- Email: [email protected]
Application of an easy and useful morphometric technique for immunohistochemistry counting
Available online at www.sciencedirect.com
Gynecologic Oncology 112 (2009) 282 – 286 www.elsevier.com/locate/ygyno
Letters to the Editor
Application of an easy and useful morphometric technique for immunohistochemistry counting To the Editor: We have been performing several scientific works related to prognostic factors of preneoplasic and neoplasic lesions of the cervix, due to the presence of post-graduation students in our department. In order to perform such studies we have been using immunohistochemistry with a certain frequency. When working with immunohistochemistry, sometimes we wish to determine the concentration of a certain cell that is being analyzed in order to compare them in different situations. Such cell count, according to a certain space unit, is called morphometry. The term morphometry is comprised by the Greek radical — morphé, which means shape, related to the Greek radical — metrikós, or to the Latin — metricu, which means act of measuring or process of establishing measurements. Thus, several computer softwares of morphometry were developed and are commercially available to use in daily clinical practice. However, in centers with scarce resources, as in our case, purchasing such softwares is not viable due to their high cost. Thus, instead of using these softwares, one of the most common ways of getting such an estimate would be through counting cells in a regular optical microscope. Due to that setback in getting such morphometry softwares, we have developed a morphometric method that is quite easy to perform, and that can be used by any research center, by making use of a Neubauer chamber, also known as Hemocytometer or Counting Chamber, which is easily found in clinical pathology labs. The Neubauer Chamber consists of a glass slide, thicker than a regular slide, where there are usually two chambers engraved in the glass (two darker parts in the center). Each Neubauer Chamber has the design of a grid, dividing it into squares with known dimensions, which are visualized only through the optical microscope. The Neubauer Counting Chamber is divided into 9 large squares of 1 mm each side by means of triple lines (the area of a large square = 1 mm2). Each large square contains 16 (4 × 4) medium size squares. Each medium size square is further divided by triple lines into 25 (5 × 5) small squares, each with side 1/5 of a mm. Thus the dimension of an individual small square of 0.05 × 0.05 mm = 0.0025 mm2 and 0.1 mm deep (Fig. 1). The system we implemented used a regular optical microscope adapted to a microcamera and screen. Thus, the image 0090-8258/$ - see front matter © 2008 Elsevier Inc. All rights reserved.
observed through the microscope lenses is captured by the video camera and showed in the chosen monitor. The Neubauer Chamber is then placed in the optical microscope, under a objective which increases 400 times the original size of the object (large magnification field), or any other increase chosen by the researcher. At last, the area of the large magnification field (400× magnification) is estimated, in mm2, by counting the total number of individual small squares of 0.0025 mm2 of the chamber present in the magnification area that was chosen. In our evaluation, the total area of a large magnification corresponded to 0.094 mm2. In such a manner, each section prepared by immunohistochemical method was evaluated under light microscope. The number of cells in study was counted at 400× magnification. A 10 field count was performed in the different situations we were evaluating. The mean number of cells present in this 10 field count represents, as mentioned before, 0.094 mm2. By means of a simple rule of three, the number of cells is converted into 1 mm2. The cells count was expressed as numerical densities, i.e. number of positive cells per square millimeter of epithelium.
Fig. 1. Schematic figure of the grid of a Neubauer chamber. The grid is divided it into squares with known dimensions, which are visualized only through the optical microscope.
Letters to the Editor
In our practice, several studies are being performed using that methodology we developed. Some of them have already been published in highly rated journals, including Gynecologic Oncology. Among them we point out the studies by Nadais et al. [1] and Campaner et al. [2,3]. Conflict of interest statement The authors have no conflict of interest to declare.
283
1980 to 2005 which included the most recent dataset submission [2]. The inclusion criteria were a diagnosis of invasive carcinoma of the vulva, with active follow up, and a known age of diagnosis. Two groups were created, b 50 and ≥ 50 years and the time period of 1980–2005 was divided into groups similar to those created by Hampl et al. The results are reported in the accompanying Table 1. The time period of 1980–89 was taken as the reference group and Chi square tests were used to compare the subsequent time periods.
