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» LymeNet Flash » Questions and Discussion » Medical Questions » The Microscopy Thread (Page 21)

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bluelyme
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tnt whats this 1 step giemsa ....

mustard when i use 3 step giemsa with phase 1200X i swear i see little myco or barts? freckle specks on edges of cells is that what you were seeing?

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Blue

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TNT
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quote:
Originally posted by bluelyme:
tnt whats this 1 step giemsa ....

mustard when i use 3 step giemsa with phase 1200X i swear i see little myco or barts? freckle specks on edges of cells is that what you were seeing?

Blue, the stuff I use is a one step Wright-Giemsa from Astral Diagnostics. No fixative step needed because it already contains the methanol and it fixes the sample the same time it stains it. Free samples at:

http://www.astraldiagnostics.com/hematology/quick-i

1-800-441-0366

I use the Quick-I "Blue" (Ha Ha), and Quick-I "Red." If you call them, they should be able to send you samples of both. Just remember to use sterile pipettes to transfer about 1ml of stain from your bottle of stain to your slide. Otherwise you contaminate the (bottle of) stain and that can introduce artifacts and contaminates to your sample when staining and to successive samples.

Contaminated stain can produce those tiny "Bart" dots en-mass on your sample, but more likely it's from unwashed slides. I find that even the "pre-cleaned" slides need to washed with soap and water before using them. I've been experimenting by placing the slides on a stainless steel slide holder and putting them through the dishwasher. But, they are still not getting clean. I am having to revert back to hand-washing with soap and water because of those "Bart" dots. It's very frustrating when a slide contains those dots because there is no way to determine if the blood sample truly shows the presence of Bartonella or Mycoplasma. That's why it's imperative to make sure the slides are thoroughly cleaned and dried before making a smear.

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bluelyme
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i'm gunna try it out tnt... they just sent some said id have to go thru distributor if i liked . they don't do private practice....1 step im 66% more efficient thanks

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Blue

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TNT
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It can happen.....IN THE U.S. : Malaria infection via BLOOD TRANSFUSION!!! And, the blood had been irradiated!

https://labmedicineblog.com/2018/04/16/blood-bank-case-study-transfusion-transmitted-malaria/

Blood Bank Case Study: Transfusion Transmitted Malaria

Case Study

A 26 year old African American female with sickle cell anemia presented to a New York emergency room with cough, chest pain, fever and shortness of breath. Laboratory results showed an increased white blood cell count, slightly decreased platelet count and a hemoglobin of 6.2 g/dl. Her reticulocyte count was 7%, considerably below her baseline of 13%. Consulting the patient’s medical records revealed history of stroke as a child and subsequent treatment with chronic blood transfusions. She was admitted to the hospital for acute chest syndrome and aplastic crisis and care was transferred to her hematologist. Two units of RBCs were ordered for transfusion.

The blood bank technologists checked the patient’s blood bank history and noted her blood type was A, Rh(D) positive, with a history of a warm autoantibody and anti-E. The current blood bank sample confirmed the patient was blood type A, RH(D) positive with a negative DAT but the antibody screen was positive. Anti-E was identified. Per request of the hematologist, phenotypically similar units were found and the patient was transfused with 2 units of A RH(negative), C/E/K negative, HgS negative, irradiated blood. The patient’s hemoglobin rose to 8g/dl and she was discharged from the hospital 3 days after transfusion.

Ten days after discharge the patient returned to the emergency room with symptoms including aching muscles, fever and chills. A delayed transfusion reaction was suspected. A type and screen was immediately sent to the blood bank. The post transfusion type and screen remained positive for anti-E, DAT was negative. No additional antibodies were identified. However, a CBC sent to the lab at the same time revealed malarial parasites on the peripheral smear. The patient was consulted for a more complete medical history and reported that she had never traveled outside of the country. A pathology review was ordered and the patient was started on treatment for Plasmodium falciparum.

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Discussion

Red Blood cell transfusions can be life saving for patients with sickle cells anemia. These patients are frequently transfused by either simple transfusion of red cell units or by exchange transfusion. Because of this, alloimmunization is reported to occur in 20% to 40% of sickle cell patients.(1) Blood bank technologists are very diligent in adhering to strict procedures and follow a standard of practice aimed to prevent transfusion reactions. While preventing immune transfusion reactions may be the most forefront in our minds when transfusing the alloimmunized patient, it is important to consider transfusion transmitted diseases as a potential complication of blood transfusions.

