microo

hello pple.
it's my turn again.
so i'm going to write about a test done in micro lab.

Wampole™ TOX A/B QUIK CHEK®
this test is a rapid immunoassay for detecting Clostridium difficile toxins A and B.
specimen: fecal specimen
suspected disease: C. difficile disease
(before proceeding with this test, we have to bring all reagents and devices to room temperature to ensure accurate results.)

Required Reagents:
- Diluent
- Congugate
- Wash Buffer
- Substrate

ASSAY PROTOCOL
1. Dilute specimen
using a small glass test tube, add 25μL of specimen to 500μL Diluent + 1 drop of Congugate. mix well.
2. Add sample
transfer 400μL of diluted sample-conjugate mixture to sample well. incubate for 15minutes.
3. Wash Membrane Device
add 300μL Wash Buffer to Reaction window
4. Add Substrate reagent
2 drops to Reaction window. incubate and read results 10 minutes later.
5. Interpretation
read from Reaction window.
two lines -> positive
1 control line -> negative
* invalid test if there is no control line. repeat test pls.
* a positive result may be interpreted at any time during the 10minute interpretation but cannot be ruled as neg or invalid until after 10 minutes.

wendy ong/0701158h

Week Nine

Section: Special Chemistry. (COBAS)

Hi, I shall talk about this section whereby the analyzer used is called COBAS c system. The tests usually done here are Acute Myocardial Infarction (AMI) Panel; CKMB mass and Troponin T. Other tests include hormones and HbA1c.

I shall elaborate more on HbA1c. HbA1c is one of the glycated hemoglobins. It is formed in a two steps non-enzymatic reactions of glucose with the N-terminal amino group of the -chain of the normal adult Hb (HbA). The amount of HbA converted to HbA1c is relative to that of the average concentration of glucose in the blood thus, HbA1c is suitable to monitor long term blood glucose control in individuals with diabetes mellitus.

This method uses a detergent in the hemolyzing reagent to eliminate interface from leukocytes. All glycated hemoglobins at the -chain N-terminus and which have antibody-recognizable regions identical to that of HbA1c are measured.
The sample which comes in EDTA tube is to check for clotted blood. If there is no clotted blood, the presence of bubbles is removed to avoid sampling error. The sample is the placed in a special HbA1c rack and placed into the machine. The machine will detect the barcode and do the test ordered. The sample (glycohemoglobin in HbA1c) is reacted with anti-HbA1c antibody to form antigen-antibody complexes. Then, polyhaptens are added to start the reaction by reacting with the excess anti-HbA1c antibodies to form an insoluble antibody-polyhapten complex which is the measure turbidimetrically. The final result is expressed as percent HbA1c. The results are then transmitted to the LIS system and the staffs allocated there will validate the results.

nyzah

Week 8: High Performance Liquid Chromatograph

Name: Eriko Wong (0700477C)

Section: Analytical Chemistry

Ok, so today I'm going to share with you about the components of HPLC and how does it work.

Here is a picture showing the different components of HPLC::)

Image source: http://www.protein.iastate.edu/hplc.html

1) Mobile Phase Supply System are to be filtered and degassed first before pass through the column so as to remove any small particles or dissolved gases. If this step is not done, it can cause the clogging of column.

2) Injector is where you would inject your sample in. This is done by a syringe and a needle. Sample is also filtered first before being carried to the column for analysis.

3) Packed column: is also known as the stationary phase. This is where the interactions between the sample, mobile phase and stationary phase occur. The different interaction of the different components in the sample will lead to the different time they elute out of the column. Often, a guard column is installed to trap unwanted materials. Column is usually housed in a oven, under a predetermined temperature to ensure a consistent interaction of the sample, mobile and stationary phase.

4) Detector: The one I'm using is Ultraviolet-visible spectrometry detector. At different wavelength, the peak area and height of the sample recorded will be different too.

An example of a chromatogram:: (x-axis: time, y-axis: the absorption by the detection)

Image Source: http://www.protein-ms.de/METHODS/HPLC/HPLC.htm

5) Recorder: This is to obtain a real time plot of the analysis. Once the analysis is done, the computer will print out the data, along with the chromatogram.

Hopefully you guys get the idea of how HPLC works.... :)

Clinical Chemistry - Urine Phase Contrast

Hey, here's Natasha again:]

I’m here to talk about urine phase contrast.

The purpose of performing urine phase contrast is mainly to differentiate isomorphic red blood cells and dysmorphic red blood cells present in the urine. Apart from that, if there are presence of white blood cells, microorganisms, epithelial cells, casts or/and crystals, they will be noted as well.

Isomorphic red blood cells, can suggest urological diseases which are non-glomerular origin. They appear bright and circular in shape. Isomorphic red blood cells still retain the haemoglobin whereas dysmorphic red blood cells have lost the haemoglobin.

Dysmorphic red blood cells, however, can suggest diseases of glomerular origin such as glomerulonephritis, etc. Dysmorphic red blood cells may appear circular or slight difference in shape and also appear as dark, thick rings.

The presence of microorganisms can indicate urinary tract infection (UTI). They may appear cocci or bacilli in shape, singly, pairs or in chains.

Crystals can be found in urine specimens that are super saturated with a crystalline compound and the formation of crystals are pH dependent. Thus a change in urine pH may cause crystal precipitation. For example, calcium oxalate maybe found in deposit of both acid/alkali urine. It may be regarded as a normal constituent of urine or may indicate renal calculi, renal disease or glycol poisoning.

The type of microscope used is the urine phase contrast microscopy. As compared to normal microscopy, the optical components of this microscope are able to change the different phases of structures into differences in light intensity.

Normal microscopy is when there is no optical contrast technique is employed. It uses transmitted light to view a specimen that contains natural contrast/colour or is stained.

The type of counting chamber used is Med-Fuchs Rosenthal counting chamber. This counting chamber is a little different from the counting chamber that we get to use in school during MCT practicals. It only consists of 16 big squares with 16 smaller squares in each of the big square. Any adjacent four squares can be used to count the isomorphic and/or dysmorphic red blood cells.

For urine phase contrast, fresh first morning mid-stream urine is collected and spin down at 2000 rpm for 10mins. The supernatant is then discarded (most of the time the cell pellet is not as visible, thus usually ¾ of the urine is discarded). The remaining urine containing the cells is then mixed well and pipetted onto the counting chamber. It is then viewed under the microscope.




Drawings are not drawn to scale.



In this picture, WBCs are the larger cells with granules inside. Isomorphic RBCs are bright ones and smaller in size than WBC. Dysmorphic RBCs are the dark, ring-shaped cells.
Picture is taken from http://lab-qa.org/u_atlas/sem01_22.htm



Med-Fuchs Rosenthal Counting Chamber. Click to have a better view:] Picture is taken from http://www.hausserscientific.com/FuchsRosenthalDirect.htm