Fecal Occult Blood Test (FOBT) and Ova, Cyst and Parasite test are two of the most common tests done in the Parasitology bench.
FOBT is done to determine the presence of hidden (occult) blood in the stool. This may be caused by undetected gastrointestinal bleeding, which could be an indicator for colorectal cancers. The test is based on immunochromatography. It uses antibodies against haemoglobin, a main component of blood. . The presence of haemoglobin will cause it to bind to the antibody, and cause a colour change in the test strip.
The stool needs to be sampled at 6 places to allow for homogenous sampling of the stool.
After the sampling vial has been inoculated with the stool, a test strip is then inserted into the test solution in the vial. The result should be visible within 5 minutes. Any result obtained after the 5 minutes window period will be considered void, as there may be a chance of false positives occurring.
The control line must be working in order to obtain a validated test result. The control line ensures that all the procedures have been done correctly. If the control line does not show, it means that the test is equivocal and cannot be used. Thus, a retest needs to be done.
In ova, cyst and parasite testing, there are two main procedures – the fixed smear and the wet mount. Fixation of the stool sample helps to preserve the state of the parasitic organisms in the stool. The wet mount enables the visualization of motile parasitic organisms in the stool.
Fixed Smear
1. Mix 1 part stool to 3 parts of Zn Poly-Vinyl Alcohol (PVA) in the provided PARA-PAK tube.
2. Let stand for 30 minutes.
3. After 30 minutes, pour some of the Zn-PVA-fixed material onto a paper towel. Let most of the excess PVA be absorbed.
4. Once most of the excess PVA has been absorbed, spread some of the fixed stool material evenly onto a glass slide. The edges of the slide must be smeared with some of the fixed material to adhere better onto the slide.
5. Once dry, heat-fix for 3 hours on a slider warmer at about 35oC.
6. After which, perform a trichrome stain.
Wet Mount
1. 10 drops of surfactant is added to the remaining Zn-PVA fixed sample and shaken vigorously. The surfactant helps to break down the larger stool particles.
2. The Macro-Con conical filter tube is then attached to the PARA-PAK tube and inverted to allow filtration of the sample.
3. Once the sample has been filtered, the empty PARA-PAK tube and filter is discarded, leaving the MACRO-CON tube full of fixed sample solution.
4. Add buffered formaldehyde (formalin) (4.4 % w/w of solution) up until the indicator line on the tube.
5. Add 5 ml of clearene.
6. Spin at 2000 rpm for 10 minutes in the centrifuge.
7. Discard the supernatant and clean the sides of the tube with a clean cotton swab to remove excess clearene and formalin.
8. Resuspend residue in equal amounts of normal saline.
9. Place a drop of the resuspended solution onto a glass slide and mount with a coverslip.
View both the fixed smear and the wet mount under a microsope.
MUNA
0703791D
Retrieved from “ http://www.nagase.com/products/oclight/oclight.pdf” on 2 August 2009.
Retrieved from http://www.cancer.net/patient/Library/Cancer.Net+Features/Treatments,+Tests,+and+Procedures/Fecal+Occult+Blood+Tests+%E2%80%94+What+to+Expect
On 2 August 2009.
Wednesday, August 19, 2009
Mycology
Cryptococcal Antigen Latex Agglutination Test
Hi! I'm posted to mycology for this week and i''ll share with you guys the test that i get a hands on with.
Cryptococcal Antigen Latex Agglutination Test is a qualitative and quantitative test. The test is used to detect capsular polysaccharide antigens of Cryptococcus neoformans in patient’s serum and cerebrospinal fluid. It is done using commercial latex particles coated with anticryptococcal globulin (detection latex). In the presence of capsular polysaccharide antigen in the patient's serum or CSF, the latex particles coated with anticryptococcal globulin will react with the antigen and form a visible agglutination. Latex particles coated with normal globulin acts as the control (control latex).
