologist, 2010; 15 (Suppl 4):5–13.
  9. Liu Z, Hou J. Hepatitis B Virus (HBV) and Hepatitis C Virus (Hq) Dual infection. Int J. Med Sci 2006; 3(2):57–62.
  10. Sanyal AJ, Yoon SK and Lencioni R. The Etiology of Hepatocellular Carcinoma and Consequences for Treatment. The Oncologist, 2010; 15 (suppl 4) 14–22.
  11. Haque S, Dilawar A and Subzwari J. Ultrasound Guided Fine-Needle Aspiration Biopsy of Metastatic Liver Disease: A Comparative Assessment of Histological & Cytological Techniques. 2012; 28:49-55.
  12. Nathan N, Narayan E, Smith M and Muuray J Cell Block Cytology: Improved preparation and its Efficacy in Diagnostic Cytology. American Society of Clinical Pathologists, 2000; 114:599-606.
  13. Evaluation of focal Liver Lesions by Ultrasound as a Prime Imaging Modality. Scholars Journal of Applied Medical Science (SJAMS), 2013; 6(6):1041-1059.
  14. Rahman F. Role of Cytology .Cell block and Immunohistochemistry in Differentiating Hepatocellular Carcinoma from MetastaticTumors in Liver.Unpublished MD thesis, 2014; BSMMU, Bangladesh.
  15. Hossain M. Characterization of Focal liver mass by computed Tomography scan with Cytopathological Correlation. Unpublished MD thesis, 2010; BSMMU, Bangladesh.
  16. Rahman AA. Prevalence of Primary HCC &Secondaries in liver in it attending Dept of Gastr&Hepatology in BSMMU. Unpublished MD thesis,2010; BSMMU, Bangladesh.
  17. Charles E, Ray MD, William S, Rilong MD (2006) Current Imaging Strategies of primary & Secondary Neoplasm of the liver. Sem: Int: Rad: Mar: 2006; 23(1):3-12.
  18. Mohammed AA Elsiddig S, Abdul Hamid M, Gasim G and Adam I. Ultrasound guided fine needle aspiration cytology and cell block in the diagnosis of focal liver lesions at Khartoum Hospital, Sudan. 2012;7:
  19. Barbhaiya M. Bhunia S, Kakka M, et al. Fine Needle Aspiration Cytology of lesions of liver and gallbladder: An analysis of 400 consecutive aspirations. J Cytol, 2014; 31(1): 20-24.

 

 

Significance of Glomerular PAS Positive Matrix Deposition in Glomerulonephritis

Significance of Glomerular PAS Positive Matrix Deposition in Glomerulonephritis

*Das RK,1 Kabir AN,2 Begum F,3 Khanam A,4 Kamal M5

Abstrat:

This study was done with the aim to calculate the glomerular PAS positive material quantitatively in glomerulonephritis and to find out its correlation with renal function. A total of 112 cases of glomerulonephritis were included in this study and morphometric measurements of the percentages of glomerular PAS positive areas were performed in a computer with a specially constructed grid. The mean of these values was scored from 1-4 according to the degree of PAS positive material deposition. With the increase of glomerular PAS positive score the levels of blood urea and serum creatinine also increased but the levels of creatinine clearance rate decreased. The percentages of glomerular PAS positive areas were correlated with renal functions expressed as levels of blood urea, serum creatinine and creatinine clearance rate. The degree of glomerular PAS positive matrix deposition showed strong positive correlation with levels of blood urea and serum creatinine (r=0.566, p<.0.001 and r=0.763, p=<0.001, respectively). Whereas creatinine clearance rate showed striking inverse relationship with PAS positive areas (r= – 0.612, p<0.001). Thus, the present study confirmed the previous results concerning a significant correlation between these parameters.

[Journal of Histopathology and Cytopathology, 2018 Jan; 2 (1):3-10]

Key words: Glomerulonephritis, PAS positive matrix, morphometry, renal function.

 

  1. *Dr. Ruhini Kumar Das, Associate Professor, Department of Pathology, M Abdur Rahim Medical College, Dinajpur. dr.ruhini@gmail.com
  2. AKM Nurul Kabir, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka.
  3. Ferdousy Begum, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka.
  4. Anisa Khanam, Associate Professor, Department of Pathology, United Hospital, Dhaka.
  5. Mohammed Kamal, Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka.

*For correspondence

Introduction
Chronic glomerulonephritis is one of the most important causes of chronic renal failure, which is the end result of a variety of human kidney diseases and is the major cause of death.1 It is clear that immune mechanism involved in most of the glomerulonephritis and initial target of injury is the glomerulus of kidney causing glomerular structural abnormalities. Other changes such as interstitial fibrosis, inflammatory infiltrate and tubular atrophy are secondary and nonspecific phenomenon, which are related to the degree of glomerular damage.2 Cell proliferation and deposition of different types of extracellular matrix components in the glomerular basement membrane (GBM) and/or in the mesangium characterizes the histology of glomerulus in glomerulonephritis.3 These changes in the glomerular basement membrane (GBM) and mesangiumare characterized by expansion of all the intrinsic extracellular matrix components due to increased production, decreased turnover or both of these components.4 Immunohistochemical techniques have demonstrated that glomerular extracellular matrix comprises laminin, fibronectin, entectin, heparansulfate, chondroitin sulfate, decorine and various types of collagen.2 Glomerular extracellular matriceses are PAS positive therefore the amount of  PAS positive material deposition can reflect the severity of glomerular injury and has been claimed important in  predicting renal function in glomerulonephritis.2 Series of histomormphometric studies established that the severity of renal failure in various types of glomerulopathy closely related with the severity of the tubulointerstitial changes, whereas the correlation with glomerular abnormalities is absent or weak.5-11 Critics of these studies were that the severity of interstitial abnormalities was evaluated by quantitative methods whereas the glomerular pathology was studied by semi-quantitative technique.2 Many investigators became puzzled by this apparent lack of correlation between glomerular abnormalities and renal failure although the glomerular pathology is the more important factor in glomerulonephritis and performed the quantitative study with particular interest to the deposition of glomerular PAS positive material. Some studies demonstrated that both glomerular and interstitial extracellular matrix accumulation contribute in impairment of kidney function. In the glomeruli PAS positive extracellular matrix deposition seems to be particularly important and correlate strongly with renal failure,2,4 whereas other failed to find such correlation.13 Therefore, the glomerular deposition of PAS positive matrix is reflected in renal failure has become a controvertial issue. This study was carried out with the aim to calculate the glomerular PAS positive material (% of total glomerular areas) by quantitative technique in glomerulonephritis and to find out its correlation with renal function to confirm this condrovertial issue.

 Methods

Patients
This study was carried out in the Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka. The renal tissues that were examined were collected for clinical purposes by percutaneous kidney biopsy by a surgical procedure. 112 patients of glomerulonephritis, where renal biopsy revealed variable degrees of increased mesangial cells, matrix, hyalinization and sclerosis were selected for this study. Renal tissues containing at least five glomeruli in haematoxylin & eosin stained sections were considered as adequate for evaluation. Blood urea, serum creatinine and creatinine clearance rate (Ccr) were done at the time of biopsy to assess the excretory function of kidney.

 Light Microscopy

Tissue for light microscopic examination was fixed in 10% formalin and processed routinely. Sections were cut 4-5 µm thickness from paraffin embedded material and stained by haematoxylin and eosin, periodic acid Schiff (PAS), Masson’s trichrome and methenamine silver (Jones) stains.

