Biomedical Science

I'm a lab tech currently working in a medium sized hospital in the hematology lab. This blog is just a little challenge for myself to keep learning about my job.

[the-art-of-medicine]

RBC Abnormal Morphology

Weiterlesen

[bmedizinischa]

Masterpost

I’ll better start this right now. ;-)

Histology - Special stains I (Giemsa, May-Grünwald-Giemsa, Ziehl-Neelson, Orcein) - Special Stain II (Auramin-Rhodamin, SFOG, Silver, Silver-Methenamin)

Microbiology - Corynebacterium - Influenza virus - Herpesviridae - Enteritic viruses

Hematology - Reference list - Overview of hematological parameters part I and part II (Part I: Analyzer, Microscopy, Special Stains / Part II: Hemostasis, Serology, Immunohematology)

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Overview of hematological parameters part II

4. Hemostasis

This part of the lab is all about blood flow and coagulation. It’s what you would consider thick blood, ergo a tendency for clots or thin blood, ergo a tendency to bleed faster.

There are two global tests aPTT (activated partial thrombinplastin time) and the Quick (prothrombin time). In our lab we often use thrombin time instead of aPTT, it’s a fairly outdated test but our doctors are used to it. There are two pathways to activate clot formation. The intrinsic pathway is monitored by the aPTT and the extrinsic pathway by the Quick.  We have tests for the activity of the factors V, VII and XIII. We don’t measure the other ones, but instead offer different tests for TDM (therapeutic drug monitoring) for Anti-Xa and Anti-IIa drugs.

Have a look at the coagulation cascade here:  

Image from here, not mine.

Other tests we offer are D-Dimer and fibrinogen. In case of damage to the blood vessels fibrinogen can be activated to fibrin which is a major component in clotting. D-Dimers are a waste product of splitting up fibrin in fibrinolysis. I’m not informed enough to confidently say any more right now.

We also have a ROTEM (rotational thromboelastometry) which is often used during an operation as it shows the momentary condition of hemostasis.

With a PFA (platelet function analyzer) you can test the functionality of the platelets and with a Multiplate you can check the impact of drugs that are supposed to inhibit certain receptors on the platelets.

5. Serology

This is a little confusing. Serology is not usually a part of an hematology lab and again for us it’s just a historical thing. In bigger labs this would be a seperate part called immunology. I don’t know that much about it yet – but I will be the next to get into that little specialized team.

The main analysis we do is called ANA (antinuclear antibody). As far as I remember from school it is a way to categorize different auto-immune diseases in a group called collagenosis (connective tissue diseases), the most known being SLE (systematic lupus erythematosus). I’ll tell you more once I know more.

6. Immunohematology --> I’ll skip right over this. I went way deeper into this part when I was an intern, so I don’t even care what we do at my current job. I’ll talk about anything I know.

[bmedizinischa]

Overview of hematological parameters part I

I’ll divide the subject of hematology into the different work stations we have in the hospital. I work in a medium sized lab, we offer analyses for our hospital and have to send away more complicated or rare analyses to bigger labs – because it’s just not cost-efficient to do it ourselves.

The parts are: Analyzer, Microscopy, Special stains / Hemostasis, Serology and Immunohematology

1. Analyzer (like XN by sysmex, Advia by siemens, DxH by beckman-coulter) CBC is most often ordered. It means complete blood count and includes five parameters: - RBC red blood count - WBC white blood count - Hb hemoglobin - Hc hematocrit - tc thrombocyte count/platelet count --> Reticulocyte count is usually ordered when the MCV (mean corpuscular volume – basically how much Hb is contained within a single erythrocyte) or the Hb are not within the normal range which is an indication for anemia and reticulocytes helps distinguish those. --> You can also order a differential, which is a differentiation of the white blood cells by the analyzer into neutrophiles, eosinophiles, basophiles, lymphocytes and monocytes. The method depends on the analyzer, I’ll get into two different methods later on. For now, let’s just say it is pretty reliable as long as the patient does not have abnormal cells. If there is any indication for abnormal cells either clinically or by the lab values, you should order a manual differentation.

