JOURNAL OF
PATHOLOGY, VOL.166: 323-326 (1992)
CRITICAL SUMMARIES FLOW CYTOMETRY The most common application of flow cytometry to be met in histopathology is the measurement of cellular DNA content. The first three papers reviewed in this article relate to one specific application of DNA flow cytometry, namely to the prognostic value of the technique in node-negative breast cancer. The second trio of papers widens the horizons to include the measurement of other parameters together with cellular DNA content. The final three papers describe wider applications of flow cytometry in cell and molecular biology. All the papers have some relevance to clinical medicine.
DNA FLOW CYTOMETRY AS APPLIED TO CARCINOMA O F THE BREAST The literature on DNA flow cytometry applied to clinical material is vast and includes many papers published during the past 18 months. DNA flow cytometry has the advantage of giving a rapid, statistically sound but crude estimate of proliferative activity in clinical lesions, as well as a measure of DNA ploidy. Breast cancer is an area in which DNA flow cytometry has been shown to be particularly useful and I have chosen three papers on this topic as examples of the large number of DNA papers which have appeared recently.
previously been shown to be strongly correlated. It is relevant to the practical value of SPF whether it adds information which is independent of tumour grade. Another minor criticism of this study relates to the length of follow-up available. The median duration of follow-up was 4 years with a range of 24-70 months. This is not totally unreasonable, but with the long clinical course of the disease a followup approaching 10 years is ideal when relating measured parameters to survival. Nevertheless, overall this paper is an example of a useful study with an adequate number of patients, good quality flow cytometric and clinical data, and a sensible Indicators of prognosis in node-negative breast multivariate analysis. The authors found that SPF cancer. H. SIGURDSSON, B. BALDETORP, A. BORG, was the strongest of the prognostic factors that they et al. N Engl J M e d 1990; 322 1045-1053. investigated. This finding agrees with a number of other published studies on node-negative breast A particular question of interest has been whether cancer, including one of our own (O’Reilly et al., J DNA flow cytometry could play a useful role in Clin Oncol 1990; 8: 2040-2046). In the present identifying those women with node-negative dis- paper, progesterone-receptor status and tumour ease, who have a relatively poor prognosis. Such size were also found to yield prognostic information women would be suitable candidates for adjuvant and the authors suggest that these be combined with chemotherapy. This paper provides an example of SPF in a ‘prognostic index’. a well-performed study aimed at addressing this question. A reasonable cohort of patients (367) was investigated with complete data available on Is DNA ploidy an independent prognostic indicator in infiltrative node-negative breast adenocarcinoma? 250 women. Flow cytometric parameters, namely S-phase fraction (SPF) and DNA ploidy, were S. KEYHANI-ROFAGHA, R. V. O’TOOLE,W. B. analysed in a multivariate analysis with clinicoJ. DECENZO FARRAR,B. SICKLE-SANTANELLO, and D. YOUNG.Cancer 1990; 65: 1577-1582. pathological factors such as patient age, oestrogen and progesterone receptors, and tumour size. The This study was also aimed at identifying a role for inclusion of tumour grade in the analysis would have strengthened the study, as grade and SPF have DNA flow cytometry in node-negative disease. 0022-341 7/92/03032344 $05.00 0 1992 by John Wiley & Sons, Ltd.
324
CRITICAL SUMMARIES
However, in this paper, the authors restricted themselves to looking at DNA ploidy. Our own experience has been (see O’Reilly et al., 1990, quoted above) that DNA ploidy is not a significant prognostic indicator, either in node-negative or in node-positive disease. The first paper described above reported the same lack of prognostic significance for DNA ploidy but there is disagreement in the literature on this point. The present study had a reasonable, if not generous, sample size of 165 patients and DNA ploidy was correlated with patient age, oestrogen receptor status, treatment, and menopausal status. Good follow-up was available on these patients (mean around 8 years, range 3-1 5 years) and no complex statistical analysis was required as the result was clear-cut. There was no trend at all for ploidy to be a prognostic marker. Although there is disagreement in the literature with some studies finding a significantly prognostic role for ploidy at least on univariate analysis or a nonsignificant trend as reported by Sigurdsson et af.(see above), this paper strengthens the overall impression that SPF is a more powerful prognostic parameter in breast cancer. A technical point touched on in this paper is the use of ‘external’ DNA standards to calibrate test samples produced from paraffin-embedded tissue. The use of typical ‘external’ standards such as normal human lymphocytes as a DNA diploid standard is contra-indicated when paraffin-embedded material is used (Schutte et af., Cytometry 1985; 6: 2630). The present authors followed the suggestion of Schutte et al. and used normal cells from other blocks from the same patient as diploid controls to check the ploidy status of their test samples. Even this practice has been suggested to be fraught with problems (Price and Herman, Cytometry 1990; 11: 845-847). It is safest to use the normal cells which must be present within any block as the ‘DNA standard’. This means only analysing a sample as DNA aneuploid if two clear G, peaks are present within the test sample.