Immunofluorescence Microscopy

Renal biopsy specimens were embedded in O.C.T. compound, rapidly frozen, then sectioned at 5µm thickness in a cryostat at -200c.  The sections were stained with FITC conjugated rabbit anti-sera against human IgG, IgM, IgA, C3 and fibrinogen. The sections were then viewed under fluorescence microscope.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. A grid superimposed on a glomerulus in the biopsy of a patient with mesangiocapillary glomerulonephritis using Adobe illustrator 8.0. Total 48 points superimposed on glomerular area within Bowman’s capsule, 9 points superimposed on PAS positive area. The resulting PAS positive area was 18.8%.

Morphometry

Histomorphometry was performed by means of an image analysis system in a computer using Adobe Illustrator 8.0. Glomerular deposition of PAS positive material (% of total glomerular area) was measured quantitatively by morphometric examination of three to five glomeruli in PAS stained sections. Tangentially cut glomeruli were excluded. A section in which the apparent diameter was <50% of maximum diameter was defined as tangential section. The quantitative examination included the areas of glomerular deposition of PAS positive material of the total glomerular area. This was performed by means of a point counting method, which has been described as an accepted standard for histomorphometric evaluation. Colour microphotographs were taken by means of an Olympus digital camera (DP 11, Japan) which was attached to an Olympus microscope (BX51TF, Japan). The original magnification was 200 X. The colour microscopic images were transferred to a computer and saved in the memory. A grid was prepared in the computer using Adobe Illustrator 8.0. The space between the intersecting points of the grid was 6.25 mm. The total number of points of the grid was 169, and total area was 5625 sq.mm. The images of a glomerului were reduced to 20% of its original size and the grid projected over it (Fig. 1). The total glomerular areas were determined by counting the numbers of intersecting points superimposed on glomerular areas within Bowman’s capsule. The percentage of PAS positive material was represented by numbers of points falling on PAS positive material as percentage of total points falling on total structure within Bowman’s capsule. To minimize the error each glomerulus was measured three times by changing the position of grid. The mean of these values for each glomerulus was calculated and scored from 1- 4 according to degree of PAS positive areas (Table I).

 

Table I:  Scoring system of glomerular PAS positive material

Glomerular PAS positive area. Score
Up to 10% of the glomerular PAS positive area 1
>10-25% of the glomerular PAS positive area 2
>25-50% of the glomerular PAS positive area 3
>50% of the glomerular PAS positive area 4

 

Statistics

The correlations of glomerular PAS staining areas with renal function were determined with Pearson’s correlation method. Results were statistically significant if p <0.05.

 Results

Of the total 112 cases, 55 (49.1%) were mesangial proliferative glomerulonephritis (Mes PGN), 45 (40.2%) were mesangiocapillary glomerulonephritis (MCGN), 8 (7.1%) were IgA nephropathy (IgAN), 3 (2.7%) were membranous glomerulonephritis (MGN) and 1 (0.9%) was chronic glomerulonephritis (CGN). The glomerular PAS positive material ranged from 8.46% to 56.73% with a mean value of 20.71 ± 7.9%. The mean values of glomerular PAS positive areas in MesPGN, MCGN, IgA nephropathy, MGN and CGN were 19.46 ± 6.3%, 22.31 ± 8.1%, 15.69 ± 3.9%, 31.05 ± 22.3% and 27.04% respectively. PAS positive areas were scored from 1 to 4 (Table II).

 Table II: Score of glomerular PAS positive areas

 

Score PAS  positive area

(Mean ± SD)

No. of patients

n = 112

        1 9.14 ± 0.77% 4
        2 18.31 ± 4.05% 85
        3 29.38 ± 5.25% 21
        4 54.88 ± 2.61% 2

 Out of 112 cases estimation of blood urea was done in 107 cases, creatinine clearance ratevin 100 cases and serum creatinine in all of the cases. It was observed that with the increase of glomerular PAS positive score the levels of blood urea and serum creatinine also increased whereas the levels of Ccr decreased (Fig. 2). Glomerular PAS positive areas showed significantly strong positive correlation with the levels of blood urea and serum creatinine (r = 0.566, p <0.001 and r = 0.763, p <0.001, respectively), whereas with Ccr the relationship was significantly inverse (r = – 0.612, p < 0.001) (Fig. 3-5).

 

 

 

 

 

 

 

Figure 2. Result of renal function tests according to score of PAS positive areas

Figure 3. Correlation between glomerular PAS positive area and blood urea

 

 

 

 

Figure 4. Correlation between glomerular PAS positive area and serum creatinine

 

 

 

 

 

Figure 5. Correlation between glomerular PAS positive area and creatinine clearance rate

 Discussion

In many of the glomerulonephritis there is deposition of PAS positive extracellular matrix in GBM and mesangium and glomerular PAS positive areas correlate with renal function expressed as blood urea, serum creatinine and creatinine clearance.

With this aim Suzuki in 1994 performed a quantitative study on diabetic nephropathy. He stained glomerular mesangial deposition of various glycoproteins with PAS and measured the positive areas by an automatic image analyzer. He correlated the percentages of glomerular PAS positive areas with serum creatinine and found significant correlation.4

 

In 1997, Vleming and co-authors performed a quantitative study on various forms of glomerular diseases to find out the correlation of glomerular PAS positive areas with renal function. They found that the percentages of glomerular PAS positive areas correlated strongly with renal insufficiency expressed as serum creatinine.2

In view of the same aim Danilewicz and Wagrowska Danilewicz in 1997 carried out a quantitative study on three forms of proliferative glomerulonephritis MesPGN, MCGN and IgA nephropathy. They failed to demonstrate any significant correlation between glomerular PAS positive matrix deposition and serum creatinine. These investigators found that correlation between these parameters tended to be positive but have not reached statistical significance. They could not agree with the earlier findings of Vleming et al 1997 and stated that in their study patients biopsies contained a mixed bag of diagnosis. Therefore, this would require external validation of their findings in a cohort of patient with one disease.13

Vleming and co-authors in 1998 performed another quantitative study with a single disease entity for confirmation of their previous findings of significant positive correlation of glomerular PAS positive material with renal function. IgA nephropathy was selected and obtained significant correlation between glomerular PAS positive areas and creatinine clearance rate at the time of biopsy.14

The present study was performed to measure glomerular PAS positive material quantitatively and to determine whether this parameter correlates with renal function. It was observed that with the increase of PAS positive scores the level of blood urea and serum creatinine also proportionately increased whereas the level of creatinine clearance rate decreased. The glomerular PAS positive areas correlated strongly with the levels of blood urea, serum creatinine and Ccr. Our results correspond to the observation of Vleming et al. 1997, Vleming et al. 1998 and Suzuki 1994. The findings of present study only differ with the study of Danilewicz et al. 1997. They did not find significant correlation between PAS positive areas and serum creatinine in MesPGN, MCGN and IgAN. Total number of cases in their study was small and they have included only less severe cases of glomerulonephritis. This may be the possible cause of insignificant correlation in their study.

Conclusion

It was concluded that the severity of glomerular PAS positive matrix accumulation in glomerulonephritis correlated with the severity of renal insufficiency and our study confirmed the results previously obtained by different observers concerning significant correlation between these parameters.

Acknowledgment

We acknowledge Bangladesh Medical Research Council (BMRC) for their support by a research grant for this study under WHO Research Programme.

