Patent Application: US-201113106275-A

Abstract:
a method and apparatus for generating an interphase chromosome profile . the method comprises obtaining a sample containing cells having chromosomes for profiling ; obtaining species specific dna probes , wherein the dna probes are capable of marking at least one chromosome at substantially equidistant locations on said chromosome ; hybridizing the sample with the dna probes with plurality of fluorescent labels ; and using visual analysis for determining the profile of the chromosome .

Description:
referring now to fig1 , each chromosome is represented by the icp color band ideogram . centromere / pericentromere areas are in blue ; short arm telomeres in green ; and long arm telomeres in red . for acrocentric chromosomes 14 , 15 and 21 , the icp ideograms are split into two parts : left and right . the left ideogram represents the color banding when the chromosome was introduced first and the right ideogram represents the color banding when the chromosome was introduced second . the terms first and second refer to the order of the respective chromosome in the acrocentric chromosome combination on slide 1 . for chromosomes 13 and 22 , only one icp color banding exists , since they are always introduced first and second , respectively . fig2 represents the traditional g - banded ideogram at 400 band level . each chromosome , such as , for example , a human chromosome can be identified in the interphase by virtue of the hybridization of specific dna sequences on the various chromosomes . these sequences can be located on the chromosome &# 39 ; s centromere or telomere , or region - specific areas that lie between the centromere and telomere . the spacing of the sequences on the chromosome is designed to be substantially equidistant based on the overall size of the short arm and long arm of the chromosome . using the substantially equidistant concept , all human chromosome arms can be classified into one of three groups : group 1 ( small ), group 2 ( medium ), and group 3 ( large ). within each group , individual bands are spaced substantially the same distance apart . this spacing coupled with the disclosed staining technique produces a unique color - banding pattern for each chromosome . the chromosomes are classified into several groups based on their size , and chromosomes within each group have the same color sequence pattern for ease of recognition . each chromosome arm can have a distinct color band “ signature .” any variations from this signature can indicate a genetic change ( i . e ., a chromosomal abnormality ). in general , the centromere of the chromosome is stained blue , and as one proceeds from the centromere towards the end of the arm , differential color bands are produced by an admixture of varying proportions of the three colors red , blue and green . by way of example , if the short arm telomere is stained green , and the long arm telomere is stained red , the color band between the centromere and a telomere can be varying degrees of mixtures yielding colors such as orange , amber , cyan etc . with this color differentiation , chromosomal variations can be easily recognizable in the interphase cell under a fluorescent microscope , without sophisticated computer software , or interferometric spectroscopy . in an alternative embodiment , the chromosome can be stained blue or other differentiating color at additional equidistant locations along the short and long arms of the chromosome between the mixed differential color staining locations . such additional staining allows for easier visual detection of genetic abnormalities by increasing the resolution as well as providing landmarks for the user to identify . as used herein the mixed color differentiating bands are referred to as major bands and the additional solid bands , such as , for example , the blue bands are referred to as the minor bands . to provide further differentiation between the short arm and the long arm of the chromosomes , the short arm and long arm minor bands can be stained to provide differing band thicknesses between the respective chromosome arms . for example , the short arm minor bands can be stained to provide minor bands having a uniform thickness smaller than the minor bands on the long arm of the chromosome . such staining can provide easier detection of an abnormality involving a single chromosome . unlike the traditional fish , the current invention relies upon improved fluorescence in situ hybridization for the detection of the hybridized sequences . according to aspects of the present invention , an admixture of specific fluorochromes conjugated to the dna probes in specific proportions can result in the production of various differential colors . this can allow for the simultaneous visualization of the entire chromosome in an interphase cell . current fish - based approaches in the art utilize specific dna probes to detect known genetic abnormalities either using metaphase chromosomes or interphase nuclei . in order to establish a diagnosis , one would start with a specific set of probes and if certain results are positive , the test is finished . however , very often the initial “ working diagnosis ” from the clinician is wrong and the laboratorian is forced to use multiple , sequential applications of dna probe sets . this is not only very time consuming , but also very expensive . most unfortunately , there may be an insufficient quantity of testing material ( i . e ., metaphase or interphase chromosomes ) available to proceed with the sequential hybridizations necessary to complete the karyotype and provide the appropriate diagnosis . classical cytogenetic analysis remain