Acknowledgements
First of all, I cannot give a word to fulfill my deeps love and thanks to (Allah) for lighting me the way not only throughout this piece of work but also throughout my whole life.
This work is dedicated to:
My husband; Dr Hussein Al Omer and my Family
I am indebted to King Saud University for support and encouragement to finish this work.
Also, I wish to express my deep thanks to:
• Prof. Dr./ Abdelfattah M. Attalah, Professor of Genetics & Immunology, George Washington University, USA (Former). Director of Biotechnology Research Center, New Damietta, Egypt, for continuous advice
• Prof. Dr./ Ahmed Abd Al Salam Settin, Professor of Pediatrics & Genetics, Faculty of Medicine, Mansoura University for his continuous help
Finally, I am indebted to all the team of the honorable Genetics Unit, Mansoura University Children Hospital, for their continuous support and encouragement
Gihan El Hussieny Gawish
January 2009
Contents
Page Title
1 Introduction
4 Review of Literature
4 I- Acute lymphoblastic leukemia
4 1-Definition
5 2-Incidence of Leukemia
6 3-Types of leukemia
6 4-Biological Classification of ALL
7 5-Causing of Leukemia
9 6-The Signs of Leukemia
11 7-Stages of Childhood ALL
11 8-Treatment of Childhood ALL
14 9-Four Phases of Treatment
15 II- Cell cycle and apoptosis
15 1-Cell cycle
19 1-1-Cell cycle and cancer
20 2-Apoptosis and its markers
21 2-1-The mechanism of apoptosis
25 2-2-Apoptosis-targeted therapies for hematologica malignancies
30 2-3-The apoptosis promoter (p53)
35 2-4-The inhibitor of apoptosis
35 2-4-1-Bcl2 proteins
38 2-4-2-C-myc oncogene
42 III-Flow cytometry
42 1-Introduction
44 2-Principles of flow cytometric instrumentation
46 2-1-Fluidic system
49 2-2-Illumination system
53 2-3-Optical and electronics system
54 2-4-Data storage and computer control system
59 3-Data analysis
63 IV- Applications of flow cytometry
65 1-Cell cycle analysis
66 1-1-Staining procedure
Page
67 1-2-Evaluation of DNA histogram
71 2-Immunophenotyping Applications
72 2-1-Erythrocyte analysis
73 2-2-HIV monitoring
73 2-3-Immunophenotyping of leukemias
75 2-4Quantification of stem cells
75 2-5-Platelet analysis
76 2-6-Testing for HLA-B27
76 3-Major applications of apoptosis analysis
77 3-1- Apoptosis light scatter
78 3-2-Apoptosis DNA analysis
80 3-3-Apoptosis cell membrane analysis
82 3-4-Apoptosis enzyme analysis
83 3-5-Apoptosis organelle analysis
83 4- Detection of apoptotic markers
86 V- Flourescence in situ hybridization
86 1-Introduction
91 2-Three different types of FISH probes
91 2-1-Locus specific probes
91 2-2-Alphoid or centromeric repeat probes
92 2-3-Whole chromosome probes
92 3-Applications of FISH
96 3-1-ALL investigation by FISH
97 3-1-1-Philadelphia
106 VI- References
144 VII- Life Flowcytometric Figures
147 VIII-Life FISH Pictures
List of Figure
Page Review of Literature
18 A schematic representation of the mammalian cell cycle Figure (2-1)
22 The intrinsic or mitochondrial pathway Figure (2-2)
24 The mechanism of apoptosis (Apoptosis triggered by external signals: the extrinsic or death receptor path way) Figure (2-3)
26 Diagram of the mitochondrial and death receptor pathways of cell death Figure (2-4)
47 Facscaliblur flow cytometry instrument Figure (3-1)
48 Flow cytometer system (Facscalibur) Figure (3-2)
50 Flow cytometers use the principle of hydrodynamics focusing for presenting cells to a laser Figure (3-3)
52 A simplified illustration of Flow Cytometry Figure (3-4)
57 Two parameter histogram and dot plot displaying FL1-FITC on the x axis and FL2-PE on the y axis Figure (3-5)
58 FlowJo program F
Acknowledgements
First of all, I cannot give a word to fulfill my deeps love and thanks to (Allah) for lighting me the way not only throughout this piece of work but also throughout my whole life.
This work is dedicated to:
My husband; Dr Hussein Al Omer and my Family
I am indebted to King Saud University for support and encouragement to finish this work.
Also, I wish to express my deep thanks to:
• Prof. Dr./ Abdelfattah M. Attalah, Professor of Genetics & Immunology, George Washington University, USA (Former). Director of Biotechnology Research Center, New Damietta, Egypt, for continuous advice
• Prof. Dr./ Ahmed Abd Al Salam Settin, Professor of Pediatrics & Genetics, Faculty of Medicine, Mansoura University for his continuous help
Finally, I am indebted to all the team of the honorable Genetics Unit, Mansoura University Children Hospital, for their continuous support and encouragement
Gihan El Hussieny Gawish
January 2009
Contents
Page Title
1 Introduction
4 Review of Literature
4 I- Acute lymphoblastic leukemia
4 1-Definition
5 2-Incidence of Leukemia
6 3-Types of leukemia
6 4-Biological Classification of ALL
7 5-Causing of Leukemia
9 6-The Signs of Leukemia
11 7-Stages of Childhood ALL
11 8-Treatment of Childhood ALL
14 9-Four Phases of Treatment
15 II- Cell cycle and apoptosis
15 1-Cell cycle
19 1-1-Cell cycle and cancer
20 2-Apoptosis and its markers
21 2-1-The mechanism of apoptosis
25 2-2-Apoptosis-targeted therapies for hematologica malignancies
30 2-3-The apoptosis promoter (p53)
35 2-4-The inhibitor of apoptosis
35 2-4-1-Bcl2 proteins
38 2-4-2-C-myc oncogene
42 III-Flow cytometry
42 1-Introduction
44 2-Principles of flow cytometric instrumentation
46 2-1-Fluidic system
49 2-2-Illumination system
53 2-3-Optical and electronics system
54 2-4-Data storage and computer control system
59 3-Data analysis
63 IV- Applications of flow cytometry
65 1-Cell cycle analysis
66 1-1-Staining procedure
Page
67 1-2-Evaluation of DNA histogram
71 2-Immunophenotyping Applications
72 2-1-Erythrocyte analysis
73 2-2-HIV monitoring
73 2-3-Immunophenotyping of leukemias
75 2-4Quantification of stem cells
75 2-5-Platelet analysis
76 2-6-Testing for HLA-B27
76 3-Major applications of apoptosis analysis
77 3-1- Apoptosis light scatter
78 3-2-Apoptosis DNA analysis
80 3-3-Apoptosis cell membrane analysis
82 3-4-Apoptosis enzyme analysis
83 3-5-Apoptosis organelle analysis
83 4- Detection of apoptotic markers
86 V- Flourescence in situ hybridization
86 1-Introduction
91 2-Three different types of FISH probes
91 2-1-Locus specific probes
91 2-2-Alphoid or centromeric repeat probes
92 2-3-Whole chromosome probes
92 3-Applications of FISH
96 3-1-ALL investigation by FISH
97 3-1-1-Philadelphia
106 VI- References
144 VII- Life Flowcytometric Figures
147 VIII-Life FISH Pictures
List of Figure
Page Review of Literature
18 A schematic representation of the mammalian cell cycle Figure (2-1)
22 The intrinsic or mitochondrial pathway Figure (2-2)
24 The mechanism of apoptosis (Apoptosis triggered by external signals: the extrinsic or death receptor path way) Figure (2-3)
26 Diagram of the mitochondrial and death receptor pathways of cell death Figure (2-4)
47 Facscaliblur flow cytometry instrument Figure (3-1)
48 Flow cytometer system (Facscalibur) Figure (3-2)
50 Flow cytometers use the principle of hydrodynamics focusing for presenting cells to a laser Figure (3-3)
52 A simplified illustration of Flow Cytometry Figure (3-4)
57 Two parameter histogram and dot plot displaying FL1-FITC on the x axis and FL2-PE on the y axis Figure (3-5)
58 FlowJo program F
Acknowledgements
First of all, I cannot give a word to fulfill my deeps love and thanks to (Allah) for lighting me the way not only throughout this piece of work but also throughout my whole life.
This work is dedicated to:
My husband; Dr Hussein Al Omer and my Family
I am indebted to King Saud University for support and encouragement to finish this work.
Also, I wish to express my deep thanks to:
• Prof. Dr./ Abdelfattah M. Attalah, Professor of Genetics & Immunology, George Washington University, USA (Former). Director of Biotechnology Research Center, New Damietta, Egypt, for continuous advice
• Prof. Dr./ Ahmed Abd Al Salam Settin, Professor of Pediatrics & Genetics, Faculty of Medicine, Mansoura University for his continuous help
Finally, I am indebted to all the team of the honorable Genetics Unit, Mansoura University Children Hospital, for their continuous support and encouragement
Gihan El Hussieny Gawish
January 2009
Contents
Page Title
1 Introduction
4 Review of Literature
4 I- Acute lymphoblastic leukemia
4 1-Definition
5 2-Incidence of Leukemia
6 3-Types of leukemia
6 4-Biological Classification of ALL
7 5-Causing of Leukemia
9 6-The Signs of Leukemia
11 7-Stages of Childhood ALL
11 8-Treatment of Childhood ALL
14 9-Four Phases of Treatment
15 II- Cell cycle and apoptosis
15 1-Cell cycle
19 1-1-Cell cycle and cancer
20 2-Apoptosis and its markers
21 2-1-The mechanism of apoptosis
25 2-2-Apoptosis-targeted therapies for hematologica malignancies
30 2-3-The apoptosis promoter (p53)
35 2-4-The inhibitor of apoptosis
35 2-4-1-Bcl2 proteins
38 2-4-2-C-myc oncogene
42 III-Flow cytometry
42 1-Introduction
44 2-Principles of flow cytometric instrumentation
46 2-1-Fluidic system
49 2-2-Illumination system
53 2-3-Optical and electronics system
54 2-4-Data storage and computer control system
59 3-Data analysis
63 IV- Applications of flow cytometry
65 1-Cell cycle analysis
66 1-1-Staining procedure
Page
67 1-2-Evaluation of DNA histogram
71 2-Immunophenotyping Applications
72 2-1-Erythrocyte analysis
73 2-2-HIV monitoring
73 2-3-Immunophenotyping of leukemias
75 2-4Quantification of stem cells
75 2-5-Platelet analysis
76 2-6-Testing for HLA-B27
76 3-Major applications of apoptosis analysis
77 3-1- Apoptosis light scatter
78 3-2-Apoptosis DNA analysis
80 3-3-Apoptosis cell membrane analysis
82 3-4-Apoptosis enzyme analysis
83 3-5-Apoptosis organelle analysis
83 4- Detection of apoptotic markers
86 V- Flourescence in situ hybridization
86 1-Introduction
91 2-Three different types of FISH probes
91 2-1-Locus specific probes
91 2-2-Alphoid or centromeric repeat probes
92 2-3-Whole chromosome probes
92 3-Applications of FISH
96 3-1-ALL investigation by FISH
97 3-1-1-Philadelphia
106 VI- References
144 VII- Life Flowcytometric Figures
147 VIII-Life FISH Pictures
List of Figure
Page Review of Literature
18 A schematic representation of the mammalian cell cycle Figure (2-1)
22 The intrinsic or mitochondrial pathway Figure (2-2)
24 The mechanism of apoptosis (Apoptosis triggered by external signals: the extrinsic or death receptor path way) Figure (2-3)
26 Diagram of the mitochondrial and death receptor pathways of cell death Figure (2-4)
47 Facscaliblur flow cytometry instrument Figure (3-1)
48 Flow cytometer system (Facscalibur) Figure (3-2)
50 Flow cytometers use the principle of hydrodynamics focusing for presenting cells to a laser Figure (3-3)
52 A simplified illustration of Flow Cytometry Figure (3-4)
57 Two parameter histogram and dot plot displaying FL1-FITC on the x axis and FL2-PE on the y axis Figure (3-5)
58 FlowJo program F
Childhood Acute Lymphoblastic Leukemia Dr ihan !L Hussieny awish" MSc" #hD $ Acknowledgements First of all, I cannot give a word to fulfill my deeps love and thanks to (Allah) for lighting me the way not only throughout this piece of work but also throughout my whole life. This work is dedicated to: My husband; Dr Hussein Al %mer and my Family I am indebted to &ing Saud 'ni(ersity for support and encouragement to finish this work. lso, I wish to e!press my deep thanks to: Prof. Dr./ Abdelfattah M. Attalah, "rofessor of #enetics $ Immunology, #eorge %ashington &niversity, &' (Former)$ *irector of +iotechnology ,esearch -enter, .ew *amietta, /gypt, for continuous advice Prof. Dr./ Ahmed Abd Al Salam Settin, "rofessor of "ediatrics $ #enetics, Faculty of Medicine, Mansoura &niversity for his continuous help Finally, I am indebted to all the team of the honorable #enetics &nit, Mansoura &niversity -hildren 0ospital, for their continuous support and encouragement
Gihan El Hussieny Gawish January 2! 1 Contents Pa"e )itle * Introduction + ,e(iew o- Literature + I. Acute lymphoblastic leukemia + 23*efinition / 13Incidence of 4eukemia 0 53Types of leukemia 0 63+iological -lassification of 44 1 73-ausing of 4eukemia 2 83The 'igns of 4eukemia ** 93'tages of -hildhood 44 ** :3Treatment of -hildhood 44 *+ ;3Four "hases of Treatment */ II. Cell cycle and apoptosis */ 23-ell cycle *2 2323-ell cycle and cancer 34 13poptosis and its markers 3* 1323The mechanism of apoptosis 3/ 1313poptosis3targeted therapies for hematologica malignancies 54 1353The apoptosis promoter (p75) 5/ 1363The inhibitor of apoptosis 5/ 2-4-1-Bcl2 proteins 56 2-4-2-C-myc oncogene +3 III.Flow cytometry +3 23Introduction ++ 13"rinciples of flow cytometric instrumentation +0 2-1-Fluidic system +2 2-2-Illumination system /5 2-3-Optical and electronics system /+ 2-4-Data storage and computer control system /2 53*ata analysis 05 I7. Applications o- -low cytometry 0/ 23-ell cycle analysis 00 1-1-Staining procedure 5 Pa"e 01 1-2-Evaluation o D!" #istogram 1* 13Immunophenotyping pplications 13 2-1-Eryt#rocyte analysis 15 2-2-$I% monitoring 15 2-3-Immunop#enotyping o leu&emias 1/ 2-4'uantiication o stem cells 1/ 2-(-)latelet analysis 10 2-*-+esting or $,"-B2- 10 53Ma<or applications of apoptosis analysis 11 3-1- "poptosis lig#t scatter 16 3-2-"poptosis D!" analysis 64 3-3-"poptosis cell mem.rane analysis 63 3-4-"poptosis en/yme analysis 65 3-(-"poptosis organelle analysis 65 63 *etection of apoptotic markers 60 7. Flourescence in situ hybridi8ation 60 23Introduction 2* 13Three different types of FI'0 probes 2* 2-1-,ocus speciic pro.es 2* 2-2-"lp#oid or centromeric repeat pro.es 23 2-3-0#ole c#romosome pro.es 23 53pplications of FI'0 20 532344 investigation by FI'0 21 3-1-1-)#iladelp#ia *40 7I. ,e-erences *++ 7II. Li-e Flowcytometric Figures *+1 7III.Li-e FISH #ictures 6 List of Figure Pa"e ,e(iew o- Literature *6 schematic representation of the mammalian cell cycle Figure 93.*: 33 The intrinsic or mitochondrial pathway Figure 93.3: 3+ The mechanism of apoptosis (poptosis triggered by e!ternal signals: the e!trinsic or death receptor path way) Figure 93.5: 30 *iagram of the mitochondrial and death receptor pathways of cell death Figure 93.+: +1 Facscaliblur flow cytometry instrument Figure 95.*: +6 Flow cytometer system (Facscalibur) Figure 95.3: /4 Flow cytometers use the principle of hydrodynamics focusing for presenting cells to a laser Figure 95.5: /3 simplified illustration of Flow -ytometry Figure 95.+: /1 Two parameter histogram and dot plot displaying F423FIT- on the ! a!is and F413"/ on the y a!is Figure 95./: /6 Flow=o program Figure 95.0: 04 nalysis pulse width versus pulse height or area we can eliminate the ma<ority of #> doublets that appear as #1 Figure 95.1: 0* *. histogram Figure 95.6: 03 *. histogram (aneuphliod population) Figure 95.2: 02 -oeffecient of ?ariation (-.?.) Figure 9+.*: 12 "ropidium iodide and T@3",@35 Figure 9+.3: 6* 'ub #2 peak by propidium iodide staining Figure 9+.5: 66 Fluoresence in situ hypridiAation Figure 9/.*: 7 List o- Abbre(iations AL cute leukemia ALL cut lymphoblastic leukemia AL) lanine amino transaminase A% cridine orange AS) spartate amino transaminase BM +one marrow CBC -omplete blood picture CD -luster of differentiation CML -hronic myeloid leukemia C7 -oefficient of variation DA#I 638 diamino 3131phenylindole DI *. inde! DMS% *imethylsulfo!ide D;A *eo!yribonucleic acid !B /thedium bromide !D)A /thyline diamine tetraacetic acid FAB French merican +ritish FACS Flow activated cell sorter FISH Fluorescence in situ hybridiAation FI)C Flourecien isothiocyanate 4<* "hase represents the gap in of *. replication time between mitosis and the start 3<M "hase represents the gap between the end of *. replication onest of mitosis HI7 0uman immunodeficiency virus HLA 0uman leukocyte antigen H#7 0uman papilloma virus LC 4iver cirrhosis MMC Mithramycin M,D Minimal residual disease #BS "hosphate buffer saline #I "ropidium iodide #S "hosphatidylserine S phase *. syntheis =BC %hite blood cells Introduction 8 -hildhood acute lymphoblastic leukemia (44) is a disease in which too many underdeveloped lymphocytes are found in a childBs blood and bone marrow. 4ymphocytes are infection3fighting white blood cells. 44 is the most common form of leukemia in children, and the most common kind of childhood cancer #Moorman et al., 2$%. cute lymphoblastic leukemia (44) represents nearly one third of all pediatric cancers. nnual incidence of 44 is about 5> cases per million populations, with a peak incidence in patients aged 137 years. lthough a small percentage of cases are associated with inherited genetic syndromes, the cause of 44 remains largely unknown #Jeffrey, 2&%$ Flow cytometry can be applied in basic research and in the clinic to identify and measure apoptotic cells. The choice of a particular flow method depends on several variables (cell system, type of flow cytometer, type of apoptosis inducer, type of information reCuired) #'o"h and Dulin", 2&%. The cell cycle was subdivided into four consecutive phases; #2 or pre3synthetic phase, ', #1 or post3synthetic phase, and M phase during which mitotic division into two daughter cells takes place. The #1 phase represents the gap in time between the end of *. replication and onset of mitosis. It is possible to discrimination between #2 vs, ' vs, #1 or M cells because of the difference in their *. content #(abino)it*h, +!!,%. The *. content of the cell can provide a great deal of information about the cell cycle. The measurement of the *. content of cells was one of the first ma<or applications of flow cytometry #Albro et al., +!!,%. 9 poptosis (programmed cell death) is a physiologic phenomenon where in the dying cell plays an active part in its own destruction #S*huler et al., +!!-%. poptosis plays a role in many diseases. There is a great potential for treatment of these diseases in developing agents that can alter the apoptotic process and change the natural disease progression. Molecules whose roles in apoptosis have been investigated include +cl31 and c3myc proteins, the p75 tumor suppressor gene and various tumor suppressor gene products #Menende. et al., 2-%. "75 is a pro3apoptotic genes present in all cells, but has special significance to cancer cells. It is a tumor repressor gene, meaning that its presence reduces the occurrence of cancer tumors by promoting apoptosis in cancer cells #Polya/ et al., +!!0%. +-41 is an important regulator of apoptosis, The oncogenic activity of the +cl1 gene is carried out via suppression of lymphocytic apoptosis or programmed cell death #1ory 2 Adams, 22 and (oumier et al., 22%. -3Myc is widely known as a crucial regulator of cell proliferation in normal and neoplastic cells 93e*hsler et al., +!!02 4a**hini and Penn, +!!5%$ The technology of flow cytometry and the discovery of a method to produce monoclonal antibodies have made possible the clinical use of flow cytometry for the identification of cell populations. Monoclonal antibodies (tagged) with the fluorescent dye are commonly used for the identification of cell surface antigens and fluorescent dyes that directly and specifically bind to certain components of the cell (i.e. *.) are used for cell cycle analysis #6han" et al., 2&%. Fluorescence in situ hybridiAation (FI'0) allows identification of specific seCuences in a structurally preserved cell, in metaphase or interphase : #1hat.imeletiou et al., 2&%. FI'0 is increasingly used for the identification of 44. FI'0 plays an important role in detecting chromosome changes #Primo et al., 2,%. lmost all the chromosome abnormalities in 44 are translocations. The most common one is "hiladelphia chromosome. It is the main product of the t(;;11) translocation. This translocation
causes a rearrangement between the proto3oncogene c3+4 and a gene
called the breakpoint cluster region (+-,). The +-,D+4 fusion gene resulting from t(;;11) translocation. FI'0 is increasingly used for the identification of +-,D+4 gene rearrangements #(udol7h et al., 2&%. >. Acute Lymphoblastic Leukemia ; cute lymphoblastic leukemia (44) is the most common form of childhood cancer. It is a type of cancer that starts from white blood cells in the bone marrow called lymphocytes. In most cases it Cuickly moves into the blood. It can then spread to other parts of the body including the lymph nodes, liver, spleen and central nervous system #Moorman et al., 2$%. 4eukemia is a cancer of the blood cells. There are several types of leukemia and these are classified by how Cuickly they progress and what cell they affect. cute leukemia is fast3growing and can overrun the body within a few weeks or months. +y contrast, chronic leukemia is slow3growing and progressively worsens over years #1arolyn et al., 22%. .ormal blood cells contain white blood cells, red blood cells, platelets and fluid called plasma. ll of these products are formed in the bone marrow, a spongy area located in the center of bones. It contains a small percentage of cells that are in development and are not yet mature. These cells are called blasts. @nce the cell has matured, it moves out of the bone marrow and into the circulating blood. The body has mechanisms to know when more cells are needed and has the ability to produce them in an orderly fashion #1arroll et al., 2,%.