 References

  1. Cotran RS, Kumar V, Collins T. (Eds). Robbins Pathologic Basis of Disease, 6th Philadephia: WB Saunders, 1999: 931-64.
  2. Vleming LJ, Baelde JJ, Westendorp RGJ, Daha MR, Es van LA, Bruijn JA and Bruijn JA. The Glomerular deposition of PAS positive material correlates with renal function in human kidney diseases. Clin Nephrol, 1997; 47: 158-167.
  3. Glick AD, Jacobson HR, Haralson MA. Mesangial deposition of type 1 collagen in human glomerulosclerosis. Hum pathol, 1992; 23: 1373-7.
  4. Suzuki D. Measurement of the extracellular matrix in glomeruli from patients with diabetic nephropathy using an automatic image analyzer. Japanese Journal of Nephrology, 1994; 36 (11): 1209-1215.
  5. Risdon RA, Sloper JC, De Wardener HE. Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerulonephritis. Lancet, 1968; 2: 363-366.
  6. Schainuch LI, Striker GE, Cuter RE, Bendin EP. Structural-functional correlations in renal diseases. II. The correlations. Hum Pathol, 1970; 1:630-641.
  7. Bohle A, Mackensen-Heam S, von Gise H. Significance of tubulointerstitial changes in the renal cortex for the excretory function and concentration ability of the kidney. A morphometric comparison. Am J Nephrol, 1987; 7: 42-433.
  8. Mackensen-Heam S, Bohle A, Christensen J, Wehrmann M, Kendjiora H, Kokot F. The consequences for renal function of widening of the interstitium and changes in the tubular epithelium of the renal cortex and outer medulla in various renal diseases. Clin Nephrol, 1992; 37:70-77.
  9. Lane PH, Steffes MW, Fioretto P, Mauer M. Renal interstitial expansion in insulin-dependent diabetes mellitus. Kidney International, 1993; 43: 661-667.
  10. Steffes MW, Bilous RW, Sutherland DER, Mauer SM. Cells and matrix components of the glomerular mesangium in type I diabetes. Diabetes, 1992; 41:679-84.
  11. Mauer SM, Steffes MW, Ellis EN, Sutherland DE, Brown DM, Goetz FC. Structural-functional relationships in diabetic nephropathy. J Clin Invest, 1984; 74:1143-1155.
  12. Thomsen OF, Andersen AR, Christiansenis A and Deckert T. Renal changes in long-term type I (insulin-dependent) diabetic patients with and without clinical nephropathy: a light microscopic, morphometric study of autopsy material. Diabetilogica, 1984; 26: 361-365.
  13. Danilewicz M and Wagrowska- Danilewicz M. The consequences for renal function of the glomerular deposition of PAS positive material in proliferative glomerulopathies. A quantitative study. Gen Diag Pathol, 1997; 143: 225-230.
  14. Vleming LJ, Fijter DE JW, Westendorp RGJ, Daha MR, Bruijn JA and Es van LA,. Histomorphometric correlates of renal failure in IgA nephropathy. Clin Nephrol,1998;49:337-344.

 

Implementing Synoptic Reporting System in Bangladesh

Journal of Histopathology and Cytopathology
Volume 2 N 1
January 2018

Editorial

Implementing Synoptic Reporting System in Bangladesh

*Talukder SI

Histopathology report is a medical document written by histopathologist. In all types of biopsy sample of tumours such as excisional, incisional, punch, curettage and needle biopsies, traditional histopathology reports of formalin fixed paraffin embedded tissue sections is followed by microscopic examination of haematoxylin and eosin stained slides is a gold standard for tumour diagnosis. Histopathologists have to write name of the organ from which the biopsy has been taken, gross description of the specimen, microscopic description of the lesion mentioning invasion, margin involvement, lymph node metastasis, grades, and diagnosis.  It may also also contain an optional comment section that is used when diagnosis is inclusive or development of cancer is unclear. Histopathologist can mention inadequacy of sample and can recommend for other tests. With the exception of a few most of the practicing histopathologists in Bangladesh usually give histopathology reports in descriptive format. Most of the oncologists in this country manage cancer patients with these traditional descriptive histopathology reports.

Many developed countries are using synoptic (structured) histopathology reports along with traditional reports in the management of cancer patients.1 Synoptic report is applicable when entire or a part of an organ with malignant tumour is removed. This report lists the most important results in a structured format. It is a clinical documentation method that uses structured checklist to help clinicians to produce more complete, consistent and valuable medical reports.2 Synoptic reports are faster to produce and easier to interpret. It includes items or fields considered most important in determining patients’ treatment options and chance of recurrence. It is a clinical template that ensures that pathologists are always prompt to report on data that is critical for clinical decision making.

Some developed countries developed electronic synoptic report writing software to digitize their process, reducing reporting time and simplifying report distribution. Histopathologists can produce consistently complete reports, using configurable template under pinhead by interoperable code such as ICD-10. Because of their consistency and the ease of their distribution, electronic synoptic reports can be compiled and searched for quality assurance and clinical research.

In Bangladesh, those who use synaptic report is usually based on customized version of the college of American Pathologists (CAP) protocol. These reports therefore vary among themselves. Bangladesh Academy of Pathology (BAP) has already taken initiative to implement synoptic reporting in histopathology reporting of cancer patient when entire tissue is removed. BAP has recently arranged a workshop on synaptic report writing among the member of this organization. They decided that these types of workshops will be continued with participants including histopathologists and clinicians.

Adoption of standardized histopathology reporting formats that suits the best in the context of Bangladesh is the first step forward. We hope, synoptic reporting will be implemented soon in Bangladesh.

 *Dr. Sadequel Islam Talukder, Assistant Professor, Department of Pathology, Shaheed Syed Nazrul Islam Medical College, Kishoreganj. Associate Editor, Journal of Histopathology and Cytopathology. sadequel@yahoo.com

References

  1. College of American Pathologists. Resources & Publications: Cancer Protocols www.cap.org/cancerprotocols.
  2. Simplifying cancer reporting with eForm. http://www.cap.org/web/home/lab/proficiency-testing/cap-eFRM?_afrLoop=987349351061638#!%40%40%3F_afrLoop%3D987349351061638%26_adf.ctrl-state%3Djimk1r9yv_30

 

 

Information for Contributors

Journal of Histopathology and Cytopathology

Information for Contributors

 General Information
The Journal of Histopathology and Cytopathology (JHC) aims in our understanding of the pathophysiological and pathogenetic mechanisms of human disease by publishing  original papers, review articles, case reports and short communications related to basic and translational fields in pathology. It serves as  bridges between basic biomedical science and clinical medicine with particular emphasis on, but is not restricted to, tissue based studies only. It is published twice a year as the Journal Committee of the Bangladesh Academy of Pathology.

Manuscript Preparation.

Manuscripts should be prepared in MS Word format in accordance with The Uniform Requirements for Manuscripts Submitted to Biomedical Journals

(see http://www.icmje.org). All pages of the manuscript should be double-spaced and numbered consecutively beginning with the Title page.  Each of the following sections should begin on separate pages: Title,  Name and affiliation of authors, Abstract and Keywords, Text, Acknowledgements, References, individual Tables and legends.  Reformatting of the accepted papers may be needed according to the Journal specifications.

Title Page

The title page should include (i) type of publication (original, review, case report etc.) (ii) the complete title of the article (iii) authors’ name in abbreviation  (iv) list of authors including full name, highest degree, signature, designation and institutional affiliation and (v) name, mailing address, email and telephone/mobile number of author responsible for correspondence.

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It should begin with full title of the article. Do not write authors name in the abstract page. The abstracts should not be more than 200 words. The abstract should state the purpose of the study or investigations, basic procedures, main findings and principal conclusion. Three to ten keywords may be provided below the abstract using terms from the Medical Subject Headings (Index Medicus, NLM, USA). Abbreviations and citations should be avoided.

