Abstract
A crucial tool in contemporary human cytogenetics is blood cell karyotyping, which offers details on abnormalities in chromosomes, their prevalence in the general public, and the connections underlying particular aberrations and phenotypes outcomes. In human cytogenetic research, an activated lymphocyte is examined following the application of spindle formation inhibitors to stop cell division during metaphase. Chromosome condensation occurs normally when the nuclear membrane disintegrates, while the chromosomes are unable to arrange oneself into a metaphase plate.Because the chromosomes appear to be free inside the cytoplasm, this look is very different from that of a real metaphase. After preprocessing and staining has occurred the chromosomes may be seen clearly.The chromosomes can be stained using one of two methods: one that results in a fairly even staining intensity, and the other that produces varied staining along the chromosome’s length. Based on the aforementioned results, it appears that the blood taken in EDTA and eparin tubes yields the same karyotyping outcomes.This type of blood collection has been not found in previous research in which depends on heparin tube collection. collection has been not found in previous research in which depends on heparin tube collection.
Keywords :Blood samples, Karyotype

Introduction

Every living thing’s genome is arranged into chromosomes, which differ in terms of size, shape, and DNA content. Mutations and rearrangements that alter the chromosomes’ construction, as well as quantity alterations (aneuploidy) brought about by spindle mistakes throughout cell division, can hinder gene flow and lower fitness, which can lead to diversity. (King, 1993). Chromosome characteristics, on the other hand, are a trustworthy standard in taxonomic and evolutionary biology since they are morphological qualities. The karyotype gives a description of how the genetic information is arranged in the chromosome and is the earliest phenotypic expression of the genotype. (Guerra ,2008). One particularly unusual feature that sticks out amid the data that can be gleaned from karyotypes is the karyotype asymmetry being which has been the topic of extensive discussion. Other data that can be taken from karyotypes includes the number and shape of chromosomes, the range of heterochromatic bands, gene placement, etc. Chromosome size and morphological changes are frequently associated with changes in karyotype symmetry. These changes are typically brought about by focal fusion/fissions or DNA sequence expansions or deletions (followed by disploidy) (Peruzzi and Erolu ,2013) .

Several cytogeneticists and cytotaxonomists used these indices for a very long period to discuss the taxonomic connections amongst various species. (Souza LGR, Crosa O and Guerra M ,2010) .It is crucial to remember that traditional karyotyping requires a lot of time—many days are needed to prepare cells for analysis. Furthermore, blood samples must reach the laboratory no later than 48 hours after sampling, ideally earlier, as live lymphocytes are necessary to ensure successful cell proliferation in culture. Selecting which test(s) to seek requires careful consideration due to the variations in resolution as well as the distinct advantages and disadvantages of each technique. The predicted symptomatic ailment or syndrome, as well as a meticulously gathered family history of a genetic illness (pedigree), will determine which test is recommended. It is recommended you speak with an expert clinical geneticist.

 According to the ACMG Practice Guidelines (2010), karyotyping is often recommended as the initial test for the following conditions:

1. 1. Common aneuploidy evaluation, such as trisomies 21, 18, or a sex chromosomal aneuploidy.
2. 2. Questionable genitalia and gender identity.
3. 3. Inappropriate secondary sexual development or tardy puberty.
4. 4. Female amenorrhea or small height.
5. 5. Singular clinical characteristics, such as cardiac disease or cleft lip.
6. Syndromes caused by chromosome breaks. Unable to conceive (Human Cytogenetics,2001).

Material and Methods

Blood samples collection

The blood samples were collected in EDTA tubes, then preserved in refrigerator at 8 C . 

Blood Lymphocyte Culture Procedure and Lymphocyte Culture

. Add 10ml of media to a plastic tube or tissue culture plate and inoculate with around 0.5ml of peripheral blood. Gently tip tubes over to combine samples.

2. 2. Give the culture a full 72 hours of incubation.
3. 3. Fill each culture tube with 0.1–0.2 ml of Colcemid Solution (Cat. No. 12-004–1). Give the culture another 15 to 30 minutes of incubation.
4. 4. Put the culture in a centrifuge tube and spin it for five minutes at 500 g.
5. 5. Take out the supernatant and put the cells back into 5–10 milliliters of hypotonic 0.075M KCl (Cat. No. 12-005-1). Incubate for ten to twelve minutes at 37°C.
6. 6. Spin for five minutes at 500g.
7. 7. Drain the supernatant, stir the cellular sediment, and gradually add 5–10 milliliters of newly produced, ice-cold fixative with 1 part acetic acid to3 parts methanol.

