Clincal Chemistry/ Laboratory Managment and Quality Assurance

Been posted to biochemistry from 22/06 to 11/07, meaning 3 weeks in biochemistry.

In biochemistry, its more like clincal chemistry, doing tests like wat we learn, example liver function test, glucose tolerance tests(GTT) etc. But most of the tests are done automated. xD

What i learnt in the first week were mainly HbA1c test, G6PD deficiency test, drug 5/drug 7 tests, GTT, daily QC and weekly maintainance. I will be talking about what i learn in biochem in these 3 weeks in a post so its easier to read. xD

HbA1c/total hemoglobin ratio

HbA1c is actually glycosylated Hb, whereby the glucose bind/attach to the Hb. Therefore, increase in blood glucose will results in increase in HbA1c. For diabetic patients, if their HbA1c is within the acceptable range, this shows that they are controlling their disease well or they are responding to the treatment given to them and vice versa.

It is to monitor glucose of the patient over a period of 3 months. Its unit is in % and the normal amount is less than 8% (if I'm not wrong). For values more than 8%, dilution has to be done, most of the time, in 1:41 (>8-12%), 1:51 (>12-15%), 1:81 (>15-20%) and 1:101 (>20%).

However, HbA1c can be increased due to high amount of glucose, so meaning that if the glucose level is higher than HbA1c, the patient is still normal, meaning that if the HbA1C value is >8% but less than 15%, I have to wait for the blood glucose level results to see if I need to do a dilution. If the glucose level is higher than the HbA1c%, the results can be accepted and key into the LIS. This is because more glucose will attach to the Hb, so will results in higher HbA1c.

If the glucose level is lower, 1:41 or 1:51 dilution has to be done accordingly. However, for HbA1c% that is more than 15%, dilution must be done immediately as it can be quite dangerous.

When the results is > 8%, dilution don't need to be done if the glucose level is higher than HbA1C, or there is previous history of the patient.

The test requires whole blood (EDTA tube) and also before testing, bubbles have to be removed because of the probe will end up taking air if there is bubbles in the blood. I was having alot of problems removing bubbles in the first few days as I have to tilt the tube to almost horizontal, then I'm so afraid to spill the blood.

The machines used by the company are ADIVA 1650 and ADIVA 1800. ADIVA 1650 is an automated machine where it can do up to 1650 tests in an hour and for ADIVA 1800, it can do up to 1800 tests per hour.

The tests are ordered manually, meaning that I have to key in the barcode no. manually, then order as BAYER, where the machine will process to add in Hb denaturant and other reagents. Then the test tube has to be inverted a few times to mix it well, after that removes the bubbles before loading into the machine. It is very important to place the barcode facing outside as it is where the machine will scan the barcode to make sure the no. is what I had keyed. The test takes around 30 mins or smth (I'm not too sure, never take note of the timing). It takes quite long as the test is run twice. After the test is completed, the results has to be checked to see if there is any need for dilution, if not, it can be key into the LIS or they call it the ultra which I dunno why. >.>

So how's the dilution done? for 1:41 dilution, it is to add 10ul of blood sample to 400ul of denaturant then incubate for 5-10 mins in room temp before putting the sample into the machine to run. Same goes for the other dilutions but the denaturant volume increase to 500ul for 1:51, 800ul for 1:81 and 1000ul for 1:101 dilution.

The principle of the test is that the concentration of HbA1c and total Hb is measured separated then the ratio is reported as HbA1c%.

G6PD deficiency

The G6PD test done is a qualitative test, meaning that it can only test for the presence of G6PD enzmye and unable to find out the amount enzyme present in the patient.

The test is fairly easy. Add 5ul of blood sample (in EDTA tube) to 100ul of G6PDS screening reagent then incubate for 10mins before adding 5ul of the solution on a filter paper (Gurthie test paper) and incubate again for 10 mins. After 10mins, read the filter paper under UV to see if there is any fluorescence of the blood sample. If there is fluorescence, it means there is presence of G6PD. No fluorescence showing G6PD deficient.

Repeat testing has to be done if there is deficient in G6PD.

However, before the test can be, the G6PD screening reagent has to be prepared beforehand. It comes in a bottle which contains freeze dried powder which is all the reagent. Then the contents are dissolved in elution/dilution buffer provided. Then the bottle is allowed to stand for 10 mins in room temp before putting on the rotator for 20 mins. After that, 100ul of the G6PDS reagent is aliquoted into test tubes. When finished aliquoting, the test tubes are capped and labelled as " G6PDS reagent, prepared by: (name of staff) and the date". The final step is to put in the -20C freezer. Then everyday around the evening, a few racks of the G6PDS reagents are taken out to thaw and then the G6PD tests can be done~! xD

The G6PDS reagent contains glucose-6-phosphate and NADP+ and when react with G6PD will produce gluconate-6-P and NADPH and H+. NADPH will fluorescence under long wave UV. So, when there is G6PD deficient, no NAPDH will be produced, and therefore there will be no fluorescent.

I had taken some pictures which shows the results of tests under UV, but blogger now having problem in uploading pics, so I will upload when I can. Pictures are of course taken with permission from my supervisor/HOD. xD

Patients's sample in EDTA tube and G6PDS reagent in the test tube.

Gurthie filter paper

10 minutes after 5ul of blood with G6PDS reagent are pipetted onto the filter paper.

When the filter is under UV light. All are G6PD present as all blood spots are fluroscence. It should be green actually, but think when taking the picture the colour will change. The UV light is blue.