 Text

The text of the original articles should be divided into following sections: Introduction,

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 References

References to literature should be numbered in Arabic numerical in superscripts consecutively in the order in which they are mentioned in the text. At the end of article the full list of references should give the name of all authors followed by the title of the article, the title of the journal abbreviated according to Index Medicus, the year of publication, volume number and first and last pages of the article. Title of the books should be followed by the edition, place of publication, the publisher, the year and the relevant pages. Examples of correct form of reference are given below: References should begin on a new page, be double-spaced and numbered in order of citation in the text, including citations in tables and figure legends. Citations that first appear in tables, figures, or supplemental data should be numbered according to the item’s first call out in the text; a separate reference list should not be prepared for supplemental data. Complete author citation is required (use of “et al” is only acceptable for sources with more than 35 authors).

References should conform to the style of the Journal.

Examples follow:

Journals: van Riel D, Leijten LM, Kochs G, Osterhaus AD, Kuiken T: Decrease of Virus Receptors during Highly Pathogenic H5N1 Virus Infection in Humans and Other Mammals. Am J Pathol 2013, 183:1382-1389

Electronic Journals: Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group: Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 2009, 6:e1000097. http://dx.doi.org/10.1371/journal.pmed.1000097

Books: Frosch MP: Central Nervous System. Robbins Basic Pathology, 9th Edition. Edited by Kumar V, Abbas AK, Aster JC. Philadelphia, PA, Saunders, 2012, pp. 811-850

Product Inserts: Cite in text only: (Affymetrix technical note: Globin Reduction Protocol: A Method for Processing Whole Blood RNA Samples for Improved Array Results. Santa Clara, CA).

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Tables

Tables should be typed written on separate numbered pages submitted after the main text on separate pages, as part of the manuscript. The preferred file format for Tables is MS Word. and should follow the reference list. All tables should be numbered consecutively using Roman numerical. Each must carry a brief descriptive heading. Tables should be planned to fit within print area. Table footnotes should use the sequential symbols: *, †, ‡, §, ¶, ∥; and abbreviations.

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Figure file formats (including those embedded in the text) are unacceptable.

Photographs and photomicrographs should be of high resolution (minimum 5 mega pixels), in original unedited form and jpg format. These should contain a legend with magnification and stain used. Figure number and name of the first author should be mentioned in each file. Legend should be given in separate page.  Patients’ identification should be hidden.

Abbreviation

Standard abbreviation should be used whenever possible. The full term for which the abbreviation stands followed by abbreviation in parenthesis should be proceed the first use of the abbreviation in the text except for standard units of measurements like 27OC and 25 mmol/L etc.

Letters to the editor

Communications with reference to an article published in the journal and current health problems in the community will be accepted as letter to the editor.

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An electronic copy (soft copy) in the form of CD must be submitted with the printed copy of the article. Electronic copy may be send by email attachment at sadequel@yahoo.com.

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Manuscript Submission

Electronic version of the manuscripts should be submitted through email to the Executive Editor.  Alternatively send DVD/CD to: The Executive Editor of the Journal of Histopathology and Cytopathology.  A cover letter to the editor must accompany the manuscript stating any,  (a) conflicts of interest (both financial and personal), (b) that the manuscript has not been published previously and is not being considered concurrently by another publication, and (c) all authors and acknowledged contributors have read and approved the manuscript. Submissions are not considered for review if previously published in any form (print or online) other than as an abstract. The editor reserves the customary right to style and if necessary shorten the material accepted for publication and to determine the priority and time of publication. Editor assumes that work based on honest observations. It is not the task of the editor to investigate scientific fraud paper.

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On receipt, manuscripts are assessed by the Editor-in-Chief, to one Associate Editor. The Reviewers’ and Associate Editor’s views are used by the Editor-in-Chief (or a Senior Editor) in reaching a decision, usually within three weeks of submission.

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Authorship credit should be based only on 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; 3) final approval of the version to be published.

 A statement outlining the specific contribution of each author to the manuscript and the work reported in it must appear after the acknowledgements section (see below).

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Conflict of interest statements

Authors must disclose all financial and personal relationships that might bias their work; to prevent ambiguity, a conflict of interest statement must appear on the manuscript title page, detailing any conflicts (or the absence thereof) for each author.

 

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Concise articles make a greater impact than long ones and are less likely to be delayed by editing to a suitable length. Full articles should be no more than 4000 words from the beginning of the Introduction to the end of the Discussion. Review articles and special features may occasionally exceed this limit by arrangement with the Editor-in-Chief.

 

Abstract (not structured and no more than 300 words)

Following the title page(s), the next page should carry an unstructured prose abstract of 300 words or less. It should clearly convey the purposes of the study, and the main procedures, findings and conclusions. It should be understandable without reference to the rest of the paper, and contain no citation to references in the reference list. Only standard abbreviations as listed below are permitted.

 

Keywords (3 to 10)

Below the abstract, authors should provide and identify as such 3 to 10 keywords or short phrases to assist indexing the article and that may be published with the abstract. MESH headings are a useful guide for authors in considering keywords.

 

Manuscript structure

Research articles are divided into sections with the headings: Abstract, Introduction, Methods, Results and Discussion. Long articles may need subheadings (especially within the Results and Discussion) to clarify their content. The sections should not be numbered. Other types of articles, such as reviews and commentaries, still need a title and abstract and should adhere as closely as possible to these guidelines.

 

 

 

A Rare Case: Non-Syndromic 46,XX Testicular DSD with Derivative Autosome

A Rare Case: Non-Syndromic  46,XX Testicular DSD with  Derivative Autosome

*Banu SG,1 Habib S,2 Islam SS,3 Bhuiyan AZ4

Abstract

46,XX testicular disorder of sex development (DSD) is a known cause of male infertility. Derivative chromosomes formed by complex rearrangements and translocations between two or more autosomes are also found to play role in male and female infertility in different studies. Combinations of sex chromosomal DSD and derivative autosomes are rare and unique. We report a case of 46,XX testicular DSD with a derivative autosome formed by rearrangement between chromosomes 2 and 3. The person was phenotypically normal (non-syndromic) with only complaint of infertility.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):162-167]

Key words: 46, XX testicular DSD, derivative chromosome, infertility

 

  1. *Dr. Sultana Gulshana Banu, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka. sgbanu.bsmmu@gmail.com
  2. Saequa Habib, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka.
  3. SM Shahedul Islam, Scientific Officer, Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka.
  4. ARM Zakaria Bhuiyan, Resident, MD Pathology, Department of pathology,Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka.

*For correspondence

Introduction

Disorder of sex development (DSD) is a congenital condition where there is a disagreement between chromosomal, gonadal and phenotypic  sex.1 The discrepancies  among  these three determinants are highly variable and depend on patients’ cytogenetic and molecular abnormalities.2 The Chicago Consensus Nomenclature (2005) divides DSD into three broad categories: sex chromosomal DSD, 46,XY DSD and 46,XX DSD. Each of these has subclassifications with defined characteristics. Non-syndromic 46XX testicular DSD is a subclass of 46,XX DSD in which the person is phenotypically male and possesses testes.3 These individuals, though are deficient of a Y chromosome, have SRY (sex-determining region on Y) gene in one of the X chromosomes. This SRY gene induces development of testes that are, however, often smaller in size with impairment of spermatogenesis of varying degrees. As a result the persons are infertile though otherwise normal.4 The mechanisms underlying SRY-positive 46,XX testicular DSD are understood by multiple studies.5,6

A derivative chromosome (der) is a structurally rearranged chromosome generated either by a rearrangement involving two or more chromosomes, or by multiple aberrations within a single chromosome. The term always refers to the chromosome that has an intact centromere.7 The structural  changes involving autosomes, in different studies, are also seen to be associated with male and female infertility.8-11

We report a case of male infertility  with a combination of 46,XX testicular DSD and a derivative autosome formed by rearrangement between chromosomes 2 and 3. To our knowledge, no report has been published so far with this unique combination.