2> *.Incidence o- Leukemia? cute lymphoblastic leukemia is the most common form of childhood leukemia where it accounts for about 97E of childhood leukemia and 17E of all pediatric cancer #8an./ows/y, 2%$ .ational -ancer Institue, -airo &niversity, 44 represents 15.5E of all pediatric malignancies and 97E of all pediatric leukemias. In a more recent research in the "ediatric 0eamatologyD@ncology &nit, in 'hams &niversity 0ospital, 44 constitutes :1E of all leukemic cases #9halifa et al., +!!!%. The global incidence of leukemias is about : to ; per 2>>,>>> people each year. ppro!imately 17>,>>> new cases occur annually worldwide. 4eukemia accounts for 1.7E of overall cancer incidence. 0owever, its incidence among children demonstrates its significance. lthough childhood cases (through 26 years of age) account for about 21E of all leukemias, childhood cancer is the second biggest killer of children (after accidents) and leukemia is the most common form of childhood cancer. The incidence of childhood 44 in the &nited 'tates has increased appro!imately 1>E over the past two decades, mostly in the >3 to 63year3old age group. @ver the course of this century, leukemia rates have also generally increased #Sandler and (oss, +!!0%. cute lymphoblastic leukemia affects slightly more boys than girls. It occurs predominantly in children, peaking at four years of age. It is seen more freCuently in industrialiAed nations, and it is slightly more common among white children and boys. 'tudies have suggested that patients who are younger than thirty five years of age far better than older patients #Jeffrey, 2&%$ 22 3.)ypes o- leukemia? +y considering whether leukemias are acute or chronic, and whether they are myelogenous or lymphocytic, they can be divided into four main types. The first one is an acute myeloid leukemia which occurs in both children and adults. The second one is an acute lymphocytic leukemia which is the most common type seen in children, but also seen in adultBs over87.The third one is a chronic myelogenous leukemia which occurs mostly in adults. -hronic lymphocytic is the fourth type which is the most often seen in people over age77, can affect younger adults, but almost never seen in children #Pui, +!!&%. In acute leukemia, the bone marrow cells are unable to properly mature. Immature leukemia cells, which are often called blasts, continue to reproduce and accumulate. In chronic leukemia, the cells can mature but not completely. They are not really normal. They generally do not fight infection as well as do normal white blood cells. @f course, they live longer, build up, and crowd out normal cells. The types of leukemia are also grouped by the type of white blood cell that is affected, leukemia that affects lymphoid cells is called lymphocytic leukemia, and leukemia that affects myeloid cells is called myeloid leukemia or myelogenous leukemia #8i*htman et al., +!!&%. 5 . Biological Classi-ication o- ALL ? cute lymphoblastic leukemia blasts are derived from either +3cell or T3cell lineages, as determined by cell surface and other markers. small percentage of the cells are either so primitive that they do not e!press enough markers to identify #(oss et al., 2, and Pullen et al., +!!!%.
21 cute lymphoblastic leukemia is categoriAed according to a system know as the French3merican3+ritish (F+) Morphological -lassification 'cheme for 44. 442 is mature3appearing lymphoblasts (T3cells or pre3+3 cells); these cells are small with uniform genetic material, regular nuclear shape, nonvisible nucleoli, and little cytoplasm. 441 is immature and pleomorphic lymphoblasts (T3cells or pre3+3cells), these cells are large, variable in siAe, varaiable genetic material, irregular nuclear shape, one or more large nucleoli and variable cytoplasm. 445 is lymphoblast(+3 cells),these are large, genetic material is finely stripped and uniform, nuclear shape is regular, there are one or more prominent nucleoli, and cytoplasm is moderately abundant #S*hra77e et al., 2%$
+.Causing o- Leukemia? The causes of the disease are not known, but e!perts believe that 44 develops from a combination of genetic and environmental factors. number of genetic mutations associated with 44 have been identified. Missing or defective genes that suppress tumors are responsible for cases of 44 #Guo et al., 2&%.
'everal things have been identified as risk factors3that is, e!posure to them puts a person at a higher risk of developing leukemia, but it is not a certainly that this e!posure will lead to leukemia. These factors include e!posure to high3energy radiation, like that released from a nuclear accident or bomb. 'ome genetic syndrome put a person at higher risk. "eople who work with the chemical benAene over a long period of time also have a greater chance of getting leukemia. 'ome scientist feel that e!posure to electromagnetic fields, like those that come from power lines, may put a person to higher risk, but this has not been proven #Pui et al., 2+%. 25 0eredity, radiation, chemical e!posures, and treatment with chemotherapeutic agents have been implicated in the development of leukemia. ?iral infection by at least one known virus, human T3cell leukemiaDlymphotropic virus type I (0T4?32), is a well3understood cause of adult T3cell leukemia #4ran*hini, +!!& and Grea)es, +!!0%. nother group of risk factors includes occupational and environmental e!posure to radiation or chemicals. The best established cause of leukemia among children is in utero e!posure to diagnostic F3rays. 4eukemia in adults is strongly associated with occupational e!posure to ioniAing radiation. There is little evidence, however, that nonioniAing radiation such as electromagnetic fields (/MF) induces leukemia. Indeed, two recent studies have shown that /MF e!posure is not a ma<or risk factor for leukemia in children or in adults. 'ome studies have reported an association between cancer and #ig# levels of electromagnetic radiation (/M,). %hether lower levels of radiation (eg, living near power lines, video screen emissions, small appliances, cell phones) play any ma<or role is uncertain but probably unlikely #8inet et al., +!!0 and :er/asalo, +!!$%. +ecause most people in the general population are not e!posed to chemotherapeutic drugs or occupationally e!posed to radiation or chemical solvents, e!posure to these agents cannot e!plain the causes of the ma<ority of leukemia cases diagnosed each year. %e conservatively estimate that the causes of at least 1>,>>> (appro!imately 9>E) of the 1:,>>> new leukemia cases that develop annually in the &nited 'tates are une!plained. Thus, the causes of leukemia remain largely unknown. lthough some success has been achieved in treating leukemias, especially in children, mortality rates have remained relatively high (appro!imately 97E in the &nited 'tates) #9a.a/ et al., +!!0%. 26 #enetic predisposition may play a ma<or role in both adult and childhood leukemia. lthough the 4eukemia 'ociety of merica emphasiAes the fact that anyone may develop the disease, an increased risk e!ists among /astern /uropean =ews, and a decreased risk e!ists among sians (differences in diet and lifestyle may play a role, however). Individuals with a family history of leukemia or lymphoma have a 7.83fold increased risk for M4. "arents of children with *own syndrome also have an increased risk of leukemia #Grea)es, +!!0 and Shannon et al., +!!2%. &p to 87E of leukemias contain genetic rearrangements, called translo*ations, in which some of the genetic material (genes) on a chromosome may be altered, or shuffled, between a pair of chromosomes. For e!ample the most common genetic in<ury in 44 is t(21;12), which means a translocation with a genetic shift between chromosome 21 and 12. It is also referred to as T/43M42 fusion and occurs in appro!imately 1>E of 44 patients. ,esearchers believe that this translocation may occur during fetal development in some patients. bout 1>E of adults and about 7E of children with 44 have a genetic abnormality called the "hiladelphia ("h) chromosome t(;;11). nother important chromosome translocation is t(6;22) involving the M44 gene on chromosome II. @ften occurring in children under one year old #9handa/ar et al., 2&%. / . )he Signs and diagnosis o- Leukemia ? The blast cells are unable to perform their normal function of fighting infection, so patients may develop fevers or infections that wonBt go away. s the number of immature cells (blasts) increases, the normal cells are crowded out. This leads to low red blood cell counts and platelets #Smith et al., +!!$%. 27 cute lymphoblastic leukemia tends to cause symptoms more rapidly than chronic leukemia. 'ome common symptoms include fever, chills, weakness and fatigue, swollen or tender lymph nodes, liver or spleen, easy bleeding or bruising, swollen or bleeding gums, night sweats, and bone pain. The abnormal cells can accumulate in the brain or spinal cord, causing headaches, vomiting, confusion, or seiAures #Ada*hi et al., 2&%.
In acute lymphoblastic leukemia, the doctor asks about medical history and conducts a physical e!am. *uring the e!am, abnormalities such as enlarged spleen, liver or lymph nodes may be detected, prompting further investigation. -omplete blood count would find blast cells present in the blood, thus suggestion a diagnosis of leukemia. This test can reveal that the patient has leukemia. sample of bone marrow is determined the type of leukemia #1ham7lin et al. +!5! and 'ur"er et al., 2,%.
complete blood cell count is the first step in diagnosing 44. This test will often show various findings, including the following: The presence of circulatory leukemic blast cells, the presence and severity of anemia and the count of a variety of blood cell types. ( high white blood cell count indicates a more severe disease.) These tests will not always show the presence of leukemic cells. +lood tests do not always detect leukemia, and about 2>E of patients with 44 have a normal blood cell count #Ada*hi et al., 2&%. If the results of the blood tests are abnormal or the physician suspects leukemia despite normal cell counts, a bone marrow aspiration and biopsy are the ne!t steps #(e.aei et al., 2,%. 28 If bone marrow e!amination confirms 44, a spinal tap may be performed, which uses a needle inserted into the spinal canal. sample of cerebrospinal fluid with leukemia cells is a sign that the disease has spread to the central nervous system. In most cases of childhood 44, leukemic cells are not found in the cerebrospinal fluid 9:ieira et al., 2&%. 0 . )reatment o- Childhood ALL ? The treatment depends on age, the results of laboratory tests, and whether or not the child has been previously treated for leukemia. &ntreated 44 means that no treatment has been given e!cept to reduce symptoms. There are too many white blood cells in the blood and bone marrow, and there may be other signs and symptoms of leukemia. ,emission means that treatment has been given and the number of white blood cells and other blood cells in the blood and bone marrow is normal that there no signs or symptoms of leukemia. ,ecurrent disease means that the leukemia has come back after going into remission. ,efractory disease means that the leukemia failed to go into remission following treatment #'assan et al., +!!0%. There are treatments for all patients with childhood acute lymphoblastic leukemia. The primary treatment for 44 is chemotherapy. ,adiaion therapy may be used in certain cases. +one marrow transplantation is being studied in clinical trials #;*/un et al., +!!0%. cute lymphoblastic leukemia patients should receive chemotherapy drugs as soon as possible after diagnosis. -hemotherapy uses strong drugs to kill leukemia cells. The goal of chemotherapy is to achieve remission (no symptoms of 44) and to restore normal blood cell production. -ommon chemotherapy drugs include do!orubicin, fludarabine and cyclophosphamide. 29 The drugs used depend on factors such as the patientBs age and the number and type of leukemia cells in the blood. &nfortunately, chemotherapy also kills normal cells, so 44 patients receiving chemotherapy may have side effects, including nausea, tiredness and a higher risk of infections #'aldu..i et al., 2&%. For most patients, chemotherapy restores normal blood cell production within a few weeks, and microscopic e!aminations of their blood and marrow samples will show no signs of leukemia cells. %hen this happens, the disease is in remission. lthough chemotherapy often brings long3lasting remissions in children, in adults, 44 freCuently returns. If the 44 returns, patients and their doctors can consider more chemotherapy or a marrow or blood cell transplant. -hemotherapeutic agents kill cancer cells by activating apoptosis,
or programmed cell death. Ma<or apoptotic pathways and the specific role of key proteins
in this response is described. The e!pression level of some of these proteins,
such as +cl1, +F, and caspase 5, has been shown to be predictive
of ultimate outcome in hematopoietic tumors. .ew therapeutic
approaches that modulate the apoptotic pathway are now available
and may be applicable to the
treatment of childhood 44 #Donadieu 2 Hill, 2+ and <a/ase et al., 2&%. ,adiation therapy uses 13rays or other high3energy rays to kill cancer cells and shrink tumors. ,adiation for 44 usually comes from a machine outside the body (e!ternal beam radiation therapy) #Durrant et al., +!!0%. +one marrow transplantation is a newer type of treatment. First, high doses of chemotherapy with or without radiation therapy are given to destroy all of the bone marrow in the body. bone marrow transplant using marrow 2: from a relative or person not related to the patient is called an allogeneic bone marrow transplant #;lri*h et al., 2+%. n even newer type of bone marrow transplant, called autologous bone marrow transplant, is being studied in clinical trials. *uring this procedure, bone marrow is taken from the patient and may be treated with drugs to kill any cancer cells. The marrow is froAen to save it. The patient is then given high3dose chemotherapy with or without radiation therapy to destroy all of the remaining marrow. The froAen marrow that was saved is thawed and given through a needle in a vein to replace the marrow that was destroyed #Sebban et el., +!!-%.