This is the kit that is used CALAS®.
http://www.cardinalhealth.com/us/en/distributedproducts/ASP/140100.asp?cat=laboratoy
Procedure
1. First, when the blood sample is received it is centrifuged at 1000 rpm for 15 minutes.
2.Then, Transfer 100ul of patient’s serum to a test tube with 100ul of pronase.Pronase is an enzyme which degrades non specific macro globulin.
3. Incubate the serum-pronase solution in a water bath for 15 minutes at 56°C.
4. Immediately boil the solution for a full five minutes to terminate the enzymatic digestion.
5. Allow the solution to cool to room temperature.
6.Then remove enough cards (see the pic above) to run the test and label one ring as detection latex with the patient's lab number (DL) and another as control latex (CL).
7. Pipette 25ul of patient serum to both CL and DL rings.
8. Holding the detection latex bottle in a vertical position, squeeze one free-falling drop of reagent to the DL ring. Do the same for the control latex bottle and drop one free-falling drop of reagent to the CL ring.
9. Place card on a rotator and rotate at 125 rpm for 5 minutes.
10. Read the result immediately.
For CSF, it does not require steps 1-3. It begins by transfering about 0.5 ml of CSF to a test tube and boiling it for a full 5 mins. The subsequence steps are the same as the blood.
Result Interpretation
Negative: No visible clumping
1+: Fine granulation against a milky background
2+: Small but definite clumps against a slightly cloudy background.
3+: Large and small clumps against a clear background.
4+: Large clumps against a very clear background.
Shameema :) 0700486D
Sunday, August 9, 2009
This is Jennifer. On the forth week of my SIP, I was posted to the blood culture station.
Blood Culture
The BACTEC Fluorescent Series
If bacteria or fungi grow in a medium that provides the essential nutrients which enhances their growth, they may cause haemolysis of red blood cells if the medium is blood. They may also cause the medium to be turbid or producing gaseous by product such as carbon dioxide production.
The BACTEC Fluorescent Series detects the production of carbon dioxide by the increasing numbers of bacteria in the blood.
If bacteria or fungi are present in the blood or other body fluids, they will break down the nutrients in these medium and release carbon dioxide. The dye in the sensor will react with the carbon dioxide produced. This reaction affects the amount of light that is absorbed by a fluorescent material in the sensor. The instrument’s photo detector measures the amount of fluorescence which responds proportionally to the amount of carbon dioxide produced by bacteria or fungi.
The instrument begins continuous automated testing and the vials’ fluorescent sensors are activated by the LED (Light Emitting Diodes) behind the vials that laminated the racks. The instrument’s detector would start taking the readings after a warm up period. The vials will be read by the detector every ten minutes and the cycle for all the racks inside the incubator will be finished after every ten minutes. If there is a positive culture, the indicator light will light up in front of the instrument and also shown on the monitor and the alarm will sound. After the positive vial was detected, the medical technologist in charge will remove the vial from the incubator and send them for blood culture for the isolation and detect the identity of the organisms present in the blood or medium.
Steps for the positive culture to be lighted up or identified:
1. Carbon dioxide was released by bacteria
2. Carbon dioxide reacts with dye in the sensor
3. LED, modulated by dye, activates fluorescent material in sensor
4. Increasing amount of fluorescent is read by photo detector
5. Raw data from detector is sent to rack microprocessor
6. To where positivity analysis is performed
7. Positive vial lamp illuminates, audible alarm sounds, positive stations are displayed
Usually, when blood specimens or other body fluids are received such as stem cells, peritoneum fluid from renal, and dialysis fluid, it comes in two bottles where one is to detect for present of aerobic bacteria while another is for anaerobic bacteria. But in some cases if there is a need to detect for the present of fungi, a third bottle will be sent together with the two bottles.
For aerobic bacteria, the cap of the bottle is in blue colour while gold colour for anaerobic bacteria.
For fungi or mycobacterium, it is in red colour.
Below is a picture that shows the BACTEC blood culture bottles with different cap colours for different types of purposes:
Procedures
The BACTEC bottles or vials are inserted into the rackets of the incubator and the racks constantