Time to clear all queries...... for cholesterol measurement!

Cheng Hong said:
Is there any other test that is done together with this test to confirm the results in case there is interferance due to bilirubin?

I say:
Pls note that a bilirubin of up to 20mg/dL do not interfere with the result. Anyway a single cholesterol measurement does not truly reflect a person's usual cholesterol concentration, it is highly recommended to perform the test twice on 2 separate occasions. The best is to encourage the participant to go to our Hospital Clinic for a thorough Liver Function check up (if the result seems weird) before a clinical diagnosis is finalised. Frankly speaking, we're trying to promote our health screen packages at the outreach program too.

Elaine said:
As you said, you went for outreach programme and did this cholesteral measurement. If the participant have the habit of eating vit. C pills everyday(is it the same as IV infusion?), does it means that he/she cannot do this test?

I say:
Since vitamin C pills are water soluble, they are flushed out of our immune system on a daily basis and do not have any significant impact on the measurement unless there is an over dosage jus before the test or having an IV infusion concurrently. This would then caused a falsely low cholesterol level.


Jue Xiu said:
hihiI just wonder as u mention the test will be affected by the 1.)IV infusion of Vitamin C2.)Elevated bilirubin (>171umol/L); Jaundice3.)Haematocrit > 55%4.)Methylaminoantiprine5.)Gentisic AcidWouldnt there be any discrepancy regarding the result? Or this test is specific for certain people due to the limitation?

I say:
helloo... I would say no, its not specific since it is used for point-of-care testing. However i would say yes for result discrepancy based on the degree of interference caused by what i've mentioned. Actually all tests have their limitations, so it's better for the particpants to have their blood serum tested too - sell health packages!

Vino said:
hey hey U mentioned abt detection reagent present in the yellow region of the test trip. wat is/ are the detection reagent?? that causes the reaction, colour change, to occur?

I say:
Sorry Vino, I could not find answer to your question as i believe the manufacturer is unwilling to reveal their secret reagent recipe. However, in parallel with the detection reagent from another brand, it was revealed that it contains microorganisms esterase and oxidase, 4-aminoantipyrine, horseradish peroxidase & substituted aniline derivatives. The reaction/colour change occurs due to liposis of the triglycerides and then measurement by photometry (refer to below, Cass's Question & my answer)


Charmaine said:
Just want to say, I learnt to use this as well!But I'm not measuring blood cholesterol level, instead I'm measuring blood glucose levels. XDCheers~Charmaine TanTG01

I say:
Oh... we too! We actually perform the finger-prick for the blood cholesterol 1st and squeeze another drop of blood for the glucose measurement as it takes a shorter time for the glucose device. While waiting for the 3 min to be up, I then measure the participant's blood pressure. Usually they would complain that their blood pressure is high due to the shock/fear that they develop for the finger-prick. All excuses.... I find it the same to measure immediately when they were jus made to sit down.

Avery said:
Hi Pei shan,i would to ask how do you select the depth of the Accu-Chek Safe-T-Pro Plus® lancet device?based on the finger thickness(hehe ^_^)?

I say:
Bingo! The lancet is usually set at a moderate depth which is sufficient to prick a person's finger. However if the person has thick skin especially the males, shift it to the deeper profile. So far that day, I only adjusted to the deepest twice, both times for men! It's better to adjust the depth than to be sorry and prick another time.

Cass said:
hi pei shan;)"The meter measures the intensity of the reaction colour based on reflectance photometry and calculates the concentration of the sample through a "lot-specific" algorithm (i.e. the data previously entered by mean of code strip), giving the cholesterol reading."what is reflectance photometry? intensity of the reaction.. meaning the darker the colour the higher the cholesterol level?

I say:
Helloooo... This is a very good question which I've lacked explaining. The reaction principle of Accutrend® Triglycerides is lipolysis of triglycerides to give free glycerol and fatty acids by the activity of a cholesterol esterase. The glycerol is phosphorylated by glycerol kinase, and the resulting glycerol phosphate is oxidized by a glycerol-phosphate oxidase. Molecular oxygen subsequently leads to the formation of hydrogen peroxide, whose oxidation equivalents are transferred by a peroxidase to an indicator, giving a blue-gray oxidation product. The intensity of the color is measured by reflectance photometry(American Association for Clinical Chemistry, Inc.) . The darker the colour, the higher the cholesterol level and the device converts this reading into a result for display.
For further reading, pls refer to http://www.clinchem.org/cgi/content/full/46/2/287

Pei Shan
TG02

Microbiology--- CAMP TEST (week 19)

hey to all!!!

I'm posting for week 19!!! TIme passed super fast...

