1. Interpretation of conventional karyotyping & ISCN Nomenclature in cytogenetics
Dr Mayur Parihar
Consultant Hematopathology and Cytogenetics
Tata Medical Center Kolkata
MBBS,MD (Path),FCCG(CMC Vellore)

2. DNA in any chromosome (50 to 250 million bases),
highly coiled and condensed
DNA from a single cell if stretched - 2m
DNA sequence for a single protein is a gene.
Each chromosome contains a few thousand genes,
Few 100 to ~2 million bases.

3. Cytogenetics
Cytogenetic analysis is an in vitro clinical laboratory procedure that evaluates the chromosomes of a cell.
Certain clinical characteristics occur consistently in association with a particular chromosome abnormality.
This phenotype-karyotype correlation is useful.

4. Methods for Identifying Chromosome Aberrations
Detected using cytogenetic and molecular methods
chromosome banding
molecular cytogenetics
Loss of heterozygosity
Microarrays
These analyses identify a broad range of chromosomal abnormalities in solid tumours including:
altered ploidy
gain or loss of individual chromosomes, deletions, amplications
structural rearrangements
Molecular techniques are considered high through put
RLGS, RDA detect allelic balances that occur by somatic recombination or copy number change
FISH/CGH - are sensitive to physical changes in genome structure or copy number

5. Molecular Cytogenetics
Powerful adjunct to conventional cytogenetic analysis
Utilizes metaphases and non-mitotic interphase nuclei
Can be applied to fixed archived tumour material
Accurate, specific

6. Molecular CytogeneticTechniques
FISH
Multi-Colour FISH (M-FISH)
Spectral Karyotyping (SKY)
Comparative Genomic Hybridization (CGH)
Microarrays

7. Cytogenetics Karyotyping All chromosomes screened Clonal Evolution
Locates areas that might contain critical genes involved in tumourigenesis
FISH
Specific part of a gene or chromosome.
Does not screen all the chromosomes for abnormalities

8. Conventional Cytogenetics
In vitro clinical laboratory procedure that evaluates the chromosomes of a cell.
Chromosomes are individually distinguishable under light microscopy only during cell division
Spontaneously proliferating cells : bone marrow, lymph nodes, solid tumors and chorionic villi.
Cultured : PB lymphocytes, tissue biopsies

9. Specimen collection and handling
Bone Marrow Aspirates
Preservative free sodium heparin
Transported at room temperature
First few millilitres of the bone marrow tap contain the highest proportion of cells
Processed without delay upon receipt to avoid cell death.

10. GTG-banded karyotype: work flow
Ann Lab Med 2014;34:

11. Protocols have to be followed!!!

13. What do you mean by bands/banding
A chromosome band is a part of a chromosome that can be distinguished from adjacent segments by appearing darker or lighter by one or more techniques.
Each chromosome has a unique pattern of light and dark bands

14. Slide Making Cell pellet dropped
Chromosome spreading depends upon temperature, relative humidity, drying time
Has to be standardized each time

17. Capturing Bone marrow : correlate with BM diagnosis, IPT
Slides are screened, looking at all metaphases
Microscopy chromosome count done of metaphases
Look out for specific abnormalities
20 metaphases are captured and analysed

18. Hyperdiploid
Normal

19. NORMAL HUMAN KARYOTYPE

20. Terminology Parts of the chromosome

22. ISCN International System for Human Cytogenetic Nomenclature
Each area of chromosome given number
Lowest number closest (proximal) to centromere
Highest number at tips (distal) to centromere

23. In designating a particular band, 4 items are required
1.The chromosome number
2.The arm symbol
3.The region number and
4.The band number within that number.
Ex: 1p31 indicates chromosome 1, short arm region3,band 1.
According to ISCN the banding levels are varied by 400,550 and 880 levels with help of banding techniques

24. Idiograms

25. ISCN
Normal male
46,XY[20]
Normal female
46,XX[10]

26. Numerical Aneuploidy Polyploidy Autosomal trisomy, 47
Sex chromosomes, 45, 47, 48, 49
Polyploidy
Whole chromosome set
Normal Human genome is diploid that is 46 chromosomes (23x2)
Triploidy, 69 (23x3)
Tetraploidy, 92 (23x4)