the standard because 1 ) it requires no prior knowledge of the disease ; 2 ) it is capable of detecting not only known , but also unknown genetic abnormalities ; and 3 ) it yields the complete karyotype in one experiment . however , this method requires culture , resulting in significant turn around time . moreover , for tissue types , such as solid tumors and products of conception , culture success rates are very low , thereby , resulting in little or no relevant information . in addition , marker chromosome identification is often very difficult and impossible with standard g - banding . the present invention overcomes these challenges by not requiring any cell culture and providing a reliable detection mechanism for all types of chromosomal changes in interphase nuclei , in less than 48 hours . icp facilitates the characterization of nearly all chromosome abnormalities through the use of a single universal probe set with no known prior knowledge of specific genetic abnormalities in a specimen . this characterization can occur one chromosome at a time , in the interphase cells , in a designated spot or well on a hybridization chamber ( slide ). by way of example , the hybridization chambers comprise a set of three slides , each containing 10 wells . slide 1 can be used for detecting robertsonian translocations ( translocations between the acrocentric chromosomes ( i . e ., chromosomes 13 , 14 , 15 , 21 , and 22 )). slide 2 can be used to characterize chromosomes 1 - 10 . slide 3 can be used to characterize chromosomes 11 , 12 , 16 - 20 , x and y . in one aspect of the invention , two wells can be left intentionally blank for further testing . the icp technique can eliminate unnecessary hybridization with numerous different probe sets thereby allowing complete karyotype information to be assembled by combining the results from the 28 wells . in one aspect of the invention , the icp technique proceeds according to the following steps : 1 ) generation of dna probes ; 2 ) in situ hybridization ; 3 ) fluorescent detection of dna hybridization ; and 4 ) microscope analysis . step 1 can comprise using dna probes from the 24 human chromosomes labeled with a fluorescent label such fluorescein , rhodamine , cascade blue and the like . the probes can be specifically generated through chromosomal micro dissection , or other method known in the art , to obtain the exact portion of the chromosome for which hybridization is desired . for example , probe generation can be performed utilizing micro dissection techniques , plasmids , cosmids , computational methods from genome information , synthesis and the like . in step 2 , interphase cells in each well can be hybridized overnight with a single set of dna probes specific for the chromosome or abnormality to be detected . hybridization can be performed using standard in situ hybridization techniques , such as pretreatment with enzymes to allow dna probes to penetrate the nuclear membrane and dna denaturation to separate the two dna strands . in step 3 , each particular label can be detected through standard fluorescent detection techniques . based on the predetermined proportion of colors at different bands along the length of the individual chromosomes , the admixture of two fluorochromes for example generates a new mixed detectable color . the various colors for the bands on the chromosomes can be predetermined and as shown in fig1 can be represented on an ideogram for ease of comparative analysis . according to step 4 , the individual color chromosome bands can be observed and understood by a human using a simple , standard fluorescent microscope . in prenatal diagnosis tat is extremely critical and there can be a significant amount of anxiety experienced by expecting parents associated with the testing . for decision making and pregnancy management , the deadline for obtaining results is within 24 weeks of gestation . in normal circumstances , the standard chromosome test is performed at around 16 - 20 weeks of gestation with results being available after 10 - 14 days . therefore , late gestation amniocentesis testing is generally undesirable . in some clinical situations , it is desirable to perform chromosome testing within the last few weeks of pregnancy . under circumstances , the results need to be obtained on an emergent basis . in early amniocentesis testing ( i . e ., 12 weeks ), results will usually take significantly longer than 10 - 14 days . this is usually due to the small number of cells available for testing . in each of these situations , the classical cytogenetics cannot generally identify the nature of marker chromosomes . as a result , special studies need to be performed which involve additional time and money . traditional fish testing can be done within 48 hours , but , it is highly limited in scope and cannot produce complete chromosome information that includes the detection of any robertsonian translocations which can potentially increase morbidity through conditions , such as , uniparental disomy ( upd ). peripheral blood chromosome testing can be performed on patients with abnormal phenotypic features , such as , for example , mental retardation or couples with infertility issues or multiple miscarriage , to establish genetic diagnosis . in general , using current methodologies , the tat for such testing is usually 5 days , however , the regular chromosomal study cannot identify marker chromosomes and unbalanced chromosome rearrangements . these situations require additional testing which increases the tat and adds to the cost . in certain conditions , abnormalities involve the ends of the chromosomes ( i . e ., subtle rearrangements ), that can be missed