Hey. I've been attached to Haematology lab for 3 weeks and i think, it's the most busiest lab. In this post, i'm going to share about Bleeding Time and Dengue Serology.
Bleeding Time
Bleeding time is done to estimate the integrity of haemostatic plug which will be formed at the site of injury to arrest bleeding. It is also used in the diagnosis of haemostatic defects and platelet dysfunction. A minor incision is made on the forearm and the time between the infliction of incision and the moment bleeding stops is measured. The formation of haemostatic plug on the site of injury depends on the adequate amount of platelets to adhere to the subendothelium and to form aggregates. A prolonged bleeding time does not itself diagnose underlying platelet disorder. It does indicate the need for further tests such as platelet count, platelet aggregation studies etc.
The items needed to conduct the test for bleeding time are sphygmomanometer, filter paper, antiseptic swab, stopwatch, plaster and single-use Surgicutt blade. The sphygmomanometer cuff is wrapped on the upper arm. Cuff will be inflated to 40mmHg and will remain at exact pressure throughout the test. The time between inflation of cuff and incision should be 20-60seconds. For a newborn, the pressure should be maintained in accordance with the weight of newborn. (<1kg-> 2 kg- 30mmHg) The forearm must be cleaned with alcohol swab before incision. Area with veins, scars or bruises should be avoided. Minimal pressure is used on Surgicutt device so that it lightly touches the skin. After incision, the timing will start. Every 30seconds, the blood is blot with the filter paper until it no longer stains the paper. For neonates lesser than 5 months, the test can be stopped at 5 minutes and bleeding is reported as more than 5 minutes. For adult and pediatrics, the test can be stopped at 20 minutes and bleeding time is reported as more than 20 minutes. The reference range based on Surgicutt’s insert, 2 to 8 minutes for adult, 1.30 to 8.99 minutes for junior (5 months to 15 years old) and 0.85 to 1.65 minutes for newborn (1 to 4 months).
The estimation of the integrity of haemostatic plug measures the interaction between the capillaries and platelets, platelet defects and vascular disorders. It measures the interaction between the capillaries and clotting mechanisms in vivo. However, the test does have limitations. It is not always positive in platelet function defects because of the mildness of the defect or variability in the condition. The results of this test alone are not sufficient to diagnose specific conditions. Further tests are needed.
Dengue Serology
Dengue serology test is performed to obtain a qualitative presumptive detection of IgG and IgM Dengue, a flavivirus, is found in large areas of the tropics and subtropics. Transmission is by mosquito, principally Aedes aegypti and Aedes albopictus. Dengue virus infection causes a spectrum of clinical manifestations ranging from asymptotic to fatal haemorrhagic disease.
When the virus is present in the patient's serum, dengue-specified IgM or IgG antibodies bind to anti-human IgM or IgG antibodies immobolized in 2 lines across the cassette membrane. Colloidal gold complexes containing recombinant dengue 1-4 antigens are captured by the bound patient's IgM or IgG to visible pink line(s).
Procedure:
1. Centrifuge the blood sample at 3000rpm for 10 minutes.
2. Sample serum should be preferably clear, non-haemolysed, non-lipaemic and non-icteric.
3. Before running the assay, allow the buffer to equilibrate to room temperature after taking it out from the fridge.
4. Remove cassette from pouch.
5. Add 10uL of serum in circular well using mechanical pipette.
6. Allow sample to absorb entirely into specimen pad within circular well.
7. Add 2 drops of buffer to square well at base of cassette. Ensure buffer bottle is held vertically.
8. Read results in exactly 15 minutes after adding buffer. All reactions are visualised as pink lines.
9. For assay to be valid, the control line must appear.
Retrieved from: http://www.panbio.com.au/download/Dengue%20Duo%20Cassette%20procedure.pdf
The picture is not very clear. So, just go to the website and see.
If only the control line appears, the test is regard negative. However, if dengue infection is strongly suspected, the doctor may consider to conduct PCR for dengue virus within 5 days.
If control and IgM lines appear, it is a primary dengue infection.
If control and IgG lines appear, it is a secondary dengue infection.
If all lines appear (control, IgM and IgG), it is a secondary infection.
Control line must appear everytime. If it does not appear, the test is regard as invalid.
Primary infection is characterised by the presence of detectable IgM antibodies 3 to 5 days after onset of infection. Secondary infection is characterised by the elevation of specific IgG antibodies 1 to 2 days after onset of infection and in majority of the cases, this is generally accompanied by an elevation of IgM antibodies.
Hope u guys understand.
Hardina Hamzah
Tg01
14 comments:
Hello Dina!
I think the bleeding time is interesting. I didn't get to do it during my attachment in Haemato. Anyway, when do the doctor order this test for the patient? Like you mentioned that even if its a prolonged clotting time, it doesnt mean anything. So is it because the patient have difficulty clotting?
You understand what I am talking about right? If not, I just wanna know why doctor order the test for the patient?
Leslie (:
Hi Leslie.
Bleeding time is done to diagnose bleeding problems.
