Prenatal Dx Lesson 4

8/13/2019 Prenatal Dx Lesson 4

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Cytogenetics


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Prenatal:

Amniotic fluid

Chorionic villous sample

Cord blood

Postnatal:

Peripheral blood

Other: Bone marrow aspirate

Solid tissue


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Ultrasound examination

picture of the baby in the uterus by using sound

waves. These sound waves are passed through the

mothers abdomen and bounce off maternal and fetal

structures to make a picture on the monitoring screen. Inan ultrasound picture, bone looks white and fluid looks

black.


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First trimester testing 10 to 13 weeks.

testing for PAPP-A (pregnancy associated plasma protein A), beta HCGand ultrasound nuchal translucency. Maternal age, weight, ethnicity,diabetes and previous clinical history are also taken into account.

Advantages much earlier in pregnancy than the triple test whichalleviates unnecessary maternal anxiety. The test is more accuratethan the triple test and results in fewer amniocenteses beingperformed.

Levels of PAPP-A are reduced in Down syndrome and trisomy 18.

Levels of beta-HCG would be raised in Down syndrome and reduced intrisomy 18.

Nuchal translucency should measure


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Triple test

AFP screening

16 weeks of pregnancy

Down syndrome, trisomy 18, spina bifida, anencephaly,and abdominal wall defects

Levels of AFP would be reduced in Down syndrome andraised in neural tube defects.

Levels of beta-HCG would be raised in Down syndrome.

Levels of estriol would be reduced in Down syndrome.

A multiple pregnancy, such as twins or triplets, also causes

an increase in AFP levels. Factors which influences the test is gestational age,

mothers age, weight, ethnicity and clinical history. Allthese factors are used to calculate the risk factor. If therisk factor is higher than 1:250-270, an amniocentesiswould be recommended


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Chorionic villus sampling (CVS)

Amniocentesis

Cordocentesis

In general, the invasive techniques are more

accurate than the non-invasive techniques

but involve greater risks to the pregnancy.


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most common test used for prenatal diagnosis ofa chromosome abnormality in a fetus

involves the removal of a small amount ofamniotic fluid, a clear straw-coloured fluid

which surrounds the baby in the amniotic sac AF comprised of fetal cells, proteins, and fetal

urine

done after 15 weeks of pregnancy

during the procedure ultrasound is used to locatethe baby and the placenta

volume of fluid withdrawn depends upon the ageof the fetus and the reason for the testing


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Amniocentesis can be performed between

the 14thto 24thweek of pregnancy under

ultrasound guidance.

Long-term culture = 10 to 18 days


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To detect abnormalities in the fetus.

To reveal chromosomal abnormalities, diseasescaused by defective genes, and certain metabolicproblems.

To detect Down syndrome when the mother will beover age 35 at due date, has a family history ofsevere birth defects or metabolic disorders, or hashad an abnormal first trimester or triple screen test.

To determine whether a baby expected to be

delivered prematurely can survive outside the womb. In Rh-negative mothers carrying Rh-positive babies,

to help evaluate the condition of the fetus and theneed for an early delivery or fetal blood transfusion.


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Advantages

It detects numerous disorders before birth.

It is virtually 100% reliable in revealing Downsyndrome and spina bifida.

Risk lowest when performed at 14-16 weeks but thesample is optimal between 18 to 20 weeks gestation.

Disadvantages

It cannot be performed as early in pregnancy as CVS,and takes longer for results to be ready.

Miscarriage rate of about 1%.

Injury to the fetus or placenta - unlikely as theneedle is guided by ultrasound and only speciallyqualified sonographers would perform the test.

Bleeding

Infection.


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The fluid is centrifuged to concentrate the

cells, which are then cultured under ideal

conditions for cell growth using specialised

media, high humidity and a CO2 controlledenvironment.