References [1] Nadais Rda F, Campaner AB, Piato S, Longo Galvão MA, dos Santos RE, Aoki T. Langerhans' cells and smoking in intraepithelial neoplasia of the cervix. Gynecol Oncol 2006;102:356–60. [2] Campaner AB, Piato S, Galvão MA, dos Santos RE, Nadais RF. Langerhans cells in cervical intraepithelial neoplasia related to smoking habits. J Low Genit Tract Dis 2006;10:223–8. [3] Campaner AB, Nadais RF, Galvão MA, Santos RE, Aoki T. Evaluation of density of Langerhans cells in human cervical intraepithelial neoplasia. Acta Obstet Gynecol Scand 2007;86:361–6.
Adriana Bittencourt Campaner Department of Obstetrics and Gynecology, Santa Casa of São Paulo Medical School, São Paulo, Brazil E-mail address: [email protected]. ⁎ Corresponding author. Fax: +55 11 2176 7211.
Table 1 Fractional distribution of invasive carcinoma of the vulva (CV) in the two age groups, stratified according to three time intervals Year groups
Age groups
Total (-n-)
p
85 81.9 81.2 82.2
2271 3874 5227 11372 ⁎
b0.001
13.5 16.7 17.2 16.3
86.5 83.3 82.8 83.7
2055 3412 4567 10034 ⁎
b0.001
33.6 33.7 35.5 34.6
66.4 66.3 64.5 65.4
134 282 423 839 ⁎
N0.05
b50 (%)
≥50 (%)
1980–89 1990–99 2000–05 Sum
15 18.1 18.8 17.8
1980–89 1990–99 2000–05 Sum 1980–89 1990–99 2000–05 Sum
Total sample
White
African American
Maria Antonieta Longo Galvão Department of Pathology, Santa Casa of São Paulo Medical School, São Paulo, Brazil
⁎ 499 patients were in unspecified, unknown or other racial groups. (p) Compares the default reference time interval of 1980–89 to the other time intervals.
24 July 2008 doi:10.1016/j.ygyno.2008.08.012
Vulvar cancer in women less than fifty in United States, 1980–2005
To the Editor: In a recent issue of Gynecologic Oncology, Hampl et al. reported their experience from 1980–2007 on invasive cancer of the vulva. Their paper highlighted the changing epidemiology of vulvar cancer, specifically, the change in location of the lesion and a shift in the age at diagnosis [1]. We found their reported increase in the fraction of vulvar cancer cases diagnosed in women under the age of 50 years, from 11% in the 1980s to 41% in the last ten years rather concerning. Further, although the overall rates and trends in this disease have been well covered recently in a number of publications many of which are cited by Hampl et al., the question regarding the fraction of vulvar cancer in women less than 50 years of age in the U.S. has not recently been reported on a large scale. We conducted a systematic analysis of the Limited use Surveillance, Epidemiology, and End Results (SEER) data from
A total of 11,372 patients were diagnosed with vulvar cancer, with 17.8% being b50 years at the time of diagnosis. As shown in the attached Table 1, the fraction of vulvar cancer in the b50 year age group increased from 15% in the 1980s to 18% in the 1990s (p b 0.001), but remained stable thereafter at 18.8% in 2000–2005 time period. Similarly, this trend remained true when only white (W) women were analyzed. Of note, there was a disparity in African American (AA) women where a much larger fraction of AA women with vulvar cancer are b50 years (34.6%; n = 839 in AA vs. 16.3%; n = 1636 in W, p b 0.001). Interestingly, the fraction of vulvar cancer occurring in b 50 year age group did not change significantly in AA (Table 1, p N 0.05), from 1980 onwards. It appears that the population based data continues to support the notion that vulvar cancer is a disease of the aged. Although we found a modest increase and considerable disparity in the racial distribution of the fraction of vulvar cancer occurring in b50 year age group; we are unable to support the claim of a drastic increase in vulvar cancer in young women, as reported by Hampl et al. [1]. Obviously, part of the difference in our results may be due to the difference in sociodemographics of the patient population in the two countries, including the prevalence of HPV as well as other risk factors. But this example reminds us that the potential for referral bias in a single institutional
Gynecologic Oncology 112 (2009) 282 – 286 www.elsevier.com/locate/ygyno
Letters to the Editor