DNA Index and S-phase fraction and their combination as prognostic factors in operable ductal breast carcinoma. H. JOENSUU, S. TOIKKANEN and P. J. KLEMI.Cancer 1990; 66: 331-340. In this paper the value of SPF and DNA ploidy in 222 cases of operable ductal breast carcinoma is reported. This number of patients, as with the previous paper, would seem adequate if not generous; however, in this instance the cases are more heterogeneous as they include both node-positive and node-negative patients. One problem that I have found in assessing the validity of published studies is to determine what constitutes an adequate sample size. Many factors will influence the decision for a given type of disease and study. A problem is that some papers quote an overall figure for patients included, which seems reasonable but on closer examination the patients may be so heterogeneous (in terms of site of lesion, stage, grade, treatment, etc.) that reasonable statistical analysis is impaired. In the present study, the data on node-negative patients, on which I will concentrate, are based on only 79 cases. Nevertheless, the overall study is carefully performed with a thorough multivariate analysis, involving the flow cytometric parameters, histologic grade, menopausal status, and a number of pathological factors. In addition, very long follow-up was available on the patients (median 25 years, range 22-35 years). As with the study of Sigurdssonetat.(1990)andourownstudiesonnodenegative disease, tumour size was found to be a powerful prognostic factor. Flow cytometric data were also found to yield independent prognostic information. However, these authors chose to combine DNA ploidy and SPF, which they found gave better discrimination than either parameter alone. This is a reasonable approach but it does make comparison with other papers more difficult. Nevertheless, there is now good agreement in the literature that SPF or a parameter including SPF is a useful prognostic tool, particularly for node-negative disease.
MULTIPARAMETRIC FLOW CYTOMETRY-DNA PARAMETERS The next three papers reviewed widen our horizons by measuring not just DNA but an additional factor too. The real power of flow cytometry is its ability to measure multiple, linked parameters simultaneously on cells. Simple to use analytical flow
PLUS OTHER
cytometers can measure five or more parameters. Thus, it is possible to detect or measure a wide range of substances on or within cells simultaneously with the measurement of DNA content. Most often, though not exclusively, use is made of monoclonal
CRITICAL SUMMARIES
antibodies tagged directly or indirectly to fluorochromes, such as fluorescein.
325
specific antibody, that G, cells could be distinguished from mitotic cells by a difference in Ki-67 staining. Mitotic cells showed the strongest staining of all. The authors also suggest that this Flow cytometric analysis of DNA content and multiparametric method may enable the distinction keratins by using CK7, CK8, CK18, CK19 and of Go and GI cells, a major goal in cell kinetic KL1 monoclonal antibodies in benign and malig- studies. Thus, this technique offers the possibility of nant human breast tumors. M . FERRERO,F. distinguishing the five basic cell kinetic compartSPYRATOS, V. LE DOUSSAL, A. DESPLACES and J. ments, namely Go,G,, S, G,, and mitosis. However, ROUESSE. Cytometry 1990; 11: 7 1G724. the evidence presented to support the discrimiA common reason for wishing to combine stain- nation between Go and GI was only preliminary ing with monoclonal antibodies and DNA has been and it is clear that the authors intend to investigate to distinguish tumour cells from non-tumour cells. this area further. The authors also make clear their This is particularly a problem with DNA diploid or realization that applying such multiparametric near-diploid tumours. A good study was published methods to clinical tumours will be harder and the in 1984 (Braylan et al., Cancer Res 1984; 44: interpretation more complex. Nevertheless, I feel 5010-5016) in which lymphocyte surface markers that this is a fascinating paper which demonstrates were used successfully to improve the discrimi- the power of multiparametric flow cytometry to nation of near-diploid abnormalities in DNA unravel the detailed kinetic make-up of complex cell content in non-Hodgkin’s lymphoma. Since then, populations. anti-keratin antibodies have been