Case Report

A 30 year old male person and his 26 year old wife attended the Gynaecology Outdoor, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka with the complaint of infertility. On query, they revealed that they had been married for four years and had not been using any contraceptive. Their past and present medical history, family history and drug history were non-contributory. The wife was examined physically in the Gynaecology Outdoor and no abnormality was detected. She was advised an ultrasonography of pelvic organs, thyroid function tests, and assay of sex hormones (oestrogen, progesterone) and their trophic hormones (follicle stimulating hormone and luteinizing hormone). The husband was advised semen analysis, ultrasonography of the testes, serum levels of testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH).

The reports of investigations of the wife were normal. Those of the husband revealed azoospermia, small-sized testes with heterogenous echotexture on ultrasonography, low serum level of testosterone and high LH. His thyroid function tests, however, were normal. The couple was next advised karyotyping to find out chromosomal abnormality if any. They were referred to the department of Pathology, BSMMU.

The wife’s karyotype was found normal with normal autosomes and sex chromosomes (46,XX). The husband was found having abnormalities in both autosomes and sex chromosomes. His karyotype showed two X chromosomes and a large derivative chromosome which appeared a long chromosome 2 with attached extra portion to its long arm. The extra portion was recognized as the long arm of chromosome 3. The karyogram also showed single normal second and single normal third chromosome. The other chromosome 3 was clearly deficient of its long arm and consisted only of the short arm. We reported the karyotype as 46, XX + der(2)t(2;3) (qter;q). The man was advised a FISH (fluorescence in situ hybridization) to study the breakpoints of the involved chromosomes; however, he did not come for a follow-up and we could not get further information.

Figure 1. Photomicrograph of a chromosome spread showing a derivative autosome formed of chromosomes 2 and 3 (black arrow), one normal chromosome 2 and one normal chromosome 3 (white arrows). The spread also shows two X chromosomes (in ellipses) and short arm of the other chromosome 3 (star).

Figure 2. Karyogram of the study case: 46, XX, der(2)t(2;3) (qter;q).

Discussion

Male infertily can result from a variety of genetic, chromosomal, developmental, hormonal and other causes like infections. There are a number of genetic and chromosomal aberrations related to male infertility. Disorders of sex development (DSD) are a group of sex chromosomal aberrations commonly associated with male and female infertility. Though the DSD categories usually show varying degrees of genital and other phenotypic  abnormalities, the 46,XX testicular DSD subgroup males are sometimes phenotypically normal with mere complaints of infertility.4,5 On examination, however, these persons often show small testes and different levels of cryptorchidism. Impairment of spermatogenesis ranges from oligo-, through astheno- and teratozoospermia to complete azoospermia.5 The testicular development in these Y-deficient individuals is instructed by the SRY (sex-determining region on Y) gene present in the paternally derived X chromosome. The SRY gene which is normally located in Y chromosome, is generally misplaced on to the X chromosome in the affected person’s father during  formation of sperm cells. This occurs in a random fashion by an abnormal exchange of genetic material between the chromosomes (translocation).4,6

 

Among the structural chromosomal abnormalities, complex rearrangements involving sex chromosomes and/or autosomes in various combinations are seen. Derivative chromosomes formed by reciprocal translocations between autosomes are found associated with both male and female infertility.7,8 These are, however, rare in humans. Relatively commoner are rearrangements within Y chromosome (deletions, inversions, insertions) in males and within chromosome 9 in both males and females.9

Other reported patterns are very rare and appear unique events. Lauricella SA, et al (2016) found an infertile mosaic woman with a karyotype  45,XX,der(18)t(18;21)(p11;q21)-21/46,XX,t(18;21)(p11;q21). 86% of her cell lines showed 45,XX-21 pattern and only 14% showed 46,XX pattern containing the derivative (18 & 21) chromosome. This is explained by the marked instability of the derivative chromosomes which can be reduced in size or disappear during karyotype evolution. The authors also explained the patient’s infertility despite having two X chromosomes by the possibility of formation of a high risk offspring affected by an unbalanced chromosomal disorder (deletion or duplication of chromosomes 18 and 21) that might had been eliminated every time of attempted conception.

The patient of the present report is already an XX male; however, the unbalanced derivative (2 & 3) chromosome he possesses can be explained similarly in having a role in his infertility. Song SH, et al (2011) found impaired spermatogenesis ranging from oligo-, astheno-, teratozoospermia to complete azoospermia in their male subjects with complex chromosomal rearrangements (CCRs) of various combinations. Among their 10 cases, there were inversions within chromosome 3, complex translocations between chromosomes 2, 7 and 4, and between chromosomes 2, 19 and 22, and also other patterns. All these cases, however, possessed normal male sex chromosomes, that is 46, XY.

Other similar studies on autosomal rearrangements producing derivative autosomes also showed association with male infertility.10,11

Conclusion

Cases of male and female infertility are best investigated through a multidisciplinary approach involving cytogenetic, molecular, hormonal, histopathological and imaging studies. With cytogenetic study, rare sex chromosomal and autosomal rearrangements forming derivative chromosomes should be kept in mind for proper evaluation.

References

  1. Chan AOK, But WM, Lee CY, Lam YY. Aetiological bases of 46, XY disorders of sex development in the Hong Kong Chinese population. Hong Kong Med J. 2015; (21): 499-510.
  2. Tian L, Chen M, Peng JH, Jhang JW, Li L. Clinical characteristics, cytogenetic and molecular findings in patients with disorders of sex development. J Huazhong Univ Sci Technol [Med Sci]. 2014; 34(1): 81-86.
  3. Hughes IA. Disorders of sex development : a new definition and classification. Best Pract Res Clin Endocrinol 2008; 22(1): 119-134.
  4. Délot EC, Vilain EJ. Nonsyndromic 46, XX testicular disorders of sex development. 2003 Oct 30 [Updated 2015 May 7]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews [Internet], Seattle (WA): University of Washington, Seattle; 1993-2018. Available from: http://www.ncbi.nlm.nih.gov/book/NBK1416/
  5. Barseghyan H, Délot EC, Vilain EJ. New genomic technologies: an aid for diagnosis of disorders of sex development. Horm Metab Res. 2015; 47: 312-320.
  6. Grinspon RP, Rey RA. Disorders of sex development with testicular differentiation in SRY-negative 46, XX individuals: Clinical and genetic aspects. Sex Dev. 2016; 10: 1-11.
  7. Simons A, Shaffer LG, Hastings RJ. Cytogenetic nomenclature: Changes in the ISCN 2013 compared to the 2009 edition. Cytogenet Genome Res. 2013; 141: 1-6.
  8. Lauricella SA, Buse M, Cavani S, Cuttaia HC, Malacarne M, Mazara MV, et al. A rare complex structural chromosomal anomaly in mosaic due to the instability of a derivative chromosome 18 in a female infertile patient. J Down Syndr Chr Abnorm. 2016; 2: 110. Doi: 10.4172/2472-1115.1000110.
  9. Kim JW, Chang EM, Song SH, Park SH, Yoon TK, Shim SH. Complex chromosomal rearrangements in infertile males: complexity of rearrangements affects spermatogenesis. Fertil Steril. 2011; 95(1): 349-352.e5.
  10. Ergul E, Liehr T, Mrasek K, Sazci A. A de novo complex chromosome rearrangement involving three chromosomes (2,13 and 18) in an oligospermic male. (e9-e12) Fertil Steril. 2009; 92: 391.
  11. Coco R, Rahn MI, Estanga PG, Antonioli G, Solari AJ. A constitutional complex chromosomal rearrangement involving meiotic arrest in an azoospermic male: case report. Hum Reprod. 2004; 19: 2784-2790.