When the cells are confluent they are ready

for in situ harvesting


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Petri Dish Method

The in situ method establishes cell growth inseveral independent culture vessels by spinning

down the amniotic fluid, resuspending the cellsin medium and plating them directly onto plasticcoverslips. As viable amniocytes attach to thecoverslips, they begin dividing forming coloniesof presumed genetically identical cells. Thecolonies are fixed in situ, that is directly on the

coverslip without disruption. Each coversliprepresents an independent culture, and thecolonies are presumed to be derived from cellsof separate origin


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Flask method Prepare 2 flasks per patient labelled A and B. Add 3ml working

culture medium to each using 10ml disposable sterile pipette. Warm inthe CO2 incubator.

Wash hands thoroughly and use PPE.Wipe gloves and bench surfaces with70% alcohol before handling samples.

Clean the culture hood. Use sterile technique throughout.

If a prenatal FISH has been requested: Under sterile conditions, mixthe amniotic fluid sample gently to distribute the cells evenly. Remove4ml of the sample into a 15ml blue capped conical tube. Label the tubewith patient name and laboratory number. Store in the main lab fridge

until required. If the sample is for FISH only, still remove 4ml AF understerile conditions in case culture is requested or needed at a later stage.

Spin the remainder of the amniotic fluid at 2000rpm for 10 minutes.

Make a note of the cell pellet size and colour, and the presence of redblood cells on the AF culture sheet.

Decant the AF supernatant into a sterile specimen jar, leaving 1ml ofamniotic fluid in the specimen tube in which to re-suspend the cells.

Take 2 flasks from the incubator labelled A and B. Label the flaskswith the patient name and laboratory number. Now write the laboratorynumber onto the patient sample tube. (This can be used as a check laterto confirm which specimen was put into the flask). Do one patient at atime.


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Use a sterile disposable plastic Pasteur pipette to re-suspend the cells.

Using the same pipette aliquot the cell suspension equally into flasks A and B.

Decant the supernatant from the sterile specimen jar back into the original specimen

tube. Store the tube in the freezer until the results have been reported.

The sterile specimen jar can be discarded in the plastic liner. This plastic liner is removedfrom the culture room at the end of each day and put into the waste disposal box in the

main cytogenetics laboratory.

Place flask A into one CO2incubator and flask B into another CO2incubator.

Checking Cell Growth for Medium Change

Leave flasks up to 5 days before checking for cell growth. Amniotic fluid samples set up onTuesday can be checked on Friday. If not ready for medium change, add 2ml of fresh

medium. Clean the inverted microscope and surrounding area with alcohol.

Check for growth using the inverted microscope. Try not to keep the cultures out of the

incubator for too long. Make a mark on the flask indicating the regions of patches of cell

growth seen.

If there are established patches or colonies of cell growth, then make a note on the

culture sheet for medium change. Also, comment on the growth e.g. small/few patches,

attaching cells. Medium should be changed Mondays, Wednesdays and Fridays. Calculate the amount of medium needed for allcultures ready for a medium change.

Aliquot this amount into a sterile flask and put into the incubator to warm up.

Use sterile technique for medium changes. For the FIRST medium change pour the

culture medium from culture A into the backup flask C. Add 3ml fresh medium to flask A

and return it to the incubator. Repeat this procedure for flask B.

Monitor flasks A and B to see when they will be ready for harvesting. If the cells are

growing but are not ready for harvesting, then change medium, by discarding the mediafrom the culture into a discard container, and add 3 ml fresh medium (warm the medium

first before use .


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Add 3 drops of colcemid with a 3ml Pasteur pipette to cultures ready for harvest.Return to 37C CO2. incubator for 1 hour.

Warm 6ml of KCL up per flask at 37C at least 30 minutes before use

Label a 15ml centrifuge tube for each sample to be used for the supernatant fromthe culture flask

Pour the culture media from the flask into centrifuge tube.

Rinse the flask with 3 ml phosphate buffer saline (PBS) and tip into centrifuge tube.

Add 2 ml Trypsin/Versene to the flask and place in the 37C incubator for 10minutes.

Tap the flask gently to dislodge the cells. Observe trypsin action under the inverted

microscope. When the cells are in suspension, transfer to the centrifuge tube. Rinse the flask with 1ml PBS. Use a plastic Pasteur pipette to collect all

available cells.