Leave in 4oC for 10 minutes.

8. 8. Go back and repeat steps 6 and 7.
9. 9. Transfer the cell pellet back to a clean slide, resuspend it in 0.5–1 ml of fresh fixative, and let it air dry.
10. At this point, Orecin or Giemsa can be used to stain the preparation. The method that is now most frequently utilized is giemsa banding.

Results and Discussion

Blood Lymphocyte Culture and Lymphocyte Culture

The EDTA blood samples were used after (7) days of collection inorder to cultured cells and karyotyping. Bellow the results obtained.Figure (1) and (2) shows cell culture results:

 Figure 1: Cell culture results

Figure 2:Cell culture results

Figure (3) indicates blood cells after harvesting:

Figure 3: Blood cells after harvesting

            Figure (4) indicates the cells before  division:

Figure 4: Cells before  division

            Figure (5) show karyotype results from EDTA blood collection tubes

Figure 5: karyotype results obtained from EDTA blood

Figure (6) shows karyotype results from heparin blood collection tubes:

Figure 6:shows karyotype results from heparin blood collection tubes

From result above it has been appeared that there is no differences in karyotyping results between EDTA and heparin tubes collected blood.

This type of blood collection has been not found in previous research in which depends on heparin tube collection. A crucial tool in contemporary human cytogenetics is blood cell karyotyping, which offers details on chromosomal aberrations, their prevalence in the general public, and the connections between particular abnormalities and phenotypic outcomes. In human cytogenetic research, a stimulated lymphocyte is examined following the application of a spindle formation inhibitor to stop cell division during metaphase. Chromosome condensation occurs normally when the nuclear membrane disintegrates, but the chromosomes are unable to arrange themselves into a metaphase plate. Because the chromosomes tend to be unbound inside the cytoplasm, this look is very different from that of a real metaphase. After preprocessing and markings, the chromosomes may be seen clearly. The chromosomes might be dyed with a method that results in varied marking across the whole length of the chromosome or in a method that produces a reasonably uniform intensity. (Nussbaum, Robert; McInnes, Roderick; Willard, Huntington , 2015).

Conclusion

The goal of this research has been achieved from result above that has been appeared that there is no differences in karyotyping results between EDTA and heparin tubes collected blood.

This type of blood collection has been not found in previous research in which depends on heparin tube collection. This make karyotyping more easy for performing and don’t restricted with time of collection.

Acknowledgment

Non .

Conflicts Interest

Non.

References

• King, M. Species Evolution: The Role of Chromosome Change; Cambridge University Press: Cambridge, UK, 1993.
• Guerra M (2008) Chromosome numbers in plant cytotaxonomy: Concepts and implications. Cytogenet Genome Res 120:339-350.
• Peruzzi L and Erolu HE (2013) Karyotype asymmetry: Again, how to measure and what to measure? Comp Cytogenet 7:1-9.
• Souza LGR, Crosa O and Guerra M (2010) Karyological circumscription of Ipheion Rafinesque (Gilliesioideae, Alliaceae). Plant Syst Evol 287:119-127.
• Weiss-Schneeweiss H and Schneeweiss G (2013) Karyotype diversity and evolutionary trends in Angiosperms. In: Greilhuber J, Dolezel J and Wendel JF (eds) Plant Genome Diversity vol. 2. Springer, Vienna, pp 209-230.
• ACMG Practice Guidelines: Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities. Manning, M and Hudgins, L. Genetics in Medicine Volume 12, Numéro 11, 742 – 745, Novembre 2010.
• Human Cytogenetics: constitutional analysis. 3e édition. E. Rooney éd. Oxford University Press 2001
• Nussbaum, Robert; McInnes, Roderick; Willard, Huntington (2015). Thompson & Thompson, Genetics in Medicine (Eighth ed.). Canada: Elsevier Inc. p. 58. ISBN 978-1-4377-0696-3.
• Hasue, F. M., Passos, M. J. D. A. C. R., Santos, T. D. C. A. D., Rocha, A. J. D. S., Vignardi, C. P., Sartorio, P. V., … & Ngan, P. V. (2013). Assessment of genotoxicity and depuration of anthracene in the juvenile coastal fish Trachinotus carolinus using the comet assay. Brazilian Journal of Oceanography, 61, 215-222.