27. Types of chromosome abnormalities
Numerical
Aneuploidy (monosomy, trisomy, tetrasomy)
Polyploidy (triploidy, tetraploidy)
Structural
Translocations
Inversions
Insertions
Deletions
Rings
Isochromosomes
ESAC

28. A 2 year old girl diagnosed as Acute Leukemia 1 month back was referred to TMC for further management.

29. Fever
No history of any treatment
Hb :10.4,TLC:10,400,Platelets:2.5 lakhs
No immature cells/blasts in the peripheral smear
Bone marrow examination : no evidence of leukemia

31. 55,XX,+X,+6,+8,+10,+14,+17,+18,+22,+22 [1]

32. CLONES
Clone is defined as a cell population derived from a progenitor.
The number of cells that constitute a clone is given in square brackets [ ] after the karyotype.
Ex: 46,XX,t(8;21)(q22;q22)[20].

33. Clone Two cells/metaphases for trisomy
Three cells/metaphases for monosomy
Two cells/metaphases for a structural abnormality
The number of cells that constitute a clone is given in square brackets [ ] after the karyotype.

34. FISH using ETV6/RUNX1 ES Probe
ETV6, Ch 12:
RUNX1,Ch21:

35. Precursor B cell ALL
Steroids the blasts dissapear

36. 45,XX,-7 Clonal ??? How many metaphases have -7?
Specimen type : Heparinized Bone Marrow Banding Resolution : 450 bphs Cytogenetic Profile Metaphases Counted : 20 Metaphases Analyzed : 20 Metaphases Karyotyped : 8 Total Chromosome Number : 45/46 Autosomes : 44/45 Sex Chromosomes : 2(XY)

37. 45,XX,-7[7]/46,XX[13]
Specimen type : Heparinized Bone Marrow Banding Resolution : 450 bphs Cytogenetic Profile Metaphases Counted : 20 Metaphases Analyzed : 20 Metaphases Karyotyped : 8 Total Chromosome Number : 45/46 Autosomes : 44/45 Sex Chromosomes : 2(XY)

38. 45,XX,-7[1]/46,XX[19] Clonality not established .
The lab should record its observation saying that monosomy 7 seen in a single metaphase/However, the clonality cannot be established
Next step to do FISH for monosomy 7 to screen more number of cells.

39. ISCN del - deletion dic - dicentric der - derivative
fra - fragile site
i - isochromosome
inv - inversion
p - short arm
r - ring
der - derivative
dup - duplication
h - heterochromatin
ins - insertion
mat - maternal origin
q - long arm
t - translocation

40. Structural Breakage in at least 1 chromosome Translocations Inversions
2 different chromosomes break and rejoin incorrectly
Inversions
2 breaks in same chromosome
Insertions
Piece of chromosome inserted
Deletions
Piece of chromosome missing

41. Translocations
Exchange of chromosome material between two chromosomes.
Balanced : No gain or loss of genomic material
Unbalanced : loss or gain of genomic material.
Denoted by t(8;21), the smaller chromosome always written first.
In case of unbalanced translocation the word derivative is used.

42. 46,XX,t(8;21)(q22;q22)[18]/46,XX[2]

43. Balanced chromosome 1 and chromosome 19 translocation
46,XY,t(1;19)(q23;p13),-9,i(9)(q10),+mar[15]/46,XY[5]

44. Unbalanced translocation chromosome 1 and chromosome 19
46,XY,t(9;22)(q34;q11.2),der(19)t(1;19)(q23;p13)[9]

45. 46,XY,+1,der(1;7)(q10;p10)[14]/46,XY[6]
Loss of 7q and partial trisomy for 1q

46. A patient of CML on Imatinib not responding
46,XY,t(9;22)(q34;q11.2),+19,+der(22)t(9;22)[12]

47. 46,XY,t(8;17;21)(q22;q25;q22),del(9)(q13q33)[4]

48. FISH showing 1F2R2G Inverted DAPI
Metaphase FISH using dual colour dual fusion RUNX1/RUNX1T1 probe
Inverted DAPI

49. Inversions Reversal of segment of chromosome Pericentric Paracentric
If too small cannot detect by karyotype
Very rare in humans
Selected against as would get reduced fertility
Pericentric
reversed segment includes centromere
Paracentric
within one chromosome arm
Paracentric inversion
main difference in karyotypes of great apes and humans so important in evolution