Treatment outcome is dependent not only on the therapy applied,
but importantly, also on the underlying biology of the tumor
and the host. /ach of these
variables must be factored into initial treatment decisions,
as well as later refinements based on initial response, and
several biological features. It is recogniAed that with improvements in therapy,
certain variables might lose their prognostic value; therefore,
risk assignment plans should be routinely reassessed. Finally
an optimal system should allow for comparison of the outcomes
of similar or identical patients, treated on different protocols #1hoi et al., 2&%. There are generally four phases of treatment for 44. The first phase, remission induction therapy, uses chemotherapy to kill as many of the leukemia cells as possible to cause the cancer to go into remission. The second phase, called central nervous system (-.') prophyla!is, is preventive therapy, it involves using intrathecal andDor high3dose systemic chemotherapy to the -.' to kill any leukemia cells present there. It is also used to prevent 2; the spread of cancer cells to the brain and spinal cord even if no cancer has been detected there. ,adiation therapy to the brain may also be given, in addition to chemotherapy, for this purpose. -.' prophyla!is is often given in con<unction with consolidation therapy. @nce a child goes into remission and there no signs of leukemia, a third phase of treatment called consolidation or intensification therapy, is given. -onsolidation therapy uses high3dose chemotherapy to attempt to kill any remaining leukemia cells. The fourth phase of treatment, called maintenance therapy, uses chemotherapy for several years to maintain the remission #Attal et al., +!!&%.
1> II. Cell cycle and Apoptosis *.Cell cycle? The concept of the cycle in its current form is introduced by Howard and Ple*, #+!&,%. They observed that *. synthesis ('3 phase) in individual cells was discontinuous and occupied a discrete portion of the cell life and was constant in duration. Mitotic division (M3phase) was seen to occur after certain period of time following *. replication. distinct phase between *. replication and mitosis was also apparent (8oo/ et al., +!!$%. -ell cycle phase of #2 was historically considered to be a time cells had little observable activity. 'ince this time precedes *. synthesis, the term #ap 2 (#2) was coined. They have diploid chromosome (1-G68 chromosome). t a certain point in the cellBs life, the *. synthetic machinery turns on. This phase of the cellBs life is labeled H'H for synthesis. s the cell proceeds through this phase, its *. content increases from 1- to 6-. t the end of ', the cell has duplicated its genome and it is in the tetraploid state. fter the ' phase, the cell again enters a phase that was historically thought to be Cuiescent. 'ince this phase is the second gap region, it is referred to as #1. In the #1 phase, the cell is producing the necessary proteins that will play a ma<or role in cytokinase. fter a highly variable amount of time, the cell enters mitosis (M). *. content remains constant at 6- until the cell actually divides at the end telophase #8iblit, +!!,%. The process of replicating *. and dividing a cell can be described as a series of coordinated events that compose a Hcell
division cycle,H illustrated for mammalian cells in Fig 93.*:$ In each cell division cycle, 12 chromosomes are replicated once (*.
synthesis or '3phase) and segregated to create two genetically
identical daughter cells (mitosis or M3phase). These events are
spaced by intervals of growth and reorganiAation (gap phases # 2
and # 1 ). -ells can stop cycling after division, entering a state
of Cuiescence (# > ). -ommitment to traverse an entire cycle is
made in late # 2 . t least two types of cell cycle control
mechanisms are recogniAed: a cascade of protein phosphorylations
that relay a cell from one stage to the ne!t and a set of checkpoints
that monitor completion of critical events and delay progression
to the ne!t stage if necessary #<asmyth, +!!$%. The first type of control involves
a highly regulated kinase family. Iinase activation generally
reCuires association with a second subunit that is transiently
e!pressed at the appropriate period of the cell cycle; the periodic
HcyclinH subunit associates with its partner Hcyclin3dependent
kinaseH (-*I) to create an active comple! with uniCue substrate
specificity. ,egulatory phosphorylation and dephosphorylation
fine3tune the activity of -*I3cyclin comple!es, ensuring well3delineated
transitions between cell cycle stages #Elled"e, +!!$%. second type of cell cycle regulation, checkpoint control, is more supervisory. It is not an essential part of the cycle
progression machinery. -ell cycle checkpoints sense flaws in critical
events such as *. replication and chromosome segregation. %hen checkpoints are activated, for e!ample by underreplicated
or damaged *., signals are relayed to the cell cycle3 progression
machinery. These signals cause a delay in cycle progression, until the danger of mutation has been averted. +ecause checkpoint function
is not reCuired in every cell cycle, the e!tent of checkpoint
function is not as obvious as that of components integral to the
process, such as -*Is #Sherr, +!!$%. 11 Figure 93.*:? A schematic representation o- the mammalian cell cycle #<asmyth, +!!$%. 15 *.*.Cell cycle and cancer? 'uperficially, the connection between the cell cycle and cancer is obvious: cell cycle machinery controls cell proliferation,
and cancer is a disease of inappropriate cell proliferation. Fundamentally,
all cancers permit the e!istence of too many cells. 0owever, this
cell number e!cess is linked in a vicious cycle with a reduction
in sensitivity to signals that normally tell a cell to adhere,
differentiate, or die. This combination of altered properties
increases the difficulty of deciphering which changes are primarily
responsible for causing cancer #Ja*/s and 3einber", +!!$%.
The first genetic alterations shown to contribute to cancer development were gain3of3function mutations. These mutations
define a set of HoncogenesH that are mutant versions of normal
cellular Hprotooncogenes.H The products of protooncogenes function
in signal transduction pathways that promote cell proliferation.
0owever, transformation by individual oncogenes can be redundant
(mutation of one of several genes will lead to transformation)
or can be cell type3specific (mutations will transform some cells
but have no effect on others). This suggests that multiple, distinct
pathways of genetic alteration lead to cancer, but that not all
pathways have the same role in each cell type #3hite, +!!$%.
More recently, the significance of loss3of3function mutations in carcinogenesis has become increasingly apparent. Mutations
in these so3 called Htumor suppressorH genes were initially recogniAed
to have a ma<or role in inherited cancer susceptibility. +ecause
inactivation of both copies of a tumor suppressor gene is reCuired
for loss of function, individuals heteroAygous for mutations at
the locus are phenotypically normal. Thus, 16 unlike gain3of3function
mutations, loss3of3function tumor suppressor mutations can be
carried in the gene pool with no direct deleterious conseCuence.
0owever, individuals heteroAygous for tumor suppressor mutations
are more likely to develop cancer, because only one mutational
event is reCuired to prevent synthesis of any functional gene
product #Mor"enbesser et al., +!!-%. It now appears that tumor suppressor gene mutations are highly likely to promote, and may even be reCuired for, a large number
of spontaneous as well as hereditary forms of cancer. 4oss
of function of the tumor suppressor gene product p,b, for e!ample,
would be predicted to liberate /1F transcriptional activators
without reCuiring phosphorylation and thus bypass a normal negative
regulation controlling entry into the cycle. 4oss of
the tumor suppressor gene product p28 would have a similar conseCuence,
liberating /1Fs by increasing p,b phosphorylation . In
addition, cell cycle progression can be halted at several points
by the tumor suppressor gene product p75, activated in response
to checkpoints sensing *. and possibly also chromosome damage;
loss of p75 would remove this brake to cycling #Symonds et al., +!!-%$ 3.Apoptosis and its markers? poptosis and necrosis are too distinct, mutually e!clusive, modes of cell death. poptosis, freCuently referred to as programmed cell death is an active and physiological mode of cell death, in which the cell itself designs and e!ecutes the program of its own demise and subseCuent body disposal. *ifferent patterns of apoptosis (early and delayed apoptosis) many cell types, cells of hematopoietic origin in particular, undergo apoptosis rapidly, to 17 within few hours following e!posure to relatively high concentration of cytoto!ic agents #Ma=ino and Joris, +!!&%. poptosis can be defined as Bgene3directed cellular self3destructionBB although this is really a phenomenon where cells are programmed to die at a particular point, e.g. during embryonic development, and even here cells may go through an apoptotic pathway. 0owever, apoptosis is certainly a distinct process from other forms of oncosis leading to necrosis #Gerbaulet et al., 2& and 3alla*h et al., +!!!%. poptosis affects individual cells, physiological induction e.g. lack of signals, phagocytosis by macrophages or other cells and there is no inflammatory response. .ecrosis affects group of cells, non physiological induction e.g. virus and poison, phagocytosis of macrophages and there is inflammatory response #3irth et al., 2&%. There are three different mechanisms by which a cell commits suicide by apoptosis. In the intrinsic or mitochondarial pathway, the outer membranes of mitochondria in a healthy cell e!press the protein; +cl1 on their surface. +cl1 is bound to a molecule of the protein paf32. Internal damage to the cell (e.g., from reactive o!ygen species) causes +cl1 to release paf32; a related protein, +a!, to penetrate mitochondrial membranes causing cytochrome c to leak out. The released cytochrome c and paf32 bind to molecules of caspase ; Fig$ 93.3:$ The resulting comple! of cytochrome c, paf32, caspase ; and T" is called the apoptosome. The apoptosome aggregate in the cytosol #<iu et al., 2& and 8am et al., 2& and 9roemer2 (eed 2%.
18 Figure 93.3:? )he intrinsic or mitochondrial pathway #8am et al., 2&%. 19 -aspase ; is one of a family of over a doAen caspases. They are all proteases. They get their name because they cleave proteins3mostly each other at aspiratic acid residues. -aspase ; cleaves and, in so doing, activates other caspases. The seCuential activation of one caspase by another creates an e!panding cascade of proteolytic activity (rather like that in blood clotting and complement activation) which leads to digestion of structural proteins in the cytoplasm, degradation of chromosomal *. and phagocytosis of the cell #3ada et al., 2&%. In the e!trinsic or death receptor pathway, Fas and the T.F receptor are integral membrane proteins with their receptor domains e!posed at the surface of the cell. +inding of the complementary death activator (Fas4 and T.F respectively) transmits a signal to the cytoplasm that leads to activation of caspase :. -aspase : (like caspase ;) initiates a cascade of caspase activation leading to phagocytosis of the cell Fig$ 93.5:. For e!ample, cytoto!ic T cells recogniAe (bind to) their target, they produce more Fas4 at their surface, this binds with the Fas on the surface of the target cell leading to its death by apoptosis. In some cases, final destruction of the cell is guaranted only withits engulfment by a phagocyte #'i=an"i et al., 2& and :e"a et al., 2&%. In the third way, neurons, and perhaps other cells, have another way to self3destruct that unlike the two paths described above, doesnBt use caspase. poptosis3 inducing factor (IF) is a protein that is normally located in the inter membrane space of mitochondaria. %hen the cell receives a signal telling it that it is time to die, IF is released from the mitochondrial, it is migrates into the nucleus and binds to *., %hich triggers the destruction of the *. and cell death #;rbano et al., 2&%. 1:
Figure 93.5:? )he mechanism o- apoptosis 9Apoptosis triggered by e@ternal signals? the e@trinsic or death receptor path way: #'i=an"i et al., 2&%$
1; *efects in programmed cell death (apoptosis) mechanisms play
important roles in the pathogenesis and progression of hematological
malignancies, allowing neoplastic cells to survive beyond their
normally intended life3spans and subverting the need for e!ogenous
survival factors. poptosis defects also serve as an important
complement to proto3oncogene activation, as many deregulated
oncoproteins that drive cell division also trigger apoptosis #E)an and 8ittlewood, +!!5%. 'imilarly, errors in *. repair
and chromosome segregation normally trigger cell suicide as
a defense mechanism for eradicating genetically unstable cells,
and thus apoptosis defects permit survival of the genetically
unstable cells, providing opportunities for selection of progressively
aggressive clones #>ono) et al., 2%. -hemotherapy and irradiation trigger apoptosis in tumor cells
and an understanding of the biochemical pathways involved in
apoptosis provides an opportunity to classify tumors based on
their response to common induction regimens. Multiple distinct
signaling pathways regulate apoptosis, but two ma<or cell death
pathways have been implicated in hematological malignancies:
the mitochondrial pathway and the death receptor pathway Fig$ 93.+: #E)ans et al., 22%. 5> Figure 93.+:? Diagram o- the mitochondrial and death receptor pathways o- cell death 9!(ans et al$" 3443:$ 52 +oth of these pathways ultimately activate members of
the caspase family of proteins that are responsible for e!ecuting
the terminal phases of apoptosis. p75 protein levels rise in
response to various cellular stresses including chemotherapy.
p75 induces the loss of mitochondrial membrane potential with
subseCuent release of cytochrome c, which forms a comple!, the
Hapoptosome,H with the adapter molecule paf32, T", and caspase3;.
This comple!, in turn, activates caspase35 #E)ans et al., 22%. nother pro!imal pathway of cell death involves death receptor
signaling at the cell surface. +inding of -*;734 and other tumor
necrosis factor (T.F) family ligands to their death inducing
receptors, -*;7D"@3 2DF' or T.F3 and T,I4 respectively, leads
to receptor trimeriAation and the recruitment of adapter molecules.
These molecules include F**DM@,T32 that in turn lead to recruitment
and activation of caspase3:. This initiator caspase also cleaves
and activates downstream caspases, including caspase35. lthough
generally described as being distinct, these two pro!imal pathways
are interconnected. For e!ample, caspase3: cleaves the pro3apoptotic
protein +I*, which results in translocation to the mitochondria
and release of cytochrome c #9ishi et al., 2,, 'lom, 2, de 4ran*his et al., 2 and Goto et al., 2+%. 'everal studies have e!amined the prognostic significance of
apoptotic protein e!pression in leukemia. *efects in the p75
pathway are distinctly rare in childhood malignancies including
44, where mutations are detected in J 7E of cases at the
time of initial diagnosis. 0owever, relapsed blasts may harbor
mutations of p75 gene much more commonly. Further, 44 blasts
at relapse have been noted to e!press high levels of the Mdm31
protein, which abrogates p75 signaling #Dir)en et al., +!!& and Pemble et al., +!!-%. 51 -ancer3associated defects in apoptosis play a role
in chemoresistance and radioresistance, increasing the threshold
for cell death, and thereby reCuiring higher doses for tumor
killing #?s*ho77 et al., +!!! and Ma/in et al., 2%. Melanoma (skin cancer) cells avoid apoptosis by inhibiting the e!pression of the gene encoding paf32. 'ome cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble (decoy) molecule that binds to Fas4, plugging it up so it cannot bind Fas. Thus cytoto!ic T cells (-T4) cannot kill the cancer cells by the mechanism of death receptor pathway. @ther cancer cells e!press high levels of Fas4, and can kill any cytoto!ic T cells (-T4) that try to kill them because -T4 also e!press Fas (but are protected from their own Fas4) #Mei=er et al., 2&%. poptosis plays a role in many diseases, such as cancer, viral infections, and autoimmune and neurodegenerative disorders. There is a great potential for treatment of these diseases in developing agents that can alter the apoptotic process and change the natural disease progression. Molecules whose roles in apoptosis have been investigated include +cl31 and c3myc proteins, the p75 tumor suppressor gene and various tumor suppressor gene products, M" kinases, and proteases #Menende. et al., 2-%. 3.*.)he apoptosis promoter 9p/5:? p75 stimulates
a wide network of signals that act through two ma<or apoptotic
pathways. The e!trinsic, death receptor pathway triggers the
activation of a caspase cascade, and the intrinsic, mitochondrial
pathway shifts the balance in the +cl31 family towards the pro3apoptotic
members, promoting the formation of the apoptosome, and conseCuently
caspase3 55 mediated apoptosis. The impact of these two apoptotic
pathways may be enhanced when they converge through +id, which
is a p75 target. The ma<ority of these apoptotic effects are
mediated through the induction of specific apoptotic target
genes. 0owever, p75 can also promote apoptosis by a transcription3independent
mechanism under certain conditions. Thus, a multitude of mechanisms
are employed by p75 to ensure efficient induction of apoptosis
in a stage3, tissue3 and stress3signal3specific manner #8inda 2 1arol, +!!$ and Susan et al., 2,%2 'ome cancer causing viruses use tricks to prevent apoptosis of the cells they have transformed. 'everal human papilloma viruses (0"?) have been implicated in causing cervical cancer. @ne of them produces a protein (/8) that binds and inactivates the apoptosis promoter p75. +inding of Fas ligand or agonistic anti3Fas antibody to the death receptor Fas can activate a caspase3cascade resulting in apoptosis. Fas cell surface e!pression was determined by flow cytometry #Hou"ardy et al., 2&%. #enes involved in apoptosis are either pro3apoptotic (promote apoptosis) or anti3apoptotic (inhibit apoptosis). "75 is a pro3apoptotic genes present in all cells, but has special significance to cancer cells. It is a tumor repressor gene, meaning that its presence reduces the occurrence of cancer tumors by promoting apoptosis in cancer cells. .ormally it induces apoptosis by activating caspases ;, :, 9, and 5. The loss of p75 decreases caspase activation and therefore the cell will not undergo apoptosis. Mutation in the p75 gene is the most common mutation in cancer; it is present in about half of all cancer tumors, :>E in all colon cancer tumors, 7>E of lung cancer tumors, and 6>E of breast cancer tumors #Polya/ et al., +!!0%.