used in a similar way, to produce cleaner DNA histograms, in carci- Progesterone receptor detection and quantification in breast tumors by bivariate immunofluorescence/ nomas from a variety of sites such as breast, DNAflow cytometry. Y. REMVIKOS, M. VUHAI,C. bladder, and head and neck. In this paper a similar LAINE-BIDRON, A. JoLLrvETandH. MAGDELENAT. finding was made in that by gating on cytokeratinpositive cells only, debris and non-epithelial cells Cytometry 1991; 12: 157-166. could be excluded from the DNA analysis. HowThis paper describes a method to measure DNA ever, the authors went further and looked at the content and progesterone receptor (PgR) simulrelative expression of the various types of keratin in taneously. This is not the first time that such a malignant vs. normal cells and diploid vs. aneuploid technique has been described (see Graham et al., tumour cells. They raise the possibility that certain Cancer Res 1989; 4 9 393k-3942) but this paper keratin phenotypes may give prognostic inforextends the application of the technique to clinical mation, though this study was too small (29 cases) breast tumours. Cell lines with known PgR levels to confirm this possibility. were used to check the sensitivity and reproducibility of the method, which was then applied to 31 Flow cytometric multiparameter analysis of prolifer- cases of breast cancer. PgR levels measured by flow ating cell nuclear antigenlcyclin and Ki-67 anti- cytometry were shown to correlate well with those gen: a new view of the cell cycle. G. LANDBERG, E. assessed by an immunoenzymatic cytosol assay. M. TANand G. Roos. Exp Cell Res 1990; 187: The authors were able to compare staining for PgR in diploid vs. aneuploid cells in aneuploid tumours 111-1 18. and to investigate the heterogeneity of staining In this study, performed on human haemato- around the cell cycle. It is clear that routine poietic cell lines, the authors have combined measurement of PgR by flow cytometry has practimeasurement of DNA content with two prolifer- cal disadvantages at present. For example, a tissue ation-linked antibodies, namely Ki-67 and an storage method is needed which is convenient and anti-PCNA antibody. Ki-67 is reputed to label the retains DNA and PgR staining. Nevertheless, majority of proliferative cells around the cell cycle, techniques such as the one described here offer the whilst this PCNA antibody labels predominantly possibility of simultaneous measurement of cliniS-phase cells. The authors were able to stain for all cally useful parameters. They also allow the varithree parameters (i.e., DNA, Ki-67, and PCNA) ation between cells to be looked at, rather than simultaneously. They showed, with elegant exper- simply measuring a crude average level of receptor, iments using metaphase-arrest agents and a mitosis- as conventional biochemical assay systems do.
326
CRITICAL SUMMARIES
WIDER APPLICATIONS OF FLOW CYTOMETRY human chromosomes. The ability to produce flow cytometric karyotypes has been developed over the past 15 years or so to a point where good quality results can be achieved from commercial flow cytometers. Individual chromosomes can be physically sorted into separate containers for further study. These techniques have wide applications in the study of a variety of cytogenetic defects and in the construction of DNA libraries. In the present study, the reproducibility of ‘normal’ flow karyoDetection and analysis by dual-laser flow cytometry types was investigated in detail. Such baseline data of bacteriophage T4 DNA inside Escherichia cofi. are important in using bivariate flow karyotyping to C. A. SANDERS, D. M. YAJKO,P. S. NASSOS, W. C. detect possibly subtle chromosomal aberrations and W. K. HADLEY. seen, for example, in tumour cells. The authors HYUN,M. J. FULWYLER Cytometry 1991; 12: 167-1 7 1. make recommendations for performing cytogenetic comparisons of tumour and normal cells from Having stressed the diversity of flow cytometric individual patients. applications, it may seem odd to have chosen this
Flow cytometric techniques can contribute to our understanding in diverse areas of contemporary biology. In this final section, I have chosen three disparate examples of such studies. I could just have well selected papers describing anything from the measurement of intracellular pH to flow cytometric applications in marine biology or water quality control.
