Dear Colleagues
Hello
Hello to everybody
In this issue we are targeting two very important procedures we do at our setup. These two procedures are described here are not in a bookish way but in a applied way so that anybody can follow them and use them in their setup. Chromosomal analysis is vast subject and can not be summed up in a news letter. Staining of chromosome and banding gives immense information regarding their numerical and structural abnormalities. A couple with previous history of Down child is scarred to have another baby. A lady with androgenic features sometimes needs chromosomal identifications for their gender. Girl with primary amennorrhoea needs to ascertain whether she is a case of Turners syndrome or not. Male with Azoospermia we always do karyotyping to see whether there is gross aneuploidy or not.
DNA PCR is another great help in a lab with infertility services and gynecology. Tuberculosis confirmation gives a hint for the definitive treatment. DNA identification for the types of Human Papillom Virus ( HPV ) in cervical discharge helps us to take action in abnormal Pap smears.
Although all the lab procedure have their own limitations and false reporting but with great care and stringent lab. protocols it becomes a great help in clinical practice . As we are reaching around 5000 practitioners ,you can place your adverts and discuss for any equipment for sale and purchase.
I wish you all the best
Dr.D’Pankar Banerji
Harvesting chromosomes from peripheral blood : Protocol used at Ideal fertility
Chromosomes are packet of DNA. In human they are packed in 23 pairs. Each pair is coming from each parent.
Gross abnormality of the DNA content can be identified by studying the chromosomes ,their numbers and their structures Why we need chromosomal analysis ? In our set up we need it for analysis of repeated abortions, to predict Down syndrome in the next child, where the lady already delivered a Down baby , to evaluate male infertility and confirming the genetic sex in intersexes . Here we are doing this test by peripheral blood.
Chromosomes are harvested only from dividing cells, but blood cells are terminal cells. So some cells have to be transformed into blast cells ( blast cells divide in culture). Lymphocytes are the cells, utilized.
Steps are :
3-5 ml Blood collected in heparin and centrifuged at 1500 rpm for 5 minutes.
Buffy coat is collected ( rich in white blood cells ) Culture media is prepared which can maintain the life of lymphocytes in vitro.
We use Ham’s F-10 HEPES buffered Blood cells are exposed to a mitogen mixed in culture media .PHA ( phytohemaggutinin ) is used in our lab as mitogen .It is a extract from red kidney beans ( Rajma ) made in-house. When T-Lymphocytes are exposed to this plant antigen they respond as they would in the body to a non-self foreign substance.
Mature T cells dedifferentiate to a T- Lymphobastic cells .It is in this immature from that the T-cell has the capacity to synthesize DNA to undergo mitosis.
Peak mitotic activity is reached after 60-70 Hours of culture and is considered to be the optimum harvesting point for chromosome studies. Precise dose of PHA is very important as too much PHA is toxic and lesser quantity yields poor results 500 micro liter of buffy coat of is mixed with 5 ml of media.1000 microliters of supernatant plasma is added in the media Tubes are placed inside the simple incubator at 37 deg.Celcius for 72 hours. At the peak of mitosis ,cells are exposed to a spindle poison called Colchicine ( 10 mic.liter in whole culture ) it is kept for 15 minute in 37 deg Celsius.
Then the content is centrifuged at 1500 rpm for 10 minutes. Pellet is separated and mixed with hypotonic solution of KCl ( 0.075M ) and it is kept for 20 minutes at room temperature.
Then 1 ml of preservative ( 3: 1 methanol, acetic acid ) is poured in the hypotonic mixture and mixed and centrifuged for 10 minutes. Three washings with the preservative done.
It is followed by slide preparation by flame dry method .The slides are either stained by Geimsa stain or they are treated by trypsin for banding.
2.Polymerase chain reaction test for detection of Mycobacterium tuberculosis
The diagnosis of tuberculosis is generally carried out through the patients’ symptoms, Checking for tuberculin ,X-ray and checking for Mycobacterium tuberculosis.
The method most commonly carried out in laboratories is the Zeil-Neelson staining that uses smear staining to test for mycobacteria . However ,although it is fast and simple , it is not possible to differentiate Mycobacterium tuberculosis from those that are not ,and the sensitivity is also rather low.