 2. Microscopy

Main thing is the manual differentiation of blood stains. Every lab has different rules for when they do a manual differentiation without an explicit order from the doctors. Most rules actually depend on the flags the analyzers give you. Both our analyzers have flags for abnormal lymphocytes and blasts, also for erythroblasts and for those flags we have to do a manual differentiation, because these are indications for a hematological disease. There are flags like left-shift – which means that there are younger neutrophiles than usual. This can be due to an infection and is not necessarily a reason to order a manual differentiation.

Microscopy is still incredibly important and although there are efforts to automatize the process, we’re not there yet. I will not write about microscopy on its own, but it will come up once I get into different diseases.

Our lab also offers different kinds of specialized microscopy, like cell count in liquor (as in cerebro-spinal fluid) and joint aspirates, also manual thrombocyte counts. We also look for crystals in the joint aspirates, seeing as those are indicators for gout or pseudo-gout.

For whatever reason our lab also offers cytological differentiation of aspirates from pleura, ascites, liquor, joints, pericard.. I think those are all. We have a pathology lab, which has a cytology section, but I think it’s a historical thing that we still do it.

We also do special stains for bone marrow differentiation – well, only the more experienced lab techs differentiate those. It takes a lot of practice and is quite different to a blood stain.

3. Special stains

This ties in closely with microscopy. I’m omitting a lot here. We have stains to look for HbF – fetal hemoglobin. We look for it in the mother’s blood and it’s not supposed to be there. It’s one of the topics I have to do some research for, but basically it means there is some kind of damaged artery connecting mother and fetus, or the placenta is damaged. Both could be an indication to deliver the baby early.

There is a stain to look for plasmodium, the pathogen that causes Malaria. I’ll cover that topic once I did the course in january. We do differentiate the different species of plasmodii.

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Hematology

So I got my diploma a year ago and now I’m working in a lab focused on hematology/immunohematology. I’m mostly writing this blog for myself and all the facts I don’t specifically credit are either from my old school scripts or based on experience.

I feel very confident about immunohematology since it is the most logical subject to me and I wrote my final paper about it. So, since this should benefit me - I’ll leave that for whenever I don’t feel like doing a lot of research. 

I’ll mostly focus on hematology, specifically the methodology in the different tests and cell morphology in microscopy. Bear with me with the images, it is hard to find any good ones online.

I’ll use these pages for reference:

Sysmex - one of the main competitors for hematology analyzers --> The case studies are especially helpful, but there are also just tables for the different cells

Onkopedia --> I think this is aimed at doctors, it’s a very clinical view

Khan-academy --> If you’re looking for online study help, you should be familiar with the Khan academy. Again, this is more of a clinical/theoretical view

Institute for tropical diseases in Switzerland --> I’ll actually do a course there in january and will revisit blood parasites then. It does have a virtual microscope for parasites in general, but that would be more interesting for microbiology.

Crash Course - Anatomy --> Crash Course has a lot of accurate videos with a nice visualization of more complex ideas. Just so happens to feature some anatomy vids too. I’d recommend the ones on ‘Blood’ and the ‘Immune System’.

I also have a playlist on youtube where I add stuff, as I stumble upon it. Right now it’s mostly Crash Course and Khanacademy. I DO watch them critically before adding anything. Any video on this list is correct as far as I can tell, might be omitting interesting details, but not giving false information. My knowledge is limited on the clinical side of things though and there might be outdated therapy suggestions etc. --> My playlist

There might be the occasional paper that inspires me to write a post. I will link to it on pubmed then. (not encouraging anyone to use scihub.. why would I do that.. that’s not nice)

[bmedizinischa]

Enteritic viruses

(Sidenote: I’m not a native speaker, so I apologize for any language mistakes. I might also have a focus centered on Europe.)

These viruses aren’t necessarily closely related, but all of them can cause watery diarrhea.