Centrifuge at 2000rpm for 10 minutes. Discard supernatant. Re-suspend the pellet.

Add 6ml of warmed hypotonic KCL and incubate at 37oC for 20 minutes.

Add 2ml fixative (30ml methanol+10ml acetic acid) slowly to the KCL and agitate tomix.

Centrifuge at 2000rpm for 10 minutes Discard the supernatant, and resuspend the pellet. Add 2ml fixative, agitate and

centrifuge at 2000rpm for 10 minutes.

Repeat step 13.

Discard supernatant. Resuspend the pellet, flood slides with fixative, tip off theexcess. Drop about 6 to 8 drops of sample per slide. Tip off excess fix. Transfer tothe 600C incubator when the slides appear grainy.

After 10 to 15 minutes, transfer the slides to the 1000C incubator for one hour.

(Slides will then be ready for staining and analysis). Store the remainder of the cell pellet at 4oC until the results have been reported.


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Can be done from the 9thweek of pregnancy

Also under ultrasound guidance

Long-term culture = 10 to 18 days


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an accepted and proven method of prenatal diagnosis

a small sample of cells (called chorionic villi) aretaken from the placenta where attached to the wallof the uterus

chorionic villi are fingerlike projections of amembrane (the chorion) that surrounds the fetus

the villi develop from the fertilized ovum and have agenetic composition similar to that of the fetus

CVS procedure can be performed transvaginally or

transabdominally, depending on the location of theplacenta

performed between 10 and 12 weeks gestation andresults are generally available within 7-10 days


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Transfer the villi into a sterile Petri dish using a sterile plastic pipette. Take as littleof the transport media (saline) as possible.

If there is a lot of blood, wash the sample with RPMI. Suck off the excess bloodymedia, and discard. Repeat until sample appears cleaner.

Tease out the chorionic villi. Check under the dissecting microscope to check thepresence of the villi and to confirm removal of maternal tissue.

Transfer the villi into a universal container, containing 5ml sterile trypsin/EDTA

0.25%. Incubate for one hour in a 370C waterbath.

Spin the universal at 2000rpm for 10 minutes. Discard the supernatant (rememberto work in a sterile environment). Be sure to remove as much trypsin/EDTA aspossible.

Add 3mls culture media. Resuspend cells by flicking the tube.

Spin the universal at 2000rpm for 10 minutes. Discard the supernatant. (This is toremove the last traces of trypsin).

Add 3mls culture media.

Transfer culture media and sample to the labelled culture flask, and leave for 2days.

After 2 days add another 2 ml culture media to the flask.

Check culture after 5-6 days. Gauge whether it is time to do a complete mediachange. There will be plenty of attached cells. If one does a media change, thenthe supernatant must be grown in flask C.

Harvest as per usual for amniocytes.


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Percutaneous umbilical cord blood sampling(PUBS) - a sample of blood is obtained from theumbilical cord while the fetus is in utero.

used for conditions in which rapid diagnosis ormanagement decisions are crucial - Rh diseasewith red blood cell isoimmunization, fetalhemolytic disease, hemoglobinopothies,coagulation defects, platelet abnormalities,evaluation of fetal acid-base status, rapidkaryotype and intrauterine infection such as

rubella, toxoplasmosis and cytomegalo virus Generally, the umbilical cord can be accessed

between 20-30 weeks gestation

Cordocentesis can be done from week 18 tillterm


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All samples will be treated as sterile and aseptic techniqueshould be used throughout.

Check and clean the air-conditioning and heating systems

Use new batch of culture media

Make sure culture tubes and flasks are sterile

Wash hands between handling of specimens Replace the culture room lab coat

Clean all working surfaces, equipment, stationary etc.with 70% alcohol

Work in a biosafety cabinet

Check incubator for any signs of contamination Work should be as sterile as possible as an amniocentesis is

a very stressful procedure for the patient and requesting arepeat specimen increase the risk of a spontaneousabortion. Therefore try to prevent infections by followingthe suggested guidelines.


