Specimen Reception and Immunology

Hi all,

Sorry for the late posting.
I have just finished Specimen Reception department in my lab and now currently at Immunology.

At the specimen reception department, it is basically about SOPs and following strictly to SOPs. As we have major clients apart from normal clinics, we have to know our clients well because the specimens received from each client are processed differently. So basically, I spent a long time just learning the procedures because we are expected to work like the other staff there.

It is also essential to know the different types of tubes and their functions because we have to inform the doctors and reject the specimen if wrong specimen types are sent for testing. (It has happened quite frequently)

Long Plain tubes:

Known as serum separator tubes when gel is added to the tube. It contains a clot activator (I don't know the exact ingredient) pre-coated in the tube. When spun down the red cells are at the bottom and the gel in the middle with the serum at the top. It is important to note that different type of gels from different materials may cause inaccuracies. For those without the gel, it only has the clot activator. Long plain tubes are usually used for serum analysis such as VDRL testing.

Heparin tubes:

Usually available as sodium, litium or ammonium salt pre-coated in the tube, it is used for trace elements screening in industrial toxicology for patients working in heavily polluting industries. It is also recommended that for potassium analysis, heparin tubes are to be used. When using the long plain, potassium would be released from the platets giving an increased in serum potassium. EDTA tubes comes with potassium salts and would cause falsely high potassium results.

Fluoride/oxalate tubes:

Oxalate will cause osmotic redistribution between the the plasma and erythrocytes, diluting the plasma as a result (water moves from erythrocytes to the plasma ). This would decrease hematocrit levels, disrupt the morphology and cell membranes of erythrocytes. When the samples arrive in our lab, "water condensation" may be observed inside the tube because of the osmotic redistribution. Therefore, fluoride/oxalate tubes are only used for ABO and antibody screening in our lab. Oxalate will also bind to calcium in the blood, causing a falsely low blood calcium level.

Citrate tubes:
Usually for coagulation studies because it preserves the coagulation factors. Citrate also causes osomotic redistribution and decreases the concentration of many analytes (like fluoride/oxalate tubes). However in our lab, citrate tubes are most commonly sent along with a EDTA for full blood count or as a follow up of an EDTA specimen usually due to possible/observed platelet clumping in certain patients (cause unknown) when the specimen is sent in EDTA.


Immunology

During my term in Immunology, the common tests run in the department are:

1. Dengue (most common but low sample volume compared to HIV and it is the only manual test.)

Run by the Centaur machine

2. HIV (most common)
3. Tumor markers ( Ca 199, Ca 125 etc)
4. Ferritin
5. Vitamin B 12 and folate acid
6. Hormones such as cortisol and testosterone

Principles of Dengue is the same as Sally's post.
Principles of HIV is the same as Lizzie's post a number of weeks ago.

As promised, I managed to find out about the alternative system to HIV testing with a much shorter testing time. As it is newly tried out in the Serology department, I don't know much about it yet. It is the Roche Modulla from Roche diagnostics.


Ca 15-3

Ca 15-3 is usually for testing of metastatic breast cancer. When a women is diagnosed with breast cancer, usually, her specimen has the presence of cancer-related antigens especially Ca 15-3. Note that for all tumor marker tests conducted, an abnormal result does not mean that ther patient has cancer. There are also conditions where abnormalities are observed in normal healthy patients. Therefore, a confirmatory test is necessary to detect cancer in patients.
Ca 15-3 can be used to monitor response to treatment of disease. In patient with known metastasis, a reduction may mean a good sign of positive response to treatment while an increase may mean the disease progressing to the next stage.

Principle of the test:
2 step sandwhich ELIZA using direct chemilluminescent.

The system will perform:

1. Dispense sample into cuvette

2. Dispense conjugate reagent and solid phase and incubate at 37 deg celcius.
The conjugate is made of monoclonal mouse antibody 115D8 labelled with fluorescein, which is specific for CA 15-3 antigens. The solid phase is made of monoclonal mouse capture antibody coupled to paramagnetic (refer to PIPC 2 notes for definition of paramagnetic) particles.

3. After incubation, the complex (conjugate and solid phase) is washed to remove unbound antibodies.

4. The complex is resuspended using the wash solution and Lite reagent is dispensed. The Lite reagent consists of monoclonal mouse antibody, DF3, specific for CA 15-3 labeled with acridinium ester.