with routine testing . currently the detection of these abnormalities requires multiple , separate fish testing to resolve whatever diagnostic issues may exist . for example , in patients with conditions such as , idiopathic mental retardation ( id ), approximately 5 - 9 % will have submicroscopic , sub - telomere rearrangements that cannot be detected by classical cytogenetics . moreover , in approximately 7 % of all patients with normal karyotypes having id , developmental disorders , will have sub - telomere rearrangements . in some cases additional cells need to be analyzed by routine testing which can significantly increase the cost . in leukemia / lymphoma cancer testing , chromosome information is not only vital in accurate diagnosis , but also critical in managing different drug regimen protocols . often a physician can be waiting for results to make critical treatment decisions . with current methodologies , tat is usually about 5 days . one major drawback to current testing is that the regular chromosome study cannot identify marker and derivative chromosomes as well as the previously mentioned subtle rearrangements that involve the telomeres of the chromosomes . these situations can require additional testing resulting in increased tat and cost . in some cases with normal / abnormal results , many more cells need to be analyzed by routine testing which can also increase the cost . unfortunately , in many cases , additional cells with chromosomes are simply unavailable for testing . in approximately 5 - 10 % of cases , no chromosome results are available at all , due to culture failure . the importance of chromosome information in solid tumor diagnosis and patient management is increasing rapidly . in cancers of solid tissues such as bladder , prostate , kidney , breast , lung and the like a regular chromosome study can take up to 30 days or more and in more than 70 - 80 % of the tests , the results cannot be obtained . this results in a high incident of study failure . similar to the leukemias and lymphomas discussed above , marker and derivative chromosome identification is not possible with regular chromosome testing . chromosome information can be obtained on miscarriage material to establish genetic diagnosis and to counsel the patient for future pregnancy decisions . using current methodologies , the tat for genetic testing on such tissue can be up to 30 - 45 days . more importantly , in 20 - 40 % of cases , results cannot be obtained by standard chromosome testing due to a compromised sample . accordingly , additional testing is generally required to properly diagnose the genetic abnormality and counsel the patient . such additional testing subsequently increases the costs and time required . icp fills the void created by the limitations of current methodologies by providing the unmet needs of the clinical cytogenetics and medical community , in a timely and cost effective manner . following are examples illustrating procedures for practicing the invention . these examples should be construed to include obvious variations and not limiting . under current methodologies 24 different colors are used to obtain a multi - color karyotype , based on metaphase chromosomes . to produce metaphase chromosomes , the material from various specimen types , such as for example , peripheral blood , bone marrow , amniotic fluid , solid mass and the like must be cultured . it is generally necessary to study 20 cells to obtain complete karyotype information . often , the amount of cells available is limited . until the present invention , no method has been available to effectively utilize the source material , so that complete characterization of the genetic changes present in the specimen presented for study , can be accomplished . there are some methods existing in the art capable of studying specific sets of chromosomes ; however , these methods are quite limited . as discussed above , suspected genetic abnormality must be known beforehand and only limited information can be gained by such testing . using the present invention with a single universal probe set , without any prior knowledge of the specific genetic changes present in a specimen , nearly all chromosome changes can be completely characterized . this can be done one chromosome at a time in interphase cells , in a designated spot or well on a hybridization chamber . in one aspect of the invention , a hybridization chamber can consist of a set of slides and as illustrated in the diagram , each slide can contain wells . by way of example , slide 1 can be a slide for detecting a special type of chromosome rearrangement called robertsonian translocation . robertsonian translocations are translocation between acrocentric chromosomes that join by their centromeres , resulting in one less centromere in the karyotype . for example , when a robertsonian translocation takes place between chromosome 14 and chromosome 21 , in a balanced form , there is one normal 21 , one normal 14 and a joined chromosome 14 / 21 in the karyotype , so that the total number of chromosomes changes from 46 to 45 . there are five acrocentric chromosomes in a human genome ( chromosomes 13 , 14 , 15 , 21 and 22 ), any one of these five can participate in this type of rearrangement , including rearrangement between both copies of the same chromosome , i . e ., chromosome resulting in 21 / 21 joined chromosome . the rearrangement can result in a balanced or unbalanced karyotype . in an unbalanced form , the total chromosome number generally remains 46 , however , there will likely be three copies of one of the acrocentric chromosomes in the karyotype . using current fish methods , on a clinical