The most sensitive cultivation methods uses a 5- 10 % CO2 partial pressure and observation takes place after sitting 4-8 weeks at 37 deg. Celsius .The problem is that it takes a long time. More recently , the Polymerase chain reaction method is being used to rapidly and accurately detect Mycobacterium tuberculosis in clinical samples. This method is reporting a sensitivity and specificity of greater than 90 % and is proving to be useful.
Steps Of PCR :
1. Samples are received at Lab : The samples may be , Sputum ( usually morning samples of three consecutive days ), Body fluids like Pleural effusions or Ascitic fluid or CSF , Endometrial biopsy in saline , urine and EDTA blood.
2. Samples are treated prior to extract the DNA of Tuberculosis. Steps are different for different samples. Mostly they are centrifuged at high speed to get a pellet which are rich in tissues or cells infected by M. tuberculosis.
3. Extraction TB DNA : Here various extraction kits are used which are commercially available . Cell proteins are lysed with Protinease K ( enzyme ). This reaction takes at higher temperature .DNA of M. tuberculosis dissolved and precipitated with alcohol ( 95-100 % ) . This precipitated DNA is isolated and dissolved in nuclease free water of TE buffer
4. There are three steps in actual PCR .These are denaturing ,annealing and extension. During denaturing the double stranded DNA of M.tub. is separated in single strand at 92 to 95 deg.Cel. for 30 sec to one minute. Then one specific part of DNA is targeted and primers are applied in the presence of T.que polymerase. This step is called annealing and usually done at the temp.56 deg.cel.for one minute Taq is an enzyme that helps to extend the synthesis of copy of particular segment in the presence of nucleotides and this is at temp 72 deg. This gives a new copy of particular and specific segment of DNA of the tuberculosis bacteria called extension.
5. This cycle is repeated number of times app. 20 to 30 times in a instrument called thermal cycler.
6. If the samples contain a tubercular bacillus then only the particular DNA of bacteria will be amplified and its presence will definitely confirm , that the person is infected by M.tub.
7. Amplified DNA is invisible to naked eye and has to be stained by a fluorescent dye.
8. Amplified DNA is then poured in small well made on agarose gel and put on in very mid electric field and left for electrophoresis.
9. Amplified DNA moves according to its weight and compared with controlled samples , negative and positive and markers .
10. After one or two hours of electrophoresis the agar plate is placed in fluorescent illuminator and bands of different DNA are visualized . If sample DNA correspond with the positive control then diagnosis is positive and if with negative then it is negative.
3. Recovery of oocyte from Pouch of Douglas during IVF—Case
Oocyte recovery is most important step in IVF and good recovery gives better number of embryos. Egg recovery is done 36 hours after a maturational injection of hCG 10000 iu .We do our pick up procedure at 11.30 –12.00 noon for our convenience. Hence most of the time the hCG injected at 11.30-12.00 at night of day before previous day.
A patient named Kaushal 35 yrs. received her hCG injection two hour prior,(around 9.30 pm), 23.4.07 . Pickup was planned as usual at 12.noon on 25.4.07. In the mean time the she had pain in her pelvic region around 11.00 am .When Sonography was done just before pickup ,we were surprised to see that out of three follicles , only one is there and there is fluid in Pouch of Douglas. Present follicle was aspirated and we aspirated the content of Pouch ,after putting her in reverse trendelenberg position for few minutes.
We were able to retrieve all the three eggs and all of them fertilized well and transferred on 29.4.2007( Day5—Three blastocysts, One good quality,see Fig. Below)
Training in IVF and Embryology
Module I : Ovulation induction and Intra Uterine Insemination ( One day ),Rs.2000
Module II : Conventional IVF and fundamentals of Embryology( Two days ), Rs.20,000
Module III : Intra cytoplasmic sperm injection, Micromanipulation ( Two days ) Rs.50,000
For details contact ,two participants per batch
Dates :
Throughout the year
Drafts should be in favor of Dr.D’Pankar Banerji, payable at Jabalpur.
Stay can be arranged in nearby hotels at an extra cost Rs.250-1000 per day
Lunch will be served during the training session.
Faculty :
Dr.D’Pankar Banerji ,Consulting Gynecologist and Infertility specialist
Dr.Mrs.Rinku Banerji,Embryologist
Certificate will be issued after the course to participants
Sale and purchase of the used equipments
* Ultrasonography machine for schimadzu SDU 350- convex & vaginal probe 2000 make. with probe converter contact +91-9998968631." Dr.kalpana khandheria
Letters to the editor:
I welcome your views and comments in this column and will be published in the forthcoming publications
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