BLOOD KARYOTYPING: WHOLE BLOOD KARYOTYPING PROCESS

Karyotyping is a technique used in cytogenetics to examine chromosomes and identify genetic abnormalities that can cause disorders or disease. It involves collecting a cell sample, treating the cells to synchronize them in metaphase, staining the chromosomes, and analyzing the number, size, shape, and banding pattern of chromosomes to create a karyotype. Abnormal karyotypes can provide information about genetic conditions like Down syndrome, Klinefelter syndrome, and Turner syndrome. The main purpose is to detect changes in chromosome number or structure that can help diagnose these genetic disorders.

1. Analyze chromosomal structures as well as to detect genetic abnormalities that may be associated with various clinical conditions of the 20 patients. BLOOD KARYOTYPING

2. When we talk about blood karyotyping, we're essentially looking at the chromosomes inside the cells of a person's blood. These chromosomes carry important genetic information, sort of like a blueprint for our bodies. By examining these chromosomes, scientists can spot any irregularities or differences in how they're structured or how many there are. This is super important because it helps doctors diagnose genetic disorders – conditions that are caused by changes in our genes. Understanding these genetic conditions is crucial because it allows healthcare providers to offer the right treatment and support. Plus, it helps families understand why certain conditions might be passed down from generation to generation. INTRODUCTION

3. CHROMOSOME STRUCTURE

4. CHROMOSOME ON THE BASIS OF CENTROMERE

5. CHROMOSOME IDENTIFICATION IN HUMAN

6. Collect 20 random blood samples from patients following proper aseptic techniques to prevent contamination. Culture lymphocytes from the blood samples to obtain a sufficient number of cells for karyotyping. Arrest cell division at the metaphase stage using appropriate chemicals to ensure chromosomes are visible under a microscope. Stain the chromosomes using Giemsa or other standard staining techniques to differentiate individual chromosomes and identify structural features. OBJECTIVE:

7. Blood Collection Supplies : Sterile syringes and needles Blood collection tubes with anticoagulant (heparin or EDTA) 20 Blood samples Alcohol swabs Tourniquet Bandages Cell Culture Reagents : Lymphoprime media (Culture Media +PHA) Phytohemagglutinin (PHA) for lymphocyte stimulation Chromosome Preparation Chemicals : Colcemid or colchicine (to arrest cells in metaphase) Hypotonic solution (0.56 M KCl ) Fixative solution (Methanol: Acetic acid, 3:1) Staining and Visualization Supplies : Giemsa stain Microscope slides and coverslips Micropipettes and tips Microscopes with 10 X & 100X resolution lenses MATERIAL AND METHODS

8. Additional Equipment : Centrifuge ( Remi ) Slide warmer (Macro Scientific) Hot air oven (Macro Scientific) Incubator set at 37°C ( Yorco ) Laminar flow hood (Bio Scientific) MATERIAL AND METHODS

9. 0.56% KCl Solution – KCl (0.56 gram), Distilled water (100 milliliter). 1:3 Acetomethanol fixative – Acetic acid (25 milliliter), Methanol (75 milliliter) Phosphate Buffer Saline (PBS) – Potassium dihydrogen phosphate (0.1 gram), Disodium hydrogen phosphate (0.46 gram), Potassium Chloride (0.1 gram), Sodium Chloride (0.4 gram), distilled water (500 milliliter). Trypsin Solution – Trypsin 1:250 lyophilized (0.1 gram), Phosphate Buffer Saline (50 milliliter). Giemsa Stain – Giemsa (3 milliliter), distilled water (50 milliliter) Neutralization Buffer – Normal Saline (50 ml), New born calf serum (2.5 milliliter). Normal Saline – Sodium Chloride (4.5 gram), Distilled water (500 milliliter). REAGENTS PREPARATION:

10. FLOW CHART

11. Collection Of Sample: First, we collect some cells that have chromosomes in them. Usually, take these cells from blood or bone marrow. So, this is the first step of karyotyping we take samples in heparin vile as the heparin is an anticoagulant which prevents blood from clotting and if blood clots it will be difficult to separate the cells from it. METHOD