Application of an easy and useful morphometric technique for immunohistochemistry counting To the Editor: We have been performing several scientific works related to prognostic factors of preneoplasic and neoplasic lesions of the cervix, due to the presence of post-graduation students in our department. In order to perform such studies we have been using immunohistochemistry with a certain frequency. When working with immunohistochemistry, sometimes we wish to determine the concentration of a certain cell that is being analyzed in order to compare them in different situations. Such cell count, according to a certain space unit, is called morphometry. The term morphometry is comprised by the Greek radical — morphé, which means shape, related to the Greek radical — metrikós, or to the Latin — metricu, which means act of measuring or process of establishing measurements. Thus, several computer softwares of morphometry were developed and are commercially available to use in daily clinical practice. However, in centers with scarce resources, as in our case, purchasing such softwares is not viable due to their high cost. Thus, instead of using these softwares, one of the most common ways of getting such an estimate would be through counting cells in a regular optical microscope. Due to that setback in getting such morphometry softwares, we have developed a morphometric method that is quite easy to perform, and that can be used by any research center, by making use of a Neubauer chamber, also known as Hemocytometer or Counting Chamber, which is easily found in clinical pathology labs. The Neubauer Chamber consists of a glass slide, thicker than a regular slide, where there are usually two chambers engraved in the glass (two darker parts in the center). Each Neubauer Chamber has the design of a grid, dividing it into squares with known dimensions, which are visualized only through the optical microscope. The Neubauer Counting Chamber is divided into 9 large squares of 1 mm each side by means of triple lines (the area of a large square = 1 mm2). Each large square contains 16 (4 × 4) medium size squares. Each medium size square is further divided by triple lines into 25 (5 × 5) small squares, each with side 1/5 of a mm. Thus the dimension of an individual small square of 0.05 × 0.05 mm = 0.0025 mm2 and 0.1 mm deep (Fig. 1). The system we implemented used a regular optical microscope adapted to a microcamera and screen. Thus, the image 0090-8258/$ - see front matter © 2008 Elsevier Inc. All rights reserved.
observed through the microscope lenses is captured by the video camera and showed in the chosen monitor. The Neubauer Chamber is then placed in the optical microscope, under a objective which increases 400 times the original size of the object (large magnification field), or any other increase chosen by the researcher. At last, the area of the large magnification field (400× magnification) is estimated, in mm2, by counting the total number of individual small squares of 0.0025 mm2 of the chamber present in the magnification area that was chosen. In our evaluation, the total area of a large magnification corresponded to 0.094 mm2. In such a manner, each section prepared by immunohistochemical method was evaluated under light microscope. The number of cells in study was counted at 400× magnification. A 10 field count was performed in the different situations we were evaluating. The mean number of cells present in this 10 field count represents, as mentioned before, 0.094 mm2. By means of a simple rule of three, the number of cells is converted into 1 mm2. The cells count was expressed as numerical densities, i.e. number of positive cells per square millimeter of epithelium.
Fig. 1. Schematic figure of the grid of a Neubauer chamber. The grid is divided it into squares with known dimensions, which are visualized only through the optical microscope.
Letters to the Editor
In our practice, several studies are being performed using that methodology we developed. Some of them have already been published in highly rated journals, including Gynecologic Oncology. Among them we point out the studies by Nadais et al. [1] and Campaner et al. [2,3]. Conflict of interest statement The authors have no conflict of interest to declare.
283
1980 to 2005 which included the most recent dataset submission [2]. The inclusion criteria were a diagnosis of invasive carcinoma of the vulva, with active follow up, and a known age of diagnosis. Two groups were created, b 50 and ≥ 50 years and the time period of 1980–2005 was divided into groups similar to those created by Hampl et al. The results are reported in the accompanying Table 1. The time period of 1980–89 was taken as the reference group and Chi square tests were used to compare the subsequent time periods.