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Sample log-in and initial setup

Tissue culture (feeding and maintaining cellcultures)

Addition of a mitotic inhibitor to arrest cells at

metaphase Harvest of cells from the culture. This step is

very important in obtaining high qualitypreparations. It involves exposing the cells to ahypotonic solution followed by a series offixative steps. This causes the cells to expand sothe chromosomes will spread out and can beindividually examined.

Chromosome are then stained and analysed todetect possible numerical and structural changes


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Phytohaemagglutinin (PHA)

a lectin found in plants

a number of effects on cell metabolism: it

induces mitosis, and effects the cellmembrane with regard to transport and

permeability to proteins

It agglutinates most mammmalian red blood

cell types In cytogenetic cell culture it is used as a

mitogen to trigger synthesis in T-lymphocytes


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RPMI 1640 Medium with L-Glutamine:

RPMI 1640 (Roswell Park Memorial Institutemedium) uses a bicarbonate buffering system

and differs from most mammalian cell culturemedia in its typical pH 8 formulation

L-Glutamine is an unstable amino acid and istemperature and pH dependant for stability

Fetal Calf Serum (FCS):

is a blood component from fetal calves whichcontains hormones, nutrients and other essential"ingredients" required to keep cells alive andgrowing in a tissue culture well

Almost always, FCS is used in conjunction with

MEM (Minimum Essential Medium).


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Colcemid:

Colcemid arrests mitotic cultured cells inmetaphase and it should be treated carefully,

since it is mutagenic, tumorigenic, andteratogenic.

Colcemid is recommended for use inchromosome analysis during lymphocytekaryotyping and amniotic fluid cell chromosome

analysis, and in cell synchronization.

Ethidium bromide

Ethidium Bromide is very poisonous! Avoidcontact with skin, especially once it is in

solution. Wear mask and Gloves when weighingout ethidium bromide.

Ethidium bromide is used to stretch thechromosomes. It intercalates between thenucleotide strands of the DNA, and makes them

longer and therefore easier to band and identify.


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Potassium Chloride (KCl) - hypotonic solution

A solution is hypotonic if it tends to lose water to areference solution (or colloidal suspension) separatedfrom it by a semi permeable membrane

This usually results from of a lower concentration ofdissolved or dispersed substances (and a highereffective concentration of water) in the hypotonicsolution

The membrane is assumed to be freely permeable to

the solute (water) but impermeable, or nearly so, tosubstances in solution or suspension in the two fluids

If a living cell is immersed in a hypotonic solution,water will move into the cell from the solution,causing the cell to expand or swell

Animal cells may eventually burst if they are unableto contain all of the incoming water

Methanol

Glacial Acetic Acid


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4 ml blood in Heparinized tube

Other coagulants toxic to cells

T-cells stimulated

Short-term culture 72 hours


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white blood cells grown in a special medium for threedays under the influence of the mitotic stimulant,phytohaemagglutinin (PHA)

After 72 hours, Colcemid, a chemical that inhibitsspindle formation, is added

The red blood cells are infused with a hypotonicsolution of potassium chloride which is thendestroyed with the addition of fixative

The remaining swollen white blood cells are fixedwith alcohol and acetic acid.

The resulting cells are dropped from a pasteurpipette onto a clean slide, causing the cells tosplatter and separate

When the slide is stained with Giemsa stain, thechromosomes of the individual white blood cells arevisible.


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All short-term culture methods are done in a

"closed" system, because the culture medium

is sufficiently buffered, at 7.2 - 7.4, to

maintain a few days worth of cell growth.

The culture medium has a pH indicator,

which will be dark pink in alkaline

conditions, and yellow in acid conditions.

Acid conditions can indicate excessive cellgrowth and subsequent media exhaustion; or

it can indicate bacterial infection (in this

case the media will also be quite cloudy).


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Bloods should be cultured within 4 days ofreceipt, and should be stored in the fridge,never frozen.

A minimum of 1ml heparinised blood is required

(sufficient for two cultures to be set up) Consider the following samples as urgent:

(mostly pre-arranged)

a) Cordocentesis

b) Blood from parents of fetus with a possibleabnormality

c) Blood from newborns with congenitalabnormalities


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Blood cultures are set up twice a week Tuesdays and Fridays

Cultures are incubated at 37oC for 3 days i.e. harvested on Mondays andFridays.

