I can't find metaphases in my blood harvest.

[Tiffany Kamila]

Hello everyone, I am having problems seeing metaphases through my
microscope. I incubate peripheral blood for 72 hours in 5% CO2 at 37
degrees. I used Gibco reagents such as PBmax and 0.075M of KCL for my
procedure. Yet, I am not seeing any metaphases. Any suggestions? Also,
I use a 10ml flask and loosen the cap, but no results. What should i
do?

[Jernej Kovač]

I saw no mention of arresting factor in your procedure description...so...cca. 30 - 20 min prior to harvest add cca. 50uL (depends on your culture volume) of colcemid to arrest cells in metaphases. 

good luck and best regards,

Jernej

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Jernej Kovač
GSM: +386 40 250 128
e-mail: jerne...@gmail.com

[Dr Muhammad Bilal Bin Majeed]

Dear Kamila,

Jernej mentioned a reasonable point. Other thing, which might be in hindrance, would be the procedure of slide making. How do you prepare the slides and stain them? Have a check again.

Best Wishes and Kind Regards,

 

Dr. Muhammad Bilal Bin Majeed
D.V.M. (RVMP)
UVAS, Pakistan
(0092) 0336 40 275 40

[Maliszewska Chris (NHS TAYSIDE)]

Hi

 

I agree with the comments about colcemid to arrest metaphase. Also didn't see that you were stimulating cultures, but maybe that was just not included. For constitutional analysis of peripheral blood use PHA, but it's not suitable for bone marrows.

 

May be telling you what you already know - but I hope it helps. A really useful source is the AGT manual - I have attached a link - lots of protocols and few nice pictures

 

http://www.agt-info.org/pdfs/Lab%20Manual%20Order%20form.pdf

 

Chris Maliszewska

Clinical Scientist (Deputy Head of Cytogenetics)

Ninewells Hospital, Dundee DD1 9SY

01382 496735

 

---

From: cytogenetics-methods...@googlegroups.com [cytogenetics-methods...@googlegroups.com] On Behalf Of Jernej Kovač [jerne...@gmail.com]
Sent: 11 February 2012 10:52
To: cytogenetics-methods...@googlegroups.com
Subject: Re: I can't find metaphases in my blood harvest.

I saw no mention of arresting factor in your procedure description...so...cca. 30 - 20 min prior to harvest add cca. 50uL (depends on your culture volume) of colcemid to arrest cells in metaphases. 

good luck and best regards,

Jernej

On 10 February 2012 16:02, Tiffany Kamila <tiffan...@gmail.com> wrote:

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Jernej Kovač
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[Roderick MacLeod]

In our experience lost metaphases are often due to over-harsh hypotonic
causing metaphases to burst. This could be due to cellular
hypersensitivity - different tissues & species each seeem to have their
own preferences - or even miscalulation. Next time try freshly prepared
hypotonic and harvest 1 min and 7 min cultures in parallel, and even
"softer" 1:1 KCl+NaCitrate.
Rod

[majd]

Sometimes the collection tube is not the correct one. CHeck it was a green top tube that contains heparin

Also, I do not see that you have used colcemide? what was the time used? should be 1microliter  per 1ml is what we do here

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[Aviv, Hana]

How about adding colcemid?

Sent from my iPhone

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[Tiffany Ajayi]

Thank you everyone for the feedbacks.  Yes, I used colcemid. I just dont understand. I am currently in Nigeria, and I worked in a cytogenetics Lab in the US, I don't know if I should alter the procedure due to difference in climate. Should I increase the volume of blood i add to the media? Also I have used PHA and still no metaphases. Could it be possible the the media is not good. Unfortunately I order my media from UK and it takes about a month to be delivered. As anyone experience this before?  the Tube might be a good suggestion, but I culture the blood soon after the blood is drawn. Lastly, if the CO2 incubator was shut off for few hours, would it affect the harvest outcome? 

--
Tiffany Titilope Ajayi
Bachelor of Science  in Neurobiology, Physiology & Behavior Science
University of California, Davis.

[AmandaH]

Hi Tiffany,

We have had problems on occasion with the Methanol used for harvest.
We had some metaphases spreads but they were very poor. When we
changed the brand of methanol the difference was enormous. We believe
that the problem was due to water in the original batch of methanol.