56 &nder normal conditions p75 is a short3lived protein. The p75
inhibitor Mdm1 (0dm1 in humans) is largely responsible for keeping
p75 in this state. Mdm1 inhibits the transcriptional activity
of p75 and, more importantly, promotes its degradation by the
proteasome #8e)ine, +!!0%. p75 mutants in tumours have a reduced affinity for *. and a reduced ability to induce apoptosis. %e describe a mutant with
the opposite phenotype, an increased affinity for some p753binding
sites and an increased ability to induce apoptosis. The apoptotic
function reCuires transcription activation by p75 #Elisabeth et al., +!!!%. /arly observations suggested that p75 may function as an oncogene, because overe!pression of p75 appeared to cause oncogenic transformation of cells. In the late 2;:>s, however, several critical discoveries defined the normal function of p75 to be anti3oncogenic. %ild3type p75 genes, when introduced into cells, were found to be growth suppressive #>sabela et al., 2%. p75 plays multiple roles in cells. /!pression of high levels of wild3 type (but not mutant) p75 has two outcomes: cell cycle arrest or apoptosis. In response to genoto!ic stress, p75 acts as an Hemergency brakeH inducing either arrest or apoptosis, protecting the genome from accumulating e!cess mutations. -onsistent with this notion, cells lacking p75 were shown to be genetically unstable and thus more prone to tumors #>sabela et al., 2%. p75 promotes cytochrome
c release through the induction of target genes encoding +053only
proteins. Importantly, p75 also induces ")"F-1 e!pression through
a response element within the ")"F-1 promoter #9annan et al.,
2+% 57 The tumor suppressor gene product p75 is clearly a central player in
many biochemical pathways that are pivotal to human carcinogenesis. The
seCuence3specific *. binding properties of this nuclear phosphoprotein
regulate the transcription of a continually e!panding number
of genes, the protein products of which regulate cell cycle progression
and apoptosis #>sabela et al., 2%. 4oss of p75 function by mutation is common in cancer. 0owever, most natural p75 mutations occur at a late stage in tumor development, and many clinically detectable cancers have reduced p75 e!pression but no p75 mutations. It remains to be fully determined what mechanisms disable p75 during malignant initiation and in cancers without mutations that directly affect p75. p75 mutants in tumours have a reduced affinity for *. and a reduced ability to induce apoptosis #<iu et al., 2&%. p75 e!pression has important
clinical implications as an indicator of prognosis and response
to chemotherapy or radiotherapy in different human tumor types.
The common effect of p75 mutations found in tumours is to inactivate p75 as a transcription factor. -onseCuently, a great deal of effort has been e!pended in trying to identify transcriptional targets of p75. "articular attention has been paid to target genes which may mediate cell3 cycle arrest and apoptosis #9o and Pri)es, +!!$ %. p75 dependent # 2 and # 1 arrest reCuires induction of the p12 cyclin3 dependent kinase inhibitor. In contrast, no single gene can e!plain p753 induced apoptosis. Many p75 target genes have been identified which function in known apoptotic pathways, regulate survival factor signalling, induce apoptosis when over e!pressed or are involved in biochemical events linked to apoptosis #'u*/binder et al., +!!0 , Miyashita and (eed, +!!& , 58 @wenAS*haub et al., +!!&, Polya/ et al., +!!0, :armehA6iaie et al., +!!0, M*1urra*h et al., +!!0 and (am7ino et al., +!!0%. p75 can activate target genes through a non3canonical seCuence.
The first such e!ample is in the p753induced gene 5 ()I33),
which has been implicated in the accumulation of reactive o!ygen
species and apoptosis induction #Polya/ et al., +!!0%. nother recently
described e!ample is the gene encoding the pro3apoptotic phosphatase
"-2, which is induced through binding of p75 to a novel palindromic
binding site #Bin et al., 2,%. novel insight into the interplay between p75 and its family
members, p85 and p95, in the induction of apoptosis has been
recently revealed #4lores et al., 22%. The effect of p85 and p95 on p75 transcriptional activity,
using a selection of knockout mouse embryo fibroblasts (M/Fs),
defined two distinct classes of target gene. %hereas p75 alone
is sufficient for the induction of p21 and 4dm2, the induction
of the apoptotic genes )E5)6 Ba7 and !o7a reCuires p75 together
with p85 and p95. This finding demonstrates an essential role
for both p85 and p95 in the efficient induction of apoptotic
target genes by p75. The mechanism of this cooperation is currently
unknown, but it may involve an enhanced binding to andDor stabiliAation
of the transcription comple! on the promoters of p75 apoptotic
target genes by the cooperative action of all three members
#;rist and Pri)es, 22%. In addition to the contribution of
p85 and p95 to the apoptotic function of p75, they play an important
role in the precise control of cell death during normal mouse
development. p95 also plays a role in the induction of cell
death in response to *. damage, a process involving cooperation
between the bl tyrosine kinase and p95 #Shaul,
2%. 59 Immunohistochemical (I0-) detection of p75 e!pression has been
established as a relatively easy and straightforward method
for fresh and archival tissues. vailable monoclonal antibodies
recogniAe both wild3type and mutant forms, but there may be
a selective detection of the latter owing to the very short
half3life of the former #Porter et al., +!!2 and Soussi et al., +!!-%. p75 is a tumor suppressor that is rarely mutated in 44 patients but whose function is freCuently altered by mutations to genes that code for proteins that regulate p75 function. ctivation of p75 occurs in response to cells that have acCuired *. damage that may be engaged in aberrant cell proliferation. Mutations to proteins that regulate p75 function, like 0*M1, p26, and p12, are freCuent findings in 44 #(oman et al., 22%. +ovine papillomavirus type 2 (+"?32)3transformed mouse fibroblast cell lines were analyAed via flow cytometry (F-M) for e!pression of p75 protein along with their *. content. 'ignificantly elevated levels of the p75 protein was present in some but not all of the transformed cell lines. Kuantitation of p75 protein in cell lines containing +"?32 *. revealed that the tumorigenic cell lines e!pressed higher levels of the p75 protein #A"rawal et al., +!!-%. The correlation between p75 abnormalities and *. aneuploidy and that analysis of p75 protein is useful for prediction of clinical course in esophageal sCuamous cell carcinoma #Gou/on et al., +!!-%. 8iu et al., (2-) evaluated changes in apoptotic proteins e!pression
that occur in response to chemotherapy in pediatric cases with acute
leukemia <ust prior to and 2, 8 and 16 hours following the administration
of multiagent 5: chemotherapy. They found great heterogeneity
in the patterns of apoptotic protein e!pression in the initial
response to chemotherapy among individual patient samples. Importantly,
no increases in p75, p12 or Mdm31 protein e!pression were seen
in leukemic blasts from the standard risk patients whose initial
treatment consisted of the non3p753dependent drugs, vincristine
and prednisone. In the subgroup of children who received at
least one p75 dependent drug, patients could be segregated into
two groups, one group that showed up3regulation of p75 protein
and its target p12, and another group that showed no increase
following therapy, thus identifying at least two distinct pathways
leading to apoptosis #1hen et al., +!!$%. 2A2A'*l2 7roteins Members of the +cl31
protein family play pivotal roles in the decision and e!ecution
phases of apoptosis in the mitochondrial pathway. To date,
16 +cl31 family members have been identified as either pro3
(e.g., +a!, +ak, +cl3F ' , +id, +ad, and .o!a) or anti3 (e.g.,
+cl31 and +cl3F 4 ) apoptotic proteins. +cl31 proteins form
homo3 and heterodimeric comple!es to regulate mitochondrial
channel formation and subseCuent release of cytochrome c from
the mitochondria #9ishi et al., 2,, 'lom, 2, de 4ran*his et al., 2, Goto et al., 2+ and 1ryns et al., +!!!%. The +cl1 family proteins are the central regulators of the mitochondrial pathway. +cl1 is an inhibitor of apoptosis. +cl1 and its human homolog introduce a new category of oncogenes that act by decreasing cell death. @ver e!pression of +cl1 promotes oncogensis by repressing cell death 5; and e!tending cell life. 0owever, overe!pression can also lead to retardation of cell cycling via prolongation of the #2 phase of the cycle #3ebb et al., 2& and Green 2 (eed, +!!5%. The +cl1 family of intracellular proteins is the central regulator of caspase activation, and its opposing factions of anti3 and pro3apoptotic members arbitrate the life3or3death decision. The oncogenic activity of the +cl1 gene is carried out via suppression of lymphocytic apoptosis or programmed cell death. #1ory 2 Adams, 22 and (oumier et al., 22%. +-41 is an important regulator of apoptosis, first identified from its involvement in follicular + cell lymphoma, where the common t(::26) translocation causes the activation of the +-41 oncogene. +-41 is now recognised as a survival factor for many types of cell, notably neurons. /!pression of +-41 is widespread during embryogenesis but is restricted to long3lived cells in the adult. critical mediator of +-41 apoptosis is interleukin32 beta3converting enAyme (I-/) a cysteine protease that processes I432 beta during the inflammatory response #(oumier et al., 22%. +-41 is a member of a multigene family (highly conserved evolutionarily with viral homologues). @ther proteins in the family (+-4F, +*, +F, +* etc) antagonise inhibition of apoptosis by binding to +-41. 0ence the balance of various members of the +-4 family determines the e!tent to which cell death is promoted or prevented. This model is consistent with the findings of high levels of +-41 in a variety of solid tumours #Jian" and Milner, 2,%. poptosis can also be induced by a variety of cytokines e.g. T#F beta family, which inhibit the proliferation of a wide variety of cell types that 6> may undergo concomitant cell death. T#F beta induced apoptosis is blocked in myeloblastic leukaemia cells by +-41 e!pressed at a level that does not block but merely delays p753induced apoptosis. This may reflect the fact that both T#F beta and p75 suppress +-41 but only p75 has the ability to activate +F, thus deflecting the e!pression pattern towards apoptosis #Se*/in et al., 22%. ctive cell suicide (apoptosis) is induced by events such as growth factor withdrawal and to!ins. It is controlled by regulators, which have either an inhibitory effect on programmed cell death (anti3apoptotic) or block the protective effect of inhibitors (pro3apoptotic). Many viruses have found a way of countering defensive apoptosis by encoding their own anti3apoptosis genes preventing their target3cells from dying too soon. ll proteins belonging to the +cl31 family contain either a +02, +01, +05, or +06 domain. ll anti3apoptotic proteins contain +02 and +01 domains, some of them contain an additional .3terminal +06 domain (+cl31, +cl3! (4), +cl3 w), which is never seen in pro3apoptotic proteins, e!cept for +cl3!('). @n the other hand, all pro3apoptotic proteins contain a +05 domain (e!cept for +ad) necessary for dimeriAation with other proteins of +cl31 family and crucial for their killing activity, some of them also contain +02 and +01 domains (+a!, +ak). The +05 domain is also present in some anti3apoptotic protein, such as +cl31 or +cl3! (4). "roteins that are known to contain these domains include vertebrate +cl31 (alpha and beta isoforms) and +cl3! (isoforms (+cl3!(4) and +cl3!(')) #Poliseno et al., 22%. ntiapoptotic + cell leukemiaDlymphoma (+-41) family proteins are e!pressed in many cancers, but the circumstances under which these proteins are necessary for tumor maintenance are poorly understood. novel functional assay that uses +cl1 homology domain (+05) peptides to 62 predict dependence on antiapoptotic proteins was e!ploiteded , a strategy, +05 profiling. +05 profiling accurately predicts sensitivity to +cl1 antagonist +T3959 in primary chronic lymphocytic leukemia (-44) cells. +05 profiling also accurately distinguishes myeloid cell leukemia seCuence 2 (M-42) from +cl1 dependence in myeloma cell lines. It was shown that the special sensitivity of -44 cells to +cl1 antagonism arises from the reCuirement that +cl1 tonically seCuester proapoptotic +IM in -44. +T3 959 displaced +IM from +cl1Bs +053binding pocket, allowing +IM to activate +F, induce mitochondrial permeabiliAation, and rapidly commit the -44 cell to death. It was demonstrated that +cl1 e!pression alone does not dictate sensitivity to +T3959. Instead, +cl1 comple!ed to +IM is the critical target for +T3959 in -44. n important implication is that in cancer, +cl1 may not effectively buffer chemotherapy death signals if it is already seCuestering proapoptotic +053only proteins. Indeed, activator +053only occupation of +cl1 may prime cancer cells for death, offering a potential e!planation for the marked chemosensitivity of certain cancers that e!press abundant +cl1, such as -44 and follicular lymphoma #Del Ga.io et al., 20%. The relationship between gene e!pression of +cl 1 and +a! and the therapeutic effect in oral cancer patients had investigated. significant correlation between +cl31D+a! gene e!pression ratio in the peripheral blood mononuclear cells ("+M-s) from the patients, and the therapeutic effect of radiation therapy These findings suggested that +cl31 and +a! gene e!pression in "+M-s may be useful as a prognostic factor in oral cancer patients #@shi/awa et al., 2$%. /pstein3+arr virus (/+?), the cause of mononucleosis and cause of +urkittBs lymphoma produces a protein similar to +cl1 and produces another 61 protein that causes the cell to increase its own production of +cl1. +oth these actions make the cell more resistant to apoptosis (thus enabling the cancer cell to continue to proliferate). /ven cancer cells produced without the participation of viruses may have tricks to avoid apoptosis #8u et al., 2&%. 'ome +3cell leukemias and lymphomas e!press high levels of +cl1, thus blocking apoptotic signals they may receive. The high levels result from a translocation of the +cl1 gene into an inhancer region for antibody production #Menende. et al., 2-%. +cl13421 contributes to the classical tumor biological features of #lioblastoma (#+M) such as intense apoptosis resistance and florid necrosis, and may provide a target for enhanced therapeutic responsiveness of this lethal cancer #Ste"h et al., 20%. 2A,1Amy* on*o"eneC The c3myc gene was discovered as the cellular homolog of the retro viral v3myc oncogene 1> years ago. The c3myc proto3oncogene was subseCuently found to be activated in various animal and human tumors. It belongs to the family of myc genes that includes +3myc, 43myc, .3myc, and s3myc; however, only c3myc, 43myc, and .3myc have neoplastic potential 93e*hsler et al., +!!0 and 4a**hini 2 Penn, +!!5%$ Targeted homoAygous deletion of the murine c3myc gene results in embryonic lethality, suggesting that it is critical for development. 0omoAygous inactivation of c3myc in rat fibroblasts caused a marked prolongation of cell doubling time, further suggesting a central role for c3myc in regulating cell proliferation #Mateya/ et al., +!!0%. 65 +ovine papillomavirus type 2 (+"?32)3transformed mouse fibroblast cell lines were analyAed via flow cytometry (F-M) for e!pression of c3myc protein along with their *. content. 'ignificantly elevated levels of the c3myc protein was present in some but not all of the transformed cell lines. Kuantitation of c3myc protein in cell lines containing +"?32 *. revealed that the tumorigenic cell lines e!pressed higher levels of the c3myc protein #A"rawal et al., +!!-%. The role of c3Myc in the cell cycle has been a confusing area due to the collection of data from different e!perimental models, although it is well established that c3myc is an early serum response gene. It should be noted that models of serum or growth factor stimulation of starved cells primarily address the # > D# 2 and # 2 D' transitions. Therefore, early studies implicated c3 Myc in the # > D# 2 transition. In cycling cells, however, the participation of c3 Myc in the cell cycle may be different. Furthermore, in anchorage3dependent cell growth, c3Myc may affect other components of the cell cycle #Amati et al., +!!5%. It is proposed that c3Myc induces apoptosis through separate Bdeath primingB and Bdeath triggeringB mechanisms in which Bdeath primingB and mitogenic signals are coordinated. Investigation of the mechanisms that underlie the triggering steps may offer new therapeutic opportunities #Prender"ast, +!!!%. The antiapoptotic effect of /pstein3+arr virus (/+?) was associated with a higher level of +cl31 e!pression and an /+?3dependent decrease in steady3state levels of c3ML- protein. lthough the /+? /+.32 protein is e!pressed in all /+?3associated tumors and is reported to have oncogenic potential, enforced e!pression of /+.32 alone in /+?3negative kata cells 66 failed to restore tumorigenicity or /+?3dependent down3regulation of c3 ML-. These data provide direct evidence that /+? contributes to the tumorigenic potential of +urkitt lymphoma and suggest a novel model whereby a restricted latency program of /+? promotes +3cell survival, and thus virus persistence within an immune host, by selectively targeting the e!pression of c3ML- #>n"rid et al., +!!!%. Much recent research on c3Myc has focused on how it drives apoptosis. c3Myc is widely known as a crucial regulator of cell proliferation in normal and neoplastic cells, but until relatively recently its apoptotic properties, which appear to be intrinsic, were not fully appreciated. Its death3 dealing aspects have gained wide attention in part because of their potential therapeutic utility in advanced malignancy, where c3Myc is freCuently deregulated and where novel modalities are badly needed. lthough its e!act function remains obscure, c3Myc is a transcription factor and advances have been made in characteriAing target genes which may mediate its apoptotic properties #Herme/in", 2,%. /ctopic e!pression of c3Myc (Myc) in most primary cell types
results in programmed cell death, and malignant transformation
cannot occur without additional mutations that block apoptosis.
The development of Myc3 induced lymphoid tumors was studied. Myc can be upregulated in acute
myeloid leukemia (M4), but its e!act role in myeloid leukemogenesis
is unclear. To study its role in M4, a murine stem
cell virus (M'-?) retroviral gene transferDtransplantation system
was used to broadly e!press Myc in the bone marrow of mice either alone
or in combination with antiapoptotic mutations. Myc e!pression
in the conte!t either of rfDInk6a loss or +cl31 coe!pression
induced a mi!ture of acute myeloid and acute lymphoid leukemias
(M4M44). In the absence of antiapoptotic mutations however,
67 all mice transplanted with 4SC%-4yc developed
M4 e!clusively. 4SC%- 4yc3induced M4 was polyclonal, readily
transplantable, possessed an intact rf3p75 pathway, and did
not display cytogenetic abnormalities by spectral karyotyping
analysis. 4astly, it was found that Myc preferentially stimulated
the growth of myeloid progenitor cells in methylcellulose. These
data provided the first direct evidence that Myc is a critical
downstream effector of myeloid leukemogenesis and suggested that
myeloid progenitors are intrinsically resistant to Myc3induced
#Hui et al., 2&%.
68 III.Flow cytometry Flow cytometry is a laser3based technology that is used to measure characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents such as nuclei and organelles #Melamed et al., +!!, ?ileney et al., +!!$ and M*1oy, 22%.
The flow cytometer is an instrument for measuring scattered and fluorescent light from single particles. The physics of the interaction of light with single particles provides the scientific foundation for the design and operation of the flow cytometer and for the critical evaluation of flow cytometric data #S*orni/ et al., +!!-%.
