CCA is a standard, well-known method, incorporated in the routine diagnosis and assessment of diseases and conditions, associated with microscopically visible genomic changes. It is currently included in ERCAHN as well as in the latest American and other guidelines [2, 10, 11]. Recognizing its key advantages and disadvantages is of essential importance in order to be consistent with current good laboratory and clinical practise standards.
CCA holds valuable advantages that render it to be an essential element of routine evaluation even in the latest guidelines. Regardless of the low resolution, CCA provides single-analysis whole-genome assessment unlike many of the newer options. It also recognises balanced changes in the genome like balanced translocations and inversions unlike comparative genome hybridisation (CGH), array CGH, single- nucleotide-polymorphism-microarray and others [12]. Additionally, examination of 20 metaphases can register mosaicism of 14% with a confidence interval of 95%, a sensitivity that other methods cannot offer [2]. CCA is also a rather inexpensive method, compared with newer ones – an advantage that is essential when it comes to routine practice in economically diverse countries.
Still, one of the main downsides of the conventional cytogenetic method is the possible failure to achieve a result. Technical challenges resulting in lack of metaphase plates could be caused by a fibrotic BM, dry tap BM aspiration, therapy induced aplastic anaemia/ myelosuppression, or bacterial contamination. Still, our experience report shows more than a sufficient success rate of 83.5%, comparable with those of other studies [13, 14]. It also complies with national standards rendered from the Bulgarian Ministry of Healthcare, with the latest one published in 2010. According to the latter, CCA with a success rate greater than 60% for BM-derived samples is considered an acceptable one [15].
Another essential disadvantage refers to the limited resolution of the method with a threshold of 5–10 MB. This characteristic refrains CCA from being appropriate for a wholesome assessment of the genetic nature. So a seemingly normal karyotype could carry significant yet unrecognisable mutations. This downside is even furtherly augmented by the typically low resolution of less than 400 bands for samples derived from BM [16]. Poor resolution is a problem that is addressed in ERCAHN as well, and there is no minimum demand since it is a well-known issue. Still, along with the incomplete success rate, it is a reason to consider and choose an additional analysis that would overcome these problems [2]. There is also the matter of timing when it comes to results. CCA has a standard turnaround time of 8–12 days for bone marrow cultures and this could be a delay that the patient cannot survive. The need of immediate therapeutic direction in some cases points out the need of an additional genetic analysis once more. The choice of this subsidiary method would depend on the diagnosis, the European recommendations and on the in-house practices and possibilities. This could be a molecular-cytogenetic one like FISH or a molecular-genetic method like Sanger sequencing, Next-generation sequencing, Polymerase chain reaction, Multiplex ligation-dependent probe amplification etc. They provide the robust guidance and clarity for methodology, good laboratory practice and quality standard, much needed with the increasing number of genetic changes. However, the necessity is not ubiquitous for it depends on the diagnosis as well as on the age group of the patients. Management is different and algorithms usually have this turnaround time well covered [17].
Noteworthy limitations are also the laborious and extensive hands-on time of sample preparation as well as the time- and effort-consuming metaphase analysis. CCA demands highly trained and experienced specialists which are well acquainted with its process and specifications. Additionally, the nature of each hematologic disease is specific and requires a certain level of knowledge regarding appropriate cultivation conditions and associated chromosomal aberrations. This necessitates the need for personnel, strictly involved and responsible for CCA of BM samples.
As per tendencies over the studied period, the first one would regard the nearly twofold increase of samples in total – 187 in 2010 to 351 in 2020 (Fig. 3). This tendency reflects National health policies that aim at improving coverage and quality of medical genetic service provision, especially at university hospitals [18]. We have also noted a marked constancy of the amount of pathology found, demonstrated as well on Fig. 3.
Regardless of the steadily increasing number of samples through the years, there is a visible plateau in respect of chromosomal abnormalities. This tendency has several likely sources. Firstly, there is a surging amount of patients, monitored after chemotherapy or after allogeneic stem-cell transplantation. Both of these groups are commonly related with loss of previously seen chromosomal mutations. This cytogenetic response is an expected and even anticipated effect, as it indicates a successful management and lack of relapse [12, 19–21]. Becoming more and more personalised, therapy inevitably reflects on the residual genetic characteristics seen during a cytogenetic follow-up. Secondly, CCA is incapable of registering submicroscopic genetic changes as previously referred in the discussion. Hence, even if more new patients are included, many of them would have a normal karyotype while probably carrying essential submiscroscopic or molecular mutations. This tendency advocates for the previously discussed need of additional analysis that is able to detect such mutations.
As seen in Table 2, there is a noticeable prevalence of (somatic) mosaicism – 58.6%, that denotes clonality, a typical characteristic of each neoplastic process. This is expected since malignancies originate from a somatic event, affecting the genetic information of a sole cell line. In a relatively smaller percentage – 13.3%, there is a presence of combined chromosomal aberrations in a single clone. This is a demonstration of the complexity, the chain of genetic events – initiating and subsidiary, which in time lead to malignization.