paper which has ‘DNA’ in its title. However, the reasons for measuring DNA and the type of DNA measured are, in this case, quite different. A complex dual-laser flow cytometer was used to measure staining with two separate DNA dyes simultaneously. The two dyes were Hoechst 33258, which binds preferentially to A-T rich regions of DNA, and chromomycin A3, which binds to G-C rich regions. A change in the ratio of staining intensities with these two dyes was used to detect the presence of viral DNA within the host bacterium. The authors were able to detect viral DNA in bacteria within 25 min of infection. The method described is rapid and could have widespread applications in the study of viral infections. A drawback is that it does require an expensive and relatively complex flow cytometer. Bivariate flow karyotyping of human chromosomes: evaluation of variation in Hoechst 33258 fluorescence, chromomycin A3 fluorescence and relative chromosomal DNA content. G. A. BOSCHMAN, W. RENS,C. H. VAN OVEN,E. M. M. MANDERS and J. A. ATEN.Cytometry 1991; 12: 559-569. This study also involved the measurement of DNA with the same two dyes used in the previous paper, but this time it was not total cellular or viral DNA that was measured but chromosomal DNA. Once again, an expensive dual-laser facility was required. This particular study is a very technical one, which looks at the standardization of the technique of measuring the DNA content of individual
Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils. S. PERTICARARI, G. PRESANI,M. A. MANCIAROTTI and E. BANFI.Cytometry 1991; 12: 687-693. This final paper is an example of an exciting type of flow cytometric application in the field of cell biology. It is possible, using a flow cytometer, to detect the presence of a receptor (or particle) and simultaneously to measure the biochemical effect of activation caused by triggering the receptor. Flow cytometric measurements can be made rapidly to look at the early stages of cellular activation and, indeed, time can be used as an additional parameter in such studies. In this paper, a two-colour fluorescent method is described which allows the simultaneous quantitation of the phagocytosis rate and the oxidative burst activation of polymorphonuclear leukocytes (PMNL). Fluorescent bacteria were used and when ingested, they trigger an oxidative burst in the PMNL. This oxidative activity leads to the conversion of a non-fluorescent precursor to a detectable fluorescent dye. The authors suggest that this technique may have an application in studying infectious diseases in chronic granulomatous disease patients.
RICHARD S. CAMPLEJOHN Richard Dimbleby Department of Cancer Research, UMDS, St Thomas’ Hospital, London SEl 7EH, U.K.
PATHOLOGY, VOL.166: 323-326 (1992)
CRITICAL SUMMARIES FLOW CYTOMETRY The most common application of flow cytometry to be met in histopathology is the measurement of cellular DNA content. The first three papers reviewed in this article relate to one specific application of DNA flow cytometry, namely to the prognostic value of the technique in node-negative breast cancer. The second trio of papers widens the horizons to include the measurement of other parameters together with cellular DNA content. The final three papers describe wider applications of flow cytometry in cell and molecular biology. All the papers have some relevance to clinical medicine.
DNA FLOW CYTOMETRY AS APPLIED TO CARCINOMA O F THE BREAST The literature on DNA flow cytometry applied to clinical material is vast and includes many papers published during the past 18 months. DNA flow cytometry has the advantage of giving a rapid, statistically sound but crude estimate of proliferative activity in clinical lesions, as well as a measure of DNA ploidy. Breast cancer is an area in which DNA flow cytometry has been shown to be particularly useful and I have chosen three papers on this topic as examples of the large number of DNA papers which have appeared recently.
previously been shown to be strongly correlated. It is relevant to the practical value of SPF whether it adds information which is independent of tumour grade. Another minor criticism of this study relates to the length of follow-up available. The median duration of follow-up was 4 years with a range of 24-70 months. This is not totally unreasonable, but with the long clinical course of the disease a followup approaching 10 years is ideal when relating measured parameters to survival. Nevertheless, overall this paper is an example of a useful study with an adequate number of patients, good quality flow cytometric and clinical data, and a sensible Indicators of prognosis in node-negative breast multivariate analysis. The authors found that SPF cancer. H. SIGURDSSON, B. BALDETORP, A. BORG, was the strongest of the prognostic factors that they et al. N Engl J M e d 1990; 322 1045-1053. investigated. This finding agrees with a number of other published studies on node-negative breast A particular question of interest has been whether cancer, including one of our own (O’Reilly et al., J DNA flow cytometry could play a useful role in Clin Oncol 1990; 8: 2040-2046). In the present identifying those women with node-negative dis- paper, progesterone-receptor status and tumour ease, who have a relatively poor prognosis. Such size were also found to yield prognostic information women would be suitable candidates for adjuvant and the authors suggest that these be combined with chemotherapy. This paper provides an example of SPF in a ‘prognostic index’. a well-performed study aimed at addressing this question. A reasonable cohort of patients (367) was investigated with complete data available on Is DNA ploidy an independent prognostic indicator in infiltrative node-negative breast adenocarcinoma? 250 women. Flow cytometric parameters, namely S-phase fraction (SPF) and DNA ploidy, were S. KEYHANI-ROFAGHA, R. V. O’TOOLE,W. B. analysed in a multivariate analysis with clinicoJ. DECENZO FARRAR,B. SICKLE-SANTANELLO, and D. YOUNG.Cancer 1990; 65: 1577-1582. pathological factors such as patient age, oestrogen and progesterone receptors, and tumour size. The This study was also aimed at identifying a role for inclusion of tumour grade in the analysis would have strengthened the study, as grade and SPF have DNA flow cytometry in node-negative disease. 0022-341 7/92/03032344 $05.00 0 1992 by John Wiley & Sons, Ltd.