Malaria is caused by a red blood cell parasite of any of the Plasmodium species. Mosquito transmitted infection is transmitted to humans through the bite of an infected mosquito. Transfusion-transmitted malaria is an accidental Plasmodium infection caused by a blood transfusion from a malaria infected donor to a recipient.

Donors, especially those from malarial endemic countries who may have partial immunity, may have very low subclinical levels of Plasmodium in their blood for years. Even these very low levels of parasites are sufficient to transmit malaria to a recipient of a blood donation. Though very rare, transfusion-transmitted malaria remains a serious concern for transfusion recipients. These transfusion-transmitted malaria cases can cause high percent parisitemia because the transfused blood releases malarial parasites directly into the recipient’s blood stream.

Blood is considered a medication in the United States, and, as such, is closely regulated by the FDA. Blood banks test a sample of blood from each donation to identify any potential infectious agents. Blood donations in the US are carefully screened for 8 infectious diseases, but malaria remains one infectious disease for which there is no FDA-approved screening test available. For this reason, screening is accomplished solely by donor questioning.(2) A donor is deferred from donating if they have had possible exposure to malaria or have had a malarial infection. Deferral is 12 months after travel to an endemic region, and 3 years after living in an endemic region. In addition, a donor is deferred from donating for 3 years after recovering from malaria. It is important, therefore, for careful screening to take place by questionnaire and in person, to make sure that the potential donor understands and responds appropriately to questions concerning travel and past infection.

Malaria was eliminated from the United States in the early 1950’s. Currently, about 1700 cases of malaria are reported in the US each year, almost all of them in recent travelers to endemic areas. From 1963-2015, there have been 97 cases of accidental transfusion-transmitted malaria reported in the United States. The estimated incidence of transfusion-transmitted malaria is less than 1 case in 1 million units.(4) Approximately two thirds of these cases could have been prevented if the implicated donors had been deferred according to the above established guidelines.(3) While the risk of catching a virus or any other blood-borne infection from a blood transfusion is very low, a blood supply with zero risk of transmitting infectious disease may be unattainable. With that being said, the blood supply in the United Sates today is the safest it has ever been and continues to become safer as screening tests are added and improved. Careful screening of donors according to the recommended exclusion guidelines remains the best way to prevent transfusion-transmitted malaria.

References

LabQ, Clinical laboratory 2014 No.8, Transfusion Medicine. Jeanne E. Hendrickson, MD, Christopher Tormey, MD, Department of Laboratory Medicine, Yale University School of Medicine
Technical Manual, editor Mark K. Fung-18th edition, AABB. 2014. P 201-202
https://www.cdc.gov/malaria/about/facts.html. Accessed April 2018
The New England Journal of Medicine. Transfusion-Transmitted Malaria in the United States from 1963 through 1999. Mary Mungai, MD, Gary Tegtmeier, Ph.D., Mary Chamberland, M.D., M.P.H., June 28, 2001. Accessed April 2018
Malaria Journal. A systematic review of transfusion-transmitted malaria in non-endemic areas. 2018; 17: 36. Published online 2018 Jan 16. doi: 1186/s12936-018-2181-0. Accessed April 2018
http://www.aabb.org/advocacy/regulatorygovernment/donoreligibility/malaria/Pages/default.aspx

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Lymedin2010
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What???? There are spirochetes in Lyme Disease victims blood....say it ain't so? [Smile]

So, we should lyse the Red Blood Cells in order to see them better and/or detect them via PCR. Sounds like what I had tried to do with the blood freezing method & found a few nice spiros sticking out like a sore thumb.

Here is one such nice spiro, with well definite spirals from my blood after it was frozen in order to lyse many/most of the red blood cells.  -

"Abstract

Lyme disease is the most frequently reported zoonotic tick-borne disease worldwide, and the number of infected humans is increasing. Lyme disease (or Lyme borreliosis) is an affection caused by the spirochete Borrelia burgdorferi, sensu lato. Lyme disease is also reported as a variety of misleading clinical symptomatologies. Infected patient’s blood serology is the most currently test used for its diagnosis. However, serology has a low sensitivity, which ranges from 34% to 70%.

