Chromosome analysis Flashcards
The criteria used for the selection of metaphase spreads suitable for analysis includes all
the following EXCEPT FOR:
Presence of an intact cell membrane(+)
Chromosome band level (length)
The quality of G-banding
The number of overlapped chromosomes
When reviewing a final karyotype a possible overlapped chromosome is suspected. In order to confirm the chromosome number of this cell, all the following information is needed EXCEPT FOR:
Patients clinical history(+) Identification number of the patient Microscope used to photograph the cell being analyzed The coordinates of the metaphase
What is the minimum number of cells that must be counted for in situ amnio analysis?
Ten cells from two coverslips Ten cells from each of four cultures Fifteen cells from one culture One cell each from fifteen different colonies from at least two coverslips(+)
Which of the following is TRUE when the banding level of chromosomes is 550-600?
There are two dark bands on chromosome 7q.
There are two dark bands on chromosome 11p.
There are two dark bands on chromosome 9q.
The first dark band on chromosome 10q21 is split into
two different bands.(+)
When an abnormal cell is detected in an in situ culture all the following should be
considered to rule out pseudomosaicism EXCEPT FOR:
Confirm abnormality in other cells in the colony.
Confirm abnormality in another culture.
Count 20 colonies from one coverslip.(+)
Confirm abnormality in other colonies on the same
coverslip.
All of the following will result in erroneous chromosome counts EXCEPT:
Nomadic intruders
Overspreading
Debris
Undertrypsinization(+)
A Cri-du-chat patient has a chromosome abnormality of
t(15;2)(q11;q21)
t(5;2)(p13;p12)
t(5;10)(q13;p22)
r(5)(p15.1q35)(+)
The peripheral blood culture of a patient suspected of having CML is 46,XY. In order to rule out the presence of the t(9;22) the following tissue would be cultured next:
Skin
Bone Marrow(+)
Another peripheral blood specimen
Peripheral blood from a family member
What is it called when two different cell lines within an organism seem to come from
different sources?
Contamination
Mosaicism
Chimerism(+)
Pseudomosaicism
Which of the following is the correct ISCN nomenclature for a male with fragile X
syndrome?
46,fra(X)(q27.3),Y
46,Y,fra(X)(q27.3)(+)
46,XY,fra(X)(q27.3)
46,X,fra(X)(q27.3)
46,XY,der(9)t(9;10)(q32;q12). Which of the following terms best describes this written karyotype?
Balanced
Unbalanced(+)
Robertsonian translocation
Reciprocal
Twenty-nine out of 30 metaphases are normal in the amniotic fluid culture of a pregnant
woman with a history of multiple miscarriages. One cell has a translocation. What does this
signify?
leukemia
cultural artifact(+)
Robertsonian translocation
derivative
Deletion (15)(q11 q13) is associated with which of the following?
Prader-Willi Syndrome(+)
Williams Syndrome
DiGeorge Syndrome
Cri-du-Chat Syndrome
I. Select Suitable Metaphases For Analysis
1.Chromosome morphology
Metaphases that are suitable for analysis:
a) Have relatively few overlaps or the overlaps do not obscure the chromosome bands.
b) Are free from cytoplasm.
c) Are banded to the degree that dark vs. light bands can be discerned.
I. Select Suitable Metaphases For Analysis
2.Suitable banding level
Chromosome length is dependent on a number of variables:
a) Tissue type
b) Colcemid exposure time
c) Stage of mitosis (e.g. Prophase, Mid-, or Late-Metaphase)
Band level refers to the number of bands that occur along the length of the chromosomes in a haploid set. Typical metaphase chromosomes, which are analyzed in clinical cytogenetic laboratories, will range from a band length of 250 to 850. When chromosomes are either longer or shorter than these band levels they become difficult to analyze.
To determine the banding level of chromosomes one should follow the following criteria (Josifek et al, 1991):
a) Determine the number of bands from several chromosomes.
b) Determine the number of bands from an entire chromosome or chromosome arm.
c) Count only the dark G-bands not including the centromere bands.
Figure 1 illustrates the difference in the number of bands on the short arm of chromosome 11’s at different band levels.