There are exceptions to the rule:

A blood sample can be harvested after 2 days if particularly urgent.

Other urgent samples can be set up on the day of receipt or FISH may be

performed at the clinicians request. Cultures can be incubated for four to five days over a long weekend.

Write up all samples to be cultured on the daily Culture Set Up Formforblood cultures/harvests. Take out the blood samples from the fridge anddouble check with the patient names and dates of birth.

Set up 2 universal culture tubes for each patient. One will be culture Aand the other in culture B. Label with name and laboratory number, A and

B. Also, mark for FISH and mark with a dot when it is a baby younger than1 month. Do not label the specimen tubes with the laboratory numberyet.

Work in the biosafety cabinet using sterile technique wash hands well,wear gloves for handling blood samples, clean the bench and pipettorswith 70% alcohol


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Culture Procedure for adults,

and children more than a month

oldCulture Procedure for infants

under one month, and

cordocentesisa) Put 1ml thawed FCS into

each tube

a) Put 5 ml RPMI into each tube.b) Add 5ml RPMI into each tubeb) Add 100ul PHA into each

tubec) Add 200ul PHA into each tube

c) Add 500ul well mixed blood

into each

tubed) Add 250 ul well mixed blood

into each tubed) Shake gently do not invert e) Shake gently do not inverte) Incubate at 37oC for 3 days f) Incubate at 37oC for 3 days


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One technologist sets up the patients of

culture A and a second technologist will set

up the tubes of culture B.

On the day of harvest, one technologist willharvest culture A and another will harvest

culture B.

Two Technologists will analyse 5 metaphase

spreads on each patient.


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Put a small bottle (10ml per tube to be harvested) of KCl into the 37oC incubator towarm up. Sign and date the blood culture/harvest sheet.

Add 100ul colcemid to each tube. Add 200ul colcemid to the culture tubes forbabies and cordocentesis specimens. Mix. Return the cultures to the 37oC incubatorfor one hour.

Collect a polystyrene container of crushed ice, to cool the fix.

Label 4 slides for each patient with the laboratory number and patient name.

I.e.: 2 slides for culture tube A and 2 slides for culture tube B. Place on slide racksin the fridge to cool down. Cold slides improve the spreading of the metaphaseplates.

Remove the cultures from the culture room incubator and spin for 10 minutes at2000rpm. Discard the supernatant into the waste bottle.

Resuspend the cells on the vortex mixer. Slowly add 10ml warm KCl to the cells.Place the culture tubes in the 37oC incubator for 20 minutes.

Spin the culture tubes for 10 minutes at 2000rpm and discard all but 0.5ml of the

supernatant into the waste bottle. Prepare the fixative (directly before use): 10ml acetic acid (dispenser on the sink)+

40ml cold methanol (in the fridge). NB Use the measuring cylinder provided for fix.Tip the fix into a small beaker, seal with parafilm. Keep the beaker in the fridge tokeep cold or place on ice.

Resuspend the cell pellet on the vortex mixer. Slowly add 5ml freshly prepared fix drop by drop.


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Spin the cells at 2000rpm for 10 minutes.Discard the supernatant into the waste bottle onthe bench. Resuspend the cells by flicking thetube.

Add another 5ml cold fix to each tube, drop by

drop. Spin at 2000 rpm for ten minutes. Discardthe fix into the waste container.

Repeat step 8 until the supernatant is clear.Usually two to three rinses in fix is all that isnecessary. (Cells can be stored at this stage,

suspended in 5mls fix, or as a cell button, in thefridge, until slides can be made).

After the last spin in fix, discard thesupernatant. Keep the cell button cool untilready to drip the slides


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Take the first set of labelled patient slides out of the fridge and arrangeon a tissue.

Flood the slides with cold fix. (10ml acetic acid + 40 ml cold methanol).Tip off the excess fix.

Suspend the cell button in the patient culture tube with 100 to 200ul ofcold fix (the volume of fix used will depend on the size of the cellbutton).