sample from a patient having down syndrome , three copies of down syndrome specific region i . e ., 21 q22 , will generally be detected . such a result is sufficient for the free form of down syndrome having three copies of chromosome , 21 , and without the presence of robertsonian translocation . however , the same clinical phenotype of down syndrome can result from the robertsonian rearrangement involving chromosome 21 and one other acrocentric chromosome . this cannot be detected using the current methods in the art . this has important clinical implications for genetic counseling and next pregnancy management as recurrence risk figures are entirely different between the free form and the robertsonian form of down syndrome . accordingly , valuable information is lost . this limitation can be overcome by the current invention . robertsonian translocation involving chromosomes other than chromosome 21 can also be clinically significant in prenatal diagnosis . a pathological condition called uniparental disomy ( upd ) exists for chromosomes 13 , 14 , and 15 . when balanced robertsonian translocations occur between acrocentric chromosomes , and the chromosome involved is 13 , 14 or 15 , the carriers have an increased risk for upd . upd in the fetus , detected in the prenatal diagnosis , contributes to severe clinical manifestations and adds significantly to the rate of morbidity . until the current invention , the only way to detect the robertsonian translocations and recognize potential upd , was by standard cytogenetics . icp overcomes this by detecting all robertsonian translocations in interphase cells , thus providing valuable information for the pregnancy management , in the prenatal diagnosis . slide 2 can comprise wells for chromosomes 1 - 10 . slide 3 can comprise wells for chromosomes 11 , 12 , 16 , 17 , 18 , 19 , 20 , x / y with additional wells left intentionally blank for further testing . by carefully plating 25 - 30 interphase cells in each well , even compromised specimens can be studied because unnecessary hybridization is eliminated and complete karyotype information can be assembled by combining the results from the wells . the probe set consists of a uniquely designed combination of dna probes for each of the 24 chromosomes . each human chromosome contains a centromere and one short arm and one long arm attached at the centromere . all chromosomes contain at the ends of the arms , specific dna sequences called telomeres unique for each chromosome . acrocentric chromosomes only have centromeres and long arms . their short arms are variable and can be absent in the genome and have no clinical significance . for this reason no probes are designed to detect the acrocentric short arms . referring now to fig1 , illustrating the color banding pattern based on the present invention . for comparison , the standard g - banding ( i . e ., the gold standard ), is included . the ideogram is the diagrammatic representation of all the bands on a chromosome . based on the current g - banding ideograms , each human chromosome was given a individual unit length . for example , chromosome 1 , the largest human chromosome , has a unit size of 73 , with short arm having a unit size of 36 and the long arm 37 ; chromosome 2 has unit size of 68 with 27 and 41 for the short and long arms , respectively . the unit sizes for all chromosomes are depicted on the ideograms . under current methodologies , the chromosome classification is generally based on g - banding patterns and chromosomes are grouped a through g and by sex chromosomes . chromosomes 1 - 3 are in group a , 3 - 4 in b , 6 - 12 and x are in c , 13 - 15 in d , 16 - 18 in f , and 19 - 20 including the y chromosome are in g . the bands on individual chromosomes are generally produced by chemical staining and have fixed locations on the chromosome and cannot be changed . therefore , identification of chromosome changes that fall between two naturally , closely spaced g - bands can be , and is often , very difficult . this problem can be overcome by the present invention by spacing the “ bands ” at substantially equidistant locations from one another based on the overall size of the short and long arms . this can allow for the easy recognition of chromosomal changes in interphase nuclei . utilizing the concept of substantially equidistant chromosomal hybridization , human chromosome arms can be classified into one of three groups : group 1 ( small ) having a unit size of 4 - 6 ; group 2 ( medium ) having a unit size of 7 - 19 ; group 3 ( large ) having a unit size of 20 - 41 . with in each group , individual bands are spaced at substantially the same distance . by way of example , the short arm can have 5 bands and the long arm can have 4 bands . with the centromere band , chromosome 1 has a grand total of 10 ( 5 + 1 + 4 ) color bands . accordingly , looking at human chromosome 1 in an interphase cell , using the current icp invention , one would observe , starting from the end of the short arm , a primary non - repeating color band , a primary repeating color band , a mixed color band , a primary repeating color band etc . until they reach the centromere and this pattern would continue until they reach the end of the long arm with a non - repeating color band . counting all bands a normal chromosome 1 would have a grand total of 19 bands . this type of resolution is equal or better than the standard g - banding pattern , which requires metaphase chromosomes , which can only be obtained after culture . by way of another example , following the above model , chromosome 18 would have a group 1 short arm and group 2 long arm ; chromosome y would have a group 1 short arm and a