324
CRITICAL SUMMARIES
However, in this paper, the authors restricted themselves to looking at DNA ploidy. Our own experience has been (see O’Reilly et al., 1990, quoted above) that DNA ploidy is not a significant prognostic indicator, either in node-negative or in node-positive disease. The first paper described above reported the same lack of prognostic significance for DNA ploidy but there is disagreement in the literature on this point. The present study had a reasonable, if not generous, sample size of 165 patients and DNA ploidy was correlated with patient age, oestrogen receptor status, treatment, and menopausal status. Good follow-up was available on these patients (mean around 8 years, range 3-1 5 years) and no complex statistical analysis was required as the result was clear-cut. There was no trend at all for ploidy to be a prognostic marker. Although there is disagreement in the literature with some studies finding a significantly prognostic role for ploidy at least on univariate analysis or a nonsignificant trend as reported by Sigurdsson et af.(see above), this paper strengthens the overall impression that SPF is a more powerful prognostic parameter in breast cancer. A technical point touched on in this paper is the use of ‘external’ DNA standards to calibrate test samples produced from paraffin-embedded tissue. The use of typical ‘external’ standards such as normal human lymphocytes as a DNA diploid standard is contra-indicated when paraffin-embedded material is used (Schutte et af., Cytometry 1985; 6: 2630). The present authors followed the suggestion of Schutte et al. and used normal cells from other blocks from the same patient as diploid controls to check the ploidy status of their test samples. Even this practice has been suggested to be fraught with problems (Price and Herman, Cytometry 1990; 11: 845-847). It is safest to use the normal cells which must be present within any block as the ‘DNA standard’. This means only analysing a sample as DNA aneuploid if two clear G, peaks are present within the test sample.
DNA Index and S-phase fraction and their combination as prognostic factors in operable ductal breast carcinoma. H. JOENSUU, S. TOIKKANEN and P. J. KLEMI.Cancer 1990; 66: 331-340. In this paper the value of SPF and DNA ploidy in 222 cases of operable ductal breast carcinoma is reported. This number of patients, as with the previous paper, would seem adequate if not generous; however, in this instance the cases are more heterogeneous as they include both node-positive and node-negative patients. One problem that I have found in assessing the validity of published studies is to determine what constitutes an adequate sample size. Many factors will influence the decision for a given type of disease and study. A problem is that some papers quote an overall figure for patients included, which seems reasonable but on closer examination the patients may be so heterogeneous (in terms of site of lesion, stage, grade, treatment, etc.) that reasonable statistical analysis is impaired. In the present study, the data on node-negative patients, on which I will concentrate, are based on only 79 cases. Nevertheless, the overall study is carefully performed with a thorough multivariate analysis, involving the flow cytometric parameters, histologic grade, menopausal status, and a number of pathological factors. In addition, very long follow-up was available on the patients (median 25 years, range 22-35 years). As with the study of Sigurdssonetat.(1990)andourownstudiesonnodenegative disease, tumour size was found to be a powerful prognostic factor. Flow cytometric data were also found to yield independent prognostic information. However, these authors chose to combine DNA ploidy and SPF, which they found gave better discrimination than either parameter alone. This is a reasonable approach but it does make comparison with other papers more difficult. Nevertheless, there is now good agreement in the literature that SPF or a parameter including SPF is a useful prognostic tool, particularly for node-negative disease.
