Microscopy question (giemsa stain)

[Josie]

Hi I’m new here.

I have 50+ symptoms, got diagnosed with Borreliosis and co-infections and I'm currently taking azithromycin, tinidazole and rifampicin.

My IgG and IgM were both negative for Babesiosis (B. divergens and B. microti).

I read in this thread about live blood microscopy: http://www.lymeneteurope.org/forum/view ... f=6&t=5763

Black and white images are insufficient for direct microscopic diagnosis of Babesiosis.
You must have a proper giemsa stained preparation

Thank you very much Alan MacDonald.

A friend (also lyme-patiënt) did a giemsa stain on my blood a while ago, see pictures.

Please can anyone help brainstorming what it might be, possibly Babesia?

Does any of you know if there's a test for Babesia Venatorum (EU1) available? (where?)

Edit to add extra picture

[inmacdonald]

Reply: testing for Babesia EU1
Each strain of Babesia requires its own blood test kit.
A kit optimized for EU 1 might be available in a European Country.

Babesia microti blood test kits are available through Commercial
Laboratories in the USA.

Do not throw away the possibilities of strains of Babesia - with
TBD vectored by European Tick vectors.
Reply : Optimization of Giemsa Stain ( or combination Wright Giemsa stains combined
For Oil immersion microscopy to detect Ring trophozoites of
Babesia inside of Parasitized Red Blood cells:
1. Prepare a Thin Bolld Smear
2. Dry the blood smear THOROUGHLY- any bubbles - ( water bubbles)
Interfere with oil immersion exam for Babesia Ring Trophozoites
3. Consider PHASE CONTRAST MICROSCOPY- it gives sharper focal planes of resolution
4. Coverslip ( glass or plastic) is not necessary for Oil Immersion exam
5. Spend at least 30 minutes per smear under oil immersion to
Detect low parasitemia Babeaia infections.
6. Use Animal inoculation with the blood of the suspect case for
An extra level of check. Babesia will proliferate in Hamster or Gerbil.
A "snip" og the tail, will provide a drop of blood for smear and exam.
Quality Control: Is your blood smear a good preparation
1. Absence of water bubbles!!!
2. The Platelets should have visible granules - if platelets look GRAY -
( I.e. - No granules seen in the human platelets) =. Poorstaining quality
3. For visible ring Trophozoites forms -- Do the Rings have a BLUE "band" and a RED
DOT- like " jewel"? Properly stained Babesia trophozoites have beautiful
Chromatin bodies ( red " jewel"). And beautiful blue ring bands.

Use Multiple focal planes for every oil immersion Babesia smear.
Constantly focus UP and DOWN. While under oil immersion.
You will be amazed at the extra "Pickups"
Best
Alan B MacDonald, MD , FCAP
Sept 21
2015

[Josie]

Dr. MacDonald,
Your reply and knowledge are much appreciated! Thank you for tips on focussing, I can’t wait to get even more amazed then I already am.

I will indeed not throw away possibilities of other strains of Babesia and keep on searching for a lab with a testkit for EU1 (and that’s also willing to cooperate), I live in the Netherlands.

Thank you for the points of extra care, I think they were all checked, but I will re-check at my friend to make sure. Also about air/water bubbles.
The smear was thin, with much care giemsa-prepared and it was far more then 30 minutes being 'eyeballed' under oil immersion.

I don’t have (access to) a phase contrast microscope to get sharper images. (unfortunately)


I don’t have a hamster or gerbil, but animal inoculation sounds nice to try and for getting some extra info.

I have a cat, likely has bartonella… I’m not sure, maybe his bacteremia surpasses mine… but do you think if it can be of any use to try a drop of his blood to inoculate? Or is it only possible with hamsters and gerbils...


These extra pictures are from another smear, from a few weeks ago. This smear was thinner than the one before.
Platelets look purple, not grey.
Is this somewhat like what you mean by visible granules?

Babesia's blue ring and red dot, I’m not sure, looks like it, or more like purple and pink, but we will put another extra focus on it on our next smear in 3-6 months. (we’re both very sick and tired and it’s a lot of work doing it carefully (as you know), we can’t be busy with microscopy as much we’d want.)

I will look deeper into it. Thanks for helping out and
keeping me motivated.

I’m sorry if my english is with flaws.