All are G6PD present. Some are different colour as the fluorescence is dimmer. As long as there is fluorescence, it means G6PD present.

G6PD deficient. There should be no fluorescence for G6PD deficient. In here is looks quite similar with those blood spot that are dimmer, but in actual fact, there is really a difference.

Drug 5/Drug 7

The test is to find out if there is any of certain drugs taken by the patient.

Drugs 5 tests for the presence of amphetamines, cocaine, marijuana, opiate and phencyclidine or drugs related to them.

It requires a urine sample from the patient and a drug device. 3 drops of urine are dropped onto the drug device whereby the urine will move up due to capillary action. If there is no presence of drug or drug present below the cut off concentration, a coloured line will appear, showing negative results. No coloured line will show that the drug present in the urine is above the cut off concentration. The coloured line is due to the Ab present in the device. Its is very similar to the pregnancy test. Their principles are the same. 3 drops of urine specimen is added into each sample well of the test device.

The princple of drug 5/7 is based on competitive binding. When corresponding drug(s) is present in urine specimen, the drug(s) will competitve against their respective drug conjugates for binding sites on the specific Ab.

When the urine samples are added into the sample wells, the urine will move upwards by capillary action. Drugs present below cut off concentration will not saturate the binding sites of their specific Ab, these allow the Ab to react with the drug-protein conjugate and resulting in a coloured line. When presence of drugs is above the cut off point, the dtugs will saturate all the Ab binding sites and thus no coloured line will be produced. When a very faint coloured line is produced, the results is still negative. This is because the drug concentration is still below the cut off point. The test is counted positive if only there is no coloured line at all.

For drug 7, additional of 2 more drugs are tests. (i will go find out what are the 7 drugs) They are barbiturates and benzodiazepines.

The reagents in the drug tests are mouse monoclonal Ab-coupled particles and the corresponding drug-protein conjugates and for the control contains goat anti-rabbit IgG polyclonal Ab and rabbit IgG.

Pictures of the test devices will be uploaded soon.

Drug 5 pack.

Drug 5 test device.

Drug 7 test devices. Benzodiazepines and barbiturates are in individual pack where as amphetamines, cocaine, marijuana, opiate and phencyclidine are in one test device.

After when 3 drops of urine are dropped into each sample well, the urine moves upwards by capillary action.

Around 5 mins later, coloured lines will appear if the drug concentration are below the cut off point. If there are no drugs above the cut off concentration, there will be total of 8 lines whereby 3 lines will belong to control. Control line must appear or else the test has to be repeated.

Test results for drug 7.

The coloured lines in the first test strip is very fade, but the results is still negative as there are still presence of coloured lines. Positive test will be absolutely no colored line at all. As long as there is some coloured line appear, it will be marked as negative.

GTT

GTT uses dipstix (glucose + ketones only) for fasting urine, 1 hour after intake of glucose and 2 hrs after intake of glucose. So same as what we had done during our cchem pract on urinanalysis, the dipstix was dip into the urine sample and excess urine is drained by the c-folded towel. Then after 2 mins, compare the colour of the dipstix with the reference on the bottle. Prescence of glucose or ketones are noted down.

Quality Control

-to be updated soon-
QC are run twice daily, one in the morning at 7.30am (and yes, i have to reach at 7.30am everday for the first week to observe the QC) and another on the in the late afternoon or evening, around 5.30 to 6pm.

So everday in the morning, (the staffs tend to reach earlier than 7.30am so they have more time to do the QC as they have to finish the QC by 9.30am) the wash solutions are checked to see if there is sufficient, if they are not enough, they will be top up.

Next, the probes will be clean using the alcohol pad, then after that the staffs will top up the reagents that are low in amount. They will then barcode scan the reagents so to update the machine the reagents in the machine and the number of tests can be done.

After that they will initalise the machine and also buffer prime (sort of washing) ISE 5 - 10 times. ISE contains electrolytes like Na, K and Cl.

After the maintainance, they will start to run QC using blank, bio-rad calbrator, bio-rad control 1 and 2, immunological control 1 and 2 (3 for certain machines), CO2 calibrator, bio-rad urine control 1 and 2 and HDL/LDL calibrator. ISE calibation involves using of serum and urine high and low. They also run QC for HbA1c where they use normal and adnormal control. The HbA1c has to do 1:41 dilution and incubate for minimum of 5 minutes before able to run the QC.

So after the QC has finished running, the QC will check if they are within the acceptable range. If it is not in the acceptable range, they have to run again until it is within the range, if not, the results for the day would not be accurate. For ISE QC, the Na, K and Cl slope will be observe for the high and low of the serum and urine. Also, if the slode is not in range, it has to be run again.

Aside of the daily QC, they also have weekly QC.

In weekly QC, they will clean the outside of the machine, and clean the ISE where they will replace Na, K and Cl with a dummy and allow the ISE to be washed. Also, the lamp of the machine is check once a week to see if the lamp is in working condition. The lamp is very important part of the machine as most tests run in the machine requires lamp to read the absorbance of the reaction. If the lamp is not in working condition, all the results can be greatly affected.

And on certain day, additional QC has to be done for ethanol tests and Apo A and B as they are run once a week as the samples are not received daily.

And for evening QC, it is more simple as they will just run the QCs of the reagents and ISE.

-the end-

and so this is almost about what i learnt in biochem. any updates will be edited in this post again.


yaNLing xD


-updated on 7 july 2009.
-updated on 12 july 2009.

Next post will be about serology~! Look forward for it okay? xD