Name:Dorene Chen
Admin no":0504156A
class: TG02
I was assigned to work in Quest Laboratories, department Clinical Chemistry/Biochemisty for the 1st 3 weeks of attachment. The department is almost fully automated except for a few tests like OGTT (Oral Glucose Tolerance Test), HbA1c (Glycosylated Haemoblobin) and Drug 5/7. For the first week, my duty is to run HbA1c using the machine. for the second week, i learnt how to test OGTT and Drug 5/7 using testing kits.
HbA1c testing :
EDTA tube is used to test for HbA1C as the whole blood is needed to test for glycoslyated haemoglobin.
Before loading sample into machine (ADVIA 1650 - Bayer) bubbles are removed to prevent the probes from taking in the bubbles. This is because the machine only required 10 microlite and difference in small volume of blood will alter the results.
the sample is then loaded into the machine and results will show in 30 mins time.
Ranges :========================Actions to be taken:
<4.5% =========Low=============Rerun test in 1:41 dilution
4.5% to 8%======Reference Range====Record results
8% to 12%====== Moderate High======Rerun sample in 1:41 dilution
12% to 15%======High============ Reun sample in 1:51 dilution
>15%==========Very high=========Rerun sample in 1:81 dilution
Dilution of the samples during rerun has to be done manually.. eg: in 1:41 dilution, 400 microlite of denatured water and 10 microlite of sample are added and incubate at room temperature for 10 mins. diluted samples will then be loaded into machine for analysis.
Prinicple of HbA1C:
Hba1c is an diagnostic tool to control, check for diabetes.
when glucose levels are persistantly high, glucose will enter RBC and form an irreversible complex with haemoglobin, to form glycosylated haemoglobin (HbA1C). this form of haemoglobin will remain in RBC until RBC dies. (RBC has a life span of 120 days).
Calculating % of HbA1c in RBC will therefore determine the glocose level that always reside in the blood. In diabetes patient, % of HbA1c is usually high. A normal level of HbA1c will show that diabetes patient has a good control of the disease.
Principle of ADVIA 1650 (Hba1C)
Uses immunoassay method. The machine first uses probe to extract 10 microlite of sample into curvette. 400 microlite of denatured water is then added.
This water causes cell lysis to allow contents in RBC to be exposed and used for experiment (sample will change from red to brown in color).
Latex coated specific antibody is then added into the curvette and this ab will bound to random glucose. An agglutinator is then added and it will bound to Ab to produce scattered light that increase the absorbance.
If there is presence of Hba1c in blood, Hba1c will competes with the agglutinator to bound to Ab and this reduces the amount of agglutinator bound to Ab which in turn reduces the scatted light and absorbance.
The amount of absorbance recorded will reflect the amount of Hba1c.
OGTT test
OGTT (Oral Glucose Tolerance Test) tests for the amount of glucose present in urine sample. Usually 3 different urine samples are needed : fasting, 1st hour, 2nd hour. Sometimes, (i dunno why) only fasting and 2nd hour are received but we can still use it to find the results. Combur test kit (dip stick) is used to detemine glucose level.
The process is quite simple: just dip the stick into urine and the colour of the reagent in stip stick will change immediately. there will be different color for different glucose level. However, the results is valid for only 3o seconds so we have to read the results immediately. This is because the reagent will react with the surroundings and change the intensity of the color.
Taken from : www.tierklinik.de/medizin.php?content=00151 , www.notfalllabor.de/Tests/20-0-10131-597-0-0-0-K.html
fasting urine : the "reference range" of glucose is usually normal or 1+ and ketone neg
1st hour urine : glucose is 4+ to 3+ and ketone neg
2nd hour urine : glucose 3+ to 2+ and ketone neg.
ketone should not be present in urine.
Eg: reference range of fasting urine is <20mg/dl, color="#ff0000" size="4">Drug 5/7 test
tests for THC (marijuana) , COC (cocaine), OPI (opiates), MET (methamphetamine), AMP (Amphetamine)
on the test kit, there are 2 lines (control and test line). The "control line" must always show when doing the test (this is to show result is accurate if no control line is seen, result is considered invalid). For the "test line" presence of this line will show neg result of this drug while absence of this line will show positive result for this drug. This test is based on chemical reactions that the reagent used will formed a colorless product with the specifc drug tested.
