Tuesday, October 30, 2007
Cholinesterase is an enzyme which catalyzes the hydrolysis of acetylcholine into choline and acetic acid, allowing a cholinergic neuron to return to its resting state after activation. It is crucial to the transmission of electrical impulses along the nerves and also indicates the liver capacity for synthesis.
There are 2 types of cholinesterase: plasma or pseudo-cholinesterase and acetylcholinesterase which are found in the blood (RBCs) and nerves.
Individuals exposed to organophosphorous compounds (e.g. insecticides) exhibit decreased levels of cholinesterase in RBCs and serum. Acetylcholine activity is a better marker for insecticide poisoning, because some individuals may have normal or near normal levels of total serum cholinesterase. However, others have recommended that the serum enzyme measurements reflect acute poisoning while RBC cholinesterase measurement is useful for long-term chronic exposure to insecticides.
Cholinesterase measurements are also used in the detection of patients with inherited abnormal variants of the enzyme. In the nerve endings, reduced cholinesterase activity results in accumulation of acetylcholine which triggers excessive stimulation.
Clinical manifestations for acute insecticide poisoning include excessive salivation, nausea, vomiting, diarrhea, abdominal cramping and asthmatic symptoms. For the cardiovascular system, bradycardia, arrthymias and pulmonary edema are some problems that may happen. Overstimulation of muscarinic sites in the CNS can cause confusion, ataxia and slurred speech. Death may occur.
Procedure for RBC cholinesterase
1. Patient’s blood in EDTA tube is poured into a glass test tube and filled to ¾ with 0.9% saline.
2. The suspension is mixed well by inverting several times and centrifuged at 3500rpm for 5 minutes.
3. Using the suction pump, the plasma and buffy coat (layer on top of RBCs) are removed.
4. 0.9% saline and spun cells are pipetted into a secondary tube to obtain a 1:1 dilution.
5. 0.5% saponin (lysing agent) and the suspension are pipetted into a new secondary tube.
6. The hemolysate is incubated in a refrigerator (2-8°C) for 10 minutes before analyzed on the Beckman Coulter LX20U analyzer.
7. The PCV (hematocrit) is measured using the suspension (obtained in Step 4). The blood is put into capillary tubes and centrifuged at 13000rpm for 5 minutes and the PCV is read off a calibrator rule.
As cholinesterase activity is expressed in activity units with respect to PCV, both enzyme activity and PCV for the hemolysate are determined. After getting the results, the formula [(Cholinesterase/PCV) X 100] is used to obtain the RBC cholinesterase level.
The reference interval for RBC cholinesterase is 15000-24000 U/L RBC.
Note: Serum cholinesterase is also measured in the laboratory, but on the Johnson & Johnson Vitros DT60 II. Information about this analyzer can be found more in 77th Medical Technologist Street blog post on the 5th week.
Sunday, October 21, 2007
via Tapval and Sedi-rate System
Erythrocyte Sedimentation Rate is helpful in the diagnosis of conditions associated with acute and chronic inflammation, including infections, cancer, and autoimmune diseases. ESR is nonspecific because the increase in ESR does not indicate exactly where the inflammation is or what is the causative agent, it just shows there is inflammation in the body. Therefore, ESR is typically used in conjunction with other tests. ESR is helpful in diagnosing two specific inflammatory diseases, temporal arteritis and polymyalgia rheumatica. Also, ESR test is used for the diagnosis and monitoring of children with rheumatoid arthritis.
Blood is collected via veni-puncture and sent to the laboratory in EDTA tube. After inserting the blood into the EDTA tube, it should be homogenously mixed to ensure all of the blood is exposed to the anti-coagulant to prevent clotting. Clotted blood is rejected as it causes increased ESR.
Tapval tube with stopper containing Trisodic Citrate (Sodium Citrate)
Pressured Pipette with inner diameter of 1.25mm
1. Patient’s particulars on the test request form are matched with the name on the blood tube.
2. Blood tube is inverted a few times to homogenously mix the blood
3. 320ml of blood is aliquot into the Tapval tube
4. The tube is flickered to attain homogenous mixture of blood with trisodic citrate
5. Pressured pipette is inserted into the tube. This must be done gently to prevent the blood from shooting up the pipette
6. After the pipette is in the tube, it further lowered into the blood until it travels to the top of the pipette
7. After ensuring there is no bubble or gap along the pipette, timer is set at 60 minutes and the pipette stands for the duration..
8. Patient’s label is then stuck to the pipette so as to prevent confusion when there’s a lot of ESR going on.
9. After 60 minutes, the level of red cells is read from its meniscus and recorded in the ESR logbook.
Sedimentation takes place in 3 stages:
A preliminary stage where rouleaux occurs and aggregates form.
Sinking of aggregates, which takes place at a constant speed.
Rate of sedimentation slows as the aggregated cells pack at the bottom of the tube.