5. Cuvette is washed to remove unbound Lite reagent.

6. Acid and base reagent is dispensed to initiate the chemiluminescent reaction.

High amount of CA 15-3 in patient's sample = high amount of relative light units (RLU) detected. This means that with an increase in CA 15-3, the intensity of chemiluminescence increases.



Once again, I appologise for posting so late.

With regards,
Yeng Ting
TG02

Cheng Hong: Histology Q&A

Qn1) How many samples are processed for H&E staining? Since it has to be done within 20mins, if something goes wrong surely they do afew samples as a contingency plan rather than banking of a lone sample right?

Ans: normally the pathologist will view at least 2 slides if the margin is not seen clearly due to the cutting, a re-cut must be done.


Qn2)example of a situation in which the doctor is waiting for the results from the lab before stitching up the patient. Sounds like 20 min is a bit rush.

Ans: all of the specimens sent for frozen sectioning are done in the specific time strain and if im not wrong, all of the patients that are waiting for the results are not stitched up yet.


Qn3: What will u do if the slide turn out to have background, pale in the nuclear and cytoplasm?

Ans: A check on the stain is done if needed the stains are changed and a QC slide will be run first before staining the re-cut sample. But normally it wont happen as every morning before the patient’s sections are stained, a QC slide will be run first and check by the senior med-tech.


Qn4:You say that it is importantt to embed the tissue in the correct orientation so i want to ask u what if the orientation is wrong? what are the actions that will be taken by you guys.....another qn is u mention that place slides into the oven set at around 82oC for 15mins(to let the tissue to “stick to the slide” to prevent it from floating off when staining)....well i remember during htech lesson everytime during staining my tissues will get washed off during staining..isit due to insufficient drying...??haf you guys ever encounter tissues being washed off during staining n if yes...wat did u guys do??

Ans:if the orientation that the pathologist request is embedded wrongly it might prevent the pathologist for analyzing the part that is doubtful. So the tissue block must be melted and re-embedded and sectioned again. yes the floating off is due to the insufficient heating and drying. There are times that the tissue might float off so the staining program must be changed eg: let the slides cool down for a minute before staining

Histology

Cheng Hong: Histology Laboratory

Receiving specimens

1) The specimens will be sent down from the operating theaters/ clinics to the lab by a porter.
2) Upon receiving the specimens the Medical Technologist(MT) must check the total number of bags sent to the lab and check the patient’s particulars and the type of specimen with the request form.
3) Initial on the receiving form and stamp the time.
4) Sign the specimen log book state the number of bags received and the time.
5) Sort out the specimens according to: Placenta/ Point Of Conception(POC)/ Specimens pending for trimming/ Others.
6) The Laboratory Assistant(LA) will then give the specimens a lab assertion number.


Tissue Processing

Specimens for trimming
1) For placenta specimen/ specimens that needs trimming the Pathologist Assistance(PA) will do the trimming and will determine the number of tissue cassettes to use.
2) The PA will dictate the measurements and the characteristics of the specimens to the LA to record
3) The cassettes are then placed in the tissue processing rack and immerse it in buffered formalin.

Product Of Conception
1) POC are stored in sealed packets(to prevent spills/leakages to protect the MT’s safety)
2) POC is processed by the MT: the POC bag is cut into half and the MT must identify the chronic villi and place into cassette.
3) The MT must record the amount of sample used, number of cassette used and if there is any reserve. (Also must indicate if there is any fetal part seen)
4) The cassettes are placed in the tissue processing rack and immerse it in buffered formalin.

Tissue Processor
1) The cassettes in the holder is then placed into the Automated Tissue Processor(Shandon Excelcior).
2) We are able to program the tissue processor to start at a specific time and we are able to choose the different programs(eg:overnight/ rapid etc)
3) The steps are processed as shown below:

1) 10% formalin 30mins(To fix the tissue & preserve cells & tissue constituents)
2) 10% formalin 30mins
3) 70% alcohol 30mins (To remove fixative and water from tissue& replace them with alcohol)
4) 95% alcohol 30mins
5) Absolute alcohol 1hr
6) Absolute alcohol 2hrs
7) Absolute alcohol 2hrs
8) Xylene 30mins (Clearing: Replacing dehydrating fluid with fluid that is miscible with dehydrating fluid & embedding medium)
9) Xylene 30mins
10) Xylene 30mins
11) Paraffin 2.5hrs (wax replacing clearing agent with embedding medium)
12) Paraffin wax 3hrs