Visible Control line (C), visible Test line (T) ==== Negative
Visible Control line (C), no Test line (T)======= Positive
No Control line (C), no Test line (T) ==========Invalid test (rerun)
Btw, urine sample is used for this test.. don't know why they want to do this test too.. well, that's all I had learnt in the 1st 2nd week =)
28 comments:
hello hello =)
Just want to know, do u have any idea if there is glycosylated haemoglobin in your blood, how it will harm our blood cells or our body in general? =)
Also, i understand that there is different color shown for different glucose level for the OGTT test. Is there like a chart to follow? Maybe u can like briefly let me know what color stands for high glucose and what for low?
Thanks!
Chen KangTing
0503331A
TG02
Hey this is about the HbA1c testing. When you said that Hba1c is a diagnostic tool to control, check for diabetes and also that by calculating % of HbA1c in RBC will therefore determine the glucose level that always reside in the blood, does it mean that the HbA1C binds to only RBC that is filled with or contains high levels of glucose? Meaning does it only bind to glycosylated haemoglobin??
And as for the reference range, what does it represent anyway? The percentage shows that whether u need to rerun or record the result but what does it really tell you? Does the percent represents the amount or percentage of glycosylated Hb??
OGTT test 1st hour urine : glucose is 4+ to 3+ and ketone neg
2nd hour urine : glucose 3+ to 2+ and ketone neg. Is this the required range or example?? If this is the range, then you plus 1 or 2 to this range? E.g if glucose is 5+ and ketone is neg., Isn’t this value abnormal??
Drug 5/7 test tests for THC (marijuana) , COC (cocaine), OPI (opiates), MET (methamphetamine), AMP (Amphetamine) and urine sample is used for this test. Does that mean that the urine contains THC, COC, OPI, and LET, etc??
Hope your doing great take care gal! Adioss
Regards
Vinodhini
TGO2
Hey,
Just two questions. First, what do you mean by denatured water? Second, why does your antibody have to be latex-coated and lastly, you mentioned a Combay test right? its method is to use dipstick or the dipstick method is actually the Combay test?
Johanna, TG02
heyhey!=)
why do u nid to carry out different dilutions for different results that fall out of range?
and i dun realli understand the part on control line used for the dip stick method.
June, TG02
hey,
is urine sample the only sample that can be tested for the Drug 5/7 test? oh then out of curiousity, why is the test named drug 5/7? is there any special meaning behind it?
thx!
-sharon ang, tgo2
hello :)
I would like to ask do you ever receive blood samples for OGTT? Do you do QC and calibrations for ADVIA 1650-Bayer? If yes, how mnay times a day? Thanks
Ci Liang TG01
Hellooo... May i know for diabetes monitoring (DM), what other test is done beside HbA1c testing. My lab include several test for DM Panel.
Pei Shan, TG02
Hey Azhar here.
For the Drug 5/7 test, when is it requested for and why? Is it to convict drug addicts, as evidence in court or something? And is there any follow up tests?
Thanks
Azhar
TG01
Hihi :)
I was just wondering what's the principle of the drug 5/7 reaction based on? Is it by chemical reaction..?
U mentioned that denatured water is used 2 lyse the cells. wat is denatured water and is it a must 2 use this or cn use DI water or anythin like tt?
thanks! hope u re doin fine! cheers!!!
shahirah, TG01
Hi kang ting =)
Actually, it is normal to have glycosylated haemoglobin (HbA1C) in blood (4.5-8%).Because as long as there is glucose in blood, some of them will enter RBC to form HbA1c. If i'm not wrong, Hba1c does not directly affect blood cells however, it causes complication for diabetes mellitus (eg: retinopathy, Nephropathy, Macrovascular disease )
For OGTT, I will post the chart as a picture in the blog cause it's quite difficult to explain =)
Dorene =D
hihi June,
Because if concentration of HbA1c is too high (eg:15%) a 1:81 dilution will determine how severe the condition of the patient is.
eg: after 1:81 dilution, the value obtained is above 8%. this means that the patient's condition is severe.. however, if concentration is below 8% it means patient's condition is not that severe compared to the value above 8%. we can see how well controlled is the disease (eg:diabetes)
For control line, I will post a picture regarding this cause it is difficult to explain without pic.. sorry for poor explanation =)
Dorene =D
Hello Sharon,
Yup, only urine sample can be tested for Drug 5/7 in my lab, because the kit requires urine sample.