Drop about six drops of the cell suspension onto each slide, from a goodheight (about 30 cm). This encourages good spreading of the

chromosomes. Tip off the excess fix. At this time note if the sample isfor FISH. Be sure not to use the entire cell pellet for making slides.

As the slides develop a grainy appearance transfer them to the 60oCincubator, to heat fix the cells.

Store the remaining cells in about 1ml fix in the universal tube. The cellsin this tube

can be used for further investigations if necessary. Place tubes for FISH

in the FISH sample box in the fridge.

Once the case has been reported the samples are discarded in the wastecontainer (red bag).

After the slides have been in the 60oC incubator for 15 20 minutes, theycan be transferred to the 100oC incubator for 1 hour to age the slides inpreparation for same day g-banding. Otherwise the slides can be left at

60o

C for a maximum of 2 days.


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Trypsin G-Banding The trypsin is thought to collapse the

chromosome structure. This process is stoppedby the rinse in cold buffer. The stain then bindspreferentially to different areas of the DNA

forming the appearance of light and dark bands.Dark bands are rich in A + T and replicate late inthe S phase. They do not seem to have manyactive genes. Depending on the stage of mitosisat which the chromosome is banded one can see

from 400 up to 850 bands. 400 bands are the minimum acceptable standard

for certain clinical referrals (numericalabnormalities).

550 bands are the recommended standard.


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Set out four clean coplin jars: Place 2 of these into the icebox with the

ice.

Into jar 1 (at room temperature) place 30ml Oxoid buffer + one mlthawed trypsin together.

Into jars 2 and 3 (in the ice box) share out the remainder of the oxoid

buffer (pH 7.3).

Into jar 4 (at room temperature) add 40ml buffer pH 6.86 and 3 ml

Giemsa stain together.

Remove the slides from the 100oC incubator and allow to cool on thebench.

Dip the slide into the first jar and agitate for about 9 seconds. The time

in the trypsin jar will vary according to how well the slide has matured,

what the humidity was on the day it was prepared and how well the

trypsin is working. Finding the correct time for each patient comes with

trial and error. Rinse the slide quickly and thoroughly in the second jar. This stops the

trypsin activity.

Rinse the slide quickly in the third jar.

Stain in the 4th jar for 4 minutes.

Rinse the slide very briefly in tap water and allow to air dry before

analysing. Label the slide G1, G2, etc.


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Sort patients from the harvest into a priority list. Take the most urgentsamples to analyse first. Consider the following patients as urgent:

a) Cordocentesis b) Parents of fetus with a possible abnormality

Babies with congenital abnormalities

Special request from a clinician

Fill in a vernier sheet with patient details. See Blood FORMS VernierSheet

Once slides are dry, scan on low power (x10 objective) to find metaphaseplates.

Move to high power (x100 objective - oil) and count the chromosomes.

Analyse the quality of the plate to see if it is suitable for photographingand karyotyping: A good plate will have:

A good metaphase will have:

A good spread so that all the chromosomes are visible.

Fewer than 5 crossovers. Good clear banding

Fairly long for higher band resolution (important for miscarriage caseswhere small translocations can be detected on longer chromosomes).

Not too spread out

The relevant chromosomes will be well banded without crossovers


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If necessary do further stains to solveuncertain findings:

QUINACRINE stain to examine the Ychromosome, and also for distinguishingbetween long satellite arms on acrocentricchromosomes and extra material on the shortarms of these chromosomes.

C-BANDING: Centromere banding is done todistinguish between inserted material at thecentromere and normal variations ofheterochromatin at the centromere region.

NOR STAINING : The nucleolus organiserregion, also used for studying the satellitearms of acrocentric chromosomes


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The chromosomes stained by quinacrine show

a series of fluorescent bands representing

the AT-rich regions which are the same as the

bands produced by G-banding. Q-banding is

useful in identifying polymorphisms on the

satellites of the acrocentric chromosomes,

and is particularly useful in identifying the Y

chromosome which shows a remarkably

bright fluorescent region at the distal end of

the long arm.