MULTIPARAMETRIC FLOW CYTOMETRY-DNA PARAMETERS The next three papers reviewed widen our horizons by measuring not just DNA but an additional factor too. The real power of flow cytometry is its ability to measure multiple, linked parameters simultaneously on cells. Simple to use analytical flow
PLUS OTHER
cytometers can measure five or more parameters. Thus, it is possible to detect or measure a wide range of substances on or within cells simultaneously with the measurement of DNA content. Most often, though not exclusively, use is made of monoclonal
CRITICAL SUMMARIES
antibodies tagged directly or indirectly to fluorochromes, such as fluorescein.
325
specific antibody, that G, cells could be distinguished from mitotic cells by a difference in Ki-67 staining. Mitotic cells showed the strongest staining of all. The authors also suggest that this Flow cytometric analysis of DNA content and multiparametric method may enable the distinction keratins by using CK7, CK8, CK18, CK19 and of Go and GI cells, a major goal in cell kinetic KL1 monoclonal antibodies in benign and malig- studies. Thus, this technique offers the possibility of nant human breast tumors. M . FERRERO,F. distinguishing the five basic cell kinetic compartSPYRATOS, V. LE DOUSSAL, A. DESPLACES and J. ments, namely Go,G,, S, G,, and mitosis. However, ROUESSE. Cytometry 1990; 11: 7 1G724. the evidence presented to support the discrimiA common reason for wishing to combine stain- nation between Go and GI was only preliminary ing with monoclonal antibodies and DNA has been and it is clear that the authors intend to investigate to distinguish tumour cells from non-tumour cells. this area further. The authors also make clear their This is particularly a problem with DNA diploid or realization that applying such multiparametric near-diploid tumours. A good study was published methods to clinical tumours will be harder and the in 1984 (Braylan et al., Cancer Res 1984; 44: interpretation more complex. Nevertheless, I feel 5010-5016) in which lymphocyte surface markers that this is a fascinating paper which demonstrates were used successfully to improve the discrimi- the power of multiparametric flow cytometry to nation of near-diploid abnormalities in DNA unravel the detailed kinetic make-up of complex cell content in non-Hodgkin’s lymphoma. Since then, populations. anti-keratin antibodies have been used in a similar way, to produce cleaner DNA histograms, in carci- Progesterone receptor detection and quantification in breast tumors by bivariate immunofluorescence/ nomas from a variety of sites such as breast, DNAflow cytometry. Y. REMVIKOS, M. VUHAI,C. bladder, and head and neck. In this paper a similar LAINE-BIDRON, A. JoLLrvETandH. MAGDELENAT. finding was made in that by gating on cytokeratinpositive cells only, debris and non-epithelial cells Cytometry 1991; 12: 157-166. could be excluded from the DNA analysis. HowThis paper describes a method to measure DNA ever, the authors went further and looked at the content and progesterone receptor (PgR) simulrelative expression of the various types of keratin in taneously. This is not the first time that such a malignant vs. normal cells and diploid vs. aneuploid technique has been described (see Graham et al., tumour cells. They raise the possibility that certain Cancer Res 1989; 4 9 393k-3942) but this paper keratin phenotypes may give prognostic inforextends the application of the technique to clinical mation, though this study was too small (29 cases) breast tumours. Cell lines with known PgR levels to confirm this possibility. were used to check the sensitivity and reproducibility of the method, which was then applied to 31 Flow cytometric multiparameter analysis of prolifer- cases of breast cancer. PgR levels measured by flow ating cell nuclear antigenlcyclin and Ki-67 anti- cytometry were shown to correlate well with those gen: a new view of the cell cycle. G. LANDBERG, E. assessed by an immunoenzymatic cytosol assay. M. TANand G. Roos. Exp Cell Res 1990; 187: The authors were able to compare staining for PgR in diploid vs. aneuploid cells in aneuploid tumours 111-1 18. and to investigate the heterogeneity of staining In this study, performed on human haemato- around the cell cycle. It is clear that routine poietic cell lines, the authors have combined measurement of PgR by flow cytometry has practimeasurement of DNA content with two prolifer- cal disadvantages at present. For example, a tissue ation-linked antibodies, namely Ki-67 and an storage method is needed which is convenient and anti-PCNA antibody. Ki-67 is reputed to label the retains DNA and PgR staining. Nevertheless, majority of proliferative cells around the cell cycle, techniques such as the one described here offer the whilst this PCNA antibody labels predominantly possibility of simultaneous measurement of cliniS-phase cells. The authors were able to stain for all cally useful parameters. They also allow the varithree parameters (i.e., DNA, Ki-67, and PCNA) ation between cells to be looked at, rather than simultaneously. They showed, with elegant exper- simply measuring a crude average level of receptor, iments using metaphase-arrest agents and a mitosis- as conventional biochemical assay systems do.