I saw ying ying's posts abt CAMP test but what i'm abt to post is a bit different from her.

Here i go...

For those who had been to micro lab, you should pretty well known with GPB/GPC/GNB/GNC.

Here i'm going to introduce you to ARCANOBACTERIUM HAEMOLYTICUM.

CHARACTERISTICS:

--- gram postive rods with pointed ends

---catalase negative

---small colonies, may be smooth, mucoid, white, dry

---B-haemolytic

---It will not grow on MacConkey agar

--- Facultative anaerobe

Certain organism such as Group B streptoccui able to produce a factor that acts synergistically with B-hemolytic of S.aureus subspec. aureus on sheep blood. The purpose of test to determine the organism's ability to produce and elaborate CAMP factor. The production of a-hemolytic(a-toxin) by clostridium perfrigen also demostrate the synergistic phenomenon with CAMP factor. If Phospholipase D is produced by organism, the CAMP reaction would be inhibited.

Medium:

TSA with 5% sheep blood agar (BAP)

Procedure:

1) preheat 35'C. Ensure plates are dry.

2) Using inoculating needle, streak B-lysin substrate organism in a straight and across centre of BAP

3) Streak QC stain ( about 2-3cm) perpendicular to S.aureus inoculum without touching the Staphylococcui

4) Incubate CO2 35'c incubator for at least 24 hours

**Note: the arrow head means +ve CAMP results
The curve head means -ve CAMP results



MY senior said that normally she will do another REVERSE CAMP test(RCT) to confirm the results from CAMP test.


PRCT-- production of a distinct crescent hemolysis at junction between streptoccus agalatiae streak and test organism.

NPRCT-- No constriction of band of hemolysis






**Note: Postive reaction

Limitation:

--> size of zone is depend on the amount of bacterial product and its diffuse rate through the medium

--> inocula should be sufficient to produce confluent growth. The extent and intensity of lysis depends on the size of the staphylocuccus inoculum. If the inocula is too light, reaction would be weak.

--> If the inocula are not perpendicular, production of arrow heads will not appear. False Negative occur.







ELaIne

SEE ya soon!!!

Research - Lab Techniques (week 18)

Hi all,

Previously I said I’d cover colony pcr and restriction digestion, but I believe those underlying principles have been covered in my first post, so it’s a bit redundant. Hence, I’ll be covering a bit on the yeast work for today.

First off, my protein interaction study is based on yeast. There are about 3 types of yeast 2 hybid assay. For my project, I’m using yeast 2 hybrid system 2. As I haven’t been exposed to the other 2 systems, I’m afraid I will not be able to cover anything on it.

Basically, the main principle of the yeast 2 hybrid is that the transcription factor in yeast, (GAL4) can be broken up into 2 pieces to form GAL4-BD (binding domain) and GAL4-AD (activation domain).
- GAL4-BD will then be subcloned into a vector for the bait gene, which is the gene of interest for study. The BD is the domain responsible for binding to the UAS (upstream activation site)
- GAL4-AD is subcloned into a vector for the prey cDNA library which is used for testing against the bait gene to see what interactions may take place. AD is the domain responsible for activation of transcription located downstream.

Hence, in event of a protein interaction between the bait and a particular prey, the 2 domains (GAL4-BD and AD) will be brought into close proximity, linking both the GAL4 UAS and transcriptional site in yeast, resulting in transcriptional activation of the beta/alpha-galactosidase, the reporter gene in yeast (MEL1, the reporter for producing alpha-galactosidase is naturally occurring in some strains of yeast, while LacZ is an E.coli enzyme that is cloned into the yeast genome).
Yeast strain that can metabolize alpha-galactosidase such as AH109, Y187 etc from clontech can metabolise x-alpha-gal (a substrate) directly as alpha-galactosidase is secreted (naturally occurring in these yeast strains); colonies can be plated on x-alpha-gal plates. However, for beta-galactosidase, LacZ gene being not naturally occurring means that the beta-galactosidase is not secreted by yeast, but rather produced in the cell upon activation. Hence, to develop blue phenotype, colony lift assay is necessary as x-gal needs to enter cells (have to permeablise yeast cells first by colony lift assay) for metabolism by beta galactosidase.
For more info on colony lift assay, please refer to www.med.unc.edu > hdohlman > lift_protocol.html

For a clearer demonstration of activation by GAL 4 transcription factor, refer to the diagram below:

Taken from: en.wikipedia.org> wiki > Two-hybrid_screening

To recap, AH109 strain of Saccharomyces cerevisiae contains 4 reporters: ADE, HIS (nutritional markers), LacZ (for beta- galactosidase, artifically cloned from E.coli), MEL1 (for alpha- galactosidase, naturally occuring in some yeast strains).