[inmacdonald]

MACDONALD'S Comments:

1. Gray platelets in the images - sub-optimal staining - you must see the fine granules inside of the platelets for optimal staining of Ring torphozoites
of Either Babesia species
Or
Of malaria Ring trophozoites.
2. Refractile -- not good --- ideally the Red Blood cells containing Babesia - will show NO REfractile change aroung the Ring trophozoites.
Please avoid consideation of Any Refractile stucture - on top of Red blood cell - It does not count
3. Only Intracelluar Ring Trophozoites count! since the Ring forms are Inside of the Red blood cell - the plane of sharp focus is identical
No REfractile material ever counts as being Intra-erythrocytic.

Suggestion:
obtain a new set of smears
Air dry - no moisture bubbles
Air dry - avoid Dust on the slide- Dust is reflective!

Good luck and keep with it.
Best,
Alan

[Josie]

Dear dr. MacDonald,
Thank you very much for your comments and suggestions!

My slow brain is giving me a hard time…. I have trouble recognising what you mean but really want to learn your directions to achieve optimal staining.
Please let me know if this is what you mean, correct me if I misunderstood or maybe if it’s not what you mean can you please pinpoint to me on a picture where I should pay attention to?

1. The picture ‘giemsa2' gives an example of an air/water bubble because for example the one in the left corner at the bottom seems not to be intracellular? And I should be able to avoid that by better air dry?

2. And for example on ‘giemsa1' the right one is likely to be air/water bubble because of refractile structure? Which can be best checked when focussing up and down? And avoided with better air dry?

3. And for example on ‘giemsa6' there’s dust on the slide because there are more different kind of things to see?
I remember I didn’t clean that one before putting my blood drop on it.

4. And the platelets don’t show as much granules as would be possible with an improved giemsa quality, trying untill the fine granules are clearly visible. And the way platelets and maybe also the wbc’s look can give extra info on if the giemsa is of okay quality to detect other stuff like ring trophozoites?

And then… after all this is solved… and we have a new smear… we can look at what’s left…

Good luck and keep with it.

Thank you very much!

Best regards & hugs,
Josie

Another picture:
Do you think this wbc shows the same issue of suboptimal staining as the platelets do?

[inmacdonald]

I see sub-optimal staining in ALL of the slides posted so far on this thread.
The platelets are Gray- [ devoid visible internal granules]
There are Water droplet type bubbles which are refractile.[ these sit "on top of" the RBC, and not Inisde of the RBC]
These are artefacts.

Babesia ring forms are never refractile.[ you MUSt image the Red chromatin dot of the Trophozoite and the blue band of the trophozoite0
They lie INSIDE of the parasitied RBC and are in the same plane of focus.
Water droplet type Refractile artefacts are never in the same sharp RBC focal plane.

You must start over.
I recommend the Quality improvement steps above.
Best to you,
Alan B.MacDonald MD, FCAP

[Josie]

Dr. MacDonald,

Thank you very much for your guidance, explanation and your time! It’s of great help to learn where to pay attention to. Of course we will start over and keep on practicing!
And first still without the phase contrast and the hamster…
and I hope I didn’t offend you with all my bloody artefacts?

What are your comments about this one on 400x darkfield, it’s my blood a while ago before I started abx:
https://www.youtube.com/watch?v=-RW5gQ2Ao_E

Spirochetes or just fibrin or so in brownian motion? Any tips for improvements?

Thank you & Best to you,

Josie

[S13]

I just wanted to jump in, because im the person who made these giemsas for Josie.

There was indeed suboptimal staining. From what i know the pH of the solution was probably not optimal. I noticed the pH of the giemsa solution greatly influences the coloration of the sample.
For josies giemsa we actually separated the fixation and coloration step. Josie did the sampling, drying and fixation with 100% methanol at home. Then the samples were shipped to my house, so i could perform the giemsa coloration step a couple of hours later. Perhaps this influenced the quality of the sample as well? Interesting to hear the bacteria like things on top of the RBC's are probably water droplets.
Ive already mentioned to Josie that i suspected that some of these objects appear not on the same level of focus as the RBC's. So that means they are on top of the RBC's. You can see that in the first picture posted in this thread: The RBC's are a bit out of focus, while the blueish objects appear in focus.
So Dr MacDonald, i think you are correct in your observations.

I think we need to modify the drying step, and i still need to figure out what goes wrong with the pH of the stain here.

Im still not exactly sure what you mean by grey platelets though? The platelets seem to be colored quite deeply, but perhaps lack a bit of detail due to limited microscope resolution? (its just an older binocular olympus microscope with a simple digital camera for image capturing)