Also, given that all your basic steps are there I would be looking at
every component you use for every step. The quality of the water used
to make the hypotonic solution can have an effect. The tube or flask
used for culture can be toxic so you could try a different tube. The
media may be a problem. We have at times had different results from
different brands of apparently identical media but I don't know what
you do if it takes a month to get to you. If you are using media with
L-glutamine already added it may be worth adding more to the media as
it has a limited life span.

It isn't likely to be the amount of blood you are adding to the
culture. You would still expect to see metaphases. We add 0.4ml to a
5ml culture for an adult if that is any help.

Unfortunately there are no easy answers. I hope you work it out.
Cheers,
Amanda

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> Tiffany Titilope Ajayi
> Bachelor of Science  in Neurobiology, Physiology & Behavior Science

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[EVANGELIDOU Paola]

We do not use a CO2 incubator for our Peripheral Blood culturing. Also do you use antibiotics in the cultures? Check that the they are not expired.

 

From: cytogenetics-methods...@googlegroups.com [mailto:cytogenetics-methods...@googlegroups.com] On Behalf Of Tiffany Ajayi
Sent: Tuesday, February 14, 2012 5:41 PM
To: cytogenetics-methods...@googlegroups.com
Subject: Re: I can't find metaphases in my blood harvest.

 

Thank you everyone for the feedbacks.  Yes, I used colcemid. I just dont understand. I am currently in Nigeria, and I worked in a cytogenetics Lab in the US, I don't know if I should alter the procedure due to difference in climate. Should I increase the volume of blood i add to the media? Also I have used PHA and still no metaphases. Could it be possible the the media is not good. Unfortunately I order my media from UK and it takes about a month to be delivered. As anyone experience this before?  the Tube might be a good suggestion, but I culture the blood soon after the blood is drawn. Lastly, if the CO2 incubator was shut off for few hours, would it affect the harvest outcome? 

[Maliszewska Chris (NHS TAYSIDE)]

If you're saying the CO2 supply was shut off for a few hours - that shouldn't be a problem - I have never used CO2 for blood cultures - and they have always been fine. If you're saying the power was shut off - yes - that is a problem, and would almost certainly spoil a harvest.

 

 

Chris Maliszewska

Clinical Scientist (Deputy Head of Cytogenetics)

Ninewells Hospital, Dundee DD1 9SY

01382 496735

 

---

From: cytogenetics-methods...@googlegroups.com [cytogenetics-methods...@googlegroups.com] On Behalf Of Tiffany Ajayi [tiffan...@gmail.com]
Sent: 14 February 2012 15:41

To: cytogenetics-methods...@googlegroups.com
Subject: Re: I can't find metaphases in my blood harvest.

Thank you everyone for the feedbacks.  Yes, I used colcemid. I just dont understand. I am currently in Nigeria, and I worked in a cytogenetics Lab in the US, I don't know if I should alter the procedure due to difference in climate. Should I increase the volume of blood i add to the media? Also I have used PHA and still no metaphases. Could it be possible the the media is not good. Unfortunately I order my media from UK and it takes about a month to be delivered. As anyone experience this before?  the Tube might be a good suggestion, but I culture the blood soon after the blood is drawn. Lastly, if the CO2 incubator was shut off for few hours, would it affect the harvest outcome? 

On Mon, Feb 13, 2012 at 3:43 PM, Aviv, Hana <avi...@umdnj.edu> wrote:

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Tiffany Titilope Ajayi
Bachelor of Science  in Neurobiology, Physiology & Behavior Science
University of California, Davis.

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[Jernej Kovač]

Its possible that your medium is not properly stored during delivery. Long exposures to high temperatures will destroy the medium. From my own experience there is no problem if the CO2 is shut for up to 8hours... the metaphases were not affected. 


Good Luck,

Jernej

[Roderick MacLeod]

HalloTiffany: I know it is tough getting reagents shipped in to certain African destinations. While culture medium is pretty robust, serum supplements contain cytokines which are labile. We generally test serum samples on fastidious cell lines for proliferation. A shockingly high percentage are dud. You may have bought a dud batch. As an alternative, I wonder if you could find a local slaughterer who would sell you FCS in bulk? Even if you have to discard batches its got to be cheaper than commercial stuff. Rod