group 1 long arm . as a further example , with respect to chromosome 1 , minor bands can be stained interstitially at locations between the color bands . such minor band staining , for example , can add eight ( 8 ) additional bands , bringing the total stained bands on the chromosome to nineteen ( 19 ). as can be seen in fig1 , the classification method is illustrated with the corresponding color banding pattern for human chromosomes . in general and for illustrative purposes only , centromere bands can be stained blue and as one proceeds from the centromere towards the end of the short arm , the differential mixed color bands can be produced by an admixture of varying proportions of primary colors . in one aspect of the invention , short arm telomere bands can be stained green and long arm telomere bands can be stained red . in another aspect of the invention , for chromosomes 1 , 5 , 9 , 16 and 19 the pericenromeric band , i . e ., the band adjacent to the centromere , can be used in lieu of the centromere . for illustrative purposes only , using chromosome 1 as an example , the centromere can be blue , the next band in the short arm can be yellow color . the next band can be amber color . the next band can be cyan color . the next band can be orange color . finally , the telomere band can be green indicating the end of the telomere . this sequence of bands , between centromere and telomere , can be reversed with respect to the primary colors for the long arm . therefore , the long arm of chromosome 1 starting with centromere blue , will have amber , cyan , orange and finally the telomere band far red . a simple read out of whole chromosome from short arm to long arm is green , orange , cyan , amber , yellow , blue , amber , cyan , orange and far red . as can be seen from this discussion each arm has its distinct color band “ signature ” and any variations from this signature would indicate a genetic change . according to aspects of the present invention , variations can be easily recognizable in the interphase cell , under a simple fluorescent microscope without inferometers , sophisticated computer software ; and the like , as are currently used in the art for fish based detection of chromosome changes . because the color bands according to the present invention are spaced more or less equidistant from each other , regardless of the size of the arm , deviations from the color sequence , missing or extra color bands , reduction or enlargement of unit size of background stain between two adjacent color bands , displacement of the color sequence would indicate chromosomal abnormalities . this method can be used for detecting both numerical and structural abnormalities of virtually any human chromosome . a few examples of chromosome abnormalities and especially marker chromosome identification using icp are discussed infra . under current methodologies , marker chromosomes could only be identified in metaphase chromosomes . utilizing the present invention , marker chromosomes can be identified in interphase nuclei . the result is a significant savings of time and money . current cytogenetic methodologies rely on natural breakpoints in a chromosome for designing probes that flank these regions . for example , to detect a leukemia specific translocation such as the translocation between chromosome 9 and 22 , probes are designed to detect the breakpoint on chromosome 9 in band 9q34 ( the abl gene ) and band 22q11 . 2 ( the bcr gene ). using two colors , red for 22 and green for 9 , a normal interphase reaction appears as two red dots and two green dots , whereas a translocation would result in one green , one red and one yellow due to the green / red fusion . by way of example , utilizing the present invention , the same translocation would appear as a displacement of chromosome 9 into two segments and chromosome 22 into two segments . the resulting color banding pattern in the chromosome 9 well on the respective slide would be : starting from short arm telomere green , light blue , orange , light blue , light blue , light blue , cyan , light blue , orange as one contiguous stretch and red away from this stretch i . e ., displacement of red which indicates a break between orange and red , where the abl gene on chromosome 9 resides . the non - separated color sequence in the same well represents the normal chromosome 9 . similarly , the color band pattern on chromosome 22 would be green separated from the light blue , amber , light blue , and yellow stretch indicating a break between green and amber . the bcr gene on chromosome 22 lies between the two color bands green and amber . again , the non - separated color pattern of green , light blue , amber , light blue and yellow indicates a normal chromosome 22 . since chromosome 22 centromere cross hybridizes with chromosome 14 , two additional separate green signals will be present . chromosome 14 hybridization wells will also have 2 extra green or light blue dots . if this were the only genetic change in that specimen , as one proceeds to read the icp slides , all wells containing chromosome 22 would exhibit the above pattern , the well containing chromosome 9 would have the above color pattern and all other chromosomes will have two stretches of contiguous color band pattern . depending on the sex of the patient , either xx or xy , chromosome color pattern would be read in the well containing the x and y chromosome probes . as can be seen from this example , prior knowledge of the disease status is unnecessary for using the icp method . in an embodiment according to aspects of the present invention , in a simple translocation like the above example of a 9 : 22 translocation , confirmation of the translocation can be accomplished by visual means . each of the individual different color bands in the genome for all human chromosomes can be available in the kit as a red or green band . as previously indicated , there can be slots or wells left empty intentionally in the hybridization chamber for optional and / or confirmatory tests . thus , selecting one of the non - centromere or non - telomere bands on either side of the breakpoint on chromosome 9 and chromosome 22 and introducing them into the hybridization chamber , any interphase cells harboring the translocation would have a color pattern of red , red / green , green . the juxtaposition of red and green would indicate the translocation . cells not harboring the translocation in this color scheme would have a reading of red , red , green and green . since centromere bands are not introduced , the cross - hybridization for chromosome 22 is therefore eliminated . by way of example , this color scheme can be used to verify virtually any simple translocation , regardless of the chromosome band involved . even complex translocations involving more than two chromosomes can be detected and diagnosed by systematic additional hybridizations using the above described method . marker chromosomes , by definition in clinical cytogenetics , indicate that by standard g - banding technique , the origin of the centromere and the additional material on that chromosome could not be identified . yet these marker chromosomes play an important role in the disease generation and / or progression . using metaphase chromosomes and the 24 - color fish techniques , one can generally identify the nature of the marker chromosomes . however , identification in the interphase nucleus is very challenging and using current methodologies , cannot be performed . an example of a marker chromosome is described as follows : having the centromere of chromosome 3 ; all of the short arm of 3 ; two bands from chromosome 7 long arm ; and a long arm telomere from chromosome 10 . this is a very complex marker and in cancer cytogenetics , especially solid tumor cytogenetics , one encounters this type of situation routinely . by utilizing the techniques of the present invention as described herein , in the icp model , hybridization is completed utilizing a three slide technology , the following results will be read : slide 1 unremarkable ( i . e ., normal ); slide 2 , 1 - 2 , 4 - 6 , 8 - 9 ( i . e ., normal ); slide 3 unremarkable ( i . e ., normal ). on slide 2 , in the hybridization well for chromosome 3 , an abnormal color band pattern would be read . in addition to the two normal contiguous color bands , an extra centromere and short arm bands will likely be present . similarly in the chromosome 7 well in addition to the two contiguous color bands , two extra , adjacent color bands will be present . finally , in the chromosome 10 well , depending on whether chromosome 10 long arm telomere composition is balanced or not , one could observe either two contiguous color bands , and an extra telomere separate band or one contiguous stretch of color bands and the displacement of the telomere band from the second color band sequence . fig6 depicts the marker chromosome in three different interphase cells . according to aspects of the present invention , in the icp model , the marker chromosomes can be “ reconstructed ”. taking the centric fragment from 3 , and the acentric fragments from 7 and 10 and hybridizing all together in an interphase cell , one can confirm whether all of these fragments formed the “ suspected ” marker . if a contiguous color band sequence was found , the results are likely positive . in addition , the corresponding bands on the normal chromosomes 3 , 7 and 10 will also likely highlight , thereby confirming the nature of the marker chromosome . fig7 depicts the “ reconstructed marker ” with the remaining color bands as discussed herein . table a represents the acrocentric chromosome combination on slide 1 according to aspects of the present invention . depending on whether the chromosome was introduced first or second , the centromere would have a light blue or green color with the remaining color sequence as depicted on the ideograms . color sequence to the left of the chromosome on the ideogram represents first and the sequence on the right represents second in the combination . for chromosomes 13 and 22 , only one color is represented because they are always introduced only either first or second . since chromosome 13 and 21 centromeres can cross hybridize , in well # 3 , all centromeres will be cyan ( light blue + green ). this holds true regardless of whether there is a translocation between these two chromosomes . when there is a translocation , in the balanced state there will be one less centromere ( i . e ., 3 in stead of 4 ) in interphase cells in this well , whereas , in the unbalanced state , the number remains 4 . the same general rule applies to chromosomes 14 and 22 as well . therefore , interphase cells in well # 7 , will act similarly to chromosomes 13 and 21 . both in the balanced and unbalanced state , there will likely be a larger contiguous stretch of colors from both chromosomes absent the centromere , representing the presence of a robertsonian translocation . in all of the wells except for # 3 and # 7 , 2 or more extra centromere bands representing the cross hybridization , will likely be present . this design ensures multiple opportunities to cross reference the genetic abnormality . according to aspects of the present invention , icp is amenable to the investigation of all types of specimen types , such as , for example peripheral blood , bone