Thus, there are numerous subsequent false-negative diagnoses despite an active clinical infection profile. Therefore, alternative and more sensitive techniques are required to detect the antigens or nucleic acids of Borrelia. Actually, the most appropriate methodological approach seems to be the polymerase chain reaction (PCR). However, PCR will detect the only “visible” part available of the targeted DNA presence in the blood of the infected patients. Consequently PCR alone will not be conclusive enough to reach the final diagnosis. Considering the ability of Borrelia to invade host cells, we hypothesize that a selective lysis of all blood cells should improve the diagnostic sensitivity of the detection of Borrelia by PCR in whole blood, and subsequently reduce the false-negative diagnostic rate, thus improving the patient’s diagnosis and therapeutic management."

https://www.sciencedirect.com/science/article/pii/S0306987718302421

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Lymedin2010
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Bluelyme, do you have any pics of your stains?

TNT, have you done any more fluoro work? Anything new?

I finally got to do some "normal blood" Acridine Orange staining & I did not find anything within the rbc's such as what look like co-infections in mine. No spirochetes either. I did find spirochetes in my wife's blood, but she did not have intra-erythrocytic parasites though. I still want to do more normal blood AO staining & to spend some more time on it. I would be more than happy if I see spirochetes in everyone's blood, then I could dismiss them as being other than Borrelia b. or artifacts altogether.

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bluelyme
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or everbody already really does have it?!...i will post some 1 step stains as they shipped me sample like tnt said...

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Blue

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TNT
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That's interesting Lymedin, but not surprising that you didn't find the intracellular objects nor spirochetes....Because chronic Lyme disease is real and many healthy people will be without the infection.

But, I also believe there are "healthy" people walking around with these things in their blood as well.

Regardless, those things are NOT ARTIFACTS!!!!!

I have less time to use my scope, but have looked at two people's blood in the past month. My acridine orange dried smears just will not turn out. The first person's samples did turn out good enough for me to get some good pics, but the more recent ones were a total flop. I'm beyond frustrated with the AO. I do everything according to the book, but still no success. I wish I knew some local lab personnel to consult about it.

If you're interested in my dried stains Lymedin, I'll try to find time to post a couple pics of them. I have not done any more work with AO wet mounts.

Blue, I can't wait to see some of your stains!!!

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BorreJaakko
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I am working on creating a deep learning model that would allow computer to automatically recognize spirochetes, first from images and later perhaps also from videos.

For this, I would need a collection of images of blood both with spiros and without spiros (with maybe some things that would resemble spiros), preferably from various sources.

I have thought about starting with darkfield non-stained samples as that is what I have as my home setup.

Do some of you wish to participate in this effort?

If you have images, that would be preferable, but videos could work too.

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Lymedin2010
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Looking forward to pics.

TNT, I have never tried dried stains, only live preps. I can look at your dried stained pics. I wish I had more energy & put more effort into looking at more normal blood.

BorreJ. that is quite a task & good luck. I sent you a link to some Giemsa/Wright stained pics on FB & that nice vid. I would only utilize proven stains or fluorescent work for references to make it official & really Borrelia. At best I think you can program a unit to take pictures of all things string-like & give a high priority notifier/count for zig-zag shapes. AO staining would make your project much easier & stand out in the identification & much easier for your program/video capture to spot.

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BorreJaakko
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quote:
BorreJ. that is quite a task & good luck. I sent you a link to some Giemsa/Wright stained pics on FB & that nice vid. I would only utilize proven stains or fluorescent work for references to make it official & really Borrelia. At best I think you can program a unit to take pictures of all things string-like & give a high priority notifier/count for zig-zag shapes. AO staining would make your project much easier & stand out in the identification & much easier for your program/video capture to spot.
Yes, saw it, thank you!

Great insights. This is most likely what I will do.

My idea was to make some part of the work a group effort, I have started to build a platform for sharing the work.

However, if not too many people are interested, what you are suggesting is a way forward for me.

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Lymedin2010
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A new cyst forming video popped up.
https://www.youtube.com/watch?v=Khj8CQBHjMg

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thatdudefromkansas1
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https://youtu.be/1A3T2uKP4HQ

New video to share

Just test.
Fresh blood draw, no culture.
Higher load that seen previously, under no treatment currently.