Paraneoplastic Pemphigus-Mixed Bullous Disease Type –Report of a Rare Blistering Condition with IgA Deposition

Paraneoplastic Pemphigus-Mixed Bullous Disease Type –Report of a Rare Blistering Condition with IgA Deposition

*Kabir AN,1 Kamal M,2  Das RK3

Abstract

A male patient of 55 years with generalized lymphadenopathy was diagnosed as Non-Hodgkin’s lymphoma. After treatment with four cycles of combined chemotherapy of Cyclophosphamide, Hydroxydaunorubicin, Oncovin and Prednisolon (CHOP) the patient developed blistering lesions all over the body. Histological examination of lesional skin showed both suprabasal and subepidermal bullae. Direct immunofluorescence (DIF) test of perilesional skin revealed linear deposition of IgA, C3 and fibrin along the basement membrane zone (BMZ); and deposition of IgG both in the epidermal intercellular substance and along BMZ. Indirect immunofluorescence (IIF) test using patient’s serum on normal human skin and Long-Evans rat urinary bladder showed linear deposition of circulating IgA along the BMZ. Protein electrophoresis on cellulose acetate membrane showed increased Gamma globulin fraction. This is a case of paraneoplastic pemphigus-mixed bullous disease type showing strong reactivity to IgA, which has not been described in literature.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):157-161]

Keywords: Pemphigus, paraneoplastic, mixed bullous disease

Introduction

The term ‘pemphigus’ refers to mucocutaneous diseases that are characterized by intraepithelial blisters, caused by a loss of normal cell-cell adhesion  (acantholysis), and are associated with autoantibodies against cell-surface proteins of stratified squamous epithelium.1 Blisters in patient with high titres of autoantibody with underlying neoplasms, most frequently lymphoma, are referred as paranneoplastic pemphigus. A case with clinical features, histologic and immunofluorescence abnormalities of pemphigoid and pemphigus with IgM paraprotein having underlying cancer was described as paraneoplastic mixed bullous disease.2 A patient with paraneoplastic pemphigus-mixed bullous disease type showing strong reactivity to IgA is presented below.

  1. *Dr. AKM Nurul Kabir, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh. kabir56@gmail.com
  2. Mohammed Kamal, Professor, Department of Pathology Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh.
  3. Ruhini Kumar Das, Professor and Head of Pathology, M Abdur Rahim Medical College, Dinajpur.

 

*For correspondence

Case History

A 55 years old man from Munshiganj district, Bangladesh was admitted at the Medicine ward of the Bangabandhu Sheikh Mujib Medical University (BSMMU) Hospital, Dhaka on the 23rd June 2001 with complaints of multiple neck and axillary swellings. Physical examination of the patient revealed mild anaemia with generalized lymphadenopathy. The lymph nodes were discrete and non-tender.

The laboratory investigations showed haemoglobin 9.5 g/dL, platelet count 300×109/L, blood urea 22 mg/dL and serum creatinine  0.7 mg/dL. X- Ray Chest (P/A view) showed left sided pleural effusion but sputum for AFB was negative and Tuberculin test also was insignificant. Ultrasonography of whole abdomen revealed lymphadenopathy. Fine needle aspiration cytology (FNAC) of cervical lymph node (June 26, 2001) showed features of non-Hodgkin’s lymphoma (NHL), small cell type, and subsequently by histopathological examination of left axillary node confirmed as NHL, low grade (July 08, 2001).

The patient was treated with four cycles of Cyclophosphamide, Hydroxydaunorubicin, Oncovin and Prednisolon (CHOP) and six month later he presented with vesiculo-bullous lesion all over the body and clinically was suspected as a case of paraneoplastic pemphigus (Figure 1).

Histopathological tests done from lesional and perilesional skin for routine examination and direct immunofluorescence (DIF) test, respectively. Routine Hematoxylin and Eosin (H & E) stained sections revealed suprabasal clefts with few acantholytic cells and a small subepidermal bulla containing fibrin, neutorphils and small number of eosinophils (Figure 2 & 3). On DIF test, cryostat sections incubated with rabbit antihuman sera conjugated with FITC made by Medic Italy, showed linear deposits of IgA (++), C3 (+), fibrin (+) and focal deposits of IgM at basement membrane zone (BMZ). Faint deposits of IgG at BMZ and in epidermal intercellular substance were also seen. In indirect immunofluorescence (IIF) test, incubation of normal human skin with serum of the patient showed strong linear deposition of IgA and faint deposit of IgM along the BMZ. IIF test on Long-Evans rat urinary bladder sections also showed linear deposition of IgA along the BMZ (Figure 4). Protein electrophoresis on cellulose acetate membrane at pH 8.8 revealed  raised gamma globulin level (27% , normal 12-18%).

 

 

Discussion

Pemphigus  is the disease, characterized by intraepithelial blisters, caused by a loss of normal cell-cell adhesion, and are associated with autoantibodies against cell-surface proteins of stratified squamous epithelia. Anhalt et al1 first described paraneoplastic pemphigus in 1990. The authors reported five patients with underlying neoplasms, who developed oral erosions and bullous skin eruptions and described as paraneoplastic pemphigus. They suggested five criteria to define paraneoplastic pemphigus 1)  Painful mucosal erosions, sometimes with a skin eruption that eventually results in blisters and erosions, in the setting of confirmed or occult malignancy 2) Histopathologic changes of acantholysis, keratinocyte necrosis, and interface dermatitis 3) DIF observation of immunoreactants, typically IgG and complement (C3) within the epidermal intercellular space as well as at the epidermal basement membrane 4) IIF observation of circulating antibodies specific for stratified squamous or transitional epithelia 5) Immunoprecipitation of a complex of proteins with molecular weights of 250, 230, 210 and 190-kd. The 250-kd and 230-kd antigens correspond with desmoplakin- I and bullous pemphigoid antigen respectively. Identities of 210-kd and 190-kd antigens were not known.  They used rodent urinary bladder epithelium for the screening of paraneoplastic pemphigus as antigens of pemphigus vulgaris and pemphigus foliaceus are not expressed in this tissue. Later in 1993 Camisa and colleague proposed major and minor criteria for the diagnosis of neoplasia induced pemphigus.3

 

These are:

Major criteria

Polymorphous muco-cutaneous eruption

Concurrent internal neoplasia.

Characteristic serum immunoprecipitation findings

Minor criteria

Positive cytoplasmic staining of rat bladder epithelium by IIF

Intercellular and basement zone immunoreactants on DIF of perilesional tissue.

Acantholysis in biopsy specimen from at least one anatomical site of involvement.

A patient should be considered to have the neoplasia induced pemphigus if all three major or two major and two or more minor criteria are met.

