Actually I am attached to Quest Lab, within the lab, we, as an intern, will rotate to different department (Hematology, Biochemistry, Immunology, Serology, Microbiology, Specimen Reception, Cytology/Histology and Paragon Lab) every three week. For these two week, I am attached to the Hematology department.
In the Hematology department, for the first week, my main duty is to do ABO blood group and Rh typing using plating method. For the second week, my main duty is to use CELL-DYN 3500/3700 to run full blood count test and malaria parasite test.
ABO blood group and Rh typing
Principle:
In the Hematology department, for the first week, my main duty is to do ABO blood group and Rh typing using plating method. For the second week, my main duty is to use CELL-DYN 3500/3700 to run full blood count test and malaria parasite test.
ABO blood group and Rh typing
Principle:
- Label the plate as below.
------A---B---AB---D---α---β---O
1
2
3
4
5
6
7
8
9
10
11
12 - Aliquot one drop of anti-A, anti-B, anti-AB and anti-D to the plate labeling A, B, AB and D respectively.
- Spin down the patient blood sample to separate the red cells and plasma.
- Aliquot one drop of plasma to the α, β, O column each.
- Dilute the red cells in saline (estimate 3-5%).
- Aliquot one drop of the diluted red cells to the A, B, AB, D column.
- Aliquot one drop of commercial A, B, O cells too the serum.
- Mix the blood with a vibrator and centrifuge it at 900rpm for 1 min 30 sec.
- Read the result manually using a stick to mix to check for agglutination.
Test Result with reference range:
- 4>agglutination>positive
- 0>no agglutination>negative
Things to take note:
- All reagent used are diluted. 1 drop of anti-A, anti-B, anti-AB, anti-D is diluted with 3 drops of saline. And one drop of commercial A, B, O cell to one drop of saline.
- If Rh negative, Du testing is needed. Add one drop of diluted patient red cells to one drop of concentrated Anti-D in a new test tube. Centrifuge. Incubate at 37°C for 15 min. Wash with saline for 3X. Add one drop of AHG and centrifuge to check for agglutination.
CELL-DYN 3500/3700 to run full blood count test and malaria parasite test
CELL-DYN3500/3700 is normally given the red cell indices, platelets, WOC (WBC optical count), WIC (WBC Impedance Count) count.
Run internal QC and control:
- Do background clean for 3-5 times to make sure the values stated on the screen is near to 0 (Stated on the PC screen connected to the machine).
- Run 3 control samples (Low, Normal, High) using open sample mode (manual). Make sure all values are within control limit.
- Run 2 Internal QC (normally is patient sample that is tested a day before and is within control limit) using closed and open mode. Make sure all values are within control limit.
Principle:
- Load the sample into the machine for it to run in closed sample mode, and then wait for result.
- For sampling error, redo the test using open sample mode.
- Check whether all the values of the result are within control limit.
- Mainly is to check for WOC, Hb, MCHC and platelets values.
Test Result with reference range:
- For malaria parasite (MP), require making a smear when the platelets are <150>
- For full blood count (FBC), to do a rerun, WOC - <2.0>25, Hb - <60>180, MCHC - <300>365 and platelets - <100> 750. Require to make a blood smear when WOC - <3>15 and platelets <150>150.
Clinical Interpretation:
- MP is test whether the patient (normally foreign worker) have malaria parasites.
- FBC is to test for any abnormal RBC, WBC and Platelets which is closely related to blood diseases such as leukemia and anemia.
- MP cells normally found in patients: Plasmodium Falciparum
Taken from http://deep6inc.com/previewher20.html
Things to take note:
- Controls and Internal QC need to be run at least 2 times a day to make sure the machines are accurate. This is to make sure that the test result for the patients sample run is reliable and accurate.
- Closed sample mode is automated method, just need to load the sample for the machine to run. Therefore, the volume of the patient blood sample must be more than 1ml.
- When using open sample mode, normally use for small amount or little sample, because is a manual method, make sure the patient sample is mix properly.
Hope you all can learn something from my post. Feel free to ask any question.
Lizzie Chew, TG01
33 comments:
Lizzie!!
Are you still very busy?? Hope you're doing well=]
In step 8 right, the centrifuge used is a special one for the plate? And what is the purpose of diluting the reagents? Wont the agglutination be too litle to be seen with our naked eyes?Will you use a microscope to view the agglutination?
Ming Boon
Tg01
hi lizzie
You posted ur blog so fast^^. Drop a question for you *tink*
"All reagent used are diluted. 1 drop of anti-A, anti-B, anti-AB, anti-D is diluted with 3 drops of saline. And one drop of commercial A, B, O cell to one drop of saline."