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Just researching and sharing.

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thatdudefromkansas1
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Should note that I have been messing with the saturation and everything in the videos.

Looking for a video sharpening tool if anyone has any suggestions.
I use Final Cut ProX but it doesn't have a native sharpening tool in editing.

So some things are very bright in the video, also compounded by the really bright light source I use and the adjustments on the objective and the condenser.

I have the objective adjusted to just below the aperture of the condenser, to the point that it is right at the cusp of losing the darkfield and being brightfield.
So there is some aberration/artefact from that.

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Just researching and sharing.

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thatdudefromkansas1
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And let's not let this thread die!

I've just been gone, and can't login to my old username anymore.

Was blocked when I was overseas from logging in.

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Just researching and sharing.

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thatdudefromkansas1
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Anyone have tips as far as AO staining?
Setup needed, and where to acquire it.

I haven't looked into it at all.

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Just researching and sharing.

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bluelyme
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glad to see you back dude ! pm tnt, he was messing with ao ...also a guy on healing well john b ...but he has some funny pics ...there maybe a post back a few pages

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Blue

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DonN
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This post went to the wrong place last night, I'm trying again.

I just found this thread today. I've been looking at my blood smear for a couple of months now and making video's. My wife says it's my new addiction. I've been on the buhner protocol for about 4 months now. In the beginning I saw these (things) everywhere in my smears, now very few. I would greatly appreciate your opinions on what it is.
https://www.youtube.com/watch?v=7bc1ou6tySo

https://www.youtube.com/watch?v=gC3ZDJF8xo0

The last one, is at the 2:00 minute mark

Thanks, Don

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Lymedin2010
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Duke: That is such an awesome video! For AO staining you can obtain from Amazon.com or Ebay.

I used .02% in this link, but I needed to dilute it down & so you can get the .01%, which is cheaper and you will probably have to dilute it down as well even with .01%:
https://www.amazon.com/Acridine-Orange-0-2-Aqueous-500ml/dp/B00K33JVKO/ref=sr_1_7?ie=UTF8&qid=1534266928&sr=8-7&keywords=acridine+orange

I explain how I diluted it down in this video description. After, I just take a drop of my diluted AO on 1 drop of blood & seal the cover slip sides with oil, even though I was only using 40x. I only seal the sides of the cover slip & not the entire cover slip. Some of the stains take right away & as time progresses more & more of the cells absorb the stain. Sometimes you have to wait 1-2 hrs, a few hours, or even the next day more will get absorbed & the stains will be brighter.
https://www.youtube.com/watch?v=qlL8ZLitiBI

Check out this vid to explain OA staining theory.
https://www.youtube.com/watch?v=zg_H_OaeTjA

Wiki:
https://en.wikipedia.org/wiki/Acridine_orange

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Lymedin2010
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DonN great video captures. In the 2nd video does it look like they are spiraling with true spirals. Hard to see clearly, but almost looks like it.

That is what we are calling spirochetes, but more testing is needed to know for sure. Best is to use DNA specific stains to know the exact species when it is a living subject.

I have also discovered that the rbc's can shed their wall & create string-like subjects as well.
https://www.youtube.com/watch?v=sPSnvQtsNkE&t=137s

What I noticed is that when I first got sick I could not find them in my blood & as I got sicker I was able to find them more & more and nowadays they are plentiful in my blood.

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TNT
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quote:
Originally posted by Lymedin2010:
Thanks TNT! I think collectively we did & learned a lot together.

Looking forward to seeing some new stuff from you.

Not long ago I did a wet mount stained with AO and was able to see quite the number of string spirochete to round body (spheroplast) conversions. But, I had started using a new camera and thought I was recording video when in fact I simply had video mode on, but was not recording. So, when I went back and looked at those videos there was only one saved from that sitting and it was only a couple-second clip that unfortunately did not catch the actual conversion. I was so disappointed. BUT, I am going to get this for you guys to see!

This is not exactly what you are hoping for, Lymedin2010 (but like I said, I'm working on that), but recently I got my acridine orange dried smears to turn out for the most part. And, very recently I got some amazing pics from one of those dried AO smears!

I repeat, pics from dried acridine orange smears.

Check these out! Let me know if you guys can't view the links.

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[ 01-04-2019, 11:13 PM: Message edited by: TNT ]

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