I was posted to the Hb electrophoresis lab for 3 weeks. Samples ranging from EDTA blood tubes, plain blood tubes and cord blood are sent here. Tests such as Kleihauer test and Sickling tests are doone here. Since quite a number of them have posted on the Kleihauer test, I shall post on the Sickling test. =)
Sickling test
USE:
Sickling test is used to demonstrate the sickling phenomenon in test samples. The technique detects both the heterozygous (AS) and homozygous (SS) forms of sickling and is a rapid screening test for the Hb S status of an individual. It is also done as a confirmatory test after alkaline and acid haemoglobin electrophoresis. Haemoglobin electrophoresis is able to differentiate between homozygous and heterozygous forms of sickling.
ADDITIONAL INFO: From the haemoglobin electrophoresis, heterozygous (AS) sickle cell trait will show 60% HbA and 40% HbS. In homozygous (SS) sickle cell anaemia, there will be a higher percentage of HbS (more than 50%) as compared to heterozygous sickle cell trait.
For sickle cell trait (AS), one of the genes is HbA, the other is HbS. In other words, the person is a carrier of the sickle haemoglobin gene but do not display symptoms of sickle cell disorder. However, one has to be careful when doing activities that require less oxygen such as scuba diving. For sickle cell anaemia, both the genes are HbS and the person will display symptoms of sickle cell anaemia such as anaemia and pain.
PRINCIPLE:
Sickling phenomenon occurs with low oxygen tension as HbS has a decreased oxygen affinity. A small drop of blood added to a reducing agent (sodium metabisulphite) to induce sickling in susceptible cells. It is then sealed between slide and cover glass and incubated at 37degrees for the sickling phenomenon to occur.
PROCEDURE:
1. 5 drops of freshly prepared reducing reagent (0.2g of sodium metabisulphite dissolved with 10mL of millipore water) is added to 1 drop of EDTA blood on a slide.
2. The reagent and blood is mixed with the pipette tip on the slide and covered immediately with a coverslip.
3. It is then sealed with petroleum jelly-paraffin wax on all 4 sides of the coverslip. It must be properly sealed to prevent false negatives.
4. The slide is then placed in a black box to ensure moisture when it is placed in the incubator at 37degrees for 2 hours or overnight. This is to prevent the preparation from drying out and obtaining false positives.
5. The slide is then examined under the microscope.
That's all for my last post for this 20-week long SIP! 3 more weeks! Yay! JIA YOU everybody. lol.
MALERIE
TG02
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10 comments:
Hi Malarie
Can u describe what will be seen under the microscope if it is positive and if it is negative?
Oh ya, also i would like to know since the electrophoresis can detect both heterozygous and homozygous form sickling, why need do the sickling test? the sickling test does not differentiate the 2 form rite? I understand it is a confirmatory test, however,i would like to know what does the sickling test shows which the electrophoresis does not show?
Thanks alotz.
Xin Ni
TG02
Hi Malarie,
can i ask if only EDTA blood can be used?can the test still be done if the blood sample is put into sodium heparin tube?in other words does the chelating effect of EDTA affects the test?
hmm...why the results will be a false negative when the slide is not properly sealed?thanks a lot;)
Rachael
Tg01
Heys:d
Why isit u have to place the slide in a black box to ensure moisture?
i mean u place it in a normal box oso it will prevent evaporating if the box is covered rite?
or isit coz its light sensitive? or mayb u said it coz ur lab juz uses a black box? lol:d
thx thx
Neela
TG02
hey malarie,
i'm sorry but i dont quite understand about the part you mentioned haemoglobin electrophoresis, heterozygous (AS) sickle cell trait will show 60% HbA and 40% HbS, how do you quantitate them? the electrophoresis result would be in the form of what? thank you
Liyanah Zaffre
0607718D
TG02
hi xin ni,
for a positive sickling test, one will be able to see sickle cells amongst the red blood cells under the microscope. if it's negative, one will be able to see wat is usually seen under the microscope in a normal person's blood such as RBCs, WBCs and platelets.
from wat i heard, over here, it is done together as a package. a sickling test is done concurrently with the haemoglobin electrophoresis. in some countries, only a sickling test is required to diagnose whether the person has sickle cell or not.
a sickling test will be able to show the shape of the RBCs - sickle cell while haemoglobin electrophoresis will be able to show the different bands of haemoglobin. for example, in alkaline electrophoresis the control used is ESFA so if the band migrates to E means the blood consists of HbE and likewise.
Malerie
TG02
hi neela,
i think any colour box will do. but my lab uses a black box. in the manual, this step is not necessary but our lab still carry out this step to prevent the preparation from drying out if it is left overnight, causing the cells to shrink and change its shape.
p.s. sickle cells are not sensitive to light.
Malerie
TG02
hi liyanah,
we dont quantitate them. the values i mentioned acts as a gauge. example, for haemoglobin electrophoresis, u'll be able to see bands. we just look at the thickness of the band and compare. if HbA looks thicker than HbS then it's heterozygous and likewise.
haemoglobin electrophoresis uses the same principle as gel electrophoresis except that haemoglobin electrophoresis is able to separate the various types of haemoglobins present in the patient's blood. the result would be in band form on the gel.
Malerie
TG02
to rachael,
no, the chelating effect of EDTA does not affect the test. only EDTA blood can be used. if heparin tube is used, the sample is rejected.
as i've mentioned in the post tt the sickling phenomenon occurs with low oxygen tension. so if the coverslip is not properly sealed, oxygen must enter, causing the sickle cell to revert back to normal, resulting in false negative results.
Malerie
TG02
Hi.. why isnt alkaline and acid haemoglobin electrophoresis suitable as a confirmatory test?
Thank you XD
Tan ZHao Rong
Tg01
hi zhao rong,
haemoglobin electrophoresis is done concurrently with the sickling test. if the sickling test is positive, haemoglobin electrophoresis should also show bands in the 'S' lane, which means HbS is present. results from both tests should tally to ensure that there is no false positive results.
Malerie
TG02
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