Bleeding problems such as unexpected bruising, cuts that bleed excessively, spots on the skin etc.
I've encountered one scenario where this 5 years old girl has greenish/reddish spots on the skin. Her mum told the doctor that her daughter sometimes suffer some bruises but they takes a long time to heal which seems unusual. For us, it just take 1 week to heal and the mark will clear eventually. But for her, the spots can last for more than a month. And sometimes may have unexpected bruising.
So the doctor send her to the lab where one of the medtech will conduct the bleeding time test.
I hope with the scenario, you can understand better.
Hardina =)
hi hardina,
under procedures for dengue serology, may i know wat is the use of the buffer and why must the buffer bottle be held vertically? thanks.
Malerie
TG02
Hi Hardina
May I know what's non-lipaemic and non-icteric?
Is there a specific type of buffer used? What's the purpose of using a buffer?
Thank you
LeeJin
TG02
hi hardina
may i know why must the buffer be at room temperature before doing the experiment? why not just take it out and use directly?
Thanks
Justina
0605950E
TG01
Hi Hardina,
1)What are the further tests requested for bleeding time?
2)Under the dengue serology (procedure),
'2. Sample serum should be preferably clear, non-haemolysed, non-lipaemic and non-icteric.'
What if that does not occur? What are the actions taken?
3)Why must the buffer be cooled to the room temperature?
4)Is bleeding time test a qualitative or quantitative test and why?
5)Why must the cuff be inflated to 40mmHg but not any higher/lower and remain at exact pressure throughout the test?
Thankz alot!
Han Yang
TG01
Hi,
You mention that test has its limitation that it will not always show positive in platelet function defects because of the mildness of the defect or variability in the condition. So is there any other cofirmatory tests that you lab does to ensure that the negative result is really negative result. If so, how?
Can you also give some examples of what kind of conditions that the bleeding test result will give a negative result rather than a positive result? For example, what kind of variability in the condition.
Thanks.
Xin Yi
TG02
Hey! I was in coagulation for a week, but didn't get to practice much there. Anyway, what is bleeding time used for and are there any controls to fun for this test? Thanks.
Debbie
TG02
Hi Malerie.
Actually, the use of a buffer is not really known. I've searched and asked but seems there is no definite answer. So here's what some medtechs and I think:
1. It's to enhance the reaction.
2. It's to allow the patient's serum to flow through the cassette.
The buffer bottle need to be held vertically as it will produce a big drop. With 2 big drops, it will be suffcient to allow the buffer and serum to flow. I've once made a mistake by not holding it vertically. Therefore, it's insufficient and will not produce any result.
Hardina =)
Hi Lee Jin.
Firstly, lipaemic means high fat content. Maybe due to high cholestrol. The serum will appear reddish as if there's haemolysis. Secondly, icteric means high bilirubin due to liver failure, jaundice etc. The serum can appear as very dark yellow to green.
So if u add "non" to the word, it means the opposite. Hehe.
I don't think there's a specific buffer used. The one the lab I'm attached to is using buffer containing 1% prolactin. The buffer comes with the dengue kit. Previously, instead of using a cassette, they used strips and they come with a buffer which contains 0.1% sodium azide.
For the purpose of the buffer, you can refer to the comment I replied Malerie.
Hardina =)
Hi Justina.
It's actually advised by the manufacturer. And what the medtech mentioned, the optimal temperature for antibodies to react is room temperature. Since the buffer is kept in the fridge, it needs to be warmed to room temperature.
One of the medtech experienced using the buffer directly and it gave a negative result. To recheck, she warmed the buffer, then the result turns out to be positive.
Manufacturer knows better.
Hardina =)
Hi Han Yang.
1. Bleeding time cannot justify any diagnosis. Therefore, further tests are needed such as platelet count or platelet aggregation studies. The test requested are depending to the doctor.
2. The blood sample will be centrifuged at a higher speed than 3000rpm. This allow the particles present in serum to be segregated and able to see a clear light yellow serum.
3. Refer to the comment I replied Justina.
4. It can't be considered as qualitative as the test doesn't involve in identification. It can be quantitative as it includes measurement but the measurement is not definite as this test is not a confirmatory test. I discussed with some medtechs, they agreed the test to be a semi-quantitative.
5. It is to ensure constant steady blood flow. As there will be an incision, it will prevent blood from gushing out.
Hardina =)
Hi Xin Yi.
As bleeding time test is not a confirmatory test, further tests are needed eventhough the result turns out normal. The request of tests depends on the doctor. The tests that can be conducted are platelet count and platelet aggregation test.
One example of condition is Von Willebrand factor. This factor is a protein that acts like glue where it helps the platelet to stick together and form blood clot. Maybe due to high amount of this factor can affect the result.
Hardina =)
Hi Debbie.
Bleeding time test is used to check how long will the platelets take to form a clot and stop the bleeding. This is mostly requested when a patient has bleeding problems(refer to comment I replied to Leslie). However, it's not a confirmatory test. Further tests are needed(described in other comments).
No control are used for this test.
Hardina =)
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