Flow cytometry uses the principles of light scattering, light e!citation, and emission of fluorochrome molecules to generate specific multi3parameter data from particles and cells in the siAe range of >.7um to 6>um diameter. -ells are hydro3dynamically focused in a sheath of phosphate buffer saline ("+') before intercepting an optimally focused light source. 4asers are most often used as a light source in flow cytometry #?albot, +!!,%. The technology of flow cytometry and the discovery of a method to produce monoclonal antibodies have made possible the clinical use of flow cytometry for the identification of cell populations. 4ight scatter is utiliAed to identify the cell populations of interest, while the measurement of fluorescence intensity provides specific information about individual cells. Monoclonal antibodies (tagged) with the fluorescent dye are commonly used for the identification of cell surface antigens and fluorescent dyes that 69 directly and specifically bind to certain components of the cell (i.e. *.) are used for cell cycle analysis #(e*/enwald, +!!, and Sha7iro, +!!&%. -ells or particles are prepared as single3cell suspension for flow cytometric analysis. This allows them to flow single file in a liCuid stream past a laser beam. s the laser strikes the individual cells. First light scattering occurs that is directly related to structural and morphological cell features. 'econd, fluorescence occurs if the cells are attached to a fluorescent probe. Fluoresent probes are typically monoclonal antibodies that have been con<ucated to fluorochromes; they can also be fluorescent stains reagents that are not con<ugated to antibodies #Par/s and Her.enber", +!5! and (e*henwald et al, +!!,%. Fluorescent probes are reacted with the cells or particles of interest before analysis; therefore, the amount of fluorescence emitted as a particle passes the light source is proportional to the amount of fluorescent probe bound to the cell or cellular constituent #(ad*liff and Jaros.es/i, +!!5%. fter acCuisition of light scattering and fluorescence data for each particle, the resulting information can be analyAed utiliAing a computer and specific software that are associated with the cytometer #(ose et al,. +!!2 and 8on"obardiAGi)en, +!!2%. There are two distinct types of flow cytometers that can be used to acCuire data from particles. @ne type can perform acCuisition of light scattering and fluorescence only. The other type is capable of acCuiring scattering and fluorescence data but also has the powerful ability to sort particles. +oth types function in a similar manner during acCuisition, for e!ample F-'can (+ecton *ickinson), this eCuipped with an air Ncooled 27 6: mw argon ion laser emitting at 6:: nm. Three fluorescence channels can be measured as well as two light scatter parameters. The F-'can is also eCuipped with a doublet discrimination module allowing the analysis of the cell cycle. The F-'can is user3operated (after instruction) and is available for use 16 hours per day #9andathil et al., 2&%. 0owever, sorting instruments have the powerful ability to physically separate particles based on light scattering andDor fluorescent emission characteristics. -ytometers were originally designed to sort, for e!ample F-' caliber 2, 1 (+ecton *ickison), this used for analysis only. &nlike the F-'can which is a dual laser system. The primary laser is an air3cooled 27 mw argon ion laser emitting at 6:: nm thus allowing two light scatter parameters and three fluorescence channels to be measured. The second laser is ared diode laser emitting at 857nm. Thus allowing for the e!citation of other dyes such as allophycocyanin or to3pro35 with power Macintosh #6 running system ;.> and cell Kuest v 5.5. Thus, cytometers that perform acCuisition without sorting are the most common of the two types #(ose et al., +!!2%. *.#rinciples o- -low cytometric instrumentation? Flow cytometers are very comple! instruments that are composed of four closely related systems. The fluidic system transports particles from a suspension through the cytometer for interrogation by an illumination system. The resulting light scattering and fluorescence is collected, filtered, and converted into electrical signals by the optical and electronics system. The data storage and computer control system saves acCuired data and is also the user interface for controlling most instrument functions. These four systems provide a very uniCue and powerful analytical tool for researchers 6; and clinicians. This is because they analyAe the properties of individual particles, and thousands of particles can be analyAed in a matter of seconds. Thus, data for a flow cytometric sample are a collection of many measurements instead of a single bulk measurement #(ad*liff and Jarose.es/i, +!!5%..
0istograms are the simplest modes of data representation. 0istograms allow visualiAation of a single acCuired parameter. Mean fluorescence and distributional statistics can be obtained based on markers that the user can graphically set on the plot. Multiple histograms can be overlaid on one another to depict Cualitative and Cuantitative differences in two or more samples. Two3parameter data plots are somewhat more complicated than histograms; however, they can yield more information. Two3parameter dot plots of F'- vs ''- allow visualiAation of both light3 scattering parameters that are important for identifying populations of interest. +ivariate fluorescent plots allow discrimination of dual3labeled populations that might remain hidden if histograms were used to display fluorescent data. Two3parameter plots that combine one light3scattering parameter and a fluorescent parameter are useful for analyAing control samples to elucidate the origin of nonspecific binding. *ata analysis is very graphically oriented. /!perience and pattern recognition become important when using two3parameter data plots for Cualitative as well as Cuantitative analysis. The techniCue of gating or drawing regions on dual parameter light3 scatter plots allows one to e!clude information and e!amine the population of interest by disallowing particles that might confound or interfere with analysis. This is one of the fundamental uses for gating #(ad*liff and Jarose.es/i, +!!5%. 7> Flow cytometers can be described as four interrelated systems which are shown in Fig$ 95.*:$ these four basic systems are common to all cytometers regardless of the instrument manufacturer and whether or not the cytometer is designed for analysis or sorting #Melamed et al., +!! and 8on"obardiAGi)en, +!!2 @wens 2 8o/en, +!!&%. +A+A4luidi* systemC The fluidic system is the heart of a flow cytometer and is responsible for transporting cells or particles from a prepared sample through the instrument for data acCuision Fig$ 95.3:$ The primary component of this system is a flow chamber. The fluidic design of the instrument and the flow chamber determine how the light from the illumination source ultimately meets and interrogates particles. Typically, a diluent, such as phosphate buffered saline, is directed by air pressure into the flow chamber. This fluid is referred to as sheath fluid and passes through the flow chamber after which it is intersected by the illumination source. Then, the sample under analysis, in the form of a single particle suspension, is directed into the sheath fluid stream prior to sample interrogations. The sample then travels by laminar flow through the chamber #@rmerod, +!!-%. 72 Figure 95.*:? Facscaliblur -low cytometry instrument$ 71 Figure 95.3:? Flow cytometer system 9Facscalibur: #@rmerod, +!!-%. 75 The pressure of the sheath fluid against the suspended particles aligns the particles in a single file fashion. This process is called hydrodynamic focusing and allows each cell to be interrogated by the illumination source individually while traveling within the sheath fluid stream #(ad*liff and Jaros.es/i, +!!5%. The flow cell is the functional core of the fluidic system because it presents cells in a single file for interrogation by the cytometer illumination system. typical flow cell Fig$ 95.5: consists of a converging noAAle in which sample is introduced at low flow rates into a larger laminar flow of isotonic saline or sheath fluid. The cells in the sample follow the converging streamlines and are hydrodynamically focused into alignment. The sample is in<ected into the center of a sheath flow. The combined flow is reduced in diameter, forcing the cell into the center of the stream. This the laser one cell at a time. This schematic of the flow chamber in relation to the laser beam in the sensing area #Phili7, 22%. +A2A>llumination systemC Flow cytometers use laser beams that intersect a cell or particle that has been hydro dynamically focused by the fluidic system. 4ight from the illumination source passes through a focusing apparatus before it intersects the sample stream. This apparatus is a lens assembly that focuses the laser emission into a beam with an elliptical cross3section that ensures a constant amount of particle illumination despite any minor positional variations of particles within the sample stream #6immermann and ?russ, +!0!%. 76 Figure 95.5:? Flow cytometers use the principle o- hydrodynamics -ocusing -or presenting cells to a laser #Phili7, 22%$
77 Laser options 4ight and fluorescence are generated when the focused laser beam strikes a particle within the sample stream. These light signals are then Cuantitated by the optical and electronics system to yield data that is inter3 prOt able by the user #Sha7iro, +!!&%. Two systems are used in flow cytometry to focus the illuminating light to the point at which it intersects the cell stream. @ne type of system uses a spherical lens to give a focal spot siAe of 5>3 8> Pm. The second system uses a pair of crossed cylindrical lenses to focus the light to a sheet about 21> Pm wide and 639 Pm deep #1ledat et al., 2-%. Most flow cytometers utiliAe a single laser, however, some systems support the simultaneous use of two or more different lasers. The most commonly used laser is an argon ion laser that has been configured to emit light in the visible range of the spectrum. 6::3 nm. 4aser emission is used for most standard applications. The ma<ority of fluorochrom that are available on the market today can be e!cited using this wavelength. Thus, laser is e!cellent e!citation sources because they provide a single wavelength beam that is also stable, bright, and narrow. 'ome type of lasers present in flow cytometers can be turned to &.?. or other wavelengths. If the e!iting is not tunable, then another laser source that emits the desired wavelength is reCuired. t the measuring point in a typical flow cytometer the stream of cells intersects a beam of light from a laser or arc lamp Fig$ 95.+:$ %hen light interacts with biological particles some of the light is scattered out of the incident beam and this scattered light may be collected over a range of angles by detectors positioned around the measuring point #Gerstner et al., 2&%. 78 Figure 95.+:? A simpli-ied illustration o- Flow Cytometry #Gerstner et al., 2&%. 79 +A,A@7ti*al and ele*troni*s systemC The e!citation optics consists of the light source and the optical components that serve to interrogate or e!cite the hydrodynamically focused sample stream in the flow cell. The 6:: nm line of the argon laser is used as a light source in many commercially available cytometers, but any light source that provides the reCuisite intensity, e.g. , the mercury vapor or the !enon are lamp can be used. @ptical components are used to e!pand, shape and focus the light which then interacts with the sample in the flow cell. The flow cell is usually made of CuartA and is designed to minimiAe diffraction and to ma!imiAe the collection of the optical signals. The light source is often a laser. 4aser is used because they provide a very concentrated and intense beam of monochromatic character of the light is especially important in making fluorescence measurements #?elford, 2-%. The amount of light that is scattered by a cell is a comple! function of its siAe, shape and refractive inde!. The sensitivity of light scattering to each of these factors is dependent upon the range of angles over which the scattered light. The light scattered at small angles (i.e. forward light scatter) could be successfully used to determine relative volume distributions for populations of cells #3an" et al., 2-%. 4ight scattered and emitted in all directions (58>Q) after the laser beam strikes an individual cell or particle that has been hydrodynamically focused. The optical and electronics system of a typical flow cytometer is responsible for collecting and Cuantitating at least five types of parameters from this scatter light and emitted fluorescence. Two of these parameters are light scattering properties. 4ight that is scattered in the forward direction (in the same direction as the laser beam) is analyAed as one parameter, and light scattered at ;>Q relative to the incident beam is collected as a second 7: parameter. Forward3scattered (F'-) light is a result of diffraction. *iffracted light provides basic morphological information such as relative cell siAe that is referred to as forward angle light scatter (F'-). 4ight that is scattered at ;>Q to the incident beam is the result of refracted and reflected light. This type of light scatters is referred to as side3angle light scatter (''-). This parameter is an indicator of granularity within the cytoplasm of cell as well as surface membrane irregularities to topographies #Phili7, 22%. Most current laboratory bench3top flow cytometers are capable of detecting fluorescence from three different regions of the visible spectrum. -utometers are optically conCuered to detect a narrow range of wave lengths in each region. Fluorescence emission is detected simultaneously along with F'- and ''- data; therefore, up to five parameters can be simultaneously measured for each analyAed sample #8on"obardiA Gi)en, +!!2% Fluorescence is detected using networks of mirrors, optics, and beam splitters that direct the emitted fluorescent light toward highly specific optical filters. The filters collect light within the range of wave lengths associated with each of the three fluorescent channels. Filtered light is directed toward photo multiplier tubes or "MTs for conversation into electrical signals #?elford, 2-%. +A-AData stora"e and *om7uter *ontrol systemC fter light scattering and fluorescence is converted to electrical signals by the optical and electronics system, the information is converted into digital data that the computer can interpret. The signals generated from cells or particles are referred to as events and are stored by the computer #(ose et al., +!!2%. 7; fter the different signals or pulses are amplified they are processed by an nalog to *igital -onverter (*-) which in turn allows for events to be plotted on a graphical scale (@ne "arameter, Two parameter 0istograms). Flow cytometry data outputs are stored in the form of computer files #(ad*liff and Jaros.es/i, +!!5%. 0istogram files can be in the form of one3parameter or two3 parameter files. 0istogram files consist of a list of the events corresponding to the graphical display specified in your acCuisition protocol. one3 parameter histogram is a graph of cell count on the y3a!is and the measurement parameter on !3a!is. ll one3parameter histograms have 2,>16 channels. These channels correspond to the original voltage generated by a specific HlightH event detected by the "MT detector. In other words, the *- assigns a channel number based on the pulse height for individual events. Therefore, brighter specific fluorescence events will yield a higher pulse height and thus a higher channel number when displayed as a histogram. graph representing two measurement parameters, on the !3 and y3a!es, and cell count height on a density gradient. This is similar to a topographical map. Lou can select 86 or 178 channels on each a!is of two3parameter histograms. "article counts are shown by dot density or by contour plot. Fig$ 95./: #(oederer et al., 2-%. 4ist3mode files consist of a complete listing of all events corresponding to all the parameters collected, as specified by your acCuisition )rotocol. This file follows a format specified by the F-' 5.> standard. ,aw list3mode data files can be opened or replayed using any program designed for analysis of flow cytometry data. Lou should keep in mind that a )rotocol serves as a template. It allows you to collect specified )arameters (i.e. F4', F42, F41, etc.), and how these parameters are displayed. )rotocols also serve 8> to determine how the data is gated, and contains all the 5egions from which your statistics will be generated. In addition, )rotocols contain other specific information that serves as direct interface between the computer workstation and the cytometer. These pertain to high voltage settings for the "MT detectors, gains for amplification of linear parameters, sample flow rates, fluorescence compensation, discrimination settings, etc. @nce your data has been collected and written into a list3mode file you can replay the file either using the specific )rotocol used for collection or any other program specifically designed for analysis of flow cytometry data. 0owever, you should keep in mind that you can only ad<ust 5egions, 3ating, and )arameters to be displayed. 'ettings such as amplification, fluorescence compensation, etc., can not be modified. Therefore, when collecting data make sure that your instrument settings are correct. Finally, if you open your listmode files using a programs such as Flow=o Fig$ 95.0:, %I.MI*I, andDor /!"@ you will have to specify parameter displays, and create 5egions and 3ating corresponding to the )rotocol used for collecting the data #M*1oy, 22%. 82 Figure 95./:? )wo parameter histogram and dot plot displaying FL*. FI)C on the @ a@is and FL3.#! on the y a@is #(oederer et al., 2-%. 81 Figure 95.0:? FlowAo program #M*1oy, 22%. 85 The number of events acCuired for each sample is always determined before analysis and is usually set using software designed to control cytometer operation. conventional acCuisition value is 2>>.>>> events per sample. 0owever this value may vary and range upward of events per sample depending on the e!perimental ob<ective #Melamed et al., +!!%. 3.Data analysis? *ata analysis is a very critical part of any e!periment that utiliAes flow cytometry. *ata is analyAed using a computer and software is usually specific to flow cytometric data and is often part of the same computer system that is used to control the instrument during acCuision. The most common display is a histogram. typical histogram data plot is shown in Fig$ 95.1" 5.6: #AbuA Absi et al., 2,%. It is also possible to display two parameters simultaneously such as F'- vs ''c or F42 vs F41. For two parameter plots, data from a population of individual particles can be displayed in the form of dots or as contours shown in Fig$ 95.2: #Par/s and Hen.enber", +!5!%. -ontour density plots display the data from a population of cells as a series of concentric lines that correlate to different cell or particle densities within the a!es. *ot3plots are probably the most common type of two3 parameter plots, and they are also the easiest to understand #(obinson, +!!,%. 86 Figure 95.1:? Analysis pulse width (ersus pulse height or area we can eliminate the maBority o- 4 doublets that appear as 3 #AbuA Absi et al., 2,%. 87 Figure 95.6:? D;A histogram #AbuA Absi et al., 2,%. 88 Figure 95.2:? D;A histogram 9aneuphliod population: #Par/s and Hen.enber", +!5!%$ 89 I7. Applications o- -low cytometry Flow cytometers are very comple! instruments that are composed of four closely related systems. They provide a very uniCue and powerful analytical tool for researchers and clinicians. Therefore, the flow cytometer is widely used in research as well as in clinical immunology and hematology to perform rapid immunophenotyping, cell sorting, and *. analysis #8on"obardiAGi)en, +!!2 and 'o"h 2 Dulin", 2&%. Flow cytometry is used for immunophenotyping and *. content of a variety of specimens including whole blood, bone marrow, serous cavity fluids, cerebrospinal fluid, urine and solid tissues. -haracteristics that can be measured include cell siAe, cytoplasmic comple!ity, *. or ,. content, and a wide range of membrane3bound and intracellular portents and sorting the cells #(e*htnwald, +!!,, @rmerod, +!!- and Assun*ao et al., 2&%. The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributed in part to the development of smaller user friendly, less e!pensive instruments and a continuous increase in the number of clinical applications as shown in )able 9+.*: #'rown and 3ittwer, 2%. Flow cytometry provides rapid analysis of multiple characteristics of single cells. The information obtained is both Cualitative and Cuantitative. %here as in the past flow cytometers were found only in larger academic 8: centers, advances in technology now make it possible for community hospitals to use this methodology #@rfao et al., +!!& and M*1oy, 22%. )able 9+.*:? Common clinical uses o- -low cytometry #'rown and 3ittwer, 2%. Field Clinical application Common characteristic measures Immunology 0istocompitability crossmatching IgM, Ig# Transplantation re<ection -*5, -I,-&4TI.# @IT5 043+19 detection 043+19 Immunodeficiency studies -*6, -*: %ncology *. content and ' phase of tumors *. Measurements of profilation markers Ii389, "-. Hematology 4eukemia and 4ymphoma phenotyping 4eukocyte surface antigens Identification of prognostically important subgroups Tdt, M"@ 0ematopiotic progenit or cell enumeration -*56 *iagnosis of systematic mastocytosis -*17,-*8; ,eticylocyte enumeration ,. utoimmuneand alloimmune disorders ntiplatelats disorders Ig#, IgM nti3neutrophils antibodies Ig# Immune comple!es -omplement, Ig# Feto3maternal hemorrge Cuantification 0emoglobin F, rhesus * 8; Immunohematology /rthrocyte surface antigens ssessment of leukocyte contamination of blood products Forward .