I. Select Suitable Metaphases For Analysis
- Suitable number of metaphases to analyze
a) Constitutional analysis
The majority of clinical cytogenetics laboratories will count 20 cells per specimen. This will rule out a mosaicism of 14% at a 95% confidence level (Hook, 1977). If one abnormal cell is detected, the number of cells analyzed should be increased to 30. This will rule out a mosaicism of 10% at a 95% confidence level. Amniocentesis coverslip cultures are the exception. In these specimens, 15 colonies from at least two coverslip cultures are counted.
Mosaicism implies that there is more than one cell line in a sample. Mosaicism is possible when an error in division or a mutation takes place during the early stages of embryonic development. Mosaicism should not be confused with Pseudomosaicism.
I. Select Suitable Metaphases For Analysis
- Suitable number of metaphases to analyze
b) Analysis of acquired abnormalities
In the analysis of leukemic bone marrow or solid tumor specimens there are often normal contaminating cells found with the neoplastic cells. Again most clinical laboratories will count 20 to 30 cells. However, in this type of analysis the cytogeneticist is analyzing the chromosomes to find the malignant cell population, rather than to rule out a constitutional chromosome abnormality.
There will be instances in the analysis of acquired abnormalities when a laboratory may choose to study more than 20 to 30 cells. For example, in studying predisposition to cancer, analysis of 50 to 100 cells is required in order to pick up rare acquired rearrangements, which may be observed in the peripheral blood. In addition, for breakage studies, 50 cells often must be analyzed to determine an accurate reading that can be compared to historical controls.
I. Select Suitable Metaphases For Analysis
-Prenatal
Typically, 15 colonies from a minimum of two coverslips are counted or analyzed.
In the analysis of in situ cultures it is important to analyze cells from a number of different colonies, coverslips and cultures. This will help rule out maternal cell contamination and pseudomosaicism. A suspected mosaicism in a prenatal sample may necessitate the analysis of 50 cells.
I. Select Suitable Metaphases For Analysis
-Oncology
Typically, 20 cells are analyzed.
In the analysis of bone marrow cultures it is important to analyze cells grown under different culture conditions. For example, most myeloid leukemias are grown in both 24 and 48 hour cultures if sufficient sample is available. Conversely, most lymphocytic leukemias and lymphomas are grown in both 24 and 72 hour stimulated cultures whenever possible. 72 hour PHA stimulated cultures are set-up and analyzed whenever T-cell disease is suspected and 72 hour LPS stimulated cultures are set-up and analyzed whenever B-cell disease is suspected.
II. Count and Analyze metaphase spreads in a Systematic Manner
1.Document modal number
The modal number is the most common chromosome count recorded from a given cytogenetic preparation.
The range is also a term referring to the number of chromosomes observed in all the cells of a population
II. Count and Analyze metaphase spreads in a Systematic Manner
2.Document karyotype
It is necessary to document pertinent information about each case studied for future references. This includes the location of the metaphase and scope, the technologist’s name, the date of analysis, and the Polaroid, negative or computer filename. All this information together with the written karyotype, prepared according to the ISCN guidelines, should be recorded within the patient report (An overview of ISCN is included in pages 48-53 of this module.
Each karyotype should be labeled with the patient name, lab number, date sample was received, and the negative reference number or computer filename.
II. Count and Analyze metaphase spreads in a Systematic Manner
3.Document slide location
Located on the stage of most compound microscopes are two vernier scales, one for the x-axis and another for the y-axis coordinates of the stage. These vernier scales can be read to the tenth degree and are highly accurate for the slide location of metaphase spreads.
II. Count and Analyze metaphase spreads in a Systematic Manner
4.Document analysis of separate colonies for in situ cultures
Amniotic fluid cultures are often grown in situ. In this type of culture system, cells are plated onto sterile coverslips and placed in petri dishes at 37oC. These cells are then allowed to attach and form colonies. When the appropriate cell density has been achieved, the coverslips are then exposed to Colcemid, hypotonic solution, Carnoy’s fixative, banding solutions and are finally analyzed under the microscope without the removal of the cells from these coverslips.
When counting and analyzing the 15 to 30 cells of an in situ culture, one should select and document cells from as many different colonies as possible. In addition, metaphase plates should be analyzed from different cultures. If an abnormality is detected, other metaphase plates from the same colony should be examined to confirm that all cells have the same abnormality within the colony. All of these precautions, if well documented, will allow interpretation of any abnormalities detected and are the most effective measure for ruling out maternal cell contamination and pseudomosaicism.