Sorry if it's too difficult but why is there a need to do dilution. Also why use 1:3 and 1:1 dilution for the reagent? Also kinda stupid question but why use saline to dilute? why no DI water?
Dorene Tg02
Hey Lizzie
Was wondering why you would need more than 1mL of sample. If the machine was completely automated, wouldn't you need less samples cause there is a very small chance that there will be pipetting errors or spills or common mistakes by human error? Also, with regards to the machine used, do you have to program it before use or do you actually just press a button and it will start processing your sample?
Johanna, TG02
hey you posted a a diagram under MP test which i believe it represent the MP test. If thats the case then, how to you identify the abnormal cells,MP cells, that are found in patients? As in any description about the morphology of the MP cell?? Like the colour or the shape? or is there any type of stain that can be used to particularly identify the MP cell?
Vinodhini
0503171A
TGO2
Hi there Lizzie...
just wondering... you said
"For malaria parasite (MP), require making a smear when the platelets are <150>"
what's the smear for? Do you actually look the parasite under the microscope or do you look for abnormal blood cells?
Cass
hi Mingboon
the centrifuge used is specially for the plate, each time it can only centrifuge 2 plate. the purpose of diluting is to enable to see the result because the hole for the plate is very small, if the reagent used is concentrated , the result might not be shown, in fact using diluted, the result is more clear and distinct. if the result is not clear need to redo on the tile to reconfirm. we only view under microscope for the Du testing to check for agglutination.
Lizzie
hi dorene,
actually is if i say like 1:3 or 1:1 some of them might be confused cos i myself also confused. DI water will cos hypotonic condition and burst the red cell whereas saline the concentration is near to our body abd give an isotonic condition.
Lizzie
hi johanna,
although the machine is automated but it is only partally automated, there is a minimum requirement for the machine for closed sample mode, it require at least 0.5ml of blood cells.
regarding to the machine, just make sure the systenm is in closed sample mode on the pc connected to the machine, then all u need to do is to press start on the machine for the machine to run by itself.
Lizzie
Hi lizzie!
What do you mind by running the IQC check by using open and closed mode?
What is open and closed mode?
Thanks! XD
Charmaine Tan~ TG01
hi cass,
a smear is needed when platelets is <150, this is a characteristic of MP. even the graph shown no abnormal sign but the platelets is low, is best to make a slide to reconfirm the person is not having MP.
under the slide, we actually look for RBC enlarge which is a characteristic ob MP and of course the MP cell which will fully confirmed that the person has contracted MP.
Lizzie
hi vino
regarding to the stain, we use a automated stainer, hus need to load the slide in and start the stainer will stain the slide. the name of the stainer is Aerospray Hematology Slide Stainer/ Cytocentrifuge. it offers rapid (3-6min) Wright Giemsa (4-8min) May Grunwald Giemsa (8-17 min) stain programs.
Morphology of MP cell is you will see alot of granules within the cells and it is stained dark purple, in fact, it is an enlarged platelets and you will also able to see ring shape in some other forms within the cells. it is distinctive from other cells.
Lizzie
Lizzie
hi charmaine
using open and closed mode is to confirm that both mode are doing well and provide accurate result.
closed mode is automated one, jus load in the sample and press start it will automatic run.
as for open mode, is manual, u need to open the cap of the tube, insert the tube to the prop, thenmake sure the tip of the prop touch the bottom of the tube for the prop the suck the sufficient volume. the volume needed is definitely smaller than the closed mode.
Lizzie
Heyy!
Your work sounds fun! A simple question for you. Can you explain WOC (WBC optical count)and WIC (WBC Impedance Count)? What are their differences and significance? Thanks!!
Kent TG01
Hi lizzie,
Using microtitre(MP) plate to do ABO phenotyping in your lab is rather interesting because I seldom do such tests in the blood bank lab that I'm attached to. Cheers!
However, I don't quite agree with your on your reply to mingboon that if the reagent (antisera) used is concentrated , the result might not be shown. In my view, I think it will give a false positive result if you could visualise how IgG and IgM binds in a microtitre plate. The commercial anti sera that you used should be of polyclonal I presume since it is anti A, B anti sera. Making a dilution thus prevent this.
Are you running ABO typing parallel with Absc in the same MP that you need to incubate at 37oC? and even if you do an Absc at 37oC are you able to detect clinically signigicant Ab? because usually IgG just sensitised the cells even at 37oC unless you add the AHG. or, you are actually doing an extended immediate spin, which I don't think is necessay for ABO typing.
Does your lab change new MP after use? because to what I know if you use a new MP you need to add antistatic solution to prevent RBC from sticking to the well.