References [1] Nadais Rda F, Campaner AB, Piato S, Longo Galvão MA, dos Santos RE, Aoki T. Langerhans' cells and smoking in intraepithelial neoplasia of the cervix. Gynecol Oncol 2006;102:356–60. [2] Campaner AB, Piato S, Galvão MA, dos Santos RE, Nadais RF. Langerhans cells in cervical intraepithelial neoplasia related to smoking habits. J Low Genit Tract Dis 2006;10:223–8. [3] Campaner AB, Nadais RF, Galvão MA, Santos RE, Aoki T. Evaluation of density of Langerhans cells in human cervical intraepithelial neoplasia. Acta Obstet Gynecol Scand 2007;86:361–6.
Adriana Bittencourt Campaner Department of Obstetrics and Gynecology, Santa Casa of São Paulo Medical School, São Paulo, Brazil E-mail address: [email protected]. ⁎ Corresponding author. Fax: +55 11 2176 7211.
Table 1 Fractional distribution of invasive carcinoma of the vulva (CV) in the two age groups, stratified according to three time intervals Year groups
Age groups
Total (-n-)
p
85 81.9 81.2 82.2
2271 3874 5227 11372 ⁎
b0.001
13.5 16.7 17.2 16.3
86.5 83.3 82.8 83.7
2055 3412 4567 10034 ⁎
b0.001
33.6 33.7 35.5 34.6
66.4 66.3 64.5 65.4
134 282 423 839 ⁎
N0.05
b50 (%)
≥50 (%)
1980–89 1990–99 2000–05 Sum
15 18.1 18.8 17.8
1980–89 1990–99 2000–05 Sum 1980–89 1990–99 2000–05 Sum
Total sample
White
African American
Maria Antonieta Longo Galvão Department of Pathology, Santa Casa of São Paulo Medical School, São Paulo, Brazil
⁎ 499 patients were in unspecified, unknown or other racial groups. (p) Compares the default reference time interval of 1980–89 to the other time intervals.
24 July 2008 doi:10.1016/j.ygyno.2008.08.012
Vulvar cancer in women less than fifty in United States, 1980–2005
To the Editor: In a recent issue of Gynecologic Oncology, Hampl et al. reported their experience from 1980–2007 on invasive cancer of the vulva. Their paper highlighted the changing epidemiology of vulvar cancer, specifically, the change in location of the lesion and a shift in the age at diagnosis [1]. We found their reported increase in the fraction of vulvar cancer cases diagnosed in women under the age of 50 years, from 11% in the 1980s to 41% in the last ten years rather concerning. Further, although the overall rates and trends in this disease have been well covered recently in a number of publications many of which are cited by Hampl et al., the question regarding the fraction of vulvar cancer in women less than 50 years of age in the U.S. has not recently been reported on a large scale. We conducted a systematic analysis of the Limited use Surveillance, Epidemiology, and End Results (SEER) data from
A total of 11,372 patients were diagnosed with vulvar cancer, with 17.8% being b50 years at the time of diagnosis. As shown in the attached Table 1, the fraction of vulvar cancer in the b50 year age group increased from 15% in the 1980s to 18% in the 1990s (p b 0.001), but remained stable thereafter at 18.8% in 2000–2005 time period. Similarly, this trend remained true when only white (W) women were analyzed. Of note, there was a disparity in African American (AA) women where a much larger fraction of AA women with vulvar cancer are b50 years (34.6%; n = 839 in AA vs. 16.3%; n = 1636 in W, p b 0.001). Interestingly, the fraction of vulvar cancer occurring in b 50 year age group did not change significantly in AA (Table 1, p N 0.05), from 1980 onwards. It appears that the population based data continues to support the notion that vulvar cancer is a disease of the aged. Although we found a modest increase and considerable disparity in the racial distribution of the fraction of vulvar cancer occurring in b50 year age group; we are unable to support the claim of a drastic increase in vulvar cancer in young women, as reported by Hampl et al. [1]. Obviously, part of the difference in our results may be due to the difference in sociodemographics of the patient population in the two countries, including the prevalence of HPV as well as other risk factors. But this example reminds us that the potential for referral bias in a single institutional