* 'ide scatter enetic disorders ".0 -*77,-*7; Functions of cells can be defined through the application of fluorochrome dyes that have an affinity for cellular components. Traditionally, common clinical applications are immunophenotyping of cells of the hematopoietic system with fluorescent3labeled antibodies raised against specific cell surface proteins #Da)is et al., 22%. @ther approaches have been used to elucidate changes in cell function and *. content. /!amples of clinical applications in eCuine patients include immune3mediated hemolytic anemia, immune3mediated thrombocytopenia (IMT), chronic inflammatory disease, and neoplasia #Da)is et al., 22%. The great advantage of flow cytometry is that its applications are highly amenable to standardiAation. The efforts that have been made for flow cytometric applications in four ma<or fields of clinical cell analysis: -*6M T3 cell enumeration, -*56M hematopoietic stem and progenitor cell 9> enumeration, screening for the 043+19 antigen and leukemiaDlymphoma immunophenotyping #9eeney et al., 2-%. The diagnosis of many primary immunodeficiency diseases reCuires the use of several laboratory tests. Flow cytometry is applicable in the initial workup and subseCuent management of several primary immunodeficiency diseases #>lloh, 2-%. *. Cell cycle analysis? The measurement of the *. content of cells was one of the first ma<or applications of flow cytometry and is still one of the biggest applications in this laboratory today #Albro et al., +!!,%. Flow cytometry offers the possibility to study several parameters simultaneously, e.g. cell cycle modulation, mode of cell death, activity of mitochondria. The phases of the cell cycle were determined and the induction of apoptosis and necrosis was demonstrated by anne!in binding, analysis of mitochondrial membrane potential and *. strand breaks #?us*h and S*hwab, 2&%. *. ploidy and proliferative activity ('3phase fraction) are the two biological parameters commonly measured by *. flow cytometric analysis. The prime purpose of most studies is the investigation of the prognostic value of *. flow cytometry in addition to the information provided by conventional clinicopathological factors known to affect disease prognosis. The general statement, for tumors in the same histopathological stage of the disease, is that diploid andDor low proliferative tumors have a more favourable prognosis than aneuploid andDor high proliferative tumors, 92 suggesting an important role of *. flow cytometry in the assessment of tumor behaviour and in the outcome evaluation of the disease. #Pinto et al., 22%. +A+AStainin" 7ro*edureC The preparation and staining of cell suspension are the ma<or factors determining the validity and reproducibility of flow cytometric analysis. There is no flow cytometric staining procedures which is universally accepted and a number of different protocols have been advocated. ll of the *. specific stains and the phenanthridinium dyes have been used for total *. staining of chromosomes. The former group has the potential disadvantage that &? e!citation is reCuired but this constitutes no problem for mercury arc lamp based system or those with a laser tunable to &? lines #Hartwell, +!!5%. The *. fluorochromes in current use were classified into groups. 'tains that intercalate with double stranded nucleic acid and include ("I), (/+) and (@);and *. specific stains that have a particular specificity for moieties in *. and include mithramiycin (MM-), ethedium bromideDmithramicin (/+DMM-), a bisbenAimidaAole derivative and 638 diamino313 phenylindole (*"I) #?aylor and Mithor7e, +!5%. "ropidium iodide is bound to polynucleotide both by means of intercalation. This is only affected to a limited e!tent by high ionic strengths and electrostatically to secondary binding sites. The binding contents of these later sites are greatly depending on the ionic strength of the medium and can be eliminated by using sufficiently high ionic strengths. nd also have optimal e!citation at a 6:: nm laser and produce good results with ,.se treatment #Hartwell, +!!5%. 91 +A2AE)aluation of D<A histo"ramC The *. histogram is a very simple data set which characteristically contains two peaks separated by a trough Fig. (632). The first peak, which is usually the larger, corresponds to cells with #>D#2 *. content and the second, which should be at double the fluorescence intensity of the first, corresponds to cells with #1MM *. content. ny cell scored in the trough has a *. content intermediate between #2 and #1MM and these usually represent cells in '3phase. In a perfect data set, which doesnBt e!ist, all #2 and #1MM cells would be scored in single channels and any cells between or immediately ad<acent to these would be in '3 phase #Henderson, +!!5%. 'ince the cell material always contained normal diploid cells such as leukocytes or normal kidney cells, these were used as an internal standard and regarded as diploid (1-) #?ribu/ati, +!5-%. %hen in doubt, lymphocytes should be added to establish the diploid *. value. 0uman Ficol3prepared lymphocytes, fi!ed in ethanol, were used as e!ternal standard. The magnitude of the signal was ad<usted so as to have the standard diploid peak in a certain channel. In necessary the illumination was ad<usted so that the coefficient of variation (-?) of the resulting lymphocyte diploid peak was less than 5E Fig$ 9+.*:$ ll cell population with #2 ma!imum deviating less than 2>E form the standard value were regarded as diploid #Gustafson, +!52%. @nly #>D#2 peak is observed in *. diploid. broad peak described by a large coefficient of variation may obscure a second peak. The coefficient of variation of the #>D#2 peak must be less than 7E for single cell suspensions prepared from freshD froAen tissues, and less than :E for 95 nuclear suspension prepared from fi!ed, paraffin embedded specimens. %here a diploid peak only is observed, one should ensure that tumor cells are present in the clinical sample analyAed #3einber", +!!$%. Full width at half ma!imum(c3a) -.?. G R SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS 61.7 E 96 "eak (or mean) channel(b) Figure 9+.*:? Coe--ecient o- 7ariation 9C$7$: #Gustafson, +!52%$ n aneuploid cell population was considered to be present when a distinct peak was found constituting at least 1.7E of the total cell material and deviating more than 2>E from the diploid standard. *. aneuploid is reported when at least two separate #>D#2 peaks are demonstrated. For some samples the diploid normal peak might be almost non e!istent; hence care should be taken to assign peaks #@rmerod, +!!-%. The degree of ploidy of this cell line was calculated by relating the #2 ma!imum of these cells to *. of the diploid #2 cells which are always present. These diploid cells can be leukocytes, fibroblasts, normal urothelial cells. 3 channel o- aneuploid peak Degree o- ploidyC DDDDDDDDDDDD Channel number o- diploid peak The degree of aneuploid was determined also by the *. inde! which represents the ratio of fluorescence intensity of aneuploid cells to the diploid cells. The *. inde! of a diploid tumor is 2.>, whereas, aneuploid tumors are designated by progressively higher indices #GDtte, 2+ and Gorden et al., 2,%. 97 /stimation of the proportion of #2, ' and #1MM cells made by automatic integration of the cells in corresponding channels in the multi channel analyAer. The values are corrected for background noise. To estimate the proportions of cells in the '3phase of aneuploid tumor cell lines where cells from the cell line coincide, the normal cell lines are subtracted. -alculation of the phase distribution reCuires a minimum of about 2>>> cells combined with a low background noise resulting from cell fragments. These calculations may also be rendered more difficult when the peaks for the deploid or aneuploid cell population, partially or completely overlap. @n the other hand, there is no ambiguity in any of these cases to establish an aneuploid cell line. n additional proof of the e!istence of an aneuploid hyperdiploid cell line is the e!istence of a #1 peak occurs to the e!treme right of the histogram without interfering with the normal cell population #'rown and 3ittwer, 2%. n aneuploid cell population with a tetraploid amount of *. was considered to e!ist when a peak e!ceeded the #1M M peak found in normal cells by three standard deviations or more. To Cuantitate the number of nuclei normally found in the 6- or #1D M peak, a number of control tissues were studied. The mean percentage of nuclei in the 6- peak were 1.96M 2.62 (standard deviation) for nuclei e!tracted from fresh normal lymphocyte. These control data provide a firm basis for using greater than 2>E of nuclei in the 6- peak as a criterion for *. polyploidy in the specimens #(abino)it*h, +!!-%. 3. Immunophenotyping Applications? The most common applications of flow cytometry are measurement of *. content in tumors and immunophenotyping of haematopoietic 98 malignancies. Flow cytometry has shown to be a suitable method for immunophenotyping of canine lymphomas #1ulmsee and <alte, 22%. Immunophenotyping of abnormal cells is now considered a fundamental tool to establish the cell lineage assignment and to obtain a more precise identification of the various cell subtypes. *iagnostic hematopathology depends on the applications of flow cytometric immunophenotyping and immunohistochemical immunophenotyping combined with the cytomorphology and histologic features of cases. The availability of monoclonal antibodies directed against the surface proteins permits flow cytometric analysis of erythrocytes, leukocytes and platelets #'rown 2 3ittwer, 2, 1hianese, 22 and Dun7hy, 2-%. Multiparameter flow cytometry with optimally selected antibody combinations has e!panded the use of this techniCue beyond traditional applications in hematopathology. +y analyAing Cualitative patterns of antigen e!pression on discrete populations or Hclusters,H one can detect immunophenotypic aberrancy in specific cell populations relative to normal and reactive populations. /valuation of patterns of antigen e!pression can also be used to supplement conventional methodologies in the diagnosis and subclassification of certain types of hematologic neoplasia. Finally, the diagnosis of some congenital disorders affecting the hematolymphoid system can be facilitated by the detection of characteristic immunophenotypic changes #9roft, 2-%. 2A+AErythro*yte analysisC Tests that appear to have the greatest potential for routine application of flow cytometry include reticulocyte and reticulated platelet enumeration, detection of erythrocyte3bound immunoglobulin, immunophenotyping of 99 leukemias and lymphomas, and bone marrow differential cell counting #'rown and 3ittwer, 2 , 3eiss, 22%. Flow cytometric methods were first applied to laboratory hematology with the improvement in reticulocyte counting and the creation of the immature reticulocyte fraction for better anemia evaluation and therapeutic monitoring #Da)is, 2+%. 9: 2A2AH>: monitorin"C More than 57 million people in developing countries are living with 0I? infection. %hile drug prices have dropped considerably, the cost and technical comple!ity of laboratory tests essential for the management of 0I? disease, such as -*6 cell counts, remain prohibitive. .ew, simple, and affordable methods for measuring -*6 cells that can be implemented in resource3scarce settings are urgently needed #Dieye et al., 2&, 3al/er et al., 2& and Pattana7anyasat 2 ?ha/ar, 2&%. 2A,A>mmuno7henoty7in" of leu/emiasC Immunophenotyping has become common in the diagnosis and classification of acute leukemias and is particularly important in the proper identification of cases of minimally differentiated acute myeloid leukemia. To evaluate the immunophenotype of adult M4, cases were studied by cytochemical analysis and by flow cytometry with a panel of antibodies #9halidi et al., +!!5%. -haracteriAation of leukemias by immunotyping is particularly helpful when the morphology is difficult to interpret. The ma<or advantage of using immune markers by flow cytometry is the identification of particular leukemia subtype, not recogniAed by morphologic criteria, which may have prognostic significance #(e.aei et al., 2,%. Flow cytometric immunophenotypic analysis allowed to establish diagnosis in cytomorphologically unclassified cases, identify acute mi!ed3 lineage leukemias (M44) with a freCuency similar to that reported in other series, and confirm the heterogeneity of acute leukemia (4) #Piedras et al., +!!0 and GDtte, 2+%. 9; Flow cytometry may be used to detect minimal residual disease (M,*) in acute lymphoblastic leukemia because leukemic cells often display aberrant phenotypes when compared to normal cells. Flow cytometry is a sensitive and specific method for detecting M,* of childhood 44, and could predict the coming relapse #6han" et al., 2&%. %ith the advent of monoclonal antibodies and a uniform nomenclature system defining antibody reactivity in terms of clusters of differentiation (-*), an independent means of characteriAing acute leukemias using cellular antigen e!pression has evolved. Immunophenotyping is usually performed using immunofluoresence techniCue and is complementary to the light microscopic based morphologic classification. This is especially true of the lymphoid leukemias where morphology and cytochemistry cannot distinguish among different lineage of lymphoid cells, such as + versus T cells. %ith Immunophenotyping lineage is assigned using a panel of monoclonal antibodies that identify the e!pression of cell surface antigens. The panel of monoclonal reagents must include antibodies reactive with both myeloid and lymphoid cells to distinguish between the two most important groups. The reactivity pattern of the leukemia cells for all reagents is then e!amined for the final assignment of lineage: +3 lymphoid, T3 lymphoid, myeloid or undifferentiated #Masla/ et al., +!!-%. -omparative studies of cell surface antigen e!pression between normal and leukemic cells indicate that most if not all leukemias e!press phenotypes that are not observed in most normal maturing cells. This aberrant e!pression of cellular antigens suggests that leukemias are not proliferations of cells arrested at one state of normal maturation; rather leukemic cells maintain a genetic program that can produce e!pression of :> antigens of any lineage. .early all laboratories performing immunofluoresence analysis use different reagents #?ersta77en et al., +!!+%. 2A-AEuantifi*ation of stem *ellsC 2>> years ago, hematopoietic stem cells were postulated as blood lymphocyte3like cells. %ithin the last 1> years, the freCuency of autologous and allogeneic transplantation of hematopoietic stem cells has increased. 0ematopoietic growth factors allow the stem cellsB mobiliAation from the bone marrow into the peripheral blood. Kuantification of these hematopoietic stem cells by means of flow cytometry can be achieved within hours #Golds*hmidt et al., 2,%. Flow cytometry has become the ma<or techniCue for the Cuality control of stem cell3containing products such as apheresis concentrates, bone marrow or cord blood #Grieson et al., +!!&%. 'tem cells can be easily identified with flow cytometry due to their uniCue characteristics. They demonstrate a medium level of -*56 e!pression, a low level of -*67 e!pression and a low forward side scattered #Jennin"s 2 4oon, +!!0 and Masla/ et al., +!!-%. 2A&APlatelet analysisC The analysis of platelets by flow cytometry is becoming more common in both research and clinical laboratories. "latelet3associated immunoglobulin assays by flow cytometry can be direct or indirect assays, similar to other platelet3associated immunoglobulin immunoassays. In autoimmune thrombocytopenic purpura, free serum antibodies are not found as freCuently as platelet3bound antibodies #Ashman et al., 2,%. :2 Immunofluorescent flow cytometry was used to measure the percentage of activated platelet populations (-*81", -*85), the percentage of plt3monocyte aggregates (pma) (-*62D-*67), and activated monocytes (-*22b, -*26, -*28) in the blood #Panasui/ et al., 2&%. 2A$A?estin" for H8AA'20C 0uman leukocyte antigen +19 (043+19) is a ma<or histocompatibility comple! class 2 molecule that is strongly associated with the disease ankylosing spondylitis. The performance of the two flow cytometric antigen assays depends on the antibody used and the positive cutoff values assigned #Sei77 et al., 2&%. flow cytometric 043+19 test is much faster than the classical microcyto!icity test #Jennin"s and 4oon, +!!0 and GDtte, 2+%. biannual e!ternal Cuality assurance scheme for flow cytometric typing of the 043+19 antigen is operational in The .etherlands and +elgium since 2;;7. For flow cytometry, the most widely monoclonal antibody used was F*9>7, followed by #'267.1 and +-3m5. The ma<ority of laboratories used more than 2 anti3043+19 monoclonal antibody for typing #Sei77 et al., 2&%. 5. MaBor applications o- apoptosis analysis? There are many ways of detecting apoptosis by flow cytometry. poptotic cells can be recogniAed by a characteristic pattern of morphological (cell shrinkage, cell shape change, condensation of cytoplasm, nuclear envelope changes, nuclear fragmentation, loss of cell surface structures, apoptotic bodies, cell detachment and phagoctosis of remains), :1 biochemical and molecular changes (free calcium ion rise, bcl1D+a! interaction, cell dehydration, loss of mitochondrial membrane potential, proteolysis, phosphatidylserine e!ternaliAation, lamin + proteolysis, *. denaturatuin, 7>35>>kb cleavage, intranucleosomal cleavage and protein cross3linking #Huban/ et al., 2- and 8iu et al., 2-%. The methods of detecting apoptosis by flow cytometry are based on the measurement of light scatter, the detection of changes in the plasma membrane, the analysis of cell organelles or the sensitivity of *. to denaturation #Sedla/ et al., +!!!%. ,A+AA7o7tosis li"ht s*atterC s cells die or become apoptotic the refractive inde! of the internal cytoplasm becomes more similar to that of the e!tracellular medium this manifests itself as a reduction in forward scatter signal. t the same time, intracellular changes and invagination of the cytoplasmic membrane lead to an increase in side (or orthogonal or ;>Q) scatter. If a dead cell discriminatory dye is added, cells that have become permeable can be identifying. In this way low level resolution of dead and apoptotic cells can be get. number of dead cell dyes are available for use and the one used will depend on any other fluorochromes that are being measured. 'ome e!amples are; 'yto! #reen (6::nm e!citation; green fluorescence emission), "ropidium Iodide (6::nm e!citation; orangeDred fluorescence emission), 93minoactinomycin3* (93 *) (6::nm e!citation; red fluorescence emission) and T@3",@35 (855nm e!citation; red fluorescence emission) #1ohen and AlA(ubeai, +!!&%. :5 ,A2AA7o7tosis D<A analysisC *uring apoptosis, calcium and magnesium dependent nucleases are activated which degrade *.. This means that within the *. there are nicks and fragmentation. %e can detect these in three ways using *. analysis to look at the sub #2 peak, using strand break labeling (T&./4) to detect broken *. or using 0oechst binding to detect *. conformational changes #Ma=ino and Joris, +!!&%. The sub3#2 Fig$ 9+.3) method relies on the fact that after *. fragmentation, there are small fragments of *. that are able to be eluted following washing in either "+' or a specific phosphate3citrate buffer. This means that after staining with a Cuantitative *. Nbinding dye, cells that have lost *. will take up less stain and will appear to the left of the #2 peak. The advantage of this method is that it is very rapid and will detect cumulative apoptosis and is applicable to all cell types #Dar.yn/iewi*., +!!0%. 0owever in order to be seen in the sub #2 area, a cell must have lost enough *. to appear there, so if cells enter apoptosis from the ' or #1DM phase of the cell cycle or if there is an aneuploid population undergoing apoptosis, they may not appear in the sub #2 peak #S*hwart. and @sborne, +!!,%. :6 Figure 9+.3:? Sub * peak by propidium iodide staining #Dar.yn/iewi*, +!!0%$ :7 lso cells that have lost *. for any other reason e.g. death by some other form of oncosis, will appear in the sub #2 region so we have to be careful about how we define the sub #2 peak #<i*oletti et al., 2+%. ,A,AA7o7tosis *ell membrane analysisC In normal cells, phosphatidylserine ("') residues are found in the inner membrane of the cytoplasmic membrane. *uring apoptosis, the "' residues are translocated in the membrane and are e!ternaliAed. In general though not always, this is an early event in apoptosis and is though to be a signal to neighboring cells that a cell is ready to be phagocytosed #(obinson, +!!,%. nne!in3? is a specific "'3binding protein that can be used to detect apoptotic cells. nne!in ?3 is available con<ugated to a number of different fluorochromes. /arly apoptotic cells are anne!in positive but "I negative. +ecause the cells arenBt fi!ed we can e!clude dead cells and it is possible to add further markers if the cytometer set up are appropriate. s with all live cell assays, we have to remember that we are only looking at a snapshot of the cells as they are at time of analysis and generally all apoptotic e!periments should be performed over a time course; Fig$ 9+.5: #?elford et al., 2-, Hombur" et al., +!!& and :ermes at al., +!!&%. :8 Figure 9+.5:? !arly apoptotic cells are anne@in positi(e but 9in this case: #I 9negati(e) #?elford et al., 2-%. :9 0oechst 55561 is a *.3binding dye that is able to Cuantitatively stain the *. of live cells. 0owever it has also been found that if the concentration of 0oechst is low, the apoptotic cells take up the 0oechst more rapidly. If we also add "I or T@3",@35 we can specifically identify the dead cells. This is a rapid and Cuantitative method but reCuires the use of a &? laser. The advantage of using T@3",@35 is that cell phenotyping using FIT-3 and "/3labelled antibodies is also possible. Thymocytes labelled with -*63 "/ and -*:3FIT- can be assessed for apoptosis using 0oechst and T@3 ",@35 #9oo7man et al., +!!-%. third way of assessing the membrane changes in apoptosis is to use L@3",@32 (Molecular "robes). s this fluorochrome emits in the green, it can be combined with propidium iodide to identify dead cells. The rationale here is that cells in early apoptosis are unable to pump out L@3",@32 but are still not permeable to other dead cells discriminatory dyes #9oo7man et al., +!!-%. ,A-AA7o7tosis en.yme analysisC Two genes (ced35 and ced36) were crucial to the process of apoptosis. The ced36 gene product has homologues in mammalian cells, especially a family of cysteine proteases that are now known as caspases. There are a number of caspases in mammalian cells that have been shown to be involved in the early stages of apoptosis e.g. (caspase1, caspase5, caspase 8, caspase ; and caspase 2>). The functions of these enAymes are not yet entirely clear but it appears that after an initial signal to the cell to undergo apoptosis, they may be responsible for the activation, amplification and e!ecution of the apoptotic cascade #1ohen and AlA (ubeai, +!!&%. :: +ecause of the central importance of the caspases in apoptosis, their detection by flow cytometry has become widespread. %e can detect the activity of enAymes implicated in apoptosis in three ways; by detecting the active form of the enAyme using a specific antibody #Smolews/i et al., 22%, by using a fluorochrome labelled peptide that binds to the active site of the enAyme #Po.arows/i et al., 2,% and by using a non3fluorescent substrate for the enAyme which yields a fluorescent product if the enAyme is active #?elford et al., 2-%. ,A&AA7o7tosis or"anelle analysisC *uring apoptosis there is often a collapse of the mitochondrial membrane potential. This can be detected in a number of ways by flow cytometry. Two dyes in particular are useful3 -MF,os (also known as Mito tracker ,ed from Molecular probes) and 4*s3972 (from /!ction). -MF,os has a chloromethyl group which allows accumulation in active mitochondria. 4ive cells that have active mitchondria are able to take up -MF,os but in cells that are undergoing apoptosis, the mitochondrial membrane potential decreases which means less dye accumulates in the mitchondria leading to a decrease in fluorescenc #1ha7man et al., +!!&%. +. Detection o- apoptotic markers? *etermination of p75 e!pression by immunohistochemistry (I0-)