12. Method For Lymphocyte Culture: We want cells that are dividing because it makes the chromosomes easier to see. So, weuse to take 5 mllymphoprime media and add 0.5 ml sample.Providing 37 ºC temperature to make the cells start dividing. We wait for the metaphase in the cell cycle. STEP 2

13. Harvesting Of the Lymphocyte Culture: Once enough cells have divided, it usually takes 3 days or 69 th hours. Then adding of colcemid to stop the division process. After 69 th hours we add 100 microlitre of colcemid and leave in a incubator for 45 min.We do this at a metaphase time when the chromosomes are really clear and easy to see. STEP 3

14. Fixation Ofthe Cells: After the incubation we vortex the tubes and centrifuge the tubes for 10 minutes in 1500 rpm. After centrifugation we add 7 ml KCL solution into it and keep it in a 37 degree for 27 mins . After that we prefix it to keep the cells and their chromosomes fixed, with the solution of acetic acid and methanol to the sample. This is done carefully we add it dropwise so the cells do not get shock. Now we will incubate this in 4 degrees for 10 mins and the we will vortex the tube and centrifuge it for 10 mins in 1500 rpm and discard the supernatant and add 7 ml fixative and store it in 4 degrees until slide preparation. STEP 4

15. Preparing Slides for Karyotyping: On the day of slide preparation, we will vortex the tube and centrifuge it for 10 min in 1500 rpm. Again, we will discard the supernatant and add 7 ml fixative and will repeat it for 1 more time until our pellet becomes white pellet. We will discard the supernatant and leave around 2 ml to dilute our pelletandmix it well with pipette now take a sample of the cell mixture and drop on a slide at a angle of 45 degree. Then they let it dry it for 1 hours at 90 ºC to harden the protein present in chromosomes. STEP 5

16. GiemsaBanding (GTG Banding): Weuse four coplin jar staining.First trypsin which digest the protein heterochromatinand euchromatin . Second neutralizing buffer is used to reduce the activity of trypsin or inhibit the activity. Third is Giemsa stain it is basically highlight the bands on chromosomes. The dye stick to the chromosomes and make them visible under a microscope. Each component in dye highlights different parts of the chromosomes. Last fourth one is distil water which is used to remove the extra stain from the slide. STEP 6

17. Analysis of Chromosome by Microscope & Cytovision Software: A trained expert looks at the slide under a bright field phase contrast microscope. They're searching for any metaphaseof cells, and capture it. STEP 7

18. Capturing Of Metaphase Pictures and Analysis: According to chromosome shapes and sizes they arrange the chromosomes. And the analysis is done through the software call cytovision . We will capture minimum 20 metaphase cells in each case. STEP 8

19. Interpretation of Karyotyping Results: We compare what they see with known patterns to figure out if there’s a genetic disorder or something else going on. STEP 9

20. RESULT S.No Case No. Sex Karyotyping Nomenclature Result 1. 1 M 46, XY, rob (14;21) (q10; q10), +21 Trisomy 21 2. 2 F 46, XX Normal Female 3. 3 M 46, XY Normal Male 4. 4 M 46, XY Normal Male 5. 5 F 46, X, iso (Xp) Isochromosome 6. 6 M 46, XY Normal Male 7. 7 F 46, XX Normal Female 8. 8 F 47, XX+21 Trisomy 21 9. 9 M 46, XY Normal Male

21. RESULT 11. 11 M 46, XY Normal Male 12. 12 M 46, XY Normal Male 13. 13 F 46, XX Normal Female 14. 14 F 46, XX Normal Female 15. 15 M 46, XY Normal Male 16. 16 M 46, XY Normal Male 17. 17 M 47, XY+21 Trisomy 21 18. 18 M 46, XY Normal Male 19. 19 F 46, XX Normal Female 20. Ankit Gusain M 46, XY Normal

22. Normal Results Females: 44 autosomes and 2 sex chromosomes (XX), written as 46, XX Males: 44 autosomes and 2 sex chromosomes (XY), written as 46,

