Q. What is the real role of the hypotonic solution in the chromosome preparation?
A. It has been both empirically and scientifically demonstrated that the hypotonic solution has a marginal role in the swelling process of the cells. Please read the article written by U. Claussen and published in 2002 (“Demistifying chromosome preparation and the implications for the concept of chromosome condensation during mitosis”, see the Bibliography section for full details) to know more about the mechanism of the hypotonic treatment. In reality, the main action of any type of hypotonic solution consists in the activation of the cellular Na+/K+ pumping system as a consequence of the physiological reaction of the living cell to the variation of the osmotic conditions.
The second remarkable effect is concerning the migration of the chromosomes towards the outer areas of the cytoplasm, which is clearly visible under the microscope (in the same article, you can see the relevant pictures).
However, what is important to notice is that each type of cell specifically reacts to a certain kind of hypotonic solution: this is the reason why our protocols recommend to use, for instance, KCl 0,075M with lymphocytes, Na Citrate 0,6% + KCl 0,1% with amniocytes, and Na Citrate 1% with chorionic villi (direct method).
Q. Why is preferable to use Colchicin instead of Vinblastin and/or Vincristin?
A. These three substances are well known and have been widely used as blocking agents to stop mitosis: the action of all of them, in fact, is based upon the depolymerization of the mitotic fuse. However, it is recommended to use Colchicin (Colcemid®) because its action is reversible whereas the link of the other two with the α and β chains of the protein called tubulin (a basic component of the microtubuli) is not. It must be noticed that the right concentration of Colchicin is mostly important; the ideal values (final concentration in the cell suspension) are as follows:
- Amniocytes/Lymphocytes/Chorionic villi: 0,05 µg/ml
- Chorionic villi (direct technique): 0,5 µg/ml
Q. Is it true that the Colchicin may “shorten” the chromosomes?
A. This is absolutely false. This phenomenon is rather an effect of a family of enzymes called “condensins”, whose action goes on despite the block of the mitosis. On the other hand, a prolonged exposure of the cells to Colchicin will result in loss of adherence, and therefore the risk is to miss a number of cells undergoing mitosis, since they will stay in suspension.
Q. How can be corrected the overspreading/underspreading of chromosomes?
A. A real “must” for any person working in a Cytogenetics lab is to know the scientific mechanisms that happen during each step of the chromosome preparation. Once this information will become part of the expertise of the technician, it will be much easier for him/her to correct a “wrong” behavior of a certain sample; generally speaking, the following actions can be taken :
- overspreading: this may be caused not only by an excess of humidity, but also by a too thick film of fixative. A possible countermeasure consists in increasing the alcohol/acetic acid ratio in the fixative solution (i.e. 4:1 instead of 3:1).
- underspreading: similarly, this can be explained not only because of a too low level of humidity, but also as a consequence of a too high quantity of protein compared to the acetic acid; hence, it is recommended to increase the contents of acetic acid.
Q. Will an instrument like “Optichrome” solve all the problems related to the variability of the ambient?
A. The “Optichrome” has been specifically designed to control humidity and temperature during the crucial phase of evaporation; therefore it will definitely minimize the adverse effect of such variables, which represent a real problem for the cytogenetics labs all over the world. Having said that, the instrument cannot obviate the most common mistakes that could be done during the different steps of the chromosome preparation, and this is the reason why it is fundamental to know the mechanisms that can lead to an optimal spreading (and banding).
Q. Amniocytes cultures are almost impossible to synchronize: why?
A. Just recently has been discovered the stem-cell like nature of these cells; hence, the structure of the membrane of this type of cells is modified in such a way that does not allow the substances normally used for synchronization to perform as usual.
Q. Which are the advantages of the synchronization, when working with lymphocytes?
A. The procedure recommended by EuroClone is based on the original paper of Webber and Garson (1983, see the Bibliography section), that in turn represents an improvement over the first experiments made by Dutrillaux (1975) and Yunis (1976). According to this revised method, the following advantages can be mentioned:
- no washing steps, and therefore less risk of contamination
- reduced number of chromosome aberration
- highest number of mitotic cells
- optimal results in terms of both spreading and banding