Sunday, 2 September 2007

Haematology - Malaria Parasite

HELLOOOOOOO ALL~~ //

Am i supposed to blog this week? Anyway, i'm gonna talk about Malaria Parasite in my entry!~ It's a hot topic in my lab right now and everyone is taught to identify and differentiate between the vivax and falciparum species during roll call and CME. I shall only focus on explaining these two species which are more common in Singapore.


Name of test - Malaria Parasite Microscopic Examination (MPME)


Introduction
  • Malaria is an infectious disease caused by the Plasmodia parasite.



  • There are 4 identified species of this parasite: Plasmodium vivax, P. falciparum, P. ovale and P. malariae.



  • It is transmitted by the female anopheles mosquito.



    (Image taken from: http://www.lawestvector.org/)


  • In fact, it can be treated in just 48 hours but it can be fatal if the diagnosis and treatment are delayed.



  • It occurs in tropical countries especially in Africa and India as the tropics provide ideal breeding and living conditions for the anopheles mosquito.



  • One child dies of malaria somewhere in Africa every 20 sec., and there is one malarial death every 12 sec somewhere in the world (quoted from the malaria site).



  • Currently there's no vaccine available; Still developing.



  • In Asia, the more common species encountered are P. vivax and P. falciparum.



  • MPME provides information to confirm the diagnosis of a MP species. The test consists of both thick and thin smears.

Procedures & Principle of MPME




  • To perform MPME, a EDTA blood tube is needed. (Alternative: finger-pricked blood)



  • Giemsa stain is a differential stain used in the diagnosis of MP.



  • It is a mixture of methylene blue and eosin and it differentially stains the RBC & platelets pink, WBC blue and MP purple.



  • #1 - Thick smear: To screen for parasite



  • A stick is used to spread the blood on the glass slide to a 50-cent coin area, yet thin enough to be seen through.



  • It is air-dried for 30 mins. -> This allows the RBC to be hemolyzed.



  • Leukocytes and any malaria parasites present are therefore, the only detectable elements.



  • The thick smear is then de-hemoglobinised in water and stained with Giemsa.



  • The WBC and MP pick up the stain and it is noticable under the microscope.



  • However, its morphology is often distorted due to the hemolysed RBC, hence it is only used to detect infection and estimate the parasite density.



  • Thick smear is more sensitive than the thin smear (as a larger volume of blood is screened), therefore it is easier to pick up low levels of infection.



  • #2 - Thin smear: To identify the parasite species



  • It is performed when thick smear shows positive MP infection.



  • The thin smear in air-dried for 10 mins and fixed in methanol.



  • The smear is covered with 10% Giemsa stain for 30 minutes, washed with distilled water, drained and dried.



  • The stain is picked up even by the RBC, hence any parsite within the cell can be identified.



  • It is used for species identification because the parasite appearance remains well preserved under the microscope.

Test Results

Thick smear - tiny purplish ring-like structure/comma observed in the hemolysed RBC -> +ve for Malaria

Thin smear - check several fields for infected RBC. Parasites in various forms can be observed e.g. early ring, late ring, early intermediate stage, late intermediate stage, presegmented, segmented (schizont), macrogametocyte & microgametocyte. More than 2 parasites can infect a cell and a person can suffer from multiple infection too. Below are some techniques to differentiate between the falciparum and vivax.



  • RBC enlarged -> vivax

  • RBC not enalrged -> falciparum

  • Many RBC infected -> falciparum

  • Schizont or growing trophozoite observed -> vivax

  • Ring form observed -> small 'comma-like', sometimes two chromatin dots (like a headphone), often multiple rings in a cell, occuring at the tip of the cell -> falciparum

  • Ring form observed -> relatively large, usually one chromatin dot with a thick blue ring (like a diamond ring) -> vivax

  • Crescent shaped, central chromatin ('banana/rod-like') -> falciparum gametocyte

  • Large central chromatin dot in RBC (without ring) -> falciparum (cerebral malaria)


Clinical Interpretation

MPME is used to screen for malaria parasite and identify the species by the use of a differential stain. Taking into consideration the limitations of the thick and thin smears, both of the smears are therefore necessary to make a definitive diagnosis. The various forms of MP require different drug therapy, hence the correct species diagnosis is vital in the recovery of a infected individual. In any case that MPME could not provide evidence to confirm the species, a Malaria Parasite PCR is done (this is not done in our lab; send-out to NUS). Meanwhile, if the patient condition is critical, the doctor in-charged may decide to dispense drug against falciparum as it is strong enough to even kill the vivax species.