What is the turn around time using MP to type? My lab uses gel cards ( and tubes) for Absc and ABO typing and seldom MP so i wish you could enlighten me more about MP since you are doing it in your lab.
Sorry.. If I frightened you with my overwhelming queries. =P
Loh Sharon, Tg 01
hi sharon,
regarding to ur question, we jus do purely ABO test, forward and reverse, and Rh typing, we do not need to incubate the plate at all because we have over hundred samples to test everyday, each plate take about 20 min to complete, therefore we will just give a one and a half minute spin to save time.
if u remember, we do use a microtitre plate in our school lab experiment, the plate is a disposable one, therefore, we will throw after each plate is used. and the plate we used is a empty one, therefore we don't need to worry about rbc sticking to it.
i don't really understand your question about Absc because my lab didn't do that test. my lab isn't that specialised (more towards commercialised), thus we only do general testing, we don't really do alot of test.
hope i answer your question.
Lizzie
hi kent,
actually, i am not so sure also, but i know that the use of optical method simultaneously with the impedance method is to analyze white blood cells. both methods will results in greater WBC count reliability. We also ascertained that NRBC and WBC interference has a greater effect on WBC impedance counts than on WBC optical counts
that's all what i know, hope i answer your question.
Lizzie
Hello Lizzie,
Are only thin films(smears) used for malarial parasite testing in your lab?
Looking forward to your reply. Thanks.
- Alex Tg02
Hi lizzie,
In your procedure section of your post, you stated that there was incubation at 37oC so I thought that you do Absc together with your ABO testing.
According to a book that I've read, it stated unless forbidden under infectious disease regulations, microplates can be kept as permanent eqipment by washing with Clorox.
Actually, my Absc question is related to the 37oC that you stated in your procedures. Since, you didn't do Absc in your lab, then it is ok.
By the way, does you lab uses gel cards for ABO, Rh testing etc?
Thank you lizzie for you prompt reply =)
Loh Sharon , Tg 01
hi alex,
actually, we use cell dyn 3700 to check for low platelets and then we make a normal smear, then we look under microscope for the mp cells, as simple as that. no further steps.
hope i answer your question.
Lizzie
hi sharon,
actually the incubation is for Du testing. my lab got no Absc test. by the way, my lab don't used gel card for convenient purpose.
hope i answer your question.
Lizzie
hihi,
regarding the abo and rh typing. how many times do you perform the test to confirm the blood group?
Juexiu
tg02
hey lizzie!!
what is α, β, O testing used for?
Ying Ying
TG01
hi jie xiu,
actually one time will do, if the forward and reverse result does not tally for the abo, we will repeat on tile to confirm.
if Rh is negative, we will do a Du testing to confirm.
hope i answer your question.
Lizzie
hi ying ying,
α, β, O are A cells, B cells and O cells which is use for reverse grouping to make sure that the forward grouping is tally with the result of the reverse.
hope i answer your question.
Lizzie
Hey,
For the MP test, how can the enlarged RBCs be differentiated from the normal RBC? And do you have to reject patient samples with less than 1ml of blood?
Thanks.
Yong Yang
TG02
hi yong yang,
good question... as a skilful lab technologist, should be able to differentiate because they are trained. of course for me, don't think i will be able to differentiate. for less than 1ml, we do not reject but in fact we will use the manual method which is more time consuming.
hope i answer your question.
Lizzie
hey lizzie.
erm may i ask, do you have to re-test for the results of malaria (for confirmation) if the first run was positive?
and how long does the machine takes to analyse the result?
is there any turnover time for the tests?
Natalie
TG01
hiya Lizzie,
May I know how do u do background clean for cell-dyn 3500/3700? Thanks ya
Michelle
Lizzie, u mentioned 'Do background clean for 3-5 times to make sure the values stated on the screen is near to 0'. Just curious to know what the uncertainty value is.and whats meant by background clean ah?
Enjoy!
Charmaine
TG01
hi natalie,
we normally do not do retest to confirm instead we look under microscope for malaria parasite cells if no MP cells we will confirm is neg.
the machine will take around 5 min to give the result for each sample.
for urgent samples we need to validate result with 2 hours.
hope i answer your question.
Lizzie
hi michelle,
the background clean is in the program just select and press start for the open sampler mode which is on the machine, the machine will do the background clean by itself. jus repeat for a few times.
for your info the open and closed sample mode has different button for "start".
hope i answer your question.
Lizzie
hi charmaine,
the value should be less than 0.1 for red indices and less than 20 for platelets. as low a possible will do.
background clean is to avoid any contamination. because if we don't do background clean for the day there might be a possiblities that the value maybe high.
hope i answer your question.
Lizzie
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