*We do not put the tissue into 100% alcohol directly as it will distort the tissue(must be a graded process)

Tissue Embedding
1) After the tissues have been processed, remove the holders and check with the log sheet if the assertion number tallies.
2) Open the cassette carefully and depending on the tissue size, choose a suitable mould size that will fit the tissue so that the entire surface will be exposed when obtaining the sections.
3) Dispense some wax into the mould and place the tissue into the mould and place on the cold plate and press the tissue evenly using a forceps or a pressing block( to ensure all surface will be exposed when sectioning).
4) Place the bottom portion of the cassette on top of the mould and dispense more wax until it fills almost half of the cassette.
5) Allow the wax to cool down for awhile before placing on the ice block(to speed up the hardening of the wax)
*It is important to embed the tissue in the correct orientation so that when the pathologist view the sections it will be in the correct orientation.

*It is also important to be quick when embedding, if the wax hardens before the the tissue is pressed out evenly, problems will arise when obtaining sections.

*For small biopsy, it is important to use a very hot mould so that we can expose the tissue as much as possible to be able to obtain good sections.


Shaving
1) After embedding, the blocks must be shaved first before cutting
2) The MT must be able to know how much to shave and to expose the tissue to prevent shaving off too much of the tissue thus not enough tissue is enough to obtain sections
3) For blocks that have blood clots we can soak them in water for about 5mins to soften the clot for easier cutting
4) For blocks that are fibrous(eg:cervix) we can soak them in softener(commercial softener/ soflant)
5) For blocks that are calcified we can do a surface decalcification by placing it in RDO
6) After steps 2 to 5 we place the blocks on the cold plate before cutting


Paraffin Sectioning
1) The aim is to obtain 3micron thick sections without folds on the tissue.
2) The blocks must be cold to obtain thin sections.
3) Insert the blade and check the thickness setting.
4) Place the block into the tissue block holder and adjust the distance from the blade using the coarse adjustment.
5) Turn the rotary knob away from you to obtain sections.
6) Slowly obtain ribbons and place on water and do any necessary adjustments(removing overlaps/folds).
7) Pick desired section and prepare a clean glass slide and fish up the section.
8) Place the section into a warm water bath(the section will spread out) and fish it up.
9) Let the slide stand for awhile before placing it in the slide holder.
10) Place the rack containing the slides into the oven set at around 82oC for 15mins(this is to let the tissue to “stick to the slide” to prevent it from floating off when staining).

*For sections that need to be cut in levels to slides must be obtain


Routine Haematoxylin and Eosin stain

1) Place the whole slide holder into the Lecia Autostainer and select the respective program
2) The steps as follows below:

(a) Dewax section in xylene for 5 minutes.
(b) Place in another xylene for another 5 minutes.
(c) Rehydrate section with absolute alcohol for 2 minutes.
(d) Place in another absolute alcohol for another 2 minutes.
(e) After then, place in 95% alcohol and 70% alcohol for 1 minute each.
(f) Rinse section with water.
(g) Stain the section with Harris Haematoxylin for 5 minutes.
(h) Rinse section with water.
(i) Differentiate staining with acid alcohol for 5-20 seconds.
(j) Blue the haematoxylin stain with running water or alkaline water.
(k) Check the differentiation microscopically.
(l) Stain the section with eosin for 1 min.
(m) Dehydrate section in graded alcohols with 70% alcohol (10 dips), 90% (10 dips) and 2 absolute alcohols for 3 min each.
(n) Clear the section in xylene for 5 min each with 2 changes.

3) After staining place the slide holder into the auto coverslipper.

*The haematoxylin will stain the nuclei blue or black and eosin will stain the cytoplasm red or pink.

*Eosin is the most suitable dye to combine with alum haematoxylin. It has the ability to distinguish between the cytoplasm of different types of cells and can also distinguish different types of connective tissue fibers.