Common traits: - cause watery diarrhea, nausea, fever... - mostly self-limiting    --> if any, the therapy would be substituting electrolytes and liquid  - Lab testing would be mostly PCR, if you do it at all - fecal-oral transmission

Norovirus

...most annoying little fuck ever... It’s the cause of a lot of hospital infections and we are not “just” talking about the patients, the employees get it too.

Clinic: - very short illness 2-3 days - main symptom is nausea - highly contagious -->transmission via aerosols possible! - incubation takes 12-48 hours - affects both adults and children annoying nosocomial pathogen, because.. - you need less than 100 particles to get infected - water can serve as a vehikel - norovirus is relatibely resistant to chlorid and to heat (good luck with your pipes) - efficent transmission between humans

Rotavirus

It mostly affects young children < 5yo It can affect adults, but it’s not serious for them, since they don’t have symptoms. Typical clinic: - starts with nausea and vomiting - fever - only afterwards the diarrhea starts and continues for up to 5 days ---> the dangerous thing is dehydration! Even today a lot of children in poor countries die because of a rotavirus - incubation 1-2 days

lab: - PCR or ELISA

A vaccination exists!

Adenovirus

affects children below 4 years old

clinic: - mild gastroenteritis - 5-12 days - self-limiting - incubation takes 8-10 days (main indicator for differentiating the infection from other enteritic viruses)

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Herpesviridae

It’s a family of viruses that all have in common that they persist after the first infection and can be reactivated. This is a family of relatively large viruses.

There are nine viruses that infect humans and five of those are fairly common.

HHV 1 & HHV 2 = Herpes simplex viruses Typ 1 mostly causese orolabial Herpes and Typ 2 mostly genital Herpes

HHV 3 = Varicella Zoster virus It causes chickenpox and can be reactivated to cause shingles

HHV 4 = Eppstein-Barr = EBV This causes kissing disease or mononucleosis

HHV 5 = cytomegalie = CMV This virus is mostly relevant because it can infect babys during birth and cause severe organ damage.

[bmedizinischa]

Influenza virus

The influenza virus is a tricky pathogen.

Its genom is segmented which means that it mutates easily. 

There’s the usual point mutation that can occur in any pathogen. More troublesome is the antigen-shift. In the replication process the whole combination can get messed up, which leads to a bigger change. It also means that the virus can adept from one host species to another. 

The whole hysteria with the bird flue? Yeah, that’s why. It’s an influenza virus and the possibility for an antigen-shift is there.

INFLUENZA-A-Virus

Influenza A can be categorized into subtypes according to their H & N - antigens (hemagglutinin and neuraminidase). For example H1N1 was the pathogen of the spanish flue in 1918.

This virus has the potential to cause a pandemic! It needs to fulfill three criterias: 1) pathogenic and virulent 2) has to be contagious from human to human 3) no or too little immunity to the pathogen in the host species.

As for the clinical picture: Incubation: 1 to 4 days Symptoms: abrupt start --> fever >38°C, myalgia, adynamia     --> complications: most dangerously pneumonia Therapy: mostly symptomatic, there are some antiviral drugs Detection: mostly a clinical diagnosis, PCR is the most reliable, but that’s tricky, coz the genome changes so quickly

[respectfulmemes]

[bmedizinischa]

Oh, I know… and I’m learning about most of them. The cells not belonging to myself as well. Looking at you, bacteria

[bmedizinischa]

Bacteriology

Genus: Corynebacterium

Microscopy: gram-positive, mostly club-shaped rods

Culture: grows on blood-infused media,some strains grow better with some kind of lipid (like Tween 80), grows in small grayish colonies 

It’s the microscopy that is specific, the culture really isn’t helping with identification. This bacterium is very rarely detected in humans, and even less often clinically relevant. 