marrow aspirate , amniotic fluid , chorionic villi , pleural effusion , lymph node biopsy , solid tumor mass , products of conception etc . of these specimen types , only the “ liquid ” specimens such as blood , bone marrow , amniotic fluid , and pleural effusion contain single cells . the remainder of the specimens are tissues comprising aggregates of cells connected together . in order to perform icp , one needs single interphase cells . thus , according to another aspect of the present invention , a method can be devised to precisely add or plate 25 - 30 cells in each well on the hybridization chamber slides . current methodologies exist to dissociate solid tissues into single cell suspensions , such as those utilizing enzymes like collegenase and trypsin . as discussed supra , liquid specimens generally do not require this type of treatment to obtain single cells . once a single cell suspension is obtained from any specimen type , the concentration of the cells is adjusted such that each “ pipette drop ” contains 25 - 30 cells . for example ; if ml of specimen contains 1000 cells and each ml contains 10 pipette drops , each drop then contains 100 cells . since each well will generally receive only one drop for icp , the specimen is appropriately diluted , in this case , 3 . 5 - 4 times so that each drop can contain the desired cell concentration . if the original volume is 10 cells per pipette drop , then the specimen is concentrated 2 . 5 - 3 times by spinning down and removing excess volume , so that each pipette drop can contain the desired cell concentration . this technique ensures that , even “ compromised ” specimens , specimens having small volumes , can be analyzed completely under the present invention , whereas other methods in the art might have failed to capture complete karyotype information . most of the fish methodologies currently only use smears prepared from the specimen with no hypotonic treatment . the discovery of hypotonic treatments such as kcl and na citrate solutions , gave birth to the modern cytogenetics field . the proper “ swelling ” of metaphase cell ensures the separation of metaphase chromosomes when they are dropped onto microscope slides . similarly , proper separation of interphase chromosomes is crucial for the successful delineation of a multi - color band sequence . by experimenting with different hypotonic solutions at different concentrations , either singly or in combination , it is likely possible to properly “ swell ” the interphase , before it is fixed onto the microscope slide in the hybridization well . 1 ) on each chromosome , the location of the dna probe hybridization is depicted on the standard g - banded ideograms . as previously disclosed , chromosome 1 can have 19 dna probes : 1 pericentromere , 4 short arm mixed colored + 5 short arm repeating color blue + 1 short arm telomere , 3 long arm colored + 4 long arm repeating color blue + 1 long arm telomere . 2 ) each chromosome band can be , for example , micro dissected or isolated using other techniques known in the art from the standard g - banded metaphase chromosome preparations . 3 ) each micro dissected chromosome band can be amplified by dop - pcr technique as generally described by telenius ( 1992 ). 4 ) repeat sequences from these probes can be removed using biotin - labelled cot - 1 dna , followed by avidin magnetic beads , as generally described by craig ( 1997 ) 5 ) repeat - free probes from the first dop - pcr cycle can be subjected to further rounds of amplification using the methodology described by liehr ( 2002 ) 6 ) using standard sonication techniques , the probe length can be adjusted to between about 200 bp - 600 bp . alternatively , smaller fragments of the probe can be created utilizing standard techniques as a kit , such as vector labs nickit kit . 7 ) the dna fragments can be labeled with fluorochromes such as for example cascade blue , alexa fluor 488 ( green ), alexa fluor 546 ( orange ), alexa fluor 647 ( far red ) using standard nick translation . 9 ) the nick translation technology for example , can incorporate the labels at multiple sites throughout the dna strand to achieve a higher sensitivity at the site of hybridization . 10 ) dna probes from the 24 human chromosomes are then ready for in situ hybridization to the interphase cells on the hybridization chamber slides . 11 ) centromere and telomere dna probes can be created or commercially available probes can be utilized . the probes can be obtained for the icp purpose without the need for a label and as described above , the appropriate label can be incorporated into the dna . 1 ) the single cell suspension can be plated onto each well on three hybridization chamber slides . the suspension is treated with hypotonic solution and fixed with a 3 : 1 methanol : acetic acid solution . 2 ) interphase cells in each well can be hybridized with a single set of dna probes specific for that well , using standard in situ hybridization techniques , such as , with the pretreatment of enzymes to allow the dna probes to penetrate the nuclear membrane , wherein dna denaturation is performed to separate the dna strands . 4 ) post hybridization washes can be done to remove excess , unhybridized probes from the slides . 7 ) all colors , both primary as well as mixed , will be detected through simple fluorescent microscope using appropriate excitation and emission filters . 8 ) a color ccd camera can be used to digitize the true colors as seen through the microscope for analysis on the computer screen . no special software manipulation of colors is needed . as previously disclosed , the acrocentric chromosome centromeres can be color stained with colors , such as , for example , light blue or green . similar accommodations can be made for certain bands on chromosome 10 , 12 and y as well as acrocentric long arm bands to ensure proper identification of all chromosome rearrangements . 