*Stain results:
Nuclei: blue
Cytoplasm: varying shades of pink
Muscle fibers: deep pinky red
Red Blood cells: orange red
Collagen: pale pinky red
Fibrin: deep red


Frozen Sectioning

1) The lab will receive a call from the Operating Theater(OT) stating the OT number and type of specimen
2) A MT will be sent up to the OT to collect the sample immediately and the pathologist will be informed
3) Upon receiving the specimen, ensure that the patient ID tallies and record the time received and immediately sent back to the lab.
4) Prepare the equipments and tools needed for the pathologist and inform them when it is ready
5) The pathologist will dictate the characteristics and measurements of the specimen and mark out the orientation of the specimens using different colour dyes.
6) About 1 to 2 samples will be cut from the specimen into size about 15mm length and with and about 3to4mm thick.
7) Squeeze some freezing medium on to the tissue holder and place the tissue on it and immerse it into liquid nitrogen for about 5to8 seconds.
8) Place the tissue holder with the frozen tissue into the freezing microtome and shave off some tissue to obtain a full face.
9) Cut the tissue and using a glass slide to pick up the section( the temperature difference of the tissue(-35oC) and the glass slide(RT) will cause the freezing medium to melt and adhere to the slide.
10) Proceed to Rapid Staining


Rapid Staining
1) Xylene 10dips
2) Wash in running water
3) Haematoxylin 1min
4) Wash in running water until no more haematoxylin runs off
5) Ammonia 3 to 5 dips
6) Wash in running water
7) Eosin 10dips
8) 70% alcohol 10dips
9) 95% alcohol 10dips
10) 100% alcohol 10dips
11) Xylene 10dips
12) Xylene 10dips
13) Mount with DEPEX
14) Slide viewed by pathologist and the pathologist will call the doctor in the OT about the diagnosis.

*The whole process must be done within 20mins as the patient in the OT is still unstitched, the doctor must know if they have removed all the tumor from the body.

Research: Lab Techniques

Hi all, we’re about 60% through with SIP! As promised, I shall continue where I left off from my previous post. Previously, I stopped at ligation. Here is a quick recap:

Steps:
1. Amplify gene 1 (PCR) and gene 2 (PCR)
2. Send amplified genes for sequencing (outsource to external company)
3. Digest vector, inserts – 1 and 2
4. Ligate inserts to vector
5. Transform ligated products
6. Colony PCR (screen for inserts in transformed cells)
7. Send positive clones from colony PCR for sequencing

I will now cover step 5.

5. Transformation
For my case, I use commercially competent TOP10 Ecoli cells, so I can use the heat shock method. Set up is as follows:

1) deactivate ligase by incubating at 65oC for 10 mins. This step is optional. Some even suggest that inactivating ligase may affect transformation efficacy. I have done both and for me there is no considerable difference.
2) Add 50ul of competent cells into 5-10ul of ligation mix. Adding too much may give rise to too many colonies, making it hard to isolate cells.
3) Ice mixture for 30 mins, before heat shock at 42oC for 2 mins. This is supposed to improve transformation efficiency.
4) Add 250ul of LB without antibiotics. Place on shaking incubator at 37oC for 1.5hrs. This is to allow the transformed cells to express the plasmid’s antibiotic resistance, in my case, kanamycin.
5) Spin down using microcentrifuge to obtain pellet. Resuspend in 100ml of LB.
6) Plate out on pre-warmed LB kan+ plates, and incubate overnight at 37oC.

Controls:
To check if transformation is working, a positive control is required. I use the original, circularized yeast vector (not digested) as a positive control. This however has its limitations. It merely tells me if my transformation process is working or not, not the efficiency I can expect. This is because cells take up supercoiled dna (smaller, vector appears as 5kbp on gel instead of it’s actual 7kbp due to it’s supercoiled state) much more easily than linearised plasmid which size has been made bigger due to the addition of the inserts (mine totals to 10kb and 8kb, and has been giving me problems). Hence sometimes I get good positive controls, but poor transformation on my recombinant cells.

Also, a negative control is necessary to ensure that kanamycin plates contain enough kanamycin concentration to prevent cells without resistance from growing – eliminate the possibility of false positive clones.

Lastly, a control using just the vector that has been double digested with restriction enzymes should be performed. There should be few or ideally no colonies present, as using 2 different enzymes will not produce sticky ends for self ligation. Colonies will indicate incomplete digestion by 1 enzyme, hence resulting in self ligation – 1 enzyme did not cut well, hence only 1 enzyme worked, producing sticky ends. To further prevent this, vector can be dephosphorylated. However, dephosphorylation can inhibit ligation and hence when 2 enzymes are used, dephosphorylation is not encouraged.