has been incorporated into routine practice and its reliability
has been consolidated. 0owever, flow cytometric (F-M) analysis
might represent an important ob<ective and rapid approach. F-M may provide important information about p75 protein e!pression
in the different subpopulations and cell cycle phases. In most breast, lung,
and colon aneuploid tumors (99E), :; p753positive cells were detected
only in the subpopulations with abnormal *. content #El)ira et al., +!!5%. +ovine papillomavirus type 2 (+"?32)3transformed mouse fibroblast cell lines were analyAed via flow cytometry (F-M) for e!pression of p75 and c3myc proteins along with their *. content. t least ;,>>>32>,>>> p75 or c3 myc protein molecules per cell were detected in the transformed tumorigenic cell lines. These results show that Cuantitative F-M can be reliably used to detect very low levels (5,>>> molecules per cell) of specific protein, and F-M is a useful tool to study the virus3induced changes in the levels of nuclear proteins within a cell population and in tumorigenesis #A"rawal et al., +!!-%. In human follicular lymphoma, nalysis of transgenic +cl1 e!pression used biotinylated
+cl132>> monoclonal antibody for the surface phenotyping of hematopoietic
cells by flow cytometry.
-ells (2> 8 per
analysis) were stained with relevant antibodies labeled with
T"/U, or cyanin 7 T-y7U) or biotin using 2E normal rat serum
to block Fc receptors. 'treptavidin con<ugated to FIT-
or "/ was used as a secondary
reagent for biotinylated antibodies. nalyses were performed
on a 4ife 'ciences ,esearch (4',) or a F-'tar II flow cytometer
(+ecton *ickinson, 'an =ose, -) #AleFander et al., 2-%. The e!pression of bcl31 was e!amined by multicolor flow cytometry in samples including lymph node or other tissue biopsy specimens containing follicular lymphoma (F4), reactive hyperplasia (,0), or other malignant lymphomas, as well as bone marrow aspirates. For all of the aforementioned reasons, a reliable flow cytometric assay for e!pression of ;> bcl31 would be a useful additional techniCue for establishing a diagnosis of F4. 0owever, the measurement of bcl31 by flow cytometric techniCues has received only scant attention. It was described a 13color flow cytometric assay using antibodies against bcl31 that demonstrated promise in the recognition of F4. #James et al., 2,%. ;2 7.Flourescence in situ hybridi8ation *.Introduction? FI'0 provides researchers with a way to visualiAe and map the genetic material in an individualBs cells, including specific genes or portions of genes. This is important for understanding a variety of chromosomal abnormalities and other genetic mutations. &nlike most other techniCues used to study chromosomes, FI'0 does not have to be performed on cells that are actively dividing. This makes it a very versatile procedure. The first step is to prepare short seCuences of single3stranded *. that match a portion of the gene the researcher is looking for. These are called probes. The ne!t step is to label these probes by attaching one of a number of colors of fluorescent dye #S*hrD*/ et al., +!!$ and 4oF et al., +!!$%. *. is composed of two strands of complementary molecules that bind to each other like chemical magnets. 'ince the researchersB probes are single3stranded, they are able to bind to the complementary strand of *., wherever it may reside on a personBs chromosomes. %hen a probe binds to a chromosome, its fluorescent tag provides a way for researchers to see its location #3hite et al., +!!& and 'loom, 2&%. Fluorescent in situ hybridiAation (FI'0) represents a modem molecular pathology techniCue, alternative to conventional cytogenetics (karyotyping). Fluorescence in situ hybridiAation (FI'0) allows identification of specific seCuences in a structurally preserved cell, in metaphase or interphase #9onto"eor"os, 2- and 1eledaet al., +!!-%. ;1 The probe, bound to the target, will be developed into a fluorescent signal. The fact that the signal can be detected clearly, even when fi!ed in interphase, improves the accuracy of the results, since in some cases it is e!tremely difficult to obtain mitotic samples Fig$ 9/.*: #Muhlmann, 22%. The power of in situ hybridiAation can be greatly e!tended by the simultaneous use of multiple fluorescent colors. Multicolor fluorescence in situ hybridiAation (FI'0), in its simplest form, can be used to identify as many labeled features as there are different fluorophores used in the hybridiAation. +y using not only single colors, but also combinations of colors, many more labeled features can be simultaneously detected in individual cells using digital imaging microscopy #(aa7 et al., +!!&%. Fluorescence, a phenomenon whereby a chemical e!cited at one light wavelength emits light at a different and usually longer wavelength, is used throughout the life sciences to study a wide variety of structures and intracellular activities. dvances in probe and microscope technology have led to the rapid development of techniCues for fluorescence over the past decade #?ras/, +!!+%. The accuracy of cytogenetic diagnosis in the management of hematological malignancies has improved significantly over the past 2> years. Fluorescence in situ hybridiAation (FI'0), a techniCue of molecular cytogenetics, has played a pivotal role in the detection of uniCue sub3 microscopic chromosomal rearrangements that helped in the identification of chromosomal loci, which contain genes involved in leukemogenesis #Amare et al., 2+%.
;5
Figure 9/.*:? Fluoresence in situ hypridi8ation #Muhlmann, 22%$
;6 The use of FI'0 is growing rapidly in genomics, cytogenetics, prenatal research, tumor biology, radiation labels, gene mapping, gene amplification, and basic biomedical research. In principle, the techniCue is Cuite straightforward #Attarbas*hi et al., 2-%. The hybridiAation reaction identifies, or labels, target genomic seCuences so their location and siAe can be studied. *. or ,. seCuences from appropriate, chromosome3specific probes are first labeled with reporter molecules, which are later identified through fluorescence microscopy. The labeled *. or ,. probe is then hybridiAed to the metaphase chromosomes or interphase nuclei on a slide. fter washing and signal amplification, the specimen is screened for the reporter molecules by fluorescence microscopy #Hohman and Gundla*h, +!!-%. FI'0 probes are commonly used to detect the presence of specific *. seCuences either when *. is condensed into metaphase chromosomes or dispersed in non dividing interphase cells. The fact that hybridiAation of probes to metaphase chromosomes is visualiAed in two dimensions while interphase targets are three dimensional has implications for both validations of assays and the development of baseline reference ranges #Pauletti et al., +!!$%. Metaphase applications generally yield clear yesDno answers while interphase applications commonly reCuire reportable reference ranges before interpreting of results. In addition to determining the presence or absence of particular seCuences in the genome, FI'0 is useful in assessing gene copy number in some disorders #Massod et al., +!!5%. ;7 nalytical uncertainty over *. probe assays also may stem from issues related to inherent population variation. The use of some repeat seCuence probes has been discontinued because of inability to detect targeted seCuences in individuals who possess very few repeats, leading to insufficient probe label in the targeted region which precludes visualiAed of the signal. 'uch probes have been eliminated #Myrata et al., +!!0 and 'ossuyt et al., +!!&%. FI'0 allows very precise spatial resolution of morphological and genomic structures. The techniCue is rapid, simple to implement, and offers great probe stability. The genome of a particular species, entire chromosomes, chromosomal3specific regions, or single3copy uniCue seCuences can be identified, depending on the probes used #Attarbas*hi et al., 2-%. &ntil recently, FI'0 was limited by the hardware, software, reagents, probe technology, and cost involved in implementing the techniCue. -ommercially available microscope hardware optimiAed for multicolor FI'0 was not available until the mid32;;>s. "rior to that, microscopes had to be customiAed for FI'0 applications. Most microscope optics was not designed to detect the low light levels inherent in FI'0 signals. s the genomic resolution of the techniCue has increased dramatically, the reCuirements on microscope optics have further increased. -hromatic aberrations among multiple wavelengths have been a problem. For multicolor analysis in particular, all lenses, including the collector lens, had to be chromatically corrected. In addition, epi3fluorescence light sources were difficult to align for uniform illumination #Amare et al., 2+%. ;8 nalysis of multicolor FI'0 images reCuires isolation of the various signals either with individual filter cubes; or utiliAation of an e!citation filter wheel with multipass dichroic and barrier filters. ,ecent developments in filter technology corrected some of the previous problems encountered through optical misalignments caused by mechanical switching of individual filter cubes. /!citation filter wheels used with multi3pass dichroic and barrier filters can be used effectively for up to three colors by employing separate e!citation filters for each color with no registration shift. +ut, for more than three colors, single3pass filters still had to be used #(a*e)s/is, 2&, >aro)aia et al., 2& and >ouro) et al., 2&%. 3.)hree di--erent types o- FISH probes? 2A+A8o*us s7e*ifi* 7robes They bind to a particular region of a chromosome. This type of probe is useful when scientists have isolated a small portion of a gene and want to determine on which chromosome the gene is located #H=almar, 2& and 3an" et al., 2&%. 2A2AAl7hoid or *entromeri* re7eat 7robes They are generated from repetitive seCuences found in the middle of each chromosome. ,esearchers use these probes to determine whether an individual has the correct number of chromosomes. These probes can also be used in combination with Hlocus specific probesH to determine whether an individual is missing genetic material from a particular chromosome #Edward et al., 2&%. ;9 2A,A3hole *hromosome 7robes They are actually collections of smaller probes, each of which binds to a different seCuence along the length of a given chromosome. &sing multiple probes labeled with a mi!ture of different fluorescent dyes, scientists are able to label each chromosome in its own uniCue color. The resulting full3 color map of the chromosome is known as a spectral karyotype. %hole chromosome probes are particularly useful for e!amining chromosomal abnormalities, for e!ample, when a piece of one chromosome is attached to the end of another chromosome #Du"an et al., 2&%. 5.Applications o- FISH? The clinical uses of FI'0 were considered in three main areas; diagnosis of individuals with birth defects and mental retardation, prenatal diagnosis and screening, and identification and monitoring of acCuired chromosome abnormalities in leukemiaD cancer. In each area the critical consideration remains a clear understanding of the capabilities and limitations of a test to provide useful information #'ossuyt et al., +!!& and Pauletti et al., +!!$%. Traditional cytogenetic analysis, detecting deletions, duplications, rearrangement and the identifications of unknown material of marker or derivative chromosomes, in individuals with birth defects andDor mental retardation has led to an understanding of the etiology of a number of syndromes. The clinical utility and limitations of these tests are both general and disease specific #8edbeteer et al., +!50, 1allen et al., +!!2, (ibeiro et al., +!!0 and 1assidy et al., +!!5%. ;: "renatal applications of FI'0 testing include both screening tests and diagnostic tests. Technical issues are few, and clinical utility raises Cuestions as to the intended use of FI'0 in testing. The application of FI'0 to prenatal screening for common autosomal trisomies and se! chromosome anomalies is becoming increasingly common. The primary considerations involve differing clinical sensitivity between the abnormalities detected by classical cytogenetic versus these detected by FI'0 based assays #E)ans et al., +!!+ and 9lin"er et al., +!!2%. mong cases ascertained via ultrasonographically identified fetal anomalies, some may be conclusive for a syndromes diagnosis and may be approached by a (diagnostic) FI'0 test. Families in which subtle or submicroscopic chromosomal abnormalities, detectable by FI'0, are known to segregate will benefit greatly from prenatal FI'0 studies #9onto"eor"os et al., 2 and 8ewin et al., 2%. Fluorescence in situ hybridiAation (FI'0) has become one of the ma<or techniCues in environmental microbiology. The original version of this techniCue often suffered from limited sensitivity due to low target copy number or target inaccessibility #6wir"lmaier, 2&%. The reagents and probes themselves were not sufficient for all applications. For instance, the efficiency of hybridiAation site detection decreased with decreasing probe siAe, creating significant limits to what could be observed via fluorescence microscopy. The number of differently colored fluorescent dyes was limited, and the photostability of the dyes was poor. +ut new developments in fluorescent dye technology and spin3off technology from the federally funded 0uman #enome "ro<ect are now having an impact. There are probes for all the human chromosomes and a ;; growing number of new gene3specific probes are available. >n situ hybridiAation kits and fluorescently labeled probes are commercially available from several companies #Sarrate et al., 2&%. The ability of FI'0 to rapidly test interphase and metaphase chromosome defects makes it especially useful in the study of cancer. In solid tumors, conventional cytogenetics is rarely used because obtaining metaphases is difficult and those cells that do proceed to mitosis may not be representative of the tumor. @ther molecular techniCues, such as "-, and 'outhern, .orthern, and %estern analysis, reCuire e!traction of the tissue. /!traction procedures net both normal and abnormal cells, so sensitivity is lower and Cuantitation less reliable than with FI'0 probes #'os*h et al., 2&%. FI'0 allows cell3by3cell analysis and thus provides for a more sensitive and reliable assessment of chromosomal aneuploidy, gene amplifications and deletions, and chromosome translocations. reliable determination of whether a gene is amplified in a specimen is often possible with evaluation of only 1> to 7> cells #@"il)ie et al., 2&%. The accuracy of cytogenetic diagnosis in the management of hematological malignancies has improved significantly over the past 2> years. FI'0 has played a pivotal role in the detection of uniCue sub3 microscopic chromosomal rearrangements that helped in the identification of chromosomal loci, which contain genes involved in leukemogenesis #Amare et al., 2+%. FI'0 was performed with specific probes to make the rapid prenatal diagnosis of *own syndrome. FI'0 was performed respectively with locus3 2>> specific probe (4'I) and centromeric probe (-/") FDL on the uncultured amniotic fluid. FI'0 is a rapid and reliable method to detect *own syndrome in uncultured amniotic fluid #3an" et al., 2&% Fluorescence in situ hybridiAation assay and to correlate the genetic findings with the pathologic grade and stage were used to investigate the chromosomal abnormalities present in bladder carcinoma #Pla*er et al., 2&%. novel application of FI'0 to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in e!tracted nuclei from paraffin3embedded tissue of human cancer with greater sensitivity and specificity than e!isting FI'0 methods. The method is applied to signal detection of the 0/,31Dneu (c3erb+31) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer #(ossi et al., 2&%. Tumor3specific chromosomal abnormalities are attracting a large interest owing to the diagnostic, prognostic, and therapeutic importance. The development of FI'0 has improved the detection of specific chromosomal abnormalities in chronic lymphocytic leukemic (-44). +y using FI'0, the problem with tumor cells with low mitotic rate is avoided since this method readily detects clonal aberrations also in nondividing, interphase cells. Three different types of probes are used centromeric probes for numerical chromosome abnormalities, whole chromosome paints, and locus3specific probes for numerical chromosome abnormalities, whole chromosome paints, and locus3specific probes. #H=almar, 2&% 2>2 FI'0 of *.3*. or *.3,. using post3mortem brain samples is one approach to study low3level chromosomal aneuploidy and selective e!pression of specific genes in the brain of patients with neuropsychiatric diseases. FI'0 could be applied to e!tended studies of chromosomal aneuploidy, abnormal patterns of chromosomal organiAation and functional gene e!pression in situ in the neurons of the brain in different psychiatric and neurodevelopmental diseases #Buro) et al, 2+%. 5.*.ALL in(estigation by FISH? To investigate patients with acute lymphoblastic leukemia (44) for T/4DM42 fusion, +-,D+4 fusion, M44 gene rearrangements, and numerical changes of chromosomes 6, 2>, 29 and 12 by fluorescence in situ hybridiAation (FI'0) and to determine the relationship and the significance of those findings #6han" et al., 2,%. Interphase fluorescence in situ hybridiAation (iFI'0) is increasingly used for the identification of +-,D+4 gene rearrangements in chronic myeloid leukemia (-M4) and acute lymphoblastic leukemia (44). FI'0 plays an important role in detecting chromosome changes, especially in some cryptic chromosome translocations and patients with culture failures #Primo et al., 2, and 6han" et al., 2,%. 44 blasts routinely contain somatically acCuired genetic abnormalities
that provide insight into pathogenesis and strongly influence
prognosis. ppro!imately one third of cases of 44 show an increase
in the modal chromosome number (e.g., hyperdiploid, V 69
chromosomes, and HhighH hyperdiploid, V 7> chromosomes) blasts
make up a uniCue biologic subset associated with increased in
vitro apoptosis and sensitivity to a variety 2>1 of chemotherapeutic
agents #Heerema et al., 2 and ?rueworthy et al., +!!2%. lmost one third of 44 blasts show chromosomal translocations
in the absence of changes in chromosome number. Four ma<or translocations
have been observed, and each defines a uniCue biological subset
of patients. The t(2;2;)(C15;C25) is a hallmark of some pre3+
(cytoplasmic PM) 44, and is characteriAed by fusion of
the E2" and )B1 genes #;*/un et al., +!!5%. *espite the adverse prognostic impact
of this translocation in older studies, recent intensification
of therapy has resulted in an improved survival for these children.