One species, C. diphtheriae, can cause diphtheria, which is a serious diagnosis and requires a therapy not only with antibiotica, but also an antitoxin. The toxin-gen however is transmitted via a bacteriophage and therefore not every strain of bacteria is toxin positive. If there is any chance that you actually found C.diphtheriae you have to let a specialized labratory handle it from there on. In a normal lab you can NOT test whether or not the strain is toxin positiv. The identification is usually done via MALDI TOF (a biochemical method). There are a few characteristics to help you in addition to that. The selective-media Tinsdale (which contains Fosfomycin), catalase positive, not mobile, partially acid-fast and reverse Camp-Test positiv.

The cell-wall structure of Corynebacteria contain a certain protein that prevent S.aureus from building a hemolysis. Since it’s a prohibition is the reverse Camp-test. The usual Camp-test would be used to confirm a kind of streptococcus, which would lead to a double hemolysis.

[bmedizinischa]

Special stains

Part II Auramin-Rhodamin

Like with the Ziel-Neelson stain, we’re looking for acid-resistant rods, so basically mycobacteria. This stain works with fluorescence though, so while doing it you have to be careful to work quickly and not exposingthe slide to the light for too long, as this would weaken the fluorescence and could cause falsely negative results. For me at work it is the only stain that I can’t control myself, because we laborants don’t have fluorescence microscopes. It’s not difficult to do though and always worked so far.

-> you only see the mycobacteria, nothing else should be stained.

SFOG

We use this stain exclusively for kidney diagnostic. It’s a trichrome stain that stains specific protein deposits in different colors.

Blue are connective tissue, basement membrane and acidic mucus. The bright orange parts are the muscles, yellowish-orange are red blood cells and blood plasma. The protein deposits are red, blueish-red if it’s amyloid. 

Silver

The main reticulum stain - often used for liver diagnostic. Against the faint redish counterstain the reticulare fibers stand out in black, while the collagenous fibers are a redish-brown.

The stain works, because the silver-ions bind to the tissue and fall-out into their elementary state. It’s critical to avoid working with any metalls while doing the stain, as that would influence the results.

Silver Methenamin Although it’s also a silver stain it does not stain the reticular fibers. Methenamin manipulates the pH so that it stains the basement membrane and Alzheimer Plaques. So it’s used either in kidney diagnostics or in the brain for Alzheimers.

--> beautifully done kidney stain

That’s it for today. I got way too annoyed searching for useable images...

[bmedizinischa]

Special stains

Part I

Giemsa - nucleus stain

We use this stain for better differentiation of the nucleus and its variations. The stain is basically just blue, no counterstain.

May-Grünwald-Giemsa

This is the usual stain in hematology, they use it for overviews and differentiating the cell types. Histology uses it for the same reason, just inside the tissue instead of the bloodstream. It’s not exactly a counterstain though. It’s one dye that reacts differently with acidic or basic structures in the tissue.

It’s more often used for cytology.

Ziehl-Neelson-Stain

This one is used to verify that there are acid-resistant bacteria in the tissue. The bacteria itself is visible as small (in relation to the surrounding tissue) red rods. The counterstain is blue. You have to use a control slide to be certain that it worked.

Second picture is not something you’d usually see in histology. It’s probably a cytology stain in a way bigger magnification.

Orcein

Orcein is primarily used for liver diagnosis. It stains Hepatitis B cells and copper remnants in the tissue. Copper could also be found in the spleen. The cells are stained coppery and you also stain the elastic fibers (but if you only wanna see those you’d rather use an elastika van gieson stain).

elastic fibers

It’s so annoying to find useable images... you just gotta believe me here I guess.

[blueberrykindoftea]

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Way easier to see the differences like that

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Karl von Frisch - The Human Body and Types of Tissue Cells, “Man and the Living World”, 1965. Upper left, nerve cell and its fibers; below, in order, muscle cells of the arm, connective tissue surrounding muscle, tough fibrous connective tissue of a tendon. Upper right, cells of cartilage; below, in order, outer cells of skin, structure of bone, and cells of the fatty tissue.

[bmedizinischa]

nice simplified visualization