My previous posts have been focusing on obtaining the bait construct. This is done by cloning in a pcr gene of interest into the bait vector that contains
1) gal4-BD
2) a nutritional factor like TRP marker for yeast selection
3) kanamycin for antibiotic selection
4) Multiple cloning site for cloning purposes.

Once bait construct has been obtained, the plasmid is transformed to yeast strain AH109, and plated on SD/-trp plates for selection of transformed yeast (yeast that is not transformed cannot grow on –trp plates) in 3 serial dilutions of 1/10, 1/100, 1/1000 for calculating transformation efficiency. Once yeast has grown, typically 3-4 days initially when incubated at 30oC, it is restreaked onto fresh -trp plate to form the master plate for mating with the cDNA library, which is commercially obtained. Alternatively, it is possible to clone your own library into a vector, though this is much more of a hassle. cDNA prey library is tittered to find amount of viable cells by plating dilutions of 1/10, 1/100, 1/1000 and 1/10,000 on SD-leu plates (Prey vector contains leu nutritional marker) and making suitable calculations - multiply cfu by plating vol and dilutional factor and divide by total volume available in mL.

Eg, 100ul of 1/10,000 dilution of prey library was plated and gave a cfu of 245 from a total cdna vol of 1ml will yield:
100ul x 10,000 df x 245cfu / 1mL = 2.45x10^8 cfu/mL

Typically, at least 2x10^7 cfu/mL is necessary to produce enough clones for screening in mating (at least 1 million diploids).

If viability of prey is high enough, proceed to mating.

*For easier reference purposes, a table below shows the comparison of bait vector and prey vector:

Mating:

After this, comes the actual mating. Grow out bait colonies in SD/-trp liquid overnight. Next, combine 1mL of bait and prey together in 2x YPDA media (50mL) and incubate at 30oC overnight at 50rpm to ensure cells do not settle down. Check for zygotes after 30hrs under microscope at 40x. If present, pellet at 700g, 5 mins and resuspend into fresh media before plating on 24.5cm x 24.5 cm SD/-trp/-leu/-ade/-his / x-alpha-gal (QDO x-alpha-gal). It is important to note that only mated colonies can grow on QDO (quadruple dropouts as prey provides leu (leucine) marker and bait has trp (tryptophan) marker and interactions between these 2 causes activation of transcriptional factors adenine (ade) and histine (his) as these 2 are under influence of the same promotor.
Within 2-3 days of incubation at 30oC, some colonies may start to turn blue. These colonies are the ones with activation of x-alpha-gal, indicating possible prey protein interaction with bait. These colonies are to be restreaked onto freah QDO x-alpha-gal plates to determine that blue phenotype is not a false positive, which typically turns white after the 2nd streak.

Rescue prey plasmid:

If many colonies are detected, a yeast colony pcr can be used to eliminate same copies of interaction. Upon confirmation, or if there are few colonies for study, yeast plasmid can be extracted by either sonication or using extraction kit (which uses lyticase to break down yeast wall). Plasmid obtain will consist of a mixture of prey, bait and yeast plasmids. To select only prey, the ampicilin selection shall be used – transform to competent cells and plate on amp plates. Bait and yeast plasmids will not be able to grow, hence resultant colonies are all from prey. Miniprep to obtain plasmid from competent cells.

Confirm interaction:

Using the rescued prey plasmid, transform to bait gene and plate to QDO x-alpha-gal plates. If colonies turn blue, use the plasmid obtained from the competent cells for sequencing to determine what protein it is. Blast results using protein query from www.ncbi.nlm.nih.gov > BLAST > blastx

That wraps basically everything in a nutshell. Controls were not discussed as they are too bothersome and the principle is the same. Feel free to ask any questions, though I suggest you guys focus on the mp reports/logbook/whatever else instead and leave me to do the same =)

Cheers,

Debra, TG02

CHEMISTRY - Measuring Cholesterol

Dear Medical Technologists, I'm going to share with you one of the point-of-care testing system that I've learnt when I was sent out for an Outreach Program few weeks ago. This is regarding cholesterol measurement using the Accutrend® GCT Meter (Roche).

Name of Test: Cholesterol (CHOL) Measurement using Accutrend® GCT Meter

(Picture taken from Divant.com)

Introduction:

Cholesterol is a major steroid in human body. Majority is free, unesterified, that form the structural component of the cell membrane. Its content in the body is dependent on the diet, synthesis within tissue cells and excretion by the liver. Proportion absorbed in the intestine is affected by the amount ingested, with less absorbance when intake is high.