Translocations between the mi!ed lineage leukemia (4,,) gene
at 22C15 and over 5> different partner chromosomes characteriAe
8E of 44 cases. 4,, translocations, most commonly t(6;22)(C12;C15),
are seen in the vast ma<ority of infant patients with 44.
recent, large series demonstrates that any rearrangement of
22C15 is associated with a worse prognosis (e.g., 1>E to 17E) #Pui et al., 22%. ,A+A+APhiladel7hia The presence
of the t(;;11)(C56;C22) translocation, commonly known as "hiladelphia
chromosome ("h), in about 5E to 7E of all children with 44
is considered as one of the molecular markers associated with
a particularly high risk for treatment failure #(ibeiro et al., +!50, 1rist et al.,+!!, Pui et al., +!!, 4let*her et al., +!!+, (eiter et al., +!!-, and 1hessells et al., +!!&% . This translocation
causes a rearrangement between the protooncogene c3+4 and a gene
called the breakpoint cluster region (+-,). %hereas the breaks
in c3+4 occur mainly in the same region (between the 2>5 e!ons a2
and a1) on chromosome ;, two different ones affect the breakpoint
cluster region on chromosome 11: the more freCuent one (appro!imately
in 1 of 5 of all cases) shows a break in the minor breakpoint
cluster region (m3 +-,) between the e!ons e2 and e1. This is predominant
in 44. In 2 of 5 of all "h M 44 cases, the ma<or (M3) +-, found between e!ons b1 and b5 or
e!ons b5 and b6 is affected. M3+-, is also found in nearly all
patients with chronic myelogenous leukemia (-M4). -himeric proteins
of 12> k* (p12>) and 2;> k* (p2;>) result from the M3+-,D+4 and
m3+-,D+4 rearrangements, respectively #9antar=ian et al., +!!+%.
These fusion proteins
cause a deregulation of protein tyrosine kinase activity. +oth
forms of the chimeric gene (+-,D+4) can be detected by polymerase
chain reaction ("-,) and fluorescent in situ hybridiAation.
#Maurer et al., +!!+, Dewald et al., +!!,, S*hlieben et al., +!!$%. Most patients with "hiladelphia ("h)3positive acute lymphoblastic leukemia (44) show evidence of secondary chromosome aberrations that may influence the course of disease and response to treatment. To better understand how these secondary chromosomal aberrations occur and to investigate whether the p2:7Dp2;> +-,3+4 fusion protein may directly induce an increased chromosomal instability and subseCuently the appearance of clonal chromosome aberrations, three +,-3+4 (p2:7D p2;>)3transduced mouse pre3+ cell lines were analyAed by spectral karyotyping and fluorescence in situ hybridiAation. The human wild3type +-,3+4 gene was e!pressed at a level comparable with that in human "h3 positive leukemias at diagnosis. ll +-,3+43transduced cell lines acCuired similar clonal chromosomal aberrations. Trisomy 7 was always present, followed by loss of the L chromosome, trisomy of chromosomes 21 and 2:, and an unbalanced translocation between chromosomes F and 21. Thus, 2>6 ectopic p2:7Dp2;> +-,3+4 e!pression, such as p12> +-,3+4, "M43 ,,, or -3ML- transduction, may induce an increased chromosomal instability leading to clonal karyotypic evolution, which may mimic secondary chromosome aberrations in human "h3positive 44 #(udol7h et al., 2&%. The "hiladelphia ("h) chromosome, the main product of the (;;11) (C56;C22) translocation, is the cytogenetic hallmark of chronic myeloid leukemia (-M4), a clonal myeloproliferative disorder of the hematopoietic stem cell; the "h chromosome is also found in a siAeable portion of acute lymphoblastic leukemia (44) patients and in a small number of acute myeloid leukemia (M4) cases. Three different breakpoint cluster regions are discerned within the +-, gene on chromosome 11: M3bcr, m3bcr, and mu3bcr #DreFler et al., +!!!%. .early all "h M 44 cell lines have the m3bcr e23a1 fusion gene (only two 44 cell lines have a b53a1 fusion) whereas all -M4 cell lines, but one carry the M3bcr b13a1, b53a1 or both hybrids. The mu3bcr e2;3a1 has been detected in one -M4 cell line. Four cell lines display a three3way translocation involving chromosomes ;, 11 and a third chromosome. dditional "h chromosomes (up to five) have been found in four "h M 44 cell lines and in 2: -M4 cell lines; though in some cell lines the e!tra "h chromosome(s) might be caused by the polyploidy (tri3 and tetraploidy) of the cells. nother modus to acCuire additional copies of the +-,3+4 fusion gene is the formation of tandem repeats of the +-,3+4 hybrid as seen in -M4 cell line I3781. +oth mechanisms, selective multiplication of the der(11) chromosome and tandem replication of the fusion gene +-,3+4, presumably lead to enhanced levels of the fusion protein and its tyrosine kinase activity (genetic dosage effect). The availability of a panel of "h M cell 2>7 lines as highly informative leukemia models offers the uniCue opportunity to analyAe the pathobiology of these malignancies and the role of the "h chromosome in leukemogenesis #DreFler et al., +!!!%. Treated children with acute lymphoblastic leukemia were analysed for chromosomal abnormalities with conventional #3banding, spectral karyotyping ('IL) and interphase fluorescent in situ hybridisation (FI'0) using probes to detect M44, +-,D+4, T/4DM42 rearrangements and I.I6 locus deletions. Three novel T/4 partner breakpoints on 2C62, :C16 and 12p21 were identified, and a recurrent translocation t(2;21)(p51;p25) was found. In addition, two cases displayed amplification (9327 copies) of M42. ,esults were demonstrated the usefulness of 'IL and interphase FI'0 for the identification of novel chromosome aberrations and cytogenetic abnormalities that provide prognostically important information in childhood 44 #<ord"ren et al., 22%. The +-,D+4 and M44DF6 fusion genes33resulting from t(;;11) (C56;C22) and t(6;22)(C12;C15) translocations, respectively33are considered as a high risk prognostic factors in children with acute lymphoblastic leukaemia (44). Their presence in malignant cells indicates patient for the most intensive antileukaemic therapy regardless of the other criteria. In contrast, the most common non3random chromosomal aberration in paediatric 4433 translocation t(21;12)(C21;C11)33is associated with a favourable prognosis. The e!amination of these rearrangements is important for the stratification of patients to the risk groups and also provides the most sensitive and specific tool for minimal residual disease (M,*) follow3up #?r/a et al., +!!! and Po7la*/, +!!,%. 2>8 7I. ,e-erences AbuAAbsi <(, 6amamiri A, 9a*mar J, 'alo"h SJ, Srien* 4, #2,%. utomated flow cytometry for acCuisition of time3dependent population data. -ytometry ., 72(1)::93;8. Ada*hi A, Sato S, Sasa/i B, Gha.i.adeh M, Maeda M, 9ai.u 9, 8iu G8, 4u/una"a B, #2&%. /lectron microscopic studies on the occurrence of activated neutrophils in peripheral blood of children with acute leukemias. = 'ubmicrosc -ytol "athol., 59(2):253:. A"rawal (S, A"rawal BP, Manti"ar)i (A, #+!!-%. Flow cytometric Cuantitation of -3myc and "75 proteins in bovine papillomavirus type 23transformed primary mouse fibroblasts. -ytometry, 2;29(5):159367. Albro J, 'auer 9D, Hit*h*o*/ 18, 3ittwer 1?, #+!!,%. Improved *. content histograms from formaline3fi!ed "araffin embedded liver tissue by protinase I digestion. -ytometry, 26:895389:. Alexander E, Alan W, Mary L, (2004). VavP-Bcl2 transgenic mice develop follicular lymphoma preceded by germinal center hyperplasia !lood, 103(6): 2276-2283 . Amare PS, 'aisane 1, Sai/ia ?, <air (, Gawade H, Ad)ani S, #2+%. Fluorescence in situ hybridiAation: a highly efficient techniCue of molecular diagnosis and predication for disease course in patients with myeloid leukemias. -ancer #enet -ytogenet., 252(1):2173 56. Amati ', Ale)i.o7oulos 9, :la*h J, #+!!5%. Myc and the cell cycle. Front +iosci., 5:*17>N*18:. 2>9 Ari*H M, :alse**hi MG, 1amitta ', #2%. @utcome of treatment in children with "hiladelphia chromosome3positive acute lymphoblastic leukemia. . /ngl = Med., 561 (26): ;;:32>>8. Ashman <, Ma*ey MG, 4an S8, A.am ;, BaIoob M, #2,%. Increased3 plateletmonocyte aggregates and cardiovascular disease in end3 stage renal failure patients. .ephrol *ial Transplant., 2:(2>):1>::3;8 Assun*ao P, Dia. (, 1omas J, de Galarreta 1M, Gon.ale.A8lama.ares @(, Po)eda J', #2&%. /valuation of Mycoplasma hyopneumoniae growth by flow cytometry. = ppl Microbiol., ;:(7):2>69376. Attal M, 'laise D, Marit G, #+!!&%. -onsolidation treatment of adult acute lymphoblastic leukemia: a prospective, randomiAed trial comparing allogeneic versus autologous bone marrow transplantation and testing impact of recombinant 4l31 after autologous bone marrow transplantation. +lood, :8(6): 282;3 281:. 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ed. &niversity "ress @!ford, p.51 1allen D, Eyre H, Bi7 M, 4reemantle 1, Haan E, #+!!2%. Molecular cytogenetic and clinical studies of 61 patients with marker chromosomes. m = Med #enet; 65: 9>;3927. 1arolyn S, (i*hard G, Samuel M, Mar/ S, Mel 4, (o"er 1, Sally H, #22%. *etecting 'mall3rea 'imilarities in the /pidemiology of -hildhood cute 4ymphoblastic 4eukemia and *iabetes +M=; 516:1:5N9 1arroll 8, Dee7a ', Don"AJoon M, Eli.abeth (, Mary (, Stella D, James (, 1eryl 8, John 1, #2,%. "ediatric cute 4emphoblastic 4eukemia. 0ematology., (2):2>13252 1assidy S, S*hwart. S, #+!!5%. "rader3 %illi and ngelman syndromes disorders of genomic imprinting. 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Multicolor fluorescent in situ hybridiAation on post3mortem brain in schiAophrenia as an approach for identification of low3level chromosomal aneuploidy in neuropsychiatric diseases. +rain *ev., 15 'uppl 2:'2:83;>. 6han" 8P, 1hen" B4, 8iu G8, 8u AD, 8iu B(, 3an" H, #2&%. The clinical significance of detecting minimal residual disease in childhood +3lineage acute lymphoblastic leukemia with flow cytometry. Zhonghua /r Ie Za Zhi., 65(9):6:237. 6han" 8, Par/hurst J', 9ern 34, S*ott 9:, <i**um D, Mul)ihill JJ, 8i S, #2,%. -hromosomal changes detected by fluorescence in situ hybridiAation in patients with acute lymphoblastic leukemia. -hin Med =., 228(;):21;:35>5. 6immerman A and ?russ 4, #+!0!%. Flow through cytophotometry. &rol. ,es., 9:2 6wir"lmaier 9, #2&%. Fluorescence in situ hybridisation (FI'0) the ne!t generation. F/M' Microbiol 4ett., 168(1):2723:. 266 7II. LIF! FL%=CE)%M!),IC FI',!S () (+) Figure 9*:? Flow cytometric analysis o- c.myc e@pression on mononuclear cells showing diagram 9A: and dot plot 9B: o- positi(ely stained cells in relation to negati(e ones$ () (+) Figure 93:? Flowcytometric analysis o- p/5 e@pression on mononuclear cells showing histogram 9A: and dot plot 9B: o- positi(ely stained cells in relation to negati(e ones$ 267 M1 M2 R1 R2 M1 M2 Diploid? *44$44F *ip #>3#2: ;5.;5 E at 55.6; *ip #13M: 6.79 E at 82.8; *ip ': 2.7> E #1D#2: 2.:6 *ip E-?: 1.:6 Diploid? *44$44F *ip #>3#2: ;>.65 E at 57.52 *ip #13M: 9.:6 E at 87.5> *ip ': 2.95 E #1D#2: 2.:7 *ip E-?: 5.29 Figure 95:? Histogram showing cell cycle parameters 9diploid: using -low cytometer FACS caliber program mod-it $ 268 Diploid? 03$46 F *ip #>3#2: 2>>.>> at55.2> *ip #13M: >.>> E at 88.12 *ip ': >.>> E #1D#2:1.>> *ip E-?: 5.28 Aneuploid *? 51$23 F neup #>3#2: ;7.59 E at67.2; neup #13M: 2.98 E at 95.;7 neup ': 1.:: E #1D#2: 2.86 neup E-?: 5.65 neup *I: 2.59 Diploid? 6/$3/ F *ip #>3#2: 2>>.>> E at 5:.71 *ip #13M: >.>> E at 99.>7 *ip ': >.>> E #1D#2: 1.>> *ip E-?: 5.61 Aneuploid *? *+$1/ F neup #>3#2: 87.26 E at 8;.97 neup #13M: 1:.91 E at 2>6.95 neup ': 8.25 E #1D#2: 2.7> neup E-?: 5.52 neup *I: 2.:2 Figure 9+:? Histogram showing cell cycle parameters diploid and aneuploid using -low cytometer FACS caliber program mod-it$ 269 7III. LIF! FISH #IC)',!S Figure A Figure B Figure C Figure D Figure 9A"B"C and D:? !ach childhood acute lymphoblastic leukemia case shows red signal which is ABL on chromosome 2 and green signal which is the breakpoint cluster region 9BC,: on chromosome 33 -or children with #hiladelphia negati(e acute lymphoblastic leukemia 9#h\ ALL:$ 26: Figure ! Figure F Figure 9! and F:? !ach childhood acute lymphoblastic leukemia case shows red signal which is ABL on chromosome 2" green signal which is the breakpoint cluster region 9BC,: on chromosome 33 and pale orange signal which is the -usion 9BC,<ABL: -or children with #hiladelphia positi(e acute lymphoblastic leukemia 9#h G ALL:$ 26;