The four main types of mosaicism:
Level I Mosaicism - Called single cell mosaicism occurs when only one colony shows metaphases with an abnormal karyotype.
Level II Mosaicism - Called Pseudomosaicism occurs when an abnormal karyotype is limited to one coverslip culture.
Confined Placental Mosaicism (CPM) - This type of mosaicism occurs when an abnormal karyotype is found only in the extraembryonic membranes of the fetus.
Level III Mosaicism - Called True mosaicism occurs when two or more colonies with the same abnormal karyotype are observed in two or more coverslip cultures.
III.Prepare accurate karyotypes from photographic prints or computer images
1. Select good quality prints or images
Photographic plates, which are to be used for karyotyping, should be of sufficient quality to allow identification of each and every chromosome. Ideally, the bands should be sharp and clear showing a clear distinction between dark and light regions along the chromosomes and there should be few overlaps.
III.Prepare accurate karyotypes from photographic prints or computer images
2.Arrange chromosomes using approved format
Autosomes are numbered from 1 to 22 according to decreasing overall length. Chromosome number 1 is the longest chromosome of the autosomes and chromosome numbers 21 and 22 are the shortest chromosomes of the autosomes (The one exception to this rule is chromosome 22 which is larger than chromosome 21.). The sex chromosomes are designated X and Y.
The human chromosomes are also grouped into groups designated by the letters of the alphabet A - G. This grouping is based on size and centromeric position.
Location of the centromere is one of the important factors in determining the identification of a particular chromosome. It is the centromere which is the landmark used to divide the chromosome into the short arm (p) and the long arm(q). Based on the proportion of the short arm to the long arm, a chromosome is described as being one of the following:
a) Metacentric - Centromere is in the middle of the chromosome and the short and long arms of the chromosome are approximately equal in length.
b) Submetacentric - Centromere is located away from the middle of a chromosome and the p arm of the chromosome is shorter than the q arm.
c) Acrocentric - Centromere is located very close to the terminal end of the chromosome and often is characterized by a secondary constriction separating the short arm from the satellite of the chromosome.
The short arm (p) of the chromosome is always oriented in the up position in the correct karyotype format.
In addition, band designation is determined by distance from the centromere. For example, bands p11 and q11 are located closer to the centromere than bands p21 and q21.
III.Prepare accurate karyotypes from photographic prints or computer images
3.Provide permanent copy of final karyotype
A permanent copy of the final karyotype should be provided with each cytogenetic report. This permanent copy does not have to be the original karyotype itself, but can be a photographic copy of the original.
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
1.Aneuploidy (numerical abnormalities)
Polyploidy - Abnormalities of the number of haploid sets in a cell. There are 23 chromosomes per haploid set (n). Two copies of the haploid set (2n) is characteristic of normal diploid cells.
Triploidy (3n) - These cells have three copies of the haploid set. Fetuses with this constitutional abnormality are often nonviable or do not live long after birth. The written karyotype of these conceptions is either 69,XXX or 69,XXY.
Two-thirds of all triploid conceptions are the result of double fertilization of the ovum by two sperm. (Remember that normally all gametes, ovum and sperm, are haploid.)
One-third of triploid conceptions arise due to fertilization of a diploid ovum by a haploid sperm.
Tetraploidy (4n) - These cells have four copies of the haploid set. Fetuses with this constitutional abnormality are also nonviable or do not live long after birth. The written karyotype of these conceptions is either 92,XXXX or 92,XXYY.
This type of abnormal conception arises due to the failure of the first cleavage division in the zygote.
Care must be taken in analyzing fetal material, which is positive for tetraploidy mosaicism. Tetraploidy is a common artifact of tissue culture and may be found in the cultures of normal fetuses.
AneuploidyTerms - This is the gain or loss of a given chromosome.
Monosomy - One copy of a given chromosome.
Disomy - Two copies of a given chromosome. This is the normal situation.
Trisomy - 3 copies of a given chromosome.
Tetrasomy - 4 copies of a given chromosome.