I checked with my colleague and found out that Drug 5/7 actually means Drug 5 and Drug 7.
Drug 5 consists of the 5 drugs that i mentioned in the blog.
drug 7 consists of the 5 drugs as well as BZO (benzodiazepines), BAR (barbiturates)
I think the name is used because of the number of drugs it tests
Dorene =D
hihi Azhar,
I'm not sure why it is being requested, perhaps by the police(not sure)..er.. maybe they are caught by police,they are in in drug rehabitation, or being doped by someone in the club then they want to verify if they took this drugs before. .. about when they can request for it, they can request for it in any clinic, cause i am working in a private laboratory, so the clinic will send the samples to us.
I'm not sure whether the results will be used in court =.= quite confidental stuff. (psss..my friend offered to call the police once when results showed positive, but my section head say no need)
Follow up test is simply to use another brand of test kit and test to detemine if the results is still +ve.
Different test kit uses different reagent so this 2nd test kit acts as confirmatory test.
Dorene =D
Hihi Shahirah =)
Drug 5/7 reaction is based on chemical reaction via use of reagent that reacts to the drugs.. the reagent will be colourless when it reacts with the specific drug (forming only 1 control line). reagent will form purple colored line if drug is not present (forming 1 control line, 1 test line)
Denatured water contains buffer that maintains the pH after cell lysis so that Hba1c will be stable after lysis of RBC.. using DI water, though causes lysis of cell, does not really maintain the desired pH.
Thanks alot for your concern =D Good luck to you too!
Dorene =D
Hey ,this is the question abt HbA1C, for this sentance"A normal level of HbA1c will show that diabetes patient has a good control of the disease"what does a normal level mean ?does it mean a consist level compare to previous result of the same patient or falls into the reference range? em~Other questions have already asked by others. So I wont repeat l:)~~
Hi Johanna,
denatured water actually means denaturation water.. it causes denaturation of cells.. but the product is called "denatured water"
I think Ab is latex coated because without it, the agglutinates formed would be very small, so it would not change the absorbance that much....
Actually, it's called Combur.. type wrongly sry~ Combur is the name of the product. Combur is the test kit that consists of dip stick. the test is called OGTT (Oral Glucose Tolerance Test).
Actually for Combur test kit, it includes pregnancy test, haemoglobin presence, WBC etc.. but for OGTT, only glucose and ketone is checked.
I will add a picture of dip stick in my blog later too, hope you can get a clearer picture of how's it look like.
Dorene =D
hihi Ci Liang =)
Currently I only uses urine sample to do OGTT. But Fluoride blood sample can also be use for OGTT.. I'm not sure how they use it to test for OGTT though, because it is automated..
*edit* I've checked with my section head and flouride blood sample is used to determine the level of glucose found in blood. So for OGTT, you can use the specific method(using 2-3 flouride sample (3 different timing) to determine glucose level or the easier method using urine *edit*
Yup, in my lab, we do QC in the monring at 7.30am and 4.30pm everyday. Caliberation is only done when there is alteration to the machine eg: change of reagents, buffers. QC and caliberation are run until all values are within range. =)
Dorene =D
Hihi Peishan
Actually for my lab, there are also many DM panels, but all of them requires HbA1c testing. Some requires glucose level test to support Hba1c results.. Abnormal Hba1c results are used to tally with glucose level..High glucose level usually means high Hba1c...
Other test includes total cholesterol level etc..
sorry to post so late..
