The Miscellaneous Bench

Hello everybody! This is Jeremy Chu (TG01) attached to bacteriology posting on...

The Miscellaneous Bench

The miscellaneous bench deals with every other specimens besides blood, stool, urine and respiratory specimens. Fluid, tissues and swabs are mainly the samples received. Some examples are heart/liver tissues, peritoneal fluid or peritoneum fluid, wound swabs and high/low vaginal swabs. Detecting bacteria at these sites are important, especially for sterile sites. There should not be any bacteria found at sterile sites such as the heart and liver or patient’s situation could be fatal.

Wound cultures are performed to isolate and identify bacteria infections causing an infection of the wound. This way, doctors can then administer the appropriate antibiotics to kill the causative agent.

Fluid culture is done, similarly to isolate and identify bacteria causing an infection. Peritoneal/Pericardial fluids are sterile fluids and any presence of bacteria is regarded as significant. Gram stain is also performed.

High vaginal swabs are cultured for the purpose of detecting group B streptococci (commonly Streptococcus agalactiae) found in the female reproductive tract. S. agalactiae can cause pneumonia and meningitis commonly in neonates and the elderly.

The different types or media used and its purposes are listed in the table below.

Media	Purpose	Types of specimens
Blood Agar	Enriched media to enhance bacteria growth	Tissues Fluids Fluid swabs Wound swabs Catheter tips/devices Vaginal swabs
MacConkey Agar	Selective media to inhibit most gram positive bacteria and differentiates lactose fermenters
Blood Agar (anaerobic)	Enriched media to enhance anaerobic bacteria growth	Tissues Fluids Fluid swabs
Cooked Meat broth	Enriched broth for cultivating bacteria	Tissues Fluids Fluid swabs Wound swabs Catheter tips/devices
LIM broth	Enriched broth used to grow group B streptococci from vaginal swabs	Vaginal swabs

Fluid culture agar plates are incubated under CO₂ conditions, while its broths in aerobic conditions. Blood, MAC, blood (anaerobic) agars and cooked meat broth are the choice of media.

Blood agar and LIM broth are used for vaginal swabs, while blood, MAC and cooked meat are used for ordinary wound cultures.

Roche Cobas e601 Analyzer

HI EVERYBODY!
stella here to post another entry!
I'm going to talk about Roche Cobas e601 Analyzer.

As i'm attached to a lab with mostly automated machines, therefore most of the tests are run on analyzers.

Roche Cobas e601 analyzer run different kinds of tests such as cardiac markers (CKMB, myoglobin, NTproBNP, Troponin T), tumour markers (AFP, CEA, CA125, CA15-3, free PSA, Total PSA) and bone markers (beta cross laps and osteocalcin)

An example of a test that is run on the Cobas e601 analyzer are cardiac markers such as Troponin T.
The purpose of performing the test is as follow:
Troponin T is useful and important in the diagnosis of myocardial damage such as myocardial infarction,

Principles of the tests : Immunoassay with Electrochemiluminescence technology

During the assay, the sample containing Troponin T is incubated with two monoclonal antibodies specific to the Troponin T. Examples of the antibodies against Troponin T are biotinylated monoclonalTroponin T-specific Antibody and a monoclonal troponin T-specific antibody labeled with a ruthenium complex. Thus, a sandwich complex is formed. Afterwhich, microparticles coated with streptavidin are added and the complex will be bound to to the solid phase through the interaction of biotin and streptavidin. The entire complex are drawn onto the electrode magnetically, with unbound components being washed away.
Electrochemiluminscent is induced by the application of a defined voltage. The photomultiplier measures the emission of light produced.

Reference interval for Troponin T:
<0.03ug/L

Troponin T can be futher divided into skeletal Troponin T and cardiac Troponin T.
Cardiac troponin T is a specific cardiac marker used for the diagnosis of myocardial damage.
It has unique cardiac specificity as it has different amino acid sequenes which are different from that of skeletal troponin T.
The presence of cardiac Troponin T in the bloodstream indicates there is myocardial damage.
In a healthy individual, no levels of troponin T can be detected in the bloodstream.
troponin T is a better cardiac marker due to its ability to stay in the bloodstream up to 2 weeks.
It rises from 4 to 6 hours, peaks at 12 to 24 hours and persist in the blood for 2 weeks.

Oral Glucose Tolerance Test

Hello everybody!

This is Hakim for another round of blogging.

NB: This is an edited entry. I have not removed anything, but have added in a few more points.