50. Inversion Reversal of segment of chromosome Pericentric Paracentric
If too small cannot detect by karyotype
Pericentric
reversed segment includes centromere
Paracentric
within one chromosome arm
main difference in karyotypes of great apes and humans so important in evolution

51. 47,XX,inv(16)(p13q22)

52. 46,XY,inv(3)(q21q26)[17]

53. Insertions
Segment of 1 chromosome inserted into another
A derA der B

54. Deletions Terminal Interstitial All result in unbalanced karyotype
loss of end of chromosome
46,XY,del(20)(q26) missing long arm of 10
Interstitial
loss of segment from within chromosome
46,XY,del(10)(q24q26) missing segment of 10
All result in unbalanced karyotype
Partial monosomy
Serious clinical effect

55. A patient of MDS
46,XY,del(20)(q11.2)[18]

56. A patient of MDS
46,XY,del(5)(q13q33)[16]

57. 46,XY,t(4;7)(q22;p22),del(7)(q21q33),dic(9;12)(p12;p11)[14]

58. Isochromosome Two copies of the same arm
Mirror image around centromere
Centromeres part in wrong plane
Monosomy for 1 chromosome arm
Trisomy for the other arm

59. A patient of ALL.Isochromosome 9q loss of 9p that houses the PAX5 gene and CDKN

60. A patient of CML in blast crisis
A patient of CML in blast crisis.Isochromosome 17q loss of 17p that houses the Tp53 gene

61. 46,XY,dup(1)(q21q32),t(-;-)

62. Questionable Identification 46,XY,?t(11;19)(q23;p13)
Metaphase FISH using MLL break apart

63. Clones and subclones A patient of CML ?blast crisis
46,XX,t(9;22)(q34;q11.2)[9]/46,idem,inv(16)(p13q22)[8]/46,XX[3]

64. Clones and subclones A patient of CML ?blast crisis
46,XY,t(9;22)(q34;q11.2)[6]/47,idem,+8[14]

65. Clones and subclones A patient of AML
46,XX,t(8;21)(q22;q22)[8]/45,X,-X,t(8;21)(q22;q22)t(17;17)(q11.2q25)[12]

66. A patient of CML on follow up not responding
46,XY,t(9;22)(q34;q11.2)[14]/47,XY,+8[2]/46,XY[4]

67. Complex Karyotype
Complex karyotype is defined as presence of three or more cytogenetic abnormalities in bone marrow not including inv(16), t(16;16), t(8;21), and t(15;17) by most of the groups
Slovak ML: German Acute Myeloid Leukemia Intergroup,Byrd JC: CALGB, Schlenk RF: SWOG;
The MRC multicenter trial defined complex karyotype as presence of 5 or more chromosomal aberrations.
Grimwade D: MRC

68. The monosomal karyotype was defined by the presence of two autosomal monosmies or one single monosomy (excluding isolated loss of X or Y) in association with a structural chromosome abnormality.

70. Patient with AML
45,XY,inv(3)(q21q26),-7[18]/46,XY[2]

71. Patient with AML
45,XX,-7,t(11;17)(q23;q12)[14]/46,XX[6]

72. 55~ 57,XX,+X,+6,+8,+10,+14,+17,+18,+22,+22 [CP10]/46,XX[10]

73. Composite Karyotype Multiple clones and subclones may be present
Karyotypic heterogeneity
Contains all clonally occurring abnormalities
Gives range of modal chromosomal number

74. 56,XXYC,+X,+4,+5,+6,+8,t(9;22)(q34;q11.2),+12,+14,+18,+21,+der(21)t(9;22)[CP14] /46,XXY[6]

75. A patient of AML with aberrant CD19
46,XY[20] nuc.ish (RUNX1x3)(RUNX1T1x3)(RUNX1conRUNX1T1x2)[190/200]

76. Karyotyping results Post Transplant
46,XY,der(11)dup(11)(q14q22)t(11;22)(q24;q11.2)[6]//46,XX[14]

77. Conclude Look for clonality of the abnormalities
In case clonality not established used other complimentary tests like FISH.
Correlate the karyotype findings with clinical picture,morphology, IPT findings, other test reports.
Communication with other members of the team essential across specialities.

78. The Cytogenetics team at the Tata Medical Center

79. THANK YOU