Dorene =D
hihi no name! (later i edit that away :P)
A normal level of Hba1c is the reference range - 4.5% to 8%.. ( FYI: Actually, the best HBa1c level is around 4.5%. )
For diabetes patient, having a consistent Hba1c level may not be good, as they can be consistently unhealthy.. eg: 12% for every routine test of Hba1c..
Another way to check if these patients controls this disease well is to compare it with previous results.. eg: 1st result = 12%, 2nd result (1 month later) = 9% .. although it is still high, but it is better controlled..
Dorene =D
hihi Vino =)
"HbA1C binds to only RBC that is filled with or contains high levels of glucose? Meaning does it only bind to glycosylated haemoglobin?? "
I don't really understand the sentence but if I guessed correctly, you got the wrong idea of what is HbA1c.
HbA1c, called glycosylated haemoglobin, is actually a complex, formed by glucose and haemoglobin. Therefore HbA1c is actually formed inside of RBC, not bound to RBC. (More detailed information is already in blog^^)
The reruns for abnormal results are just used as a confirmatory test, to verify that the results is correct.. eg: in first run, i got 15%. In 1:81 dilution, i get the result of 9% (2nd result is often still not within range). The results from rerun is still higher than average. Therefore, the patient has abnormal level of HbA1C.
the reference value tells you the percentage of HbA1c existed in RBC. High abnormal reference range means that there is prolonged high glucose level in blood which can lead to eg: diabetes.
For the next question, the value for glucose is only neg, +1, +2,+3,+4. As glucose level in people differs from one to another, there is no standard reference range (eg: if referece range for fasting urine is <20mg/dL, a +2 result, ~40mg/dL, is still acceptable)...gluose level is supposed to be low during fasting, higher during 1h (after eating) and slightly or remains the same in 2h. but if you see a result that shows, 3+ for fasting, 3+ for 1h, 4+ for 2nd h, it shows that the value is abnormal.
For the final question, these drugs are not supposed to be in people, unless they consume illegal drugs... therefore if the test is positive, well good luck to them =)
take care too! Hope you're doing well in your attachment
Dorene =D
hihi
you mention about the machine ADVIA 1650 - Bayer. other then it run Hba1c, what other test the machine run? any control need to be run? at my attachment there is also a machine(cobas) for the hba1c but it run more then 1 type test.
could you update us when you know what the purpose of the drug 5/7 testing? as u mention you don't know why they do this test.
Juexiu
tg02
Hi Dorene, I understand that only when the glucose level is high in the body,glucose will bind to RBC and form an irreversible complex with haemoglobin, to form (HbA1C). However, can you explain what do you mean by 'A normal level of HbA1c will show that diabetes patient has a good control of the disease' ? Thanks :)
Sasi
050380G
TG02
Hi Dorene, I understand that only when the glucose level is high in the body,glucose will bind to RBC and form an irreversible complex with haemoglobin, to form (HbA1C). However, can you explain what do you mean by 'A normal level of HbA1c will show that diabetes patient has a good control of the disease' ? Thanks :)
Sasi
0503804g
TGO2
hello Juexiu
The machine runs almost all tests in biochemistry.. the no. of test available is around 3 pages.. =.=" so can't list them all but here are some of the common tests..lipid profile (cholesterol, LDL, HDL, triglycerides), diabetic profile ( glucose,Hba1c) alcohol serum, creatinine, CK, etc etc..
No controls are used actually, cause everyday there are around 4000 samples to be run.. with control = 8000 *shivers* but we have QC.. but I'm not sure with other departments though =)..
the purpose of drug5/7 is to check for illegal drug intake.. er..It's actually on the first sentence..maybe i didn't explain well^^ paiseh
" tests for THC (marijuana) , COC (cocaine), OPI (opiates), MET (methamphetamine), AMP (Amphetamine) "
that last sentence mean i don't know why the people requested those test^^.. kinda stupid (opps) cause some of them are requested from clinics..sry for misleading..
Dorene =D
hihi sasi
the normal results actually means reference range.. ^^ maybe i phrased that abit weird...
If the patient Hba1c % is in reference range, it shows that he has a good control of the disease
Dorene =D
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