PURPOSE OF OGTT
The oral glucose tolerance test (OGTT) is a test performed to determine if the patient has diabetes. In my lab, majority of the patients who undergo this test is pregnant. This is because pregnant women could acquire gestational diabetes, which is a form of type II diabetes that is first diagnosed or begins during pregnancy. This condition usually ends soon after the person gives birth.

In a nutshell, the patient would have to give a urine sample and have their blood drawn out. After this, the patient is given a large dose of glucose and subsequently, urine is collected and blood is drawn after a period of time (depending on the GP's request). After the final urine collection and blood drawing, the urine samples are tested for glucose and ketones by using the dipstick. The blood tubes are centrifuged and its plasma is put in an analyzer to test for glucose levels.

PRINCIPLE OF TESTING PLASMA GLUCOSE LEVELS
Our lab is using the COBAS Integra 400 plus. It uses the principles of light absorbance and colorimetry. Glucose in the plasma is oxidised by glucose oxidase, producing gluconic acid and hydrogen peroxide. Peroxidase converts the hydrogen peroxide into oxygen and water. The oxygen liberated in the reaction is taken up by 4-aminophenazone with phenol and forms a pink-coloured substance. The substance can be measured at a wavelength of 515mm. A halogen lamp will produce a light beam and passes the cuvette. A photodetector would sense the amount of light passed through the cuvette (and also the amount of light absorbed by the substance). It is important that calibration is done frequently so that the results would be accurate.

PROCEDURE OF OGTT
It is important to note that the patient is required to fast for 8 hours before the test commences. This is done so as to prevent any pre-analytical variations. For example, a high plasma glucose after a meal could misdiagnose the patient as having impaired glucose tolerance (IGT). The patient is allowed to drink water during the fasting, but they are advised to try to drink as little as possible as the blood could be diluted if they take in large amounts of water. Also, the tube collected is a fluoride tube so that it will reduce the rate of glycolysis by the red blood cells so as to stabilize the levels of glucose. So after the first round of specimen collection, the patient is given a 75 gram glucose drink to drink. She has to finish the whole bottle. It tastes like concentrated F&N Orange. From my own experience, the drink is extremely sweet. I couldn't even finish the whole bottle (which is about 350mL) so I'm not sure which is more painful for the pregnant woman; childbirth or finishing up the whole bottle. There are 2 different timeframes for blood to be drawn, depending on the GP's request. It's gonna be quite confusing, so let's give an example. - Patient A is requested to have her blood and urine collected 1 hour AND 2 hours after drinking the glucose - Patient B is requested to have her blood and urine collected only 2 hours after drinking the glucose So after both patients finish the bottle, she comes back in either 1 hour (for patient A) or 2 hours time (for patient B), depending on the GP's request. During this time, she still cannot eat or drink anything (other than water, but in small amounts). Patient A comes back in 1 hour to have her blood and urine collected. At 2 hours after drinking the glucose, BOTH Patients A & B comes back to have their blood and urine collected. Only after this is done, they are able to eat and drink. For urine sample: The dipsticks are dipped into the urine to check for glucose and ketone levels. A high level of glucose in the urine would mean that there is a high level of glucose in the patient's bloodstream and the levels have exceeded the kidney's threshold. So the kidneys aren't able to reabsorb any more glucose and is then excreted through the urine. Ketone bodies are a result of lipid metabolism. This is caused by starvation. Ketone bodies would be present in diabetics. For blood sample: The blood tubes are centrifuged. The blood tubes MUST be labeled (0 hr, 1 hr, 2 hr) so as to prevent any confusion. The tubes are then put in an analyzer machine. For our lab, we are using the COBAS Integra 400 plus.

Note: For some reason, I can't upload pictures, so here is the link to the website that contains the picture of the analyzer.
http://labsystems.roche.com/content/products/integra_400plus/introduction.html
REFERENCE RANGE OF OGTT If the levels of plasma glucose is higher than normal, then we can say that the pregnant woman is suffering from gestational diabetes. There are many diagnosing criteria for OGTT. The World Health Organization's (WHO) guidelines for diagnosing GDM is ≥5.3 mmol/L for 0-hour plasma glucose, OR ≥10.0 mmol/L for 1-hour plasma glucose, OR ≥8.6 mmol/L for 2-hour plasma glucose. However, some doctors do not request for a 1-hour plasma glucose because it is not as clinically significant as the 0-hour and 2-hour plasma glucose levels.

They are advised to maintain a healthy lifestyle and have a proper suitable diet. They can also take insulin.

Ok that's all from me. If you have any questions for me, I'd be glad to answer them as fast as possible.

Happy SIP-ing!
Hakim
0703555C

13 more weeks!!!