Cholesterol is insoluble in water, thus before being absorbed, unesterified cholesterol is first solublized. It is excreted unchanged or as bile acids in bile. Transportation in plasma and its metabolism are related to lipoprotien metabolism. 2/3 of the plasma cholesterol is in the esterified form.


Materials

• Accutrend® GCT Meter



• Accu-Chek Safe-T-Pro Plus® lancet device
  
  
  

(Picture taken from: Accu-Chek.ca)


• Cholesterol Test Strip



• Code Strip (found in every new pack of test strips, for calibration purpose)



• Control CH1 (for QC purpose)



• Alcohol Swab



• Cotton ball

Procedure (from finger-prick to measurement):

• Switch on the Accutrend® GCT Meter and insert the test strip



• Tally the participant's name with the LIS



• Select the depth of the Accu-Chek Safe-T-Pro Plus® lancet device and the puncture site



• Clean the puncture site with alcohol swab



• Twist off the sterility cap and gently press the lancet against the side of the finger tip



• Apply a hanging drop of blood to the yellow test field on top of the test strip



• Close the meter flap immediately and wait for 180s countdown



• Stop the bleed using a cotton ball



• Read measurement



• Enter into LIS

Principle of Analysis: Reflectance Photometry

The yellow region of each test strip contains detection reagent which reacts with the blood and causes a colour change. The meter measures the intensity of the reaction colour based on reflectance photometry and calculates the concentration of the sample through a "lot-specific" algorithm (i.e. the data previously entered by mean of code strip), giving the cholesterol reading.

Test Results:

Range: 3.88 - 7.76mmol/L

LO indicates value is below 3.88; HI indicates value is above 7.76

In either cases, turn the strip over and check if the test region is uniformly covered by the blood. If partially coloured, repeat test.

Desirable: < 5.12 mmol/L
Borderline High: 5.12 - 6.18 mmol/L
High: > 6.18 mmol/L

Advantages:

• Can be performed on non-fasting individual's blood



• Uses dry chemistry methodology outside a lab setting (suitable for Public screening)



• Low cost



• Convenient; Portability



• Fast results (within 180s)



• Minimal blood for testing (requires only 1 drop)



• Easy to operate for the medical worker



• Gives a relatively accurate result
  

Disadvantages:

Test is affacted by...

• IV infusion of Vitamin C



• Elevated bilirubin (>171umol/L); Jaundice



• Haematocrit > 55%



• Methylaminoantiprine



• Gentisic Acid
  

Clinical Significance:

There is a good correlation between cholesterol level and incidence of coronary artery diseases. Hence, by offering this fast and convenient point-of-care testing, any risk of atherosclerosis can be detected early. However, further investigation is required to justify the finding.

Questions for me???


PEI SHAN, TG02

Haematology

Osmotic Fragility Test

It is used in the diagnosis of hereditary spherocytosis and the screening for thalassemia.
Hereditary spherocytosis is a disorder in which red blood cells are defective because of their round, ball-like (spherical) shape. These cells are more fragile than normal.

Principle:
Red cells will be haemolysed when placed in a hypotonic saline solution. Time and extend of complete haemolysis will depend on the red cell membrane resistance and size and shape of the red cells. Spherocytes will lysed quite quickly in slightly hypotonic solution. Hypochromic microcytic cells will be more resistant to hypotonic solution.

Procedure:
1) Deliver 5ml of 0.9, 0.75, 0.70, 0.65, 0.6, 0.55, 0.50, 0.45, 0.4, 0.35, 0.30, 0.25, 0.2, 0.1% of hypotonic saline solutions into test tubes.
2) 50μl of blood to each tube and mix.
3) Stand tubes for 30mins at room temperature and centurifuge.
4) Pipette the supernatants to cuvette and read absorbance at 540nm.
Note: Supernatent from 0.90% NaCl is used as a blank.

Interpretation of results:
- In normal subjects an almost symmetrical sigmoid curve results.
- Subject with decrease resistance to lysis will show fragility curve that shift to the left.
- Subject with increase lysis will show fragility curve shift to right


(taken from http://www.suite101.com/article.cfm/medical_student/40859)

Clinical Significance
The ability of normal red cell to withstand hypotonicity results from its biconcave shape which allows the cell to increase its volume by about 70% before the surface membrane is stretched, and once this limit is reached lysis occurs.

Spherocytes have an increased volume to surface area ratio; their ability to take in water before stretching the surface membrane is thus more limited than normal and therefore is more susceptible to osmotic lysis.

Decreased osmotic fragility indicates the presence of flattened red cells in which the volume to surface area ratio is decreased. Such a change occurs in iron deficiency anemia and thalassemia in which the red cells with low MCH and MCV are resistant to osmotic lysis.

Eunice

0503245C