Kanamycin plates:
Usually made up with kanamycin concentration of 50ug/ul.
1) Warm up 400mL of LB + agar mixture using microwave at 30% power. The mixture heats up unevenly and has a tendency to boil over if not careful.
2) Allow mixture to cool at room temperatute in it’s liquid state.
3) Add 400ul of 50ug/ul kanamycin when mixture has cooled. This is so that the antibiotic will not be killed off by the heat. To be done in laminar flow hood to avoid possible contamination.
4) Mix bottle well, and pour onto empty plates. 400mL can make up 20 plates nicely. No specific amount needs to be poured, it is at user discretion. In other words, guesstimate. Best to keep as equal as possible though qualitatively, from plate to plate.
5) Allow plates to solidify in 37oC incubator. When solid, open cover to allow excess water due to condensation to evaporate, before storing in fridge for future use.

That’s about it for transformation process. Any queries, questions, comments don’t hesitate. I shall cover colony PCR and other techniques like restriction digestion and sequencing in the next update, week 18.

Till then,

Debra, TG02

immunoassay__coba

hello everyone! how was yr week? weenkend le.. relax.. i have been dreaming of works everynight... stress.


i'm going to blog abt CODA open microplate technique. This machine is used for ANA( anti-nuclear antibodies) screening test and Anti-ds DNA. i think someone blog this before but not using CODA. let see whether it will be the same. Normally, the doctor will only order ANA screening if he suspect the patient suffer from auto-immune diseases. ANA screening is not 100% accurate.


Introduction:

Refer to: http://www.bio-rad.com/

CODA is an interagated immunoassay analyzer intented for the automation of microplate based assays for in vitro diagnostic use. The system is open, such that a variety of microplatedbased (8wells X 12 strips) enzyme immunoassays (ELAs) can be programmed and run on the instrument. The sample and reagent pipettes, incubator, washer, reader and robotics are housed in the compact bench top unit.


System overview:

-->pipette all standards, calibrators. control, and samples

--> pipette reagents

-->shake & wash the microplate

-->incubate the plate @ ambient temperature or in a heated incubator

--> Read the optical density of the microtiter plate

--> Calculate the curve fitting and patient results

--> Print the run report, including interpretion of results


Principles of this test is the almost the same as normal immunoassay (Ab-Ag binding plus substrate). The principles for both ANA screening & ANti-ds DNA test is different, but both tests use the ELAs technique.

ANA screening test:

it is used to screen for the presence of antinuclear antibodies in the human serum as an aid in the diagnosis of certain systemic rheumatic diseases. This assay collectively detects, in one well, total ANAs against double stranded DNA (dsDNA, nDNA) histones, SS-A/Ro, SS-B/La, Sm, immunofluorescent (IFA) Hep-2 ANAs.

Anti-dsDNA:

To screen for the presence of dsDNA antibodies in human serum as an aid in the diagnosis of systemic lupus erythematosus (SLE).

Workflow before the test begin:

1) Allow wash buffers, reagents and samples to warm up to room temperature

2) Install wash buffers onto Coda, Refill pipette wash

3) Switch on Coda and Computor

4) Prime wash bottles 1 & 2

5) Perform chamber adjust (-1000)



Procedure:

1. Apply 100ul diluted samples and controls to wells:

• controls-apply 100ul of diluted controls (1:40) to assigned wells. add 100ul of sample diluent as a blank control.


• patient sample- apply 100ul of diluted patient serum (1:40) to assigned wells


• apply 100ul sample diluent to blank control well
  

2. Shake plate gently, then incubate for 30mins @ r.t.p

3. incubated samples are discarded to the waste bottle.

4. Gently fill 5X with 100ul of wash solution and discard. Remove all liquid before proceeding

5.adding 100ul conjugate to all wells. Discard excess liquid.

6. Incubate wells @ r.t.p for 30mins

7. Discard conjugate by flicking

8. Wash wells again 5X

9. add 100ul of substrate to each well. Discard excess transferred substrate after use

10. incubate @r.t.p for 30 mins

11. Stop color development by adding 100ul stop solution to each well.

12. REad the results with ELAs reader @ 450nm.

Results:

ANA screening: <1.0>

Positive Control: appear dark yellow

If the result is Negative, it will appear light yellow or colorless

Anti-ds DNA:

Elaine

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-