8 ) the slides can be counter - stained with dapi which gives good contrast to all the primary and mixed colors . 9 ) based on the predetermined proportion of colors at different bands along the length of the individual chromosomes , the admixture of primary colors generates a new detectable mixed differential color . an example of the color scheme for all bands on all human chromosomes is depicted in fig1 . 1 ) upon color development of individual chromosome bands , the interphase chromosomes can be observed by using a simple , standard fluorescent microscope with appropriate filters for excitation and emission . ccd cameras can be used as a visual aid , however , are not necessary . 2 ) starting with slide 1 the color pattern in each interphase cell can be recorded . based on the expected normal color pattern from the ideograms , one can determine the normal or abnormal status of the respective acrocentric chromosomes . in general one could score 20 interphase cells from each well . 3 ) repeating the same analysis for the remainder of the chromosomes on slide 2 and 3 , i . e ., recording the normal and abnormal status of each chromosome using 20 interphase cells , one can combine the results to get a complete karyotype profile of the individual tested . 4 ) if additional analysis is indicated , for example , suspicion of mosaicism — normal and abnormal cells , clarification of simple or complex translocations , marker chromosomes or the like , the empty wells on a , such as , slide # 3 can be used for confirmatory experiments . if needed , an entire extra slide can be used depending on the design of the confirmatory experiment . 5 ) the banding pattern of any chromosome can be documented by simple digital photography . 6 ) simple mosaicism can be easily detected on microscope analysis . even complex , apparently related clonal mosaicism can be clarified . the following example is illustrative of the clarification procedure according to aspects of the present invention . as an example , a karyotype with the following results is analyzed : + 8 / t ( 9 ; 22 )/ t ( 9 ; 22 ),+ 8 / normal . in this case of chronic granulocytic leukemia there are two clonal abnormalities : the t ( 9 ; 22 ) as discussed supra the characteristic change for this disease . trisomy 8 (+ 8 ) signifies advanced disease progression . however , trisomy 8 can exist alone as an “ unrelated ” clone . thus , establishing whether the same clone has both the t ( 9 ; 22 ) and + 8 is clinically very important . by way of example , on the first icp routine reaction , there were 10 cells with t ( 9 ; 22 ); 5 cells with + 8 , cells normal for chromosome 8 and 10 cells normal for chromosomes 9 and 22 . as explained everywhere , the clarification of the translocation can be done by the red : blue experiment , where the red : green juxtaposition indicated a translocation and separate red and green indicate no translocation . combining that result with the probe for the chromosome 8 centromere ( blue ), the results are likely as follows : there were likely 5 cells with t ( 9 ; 22 ); 5 cells with t ( 9 ; 22 ) and + 8 ; 10 cells normal for chromosomes 8 , 9 and 22 . these results would clearly indicate that + 8 was part of the disease progression , not an isolated event . if it were an isolated event , no cells with the combination result will be observed . referring now to fig1 , a hybridization slide according to aspects of the present invention is illustrated and generally referred to by the reference numeral 10 . the slide 10 can comprise a body 20 , a plurality of wells 22 and an identification label area 24 . the body 20 can comprise glass or any other translucent material sufficient for performing hybridization thereon . the wells 22 can comprise areas printed for separating the wells 22 thereon . the wells can also comprise ground out indentations , molded indentations or the like for receiving and hybridizing a sample . the identification label area 24 can comprise a clear area or frosted area for receiving a label or other identification means . the following tables provide a quick reference to the user to compare results obtained utilizing the techniques disclosed herein to techniques utilizing the gold standard g - banding . the tables below illustrate the interphase chromosome profile ( icp ) banding as compared to the standard g - banding methods . for ease of reference , the following abbreviations are provided : q = long arm ; p = short arm ; p = proximal to centromere ; q = distal to centromere ; ter = telomere ; ma = major band ; and mi = minor band . inasmuch as the preceding disclosure presents the best mode devised by the invention for practicing the invention and is intended to enable one skilled in the pertinent art to carry it out , it is apparent that methods incorporating modifications and variations will be obvious to those skilled in the art . as such , it should not be construed to be limited thereby but should include such aforementioned obvious variations and be limited only by the spirit and scope of the following claims . babu v r and wiktor a . a fluorescence in situ hybridization technique for retrospective cytogenetic analysis . 1991 , 57 : 16 - 17 . berend sa , horwitz j , mccaskill c , shaffer l g . identification of uniparental disomy following prenatal detection of robertsonian translocations and isochromosomes . am j hum genet . 2000 , 66 ( 6 ): 1787 - 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