1-2 day = 0mm to 4mm
3days - 1month = 0mm to 10mm
2 months – 12 years old = 0mm to 12mm
> 12 years old = 0mm to 12mm
Elevated ESR may be indicative of:
· Connective tissue diseases: systemic lupus erythematosus (SLE), giant cell arteritis and systemic sclerosis.
· Polymyalgia rheumatica (PMR): a condition particularly common among elderly women which causes chronic aching and stiffness of the muscles, especially in the shoulders and upper arms.
· Rheumatoid arthritis.
· Kidney disease.
· Sarcoidosis : disease of unknown cause in which inflammation occurs in the lymph nodes, lungs, liver, eyes, skin, or other tissues.
· Severe or chronic infection.
However, there are some factors that contribute to increased ESR which are independent of inflammation or infection. For example, increased fibrinogen, increased globulin, anaemia, decreased albumin and packed cell volume.
Also, females tend to have higher ESR, menstruation and pregnancy can cause temporary elevations. Drugs such as dextran, methyldopa (Aldomet), oral contraceptives, penicillamine procainamide, theophylline and vitamin A can increase ESR while aspirin, cortisone, and quinine may decrease it.
Tuesday, October 16, 2007
Glucose is the major energy source for the human body. When the total daily energy intake exceeds daily expenditure, the excess is converted to fat and stored in adipose tissue. When daily energy needs are not met by sufficient calorie intake, glucose is produced from the berakdown of carbohydate stores and from non-carbohydrate sources (eg. gluconeogenic amino acids, lactate and glycerol) the glucose level in blood is kept within a fairly narrow range through a variety of influences. Feeding and fasting cause variation in the blood glucose levels..
Glucose measurement is performed for the diagnosis and follow-up of carbohydate metabolism abnormalities (eg. diabetes mellitus, hypoglycemia) and other conditions (such as cerebrospinal fluid glucose in suspected meningitis).
Glucose levels in the body increase due to:
- Diabetes Mellitus including: Hemochromatosis, Cushing's syndrome, Acromegaly and gigantism
- Increased circulating epinephrine: Adrenal injection, pheochromocytoma, stress (emotions, burns, shock)
- Acute pancreatitis
- Chronic pancreatitis
- Wernicke's encephalopathy
- CNS lesions
- Effects of drugs
Glucose levels in the body decrease due to:
- Pancreatic disorders: islet cell tumors
- glucagon deficiency
- carcinoma of adrenal gland
- carcinomaof stomach
- Hepatic Disease: Poisoning, hepatitis, cirrhosis, primary/ metastatic
- Endocrine Disorder: Hypopituitarism and Addison's disease, hypothyroidism
- functional disturbances: post gastrectomy. gastroenterostomy, antonomic nervous system disorder
- Pediatric Anomalies:
-prematurity,infant of diabetic mother, ketotic hypoglycemia, Zetterstrom's syndrome, idiopathic leucine sensitivity, spontaneous hypoglycaemia in infants
- Enzyme diseases:
-Von Gerke's disease, galactosemia, maple syrup urine disease, fructose intolerance
The Analyser used for the glucose measurement is the Beckman Coulter LX20 PRO analyser, besides this test, the analyser can also be used to measure various electrolytes (eg. sodium, potassium) and some liver enzymes (GGT, ALT,AST etc)
The samples usually collected for the glucose test are serum, plasma, urine or cerebrospinal fluid.
Principle of Analysis
The analyser determines the glucose concentration by using a glucose reagent in conjunction with calibration standards done by the med tech. when the reagent is input into the machine.
The Analyser measures the oxygen consumption rate of the sample.
10 microlitre of sample is injected into the reaction cup containing the glucose oxidase solution. The ratio used is one part sample to 76 part reagent. The peak of the oxygen consumption is directly proportional to the concentration of glucose in the sample.
The machine then prints out the readout when the test has finished.
Saturday, October 6, 2007
Identification and Quantitation of Stenotrophomonas maltophilia periplasmic proteins using 2-D Gel Electrophoresis and MALDI
For my project, Stenotrophomonas maltophilia (S. maltophilia) was the bacteria used. It is a gram negative bacteria thus it has a relatively large periplasmic space(periplasm) as compared to gram positive bacteria.
S. maltophilia was streaked on the nurtient agar plate and incubated in a 37ºC incubator for 24 hours. After which all colonies will be scraped off the agar plate and reusepened in 5mL of PBS and the cell density is taken. 5X107 cells will then be inoculated into each LB broth and grown at 28ºC and 37ºC in 20mL LB- broth for 18 hours. Cells will be subjected to washing using PBS for the purpose of removing dead cells and proteins that are released due to cell lysis.