Pictures (produced with permission from haematology section head)

-From Mr. J-


Mixed infection of vivax and falciparum.



The largest RBC is a vivax macrogametocyte. Schizont suggests vivax infection as it is absent in falciparum PBF.


Mostly falciparum parasites infection here.


Actual Diagnosis of Mr. J: Falciparum & Vivax Infection

---------------------------------------------------------------------------------------------------------------------------------

-From Miss. M-

Enlargement of RBC due to vivax infection is difficult to spot in anaemic patients. Hence no concluding statement could be made. Note that the RBC are anisopoikilocytes.


The RBC in the middle appears as enlarged, but it may be due to infection of a normal RBC in an anaemic patient. Therefore, it is better to check other fields for other signs or send-out the sample for a PCR testing.


Actual Diagnosis of Miss M: Vivax Infection

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Questions? ^_^
Reminder: Close all your windows between 5-10pm and early morning to prevent from being a MP victim!


-Pei Shan, TG02-

16 comments:

first6weeks said...

Hi Pei Shan.

You mentioned that hemolyzed RBCs may cause WBCs and MPs to appear distorted under the microscope.

When I was in Cytology, we used Clarke's Solution on bloody smears. Basically, it contains 900ml of 95% ethanol and 100ml of acetic acid. As a haemolytic agent, it reduces the effect of blood contamination obscuring cellular material. Does your lab also use Clarke's solution; If so, why not?

Desmond Heng
TG02
0503179D

ALsubs said...

hi peishan

why does the female mosquito carrying the parasite only infect rbcs? thanks..

Shu Hui
TG02

MedBankers said...

Hi Desmond,

Your question set me thinking for a long long time. Somehow, I came up with this answer...

1stly, for a MPME, two blood films will be prepared, a thick and a thin one. Since the purpose of the thick blood film is to only screen for parasite presence, it doesn't matter if the other cell morphology is distorted. The confirmatory of the diagnosis is based on the thin blood film which is well-preserved through methanol fixation. Therefore the identification of the species is also done when reading the thin film slide (clear morphology).

I don't know whether my lab uses Clarke's Solution in cytology ('cus i hav not been there yet), but not in haematology. I guess methanol is sufficient to fix the thin film and more cost effective too.

Hope I've clear your doubts!

Pei Shan

MedBankers said...

Hi Shu Hui,

Thank you for your question; I'm wondering why too!

The female anopheles mosquitoes choose RBC as the host b'cus they sort of rely on hemoglobin (Hb) to replicate. The RBC-infective MP are called merozoites. They grow and multiply within human RBC by ingesting Hb. The Hb is then degraded and heme is released. The toxic heme is in turn detoxified by its heme polymerase and sequestrated as hemozoin (malaria pigment). The RBC is then lysed & the parasite infects other RBC.

Cunning right!

-Pei Shan-

Anonymous said...

Ms Chew here!

Can someone speak to Elaine and let me know why she has not been participating in the blog? So far, i only see her posting once but she has not been asking others questions.

BloodBank.MedMic.Haematology said...

Hi Pei Shan

May I know what is the difference in procedure to prepare a thick and thin slide? You only mention about thick slide to spread to a size of fifty cent coin. Do you do the same for the thin slide? Just that you preserve the morphology of the cells properly in the thin slide?

Also, Giemsa stain takes 30 minutes to penatrate and stain the inner of RBC?