326
CRITICAL SUMMARIES
WIDER APPLICATIONS OF FLOW CYTOMETRY human chromosomes. The ability to produce flow cytometric karyotypes has been developed over the past 15 years or so to a point where good quality results can be achieved from commercial flow cytometers. Individual chromosomes can be physically sorted into separate containers for further study. These techniques have wide applications in the study of a variety of cytogenetic defects and in the construction of DNA libraries. In the present study, the reproducibility of ‘normal’ flow karyoDetection and analysis by dual-laser flow cytometry types was investigated in detail. Such baseline data of bacteriophage T4 DNA inside Escherichia cofi. are important in using bivariate flow karyotyping to C. A. SANDERS, D. M. YAJKO,P. S. NASSOS, W. C. detect possibly subtle chromosomal aberrations and W. K. HADLEY. seen, for example, in tumour cells. The authors HYUN,M. J. FULWYLER Cytometry 1991; 12: 167-1 7 1. make recommendations for performing cytogenetic comparisons of tumour and normal cells from Having stressed the diversity of flow cytometric individual patients. applications, it may seem odd to have chosen this
Flow cytometric techniques can contribute to our understanding in diverse areas of contemporary biology. In this final section, I have chosen three disparate examples of such studies. I could just have well selected papers describing anything from the measurement of intracellular pH to flow cytometric applications in marine biology or water quality control.
paper which has ‘DNA’ in its title. However, the reasons for measuring DNA and the type of DNA measured are, in this case, quite different. A complex dual-laser flow cytometer was used to measure staining with two separate DNA dyes simultaneously. The two dyes were Hoechst 33258, which binds preferentially to A-T rich regions of DNA, and chromomycin A3, which binds to G-C rich regions. A change in the ratio of staining intensities with these two dyes was used to detect the presence of viral DNA within the host bacterium. The authors were able to detect viral DNA in bacteria within 25 min of infection. The method described is rapid and could have widespread applications in the study of viral infections. A drawback is that it does require an expensive and relatively complex flow cytometer. Bivariate flow karyotyping of human chromosomes: evaluation of variation in Hoechst 33258 fluorescence, chromomycin A3 fluorescence and relative chromosomal DNA content. G. A. BOSCHMAN, W. RENS,C. H. VAN OVEN,E. M. M. MANDERS and J. A. ATEN.Cytometry 1991; 12: 559-569. This study also involved the measurement of DNA with the same two dyes used in the previous paper, but this time it was not total cellular or viral DNA that was measured but chromosomal DNA. Once again, an expensive dual-laser facility was required. This particular study is a very technical one, which looks at the standardization of the technique of measuring the DNA content of individual
Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils. S. PERTICARARI, G. PRESANI,M. A. MANCIAROTTI and E. BANFI.Cytometry 1991; 12: 687-693. This final paper is an example of an exciting type of flow cytometric application in the field of cell biology. It is possible, using a flow cytometer, to detect the presence of a receptor (or particle) and simultaneously to measure the biochemical effect of activation caused by triggering the receptor. Flow cytometric measurements can be made rapidly to look at the early stages of cellular activation and, indeed, time can be used as an additional parameter in such studies. In this paper, a two-colour fluorescent method is described which allows the simultaneous quantitation of the phagocytosis rate and the oxidative burst activation of polymorphonuclear leukocytes (PMNL). Fluorescent bacteria were used and when ingested, they trigger an oxidative burst in the PMNL. This oxidative activity leads to the conversion of a non-fluorescent precursor to a detectable fluorescent dye. The authors suggest that this technique may have an application in studying infectious diseases in chronic granulomatous disease patients.
RICHARD S. CAMPLEJOHN Richard Dimbleby Department of Cancer Research, UMDS, St Thomas’ Hospital, London SEl 7EH, U.K.