23. MALE METAPHASE

24. MALE KARYOTYPE

25. FEMALE METAPHASE

26. FEMALE KARYOTYPE

27. In our study of blood karyotyping of 20 randomly selected samples, we identified three cases of trisomy 21, commonly known as Down syndrome, and one case of isochromosome . This finding underscores the importance of karyotyping in detecting chromosomal abnormalities that have significant clinical implications. DISCUSSION

28. The results of our study highlight the critical role of blood karyotyping in identifying chromosomal abnormalities. The detection of three cases of trisomy 21 and one case of isochromosome among 20 samples demonstrates the method's effectiveness in diagnosing genetic conditions that can have profound health impacts CONCLUSION:

29. Finding Genetic Problems: Karyotyping helps doctors see if there are any issues with a person's chromosomes. This can show conditions like Down syndrome or other genetic disorders. Parental screening: It's used during pregnancy to check if a baby has normal chromosomes. This helps parents and doctors prepare for any special needs the baby might have. Finding Cancer Clues: Karyotyping can also help doctors understand certain types of cancer. It shows if there are abnormal chromosomes in cancer cells, which helps in treatment decisions. APPLICATIONS OF KARYOTYPING

30. Learning About Genes: Researchers use karyotyping to study how genes work and why some diseases happen. This helps them find new treatments and understand how our bodies work. Helping with Family Decisions: Karyotyping gives important information for families. It helps them understand if a genetic condition runs in the family and what they can do to plan for it. Checking Embryos in IVF: During fertility treatments like IVF, karyotyping helps doctors choose healthy embryos to increase the chances of a successful pregnancy. CONT..

31. Whole picture of chromosomes Balanced translocation Numerical finding Structural abnormalities Mosaicism ADVANTAGES

32. Invasive Procedure: Karyotyping often requires a sample of cells, which is typically obtained through procedures blood draw. These procedures carry a small risk of complications such as infection or discomfort. Limited Resolution: While karyotyping can detect large-scale chromosomal abnormalities, it may miss smaller genetic changes that can also affect health. This means it may not provide a complete picture of someone's genetic condition. Time-consuming: The process of preparing and analysing karyotype results can take several days to weeks. During this time, patients and healthcare providers may experience uncertainty or anxiety while waiting for results. DISADVANTAGES

33. Interpretation Challenges: Sometimes, karyotyping results may be complex or unclear. Interpreting these results accurately requires specialized knowledge and experience, which may not always be readily available. Ethical and Emotional Considerations: Learning about genetic abnormalities through karyotyping can raise difficult ethical and emotional questions for individuals and families. It may influence decisions about pregnancy, family planning, and medical care in profound ways. Limited Information: Karyotyping provides information about chromosomes but does not reveal details about specific genes or mutations that may be responsible for certain conditions. Additional genetic testing may be needed to understand these aspects fully. CONT..

34. 1. Shaffer, L. G., McGowan-Jordan, J., & Schmid , M. (Eds.). (2013). ISCN 2013: An international system for human cytogenetic nomenclature. Karger Medical and Scientific Publishers.   2. Haddad, R., & Brunetti-Pierri , N. (2016). The changing landscape of molecular diagnostic testing: Implications for academic medical centers. JAMA, 316(7), 712-713.   3. Rowley, J. D. (2001). Chromosome translocations: Dangerous liaisons revisited. Nature Reviews Cancer, 1(4), 245-250.   4. Nature Publishing Group. (2001). Chromosome banding revealed by different staining techniques. Nature Genetics, 29(3), 253.   5. Schematic diagram of karyotyping (Mueller et al. 2001)    6. National Human Genome Research Institute. ( n.d .). Learning About Turner Syndrome. Retrieved from https://www.genome.gov/Genetic-Disorders/Turner-Syndrome .   7. National Down Syndrome Society. ( n.d .). What is Down Syndrome? Retrieved from https://www.ndss.org/about-down-syndrome/down-syndrome/ REFERENCES