I use the Diff-Quik stain in my lab. For a normal PBF, the slides are dipped into the fixative solution(containing Fast Green 0.002g/l in Methanol) 5 X 1sec, then the Stain Solution 1(contains eosin Y 1.22g/l in phospate buffer pH 6.6 and 0.1% sodium azide as preservative) for 5 X 1sec, then stain solution 2(contains Thiazine Dye 1.1g/l in phosphate buffer pH6.6) 5 X 1sec.

So altogether only 15 seconds. It is classified as Romanowsky-type stain, the same catgory of stains as Giemsa stain. Can Diff-Quik be used for this? 15 sec and 30 mins quite big difference huh..

Douglas
TG01
0503224H

MedBankers said...

Hi Ms Chew,

I'll remind Elaine!

Pei Shan.

MedBankers said...

Hi Douglas,

Sorry for not stating clear in my entry. The difference is thin film, as the name implies, uses only a drop of blood, like the amount we use in a normal PBF. Yes, for thin film the morphology is preserved but not so much of importance in a thick film (only for screening purpose).

The 30 minutes incubation time is to allow the parasite present to pick up the stain.

As to why not use Diff-Quik solution instead, eh... i'll explain based on my knowledge. 1stly, Giemsa stain is the 'gold standard' stain used in the PBF of MPME. Many advantages have been reported e.g. deep-staining of parasites, resist fading, contrasting stain of morphology structures for identification (esp. useful in thin film). Diff-Quik solution is a modified version of the Giemsa stain. It is often used in rapid staining to save time but it produces poorer slide quality which may somehow hinder the identification of MP strains (although personally, i think the quality is still acceptable for a well-trained med. tech). The next consideration is cost-effectiveness. Giemsa stain is way more cheaper than Diff-Quik Solution (Only about 1/3 the price of it). So to save cost...

You mean your lab do Malaria Parasite screening for horses too? and you're using Diff-Quik Solution?? I'll check my answer with my senior tomorrow... thanks for your question~

Pei Shan.

MedBankers said...

hey pei shan,

thanks for the reminding. sorry miss chew!i will start commenting.

ps:
in my lab, very few cases of malaria parasite screening here, thus i almost have no opportunity to view the screening process.

Here a extra information.

when the lab tech found out that the patient have malaria parasite. They will enter the patient's particulars & results into malaria parasite positive book. Then, they will fill up a form and fax to Ministry of health. 2 thick & thin smears will be prepare and fix in methanol. slides will NOT be stain. The slides will be send to National MAlaria Reference Centre

elaine

BloodBank.MedMic.Haematology said...

Eh.. Nv test for MP leh.. Just that we use Diff-Quik for most of our samples, and I happen to know that they both are of the same type of stain and that Diff Quik so much faster. Ya I think it's true that it save cost. But apparently my lab seems like they are very rich to splunge on such stuff. hahas.. =)

Douglas
TG01
0503224H

The Lab Freaks said...

PEI SHAN!

Wah, i see you have used the knowledge we learnt abt malaria parasites very well. Cheers! =)

Sharifah

MedBankers said...

Hi elaine!!

Eh... no leh... only thin film is fixed. Thick films should not be fixed! Pls note. So your lab send-out... OooH..

Pei Shan.

MedBankers said...

Hi Douglas,,

I'll checked with my senior. According to what he says, Giemsa stain is the recommended stain by CAP. During Proficiency Testing, Giemsa stain is used to perform MPME. So after being CAP accreditated, our lab continue to use Giemsa stain. I was told that previously Field's stain was used instead. It is a rapid stain 5-10sec only. But the slide quality is rather bad - even some bacteria is stained, thus interfering with the reading of slide.

So simply, my lab want to follow the 'gold standard' set by CAP. ^_^

Pei Shan.

MedBankers said...

Hi Sharifah,

Hee.. you know a lot too! Have fun in haematology this week!!! I cant wait for my turn...

Pei Shan.

MedBankers said...

hey pei shan,

yup! only thin smears is fixed with methenol. sorry!

cheers, wish u have a great time @ haema.
elaine

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