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Switch on the fluorescent microscope. Makesure that the filter block number 1 (U-MWBV2) isin position.

The quinacrine stain is kept in a Coplin jar in thefridge. Stain the slide for 4 minutes at room

temperature. Rinse off the stain under running tap water.

Mount a rectangular cover slip onto the slideusing the phosphate buffer pH 6.86 solution.

Squeeze out any excess buffer.

Screen the slide under the fluorescentmicroscope, using the low power objective.When a metaphase spread is seen, use oil and goto the high power objective (X100) to analyse.


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Can be used to confirm the presence of the Y chromosome.The Y chromosome has a very bright heterochromaticregion at the telomeric end of the q arms. This region canvary in size, and although it is usually quite obvious withthe quinacrine stain, one should still analyse the samplecarefully before ruling out the presence of a Y.

If no Y is seen, then at least 10 cellsshould be screened torule out the Y chromosome in the event of female cellspresent due to maternal contamination e.g. in amnioticfluid.

Note the presence of large satellites and distinguishbetween satellites and extra chromosomal material on the

arms of acrocentric chromosomes. Bear in mind that there are cases when a part of the Y

chromosome is deleted and then it has no fluorescence.This is known as a non-fluorescing Y.

Write all findings on the vernier sheet and sign


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This stain produces selective staining of the

heterochromatin localised in the

pericentromeric regions of all chromosomesand also on the distal long arm of the Y.

C-banding is useful in confirming pericentric

inversions, and for identifying polymorphisms

of the centromeric regions of chromosomes1, 9, and 16.

It can also be used to confirm translocations

involving the Y.


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Preheat the 2 X SSC in the 60oC incubator. Rinse the slides in 95% alcohol.

Rinse the slides again in fresh 95% alcohol.

Wash the slides in Normal saline until they are clean andno longer have a greasy look.

Place the slides in a coplin jar with the saturated bariumhydroxide at room temperature for 10 minutes. Do notre-use the barium hydroxide.

Rinse the slides in 70% alcohol.

Wash in normal saline.

Place the slides in the hot 2 X SSC solution in theincubator and leave for three and a half hours at 600C.

Wash the slides well in hot tap water.

Stain the slide flat with the buffered Giemsa for threeminutes. Rinse in tap water. Air dry and examine underoil.


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This is a silver stain that stains the nucleolus

organiser region (NOR).

It is useful for studying variations in the size

of the stalks and satellites of the acrocentricchromosomes.

The NOR region is responsible for the

formation and maintenance of the nucleolus

in interphase nuclei. It is made up ofmultiple copies of DNA sequences for

ribosomal RNA.


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WEAR GLOVES: silver nitrate stains the skin!!

Place unstained slide in coplin jar filled with distilled water. Place in 37Cincubator or water bath for 2 hours. Remove slide and allow to air dry.

Prepare a moist chamber by placing wet paper towels at the bottom of a plasticbox. Create a raised platform using a glass staining dish lid, to ensure the slides arelevel and raised. Seal the box with a lid.

Place 5 7 drops of freshly prepared silver nitrate solution near the label end ofslide using a syringe.

Using a pair of forceps, lower one end of pre-cleaned cover slip into the drops ofsilver nitrate solution on the slide and gently lower cover slip to avoid trapping airbubbles.

Transfer the treated slide to a moist chamber and support on platform above wetpaper. Seal the box.

Carefully place moist chamber in the 37C incubator. Ensure that treated slide ishorizontal in the incubator.

Incubate overnight at 37C.

Carefully remove the slides from the moist chamber and using a squirt bottle ofdistilled water, wash the cover slip and silver nitrate solution into the sink (with tapwater running). Discard the cover slip into the sharps container using forceps. Donot dismantle the moist chamber.

Rinse the slide in a coplin jar of distilled water. Allow to dry.

Check the treated slide under 10X phase and 60/100X phase to judge effectivenessof first treatment.

If stained NORs are not apparent, repeat steps 3-10.

Repeat treatment at hourly intervals two or three times if necessary.

Counterstaining is not necessary.


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Prenatal Dx Lesson 4