Mechanisms of Aneuploidy
Nondisjunction - Nondisjunction is the failure of paired chromosomes (Meiosis I) or sister chromatids (Meiosis II or Mitosis) to separate at anaphase. If nondisjunction occurs during Meiosis I or during Meiosis II, this abnormal division can result in a constitutionally abnormal individual. If nondisjunction occurs during Mitosis, this abnormal division will result in a mosaic individual.
Anaphase lag - This mechanism of aneuploidy refers to when a chromosome lags behind as the spindle separates the chromosomes to opposite poles.
Aneuploidy is the most common karyotypic anomaly encountered in phenotypically abnormal live births. It is estimated that 20 percent of all human conceptions are aneuploid.
Constitutional autosomal monosomies are not compatible with life. Constitutional autosomal trisomies are limited to the smaller chromosomes. Clinical examples of these aneuploidies will be given on page 31 of this module.
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
- Structural rearrangements
- Reciprocal Translocations
Chromosome breakage is not a rare event. Daily our cells undergo assault by external and internal clastogens. Each cell has numerous enzymes, which repair broken DNA strands. This repair goes on throughout the life of each cell. Most of the time the DNA is repaired correctly, but occasionally errors will occur. When these rearrangements occur in germ cells, they can give rise to offspring with constitutional chromosomal abnormalities. Constitutional abnormalities are present in all cells of an individual. If these rearrangements are acquired after zygote formation, they give rise to individuals with nonconstitutional chromosomal abnormalities seen in only a proportion of cells.
Chromosomal structural rearrangements can be divided into two groups.
- Interchromosomal rearrangements
Reciprocal Translocations
These are balanced exchanges of genetic material between two or more chromosomes. In other words, no genetic material is lost during the exchange. These rearrangements can occur when two or more chromosomes simultaneously break. Whenever chromosomes break, they have “sticky ends” which can readily unite with another “sticky end.”
Approximately one out of 1,000 individuals is a balanced translocation carrier. Phenotypically these individuals are normal; however they are at risk for having miscarriages or abnormal children depending on the breakpoints and chromosome involved in rearrangement. The actual risk for an abnormal outcome due to abnormal meiotic configurations is 11-12%. Twenty percent of individuals with multiple miscarriages will be balanced translocation carriers.
If alternate segregation of the chromosomes takes place, balanced gametes will be formed. These gametes will contain either the normal copies of the chromosomes or the balanced translocation.
If adjacent segregation of the chromosomes takes place, unbalanced gametes will be formed. In most instances this will lead to fetal demise. The smaller the segment of DNA involved in translocation, the more likely those abnormal fetuses will come to term.
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
- Structural rearrangements
- Robertsonian translocations
These translocations occur when there is a fusion in the centromere region between two acrocentric chromosomes. Individuals with Robertsonian translocations and only one other normal homolog of each of the chromosomes involved are phenotypically normal. They have normal phenotypes because they still have only two copies of all the genes except for the short arms of the two acrocentric chromosomes involved. (The short arms contain ribosomal genes located on all acrocentrics and highly repetitive satellite DNA; the loss of these regions is well tolerated). However, these individuals will have unbalanced gamete formation and have a 2/3 theoretical risk of having abnormal conceptions if mated with a normal individual.
Approximately one out of 1,000 individuals are carriers of Robertsonian translocations. Der(13;14)(q10;q10) is the most commonly observed Robertsonian translocation followed by der(14;21)(q10;q10).
Even though the theoretical risk of a translocation Down’s fetus is 1/3 in individuals who are carriers of der(14;21)(q10;q10) translocations, the actual empirical risk is 10-15% if the mother is the carrier and 0-2% if the father is the carrier.
Robertsonian translocations form a trivalent configuration. Alternate segregation produces roughly equal frequencies of balanced gametes. That is, gametes with either normal chromosomes or the Robertsonian translocation.
Adjacent segregation produces unbalanced gametes, three which are lethal (monosomy 21, monosomy 14, and trisomy 14) and one which results in translocation Down Syndrome.
IV.Identify Chromosome Abnormalities and Evaluate Clinical Implications
- Structural rearrangements
- Deletions
Deletions result in the loss of chromosomal material. This results in functional monosomy of the segment involved.
Terminal deletions are the result of one break with the subsequent loss of the acentric fragment during cell division due to anaphase lag.
Interstitial deletions result from two breaks within the same chromosome arm. Again the acentric fragment is lost during the following cell divisions.