Picture taken from: http://rds.yahoo.com/_ylt=A9ibyiCO_gZHbVkAowyjzbkF/SIG=1300lqsrd/EXP=1191727118/**http://www.cofc.edu/~delliss/virtuallabbook/StreakPlates/StreakExamples.html
This is the procedure for cell washing:
1. Take out the cells from the incubator and spin at 3000g for 20 minutes at 10ºC
2. Discard the supernatant into a waste bottle
3. Pipette 10mL of PBS
4. Pipette well and vortex at low speed to resuspend the cells
5. Spin down centrifuge tubes at 3000g for 20 minutes at 10ºC
6. Repeat step 2 to step 5 twice
7. Resuspend the cells in 10mL of PBS
8. Pipette well and vortex at low speed to resuspend the cell pellet
Density of the live cells will then be determined. 6 eppendorf tubes cotaining each
10 to the power of 10 cells will be subjected to extraction using chloroform
This is the procedure for chloroform shock:
1. Spin down cells at 3000g for 20 minutes at 10oC
2. Resuspend pellet in remaining medium by brief vortexing
3. Add 20uL of chloroform to each eppenforf tube
4. Incubate at room temperature for 15 minutes
5. Combine the 6 eppendorf tubes together
6. Add 400uL of 0.1M Tris-HCl to the combined eppendorf tubes
7. Spin at 6000g for 20 minutes
8. Pipette out supernatant in a new eppendorf tube
9. Spin at 14000g for 10 minutes
10. Pipette supernatant in a new eppendorf tubes
Once periplasmic proteins are extracted, microcon purification is done. The purpose of microcon purification is to further concentrate the protein samples
Micron picture. Taken from: http://www.life.sci.qut.edu.au/epping/LSB607OLT/607concentrators.html
Then Bradford assay will be done to give a estimation of the protien concnetration
Taken from: http://www.biocompare.com/review/926/Quick-Start(tm)-Bradford-Protein-Assay-Kits-From-Bio-Rad.html
Once the protien concentration is determined, 2D gel electrophoresis will be done. Basically protiens will be 1st separated according to their pI by isolecetric focusing (IEF) after which according to their molecular weight (for information on how 2D gel electrophoresis is done, refer to Jia xin's blog on Aug 4, 2007)
BIO-RAD PROTEAN IEF CELL used for IEF (picture taken with permission)
BIO-RAD Criterion Dodeca™ Cell used to separate the protein sample according to molecular weight (picture taken with permission)
Once the proteins are separated, the gel is stained with SYPRO RUBY stain for 16 hours and scanned with PharosFX™ Plus Molecular Imager (for information pls refer to MIng Boon's 17th Aug posting)
This is how a SYPRO RUBY stained gel looks like ( picture taken with permission)
This is the PharosFX™ Plus Molecular Imager which I used ( picture taken with permission)
Once the gel is sacnned, quantitation will be done using a software program:PDQuest. PDQuest will allow us to quantitate the gel spots (for this research project, we want to quantify protein spots present in 28ºC and 37ºC with 2-folds) After Quantitation, gel spots will be slected, cut and automatic process by Xcise ( I won't dicsuss about how Xcise works, it was discussed in Ming Boon's posting on 07/07/07)
Xcise machine from Shimadzu Biotech, proteome systems, Japan (Picture taken with permission)
After processing by Xcise, the protein will be spotted on the MALDI plate and subjected to MALDI analysis. The picture below is the MALDI machine used
(picture taken with permission by my supervisor)
end of story=).......
...feel free to ask any question
Biosafety Level II Cabinet
Tuesday, October 2, 2007
In order to prevent graft rejection, transplant recipients have to take immunosuppressive drugs for their whole life. Immunosuppressive drugs are intended to interrupt or prohibit the immunological mechanisms occurring during graft rejection by interference with the function and metabolism of activated cells.
Name of test: Cyclosporin testing
Cyclosporin is a potent immunosuppressive drug got combating tissue rejection following organ transplant. It has serious toxic side effects and is recommended to have regular drug monitoring. Cyclosporine has increased both the short- and long-term survival rates for transplant patients, especially in heart and liver operations. The rejection of grafted tissues occurs when white blood cells (lymphocytes) called T-helper cells stimulate the activity of cell-destroying (cytotoxic) T-killer cells. These T cells, along with other white blood cells like monocytes and macrophages, cause the tissue rejection of the implanted organs. Cyclosporine reduces the ability of accessory cells to produce interleukins, resulting in decreased replication of helper and killer T cells.
Testing cyclosporine levels in the blood can help ensure that drug levels are in a range that will be therapeutic for you.
Procedure of test:
Pre-treatment is needed. The drug is coated on RBCs, therefore the solubilization reagent is added to lyse the RBC and release it. Precipitation reagent is added to suspend the protein. It is then vortexed to ensure through mix and centrifuged to obtain clear supernatant. The clear supernatant is then pipetted out and place into a sample cup which is then placed into the TDX analyzer. The reagents needed are also placed inside the analyzer. TDX is used for quantitative measurement of drugs. It uses the Fluorescence Enzyme Immunoassay (FPIA) technology.
If the level is too low, organ rejection may occur.
If the level is too high, it will result in toxicity. Toxic side effects produced are primarily nephrotoxicity and hepatotoxicity.
Loh Mun Jo-anne
Clinical Diagnostics, Chapter 47: Therapeutic drug monitoring, page1155-1156
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