In paraneoplstic pemphigus the tumour antigens evoke an immune response that is primarily humoral. The neoplasm does not appear to produce the autoantibodies in paraneoplastic pemphigus. Non-neoplastic B lymphocytes are probably responsible,  as invivo-bound immunoglobulins are polyclonal.3

In the present case the lesions were polymorphous, crusted and vesicular eruptions all over the body and developed after treatment of Non-Hodgkin’s lymphoma. The lesions were both suprabasal with acantholytic cells and subepidermal representing mixed bullous lesion of pemphigus and   pemphigoid. Bystryn and colleagues described a case with mixed bullous disease exhibiting combined features of cicatrical pemphigoid and pemphigus and associated with a B-cell lymphoma producing IgM paraprotein.2 In DIF testing of present case faint deposition of IgG was seen in intercellular substance and along BMZ.  Deposition of IgA was strong but it was seen along BMZ. The combination of intercellular and subepidermal deposition of immunoreactants is a clue to the diagnosis of paraneoplastic pemphigus.4 Because circulating antibodies that bind to the cell surface of stratified squamous epithelia are common to all forms of pemphigus, other substrates, such as rodent bladder, is useful in distinguishing paraneoplastic pemphigus from pemphigus vulgaris or pemphigus foliaceus. Binding to rat bladder transitional epithelium is specific for circulating autoantibodies from patients with paraneoplastic pemphigus with a specificity of 83%. However, testing on rat bladder has a sensitivity of only 75%4. In present case the important antibody deposition was IgA and located along BMZ of rat urinary bladder, but no deposit is seen at intercellular space of bladder epithelium. These features are indicative of new entity not yet described previously. Bystryn and colleagues found that the IgM  paraprotein was deposited to intercellular antigen of human skin but did not react to mammalian bladder in their case. In the present case two major and almost three minor criteria, for the diagnosis of paraneoplastic pemphigus proposed above, are met. We believe that this condition represents a novel bullous disease, and diagnosed as paraneoplastic pemphigus (mixed bullous disease type).  Though the immunoprecipitation analysis is a standard diagnostic procedure for paraneoplastic pemphigus because it has higher specificity and sensitivity than IIF testing,4 unfortunately as it is not widely available, is not done in this case.

Conclusion

Paraneoplastic pemphigus may present with variable features. The present case of paraneoplastic pemphigus has distinct features with autoantibody – IgA to some components of BMZ. Strong reactivity of IgA to BMZ illustrates a distinct bullous disease associated with paraneoplastic syndromes and at least one possible mechanism for such eruption is the production of anti-skin antibodies in patient with malignant B cells. However, definite nature of antigens remains to be explored and the full spectum of bullous disease associated with underlying cancers remains to be determined. As possibility of underlying malignancy including lymphoma is present in a small proportion of patients of pemphigus, complete physical examination and laboratory investigations are mandatory in cases of blistering disease5 and IIF test with rat urinary bladder may be included in the suspected cases.

References

  1. Anhalt GJ, Stanley JR et al. Paraneoplastic pemphigus. An autoimmuno mucocutaneous disease associated with neoplasia. N. Eng. J. Med. 1990; 323:1729.
  2. Brystryn JC, Hodak E, Gao SQ et al. A paraneoplastic mixed bullous skin disease associated with anti-skin antibodies and a B-cell lymphoma. Arch Dermatol 1993;129:870.
  3. Camisa C, Helm TN. Paraneoplastic pemphigus is a disninct neoplasia-induced autoimmune disease. Arch Dermatol. 1993; 129:883.
  4. Goldberg L, Nisar N. Pemphigus paraneoplastic. Boston University eMedicine online [internet]. 2003 January (Cited 2004), Available from http// www. eMedicine.com/dem/pemphigus/paraneoplastic.
  5. Schofield OMV, Hunter JAA. Disease of skin in: Haslett C, Chilvers ER, Hunter JAA et al, Davidson’s Principles and Practice of Medicine (18th eds). Edinburgh, Churchill Living stone 1999; pp877-921.

Balanced X-Autosome Translocation in Infertile Woman: Report of Two Cases

Balanced X-Autosome Translocation in Infertile Woman: Report of Two Cases

*Habib S,1 Islam SS2

Abstract
Individuals with apparently balanced translocations, often, show no clinical findings. X-chromosomal translocations involving different autosomes have been reported. The phenotypic manifestations of these translocations depend on several factors. X-autosome translocations can also affect fertility where chromosomal changes result in inactivation of genes governing reproduction. This report is described two cases of phenotypically normal Bangladeshi women with the complaint of primary infertility associated with secondary amenorrhea and streak ovaries. Chromosomal analysis revealed an apparently balanced reciprocal translocation involving the long arm of the X chromosome (q2) with the short arm of chromosome 1(p3) and the long arm of chromosome 19(q13) in all the cells with the karyotype 46,X,t(X:1)(q22:p32) and 46,X,t(X:19)(q22:q13.1). Studies examining X-chromosome deletions have predicted that Xq aberrations within the Xq13–Xq27 region can result in gonadal failure. Reciprocal translocations between autosomes and gonosomes contribute significantly to primary infertility.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):151-156]

Keywords: Balanced X-autosome translocation, phenotype, Primary infertility

Introduction

X-autosome translocations are rare and associated with different phenotypes. There are balanced and unbalanced X-autosome translocation. Balanced type is usually associated with normal phenotype whereas the unbalanced one is with various congenital anomalies. Phenotypic effects of balanced X-autosome translocations in females: a retrospective survey of 104 cases reported from UK laboratories by Water JJ et al.1 Their cases were: multiple congenital abnormalities and/or developmental delay (MCA/DD): 26 (42%); gonadal dysfunction: 22 (35%); phenotypically normal with or without recurrent miscarriage (NRM): 9 (15%); recognized X-linked syndrome: 5 (8%). X chromosome translocations are frequently associated with primary or secondary amenorrhea. In this report, the clinical, biochemical and cytogenetic aspects of two healthy infertile women with balanced X-autosome translocation between chromosome X and two different autosomes: chromosome 1 and 19 were presented.

 

  1. *Dr. Saequa Habib MBBS MD (Pathology) Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University. saequa20@yahoo.com
  2. SM Shahedul Islam, B Sc, M Sc (Biochemistry & Molecular Biology) Scientific Officer, Department of Pathology, Bangabandhu Sheikh Mujib Medical University.

 

*For correspondence

 

Case Presentation

Case 1

A 27 year-old female with the complaint of primary infertility, was referred to the department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh for cytogenetic evaluation. She was born to a nonconsanguineous parent and her mother had no menstrual problem. Her siblings were healthy. Her menarche was at 13 years of age but she had irregular menstruation with 4-8 months interval. She was married for 7 years. Her husband lived abroad. Her physical examination revealed normal height and weight and showed normal intelligence. On per vaginal examination small uterus was found with no abnormality of the external genitalia. Her luteinizing hormone and TSH level were normal but follicle stimulating hormone and anti-Mullerian hormone were at postmenopausal level. Ultrasound examination of the pelvis revealed normal uterus and streak ovaries.

Cytogenetic analysis of the peripheral blood lymphocytes was carried out according to the standard karyotyping technique. Peripheral blood lymphocytes were stimulated with phytohemagglutinin and harvested at 72 hours with colchicine. Hypotonic treatment was given to the cells and then they were fixed with Carnoy’s fixative. Standard GTG banding was done.2 Karyotype analysis of 100 metaphases revealed a pattern of 46, X, t(X;1)(q22; p32), suggestive of a balanced sex autosome translocation involving the long arm of chromosome X and short arm of chromosome 1 (Figure 1). ISCN guidelines for the chromosomal nomenclature (2016) were followed for the karyotype analysis and analysed by using Leica DM6000 B Motorized microscope and Leica Cytovision software.3 Parental and siblings karyotypes were not done.

Case 2

A 32 year-old female was referred with the complaint of primary infertility. She had two sisters and they had children. She had delayed puberty with menarche at the age of 15 years. After then menstruation occurred only after taking pills. She was married for 16 years. Her height was 5 feet 3 inch and weight was 60 kg. She was graduated and was a health worker. She had complaint of decrease libido. On per vaginal examination small uterus was found with no abnormality of the external genitalia. Her luteinizing hormone, thyroid stimulating hormone, prolactin and testosterone level were normal but follicle stimulating hormone was at postmenopausal level. Ultrasound examination of the pelvis revealed hypoplastic uterus and streak ovaries.

Cytogenetic analysis of the peripheral blood lymphocytes was carried out and standard GTG banding was done.2 Karyotype analysis of 100 metaphases revealed 46, X, t(X;19)(q22; p13.1), suggestive of a balanced sex autosome translocation involving the long arm of chromosome X and long arm of chromosome 19 (Figure 2).

Table  I: Previously reported cases of balanced X; 1 and X; 19 translocations

Symptoms Karyotypes Data by
Developmental delay 46, X, t (X; 1)(p22.1;p31) de novo Waters JJ et al1 (2001)
Recurrent miscarriages 46, X, t (X; 1)(p22.1;p32) de novo
Learning difficulties 46, X, t (X; 1)(p11.4;p36.3) de novo
Mother: abnormal scan 46, X, t (X; 1)(q26;p22) de novo
Multiple congenital anomalies/developmental delay 46, X, t (X; 1)(q26;p22) mat
Primary amenorrhea 46X: t (X; 1) (q21;p32) de novo Venkateshwari A et al9 (2015)

 

Primary amenorrhea 46,X,t(X; 1)( q22;p13) Razavi Z and   Momtaz HE10 (2017)
Primary amenorrhea 46,X,t(X;19)(q28;p13.1) Shetty DL et al11 (2014)

 

 

 

 

 

 

 

 

 

Figure 1. Photomicrograph of a karyotype showing translocation between chromosome X and chromosome 1 [46, X, t(X; 1) (q22; p32)] (Giemsa stain).

 

 

 

 

 

 

 

 

 

Figure 2. Photomicrograph of a karyotype showing translocation between chromosome X and chromosome 19 [46, X, t(X; 19) (q22; p13.1)] (Giemsa stain).

Discussion

Chromosomal conditions involving the sex chromosomes often affect sex determination (whether a person has the sexual characteristics of a male or a female), sexual development, and the ability to have children (fertility). The signs and symptoms of these conditions vary widely and range from mild to severe. They can be caused by missing or extra copies of the sex chromosomes or by structural changes in the chromosomes.

X-autosome translocations are rare, being estimated to occur in about 1/30,000 live births.4   In cases of balanced X-autosome translocation in female carriers, the normal X chromosome is usually inactivated, leaving the derivative X chromosome in the active state. The present cases revealed sex autosome translocation in phenotypically normal female with secondary amenorrhea. Cytogenetic analysis revealed 46, X, t (X; 1) (q22; p32) and 46, X, t(X; 19) (q22; p13.1) karyotype indicating its possible association with irregular menstruation and abnormal hormone level. Most carriers of an X-autosome translocation are phenotypically normal.5,6,1 In female carriers, gonadal dysgenesis may occur, and ∼9% may have multiple anomalies and/or mental retardation.7 Since the 2 copies of the X chromosome are necessary for ovarian development and integrity, the gonadal dysgenesis with infertility in our patient can be attributed to the partial loss of Xq, which contains various genes necessary for a normal ovarian reproductive function. MG Mattei et al concluded that  in X-autosome translocation 50% women will be sterile.8 There are reported cases of balanced X; 1 and X;19 translocations with different clinical manifestations including infertility showed in Table I.

Balanced X-autosome translocations show exchange between long arm segments of an X chromosome to an autosome with larger number of breakpoints. Infertility because of gonadal dysgenesis is common among those women in whom the breakpoint in the derivative X-chromosome involves the critical region Xq13–q26.1,5,6 X-autosome translocation causing gonadal dysgenesis with bilateral streak gonads as well as aberrant ovarian and sex development has been demonstrated by numerous studies.12, 13 Translocations involving the long arms of the X-chromosome and several autosomes (1–4, 6–9, 11, 12, 14, 15, 17, 19, 21, and 22), resulting in various degrees of gonad dysfunction, have also been reported.14

X-autosomal translocations are generally of maternal in origin or may arise in de novo.6 Fertility effects of a balanced X-autosome translocation vary depending on the sex of the carrier, the position of the translocation breakpoints and the pattern of X-inactivation.5,6,7 In the reported cases X autosome translocation may be de novo as their siblings had no menstrual abnormality. To conclude that balanced X-autosome translocation can be a cause secondary amenorrhea associated with infertility and should be investigated by cytogenetic analysis followed by genetic counseling.

References

  1. Waters JJ, Campbell PL, Crocker AJ, Campbell CM. Phenotypic effects of balanced X-autosome translocations in females: a retrospective survey of 104 cases reported from UK laboratories. Hum Genet 2001; 108 (4): 318– 27.
  2. Verma RS and Babu A. Human chromosome: manual of basic techniques.1st New York, USA. 1989: pp 4-44, 152-165.
  3. ISCN: an international system for human cytogenomic nomenclature .In Jean McGowan-Jordan, Annet Simons, Michael Schmid eds. Cytogenetic and Genome Research. New York, Karger. 2016: Vol. 149, No. 1-2.
  4. Sharp AJ, Spotswood HT, Robinson DO, Turner BM, Jacobs PA. Molecular and cytogenetic analysis of the spreading of X inactivation in X;autosome translocations. Hum Mol Genet. 2002; 11 (25): 3145– 56.
  5. Madan, K. Balanced structural changes involving the human X: effect on sexual phenotype.  Genet.1983; 63: 216–221.
  6. Kalz‐Füller B, Sleegers E, Schwanitz G, Schubert R. Characterisation, phenotypic manifestations and X‐inactivation pattern in 14 patients with X‐autosome translocations.  Genet.1999; 55: 362–366.
  7. Schmidt, M. and Du Sart, D.) Functional disomies of the X chromosome influence the cell selection and hence the X inactivation pattern in females with balanced X‐autosome translocations: a review of 122 cases. J. Med. Genet1992; 42:161–169.
  8. Mattei MG, Mattei JF, Ayme S Giraud F. X-Autosome translocation: cytogenetic characteristics and their consequences. Hum Genet 1982; 61:295-309.
  9. Venkateshwari A, Srilekha A, Veena K, Sujatha M, Jyothy A. A Rare De Novo Balanced 1X; 1 Translocation in an Indian Female with Primary Amenorrhea. J Reprod Infertil. 2015; 16: 171–3.
  10. Razavi Z and Momtaz HE. Balanced Reciprocal Translocation t(X; 1) in a Girl with Tall Stature and Primary Amenorrhea Iran J Med Sci. 2017 Mar; 42(2): 210–214.
  11. Shetty DL, Kadam AP, Koppaka NT, Dalvi RC, Chavan DS, Das BR, Mandava S. X-autosome translocations in amenorrhoea: a report of a three way translocation from Indian Population. Gynecol Endocrinol, 2014 Early Online: 1–5.
  12. Mohandas T, Geller RL, Gerald P, Yen H. Cytogenetic and molecular studies on a recombinant human X chromosome: implications for the spreading of X chromosome inactivation. In Proceedings of the National Academy of Sciences of the United States of America, 1987;84(14):4954–4958.
  13. Carpenter NJ, Say B, Browning D. Gonadal dysgenesis in a patient with an X; 3 translocation: case report and review. Journal of Medical Genetics, 1980; 17(3): 216–221.
  14. Madan KP, Hompes GA, Schoemaker J, Ford CE. X-autosome translocation with a breakpoint in Xq22 in a fertile woman and her 47, XXX infertile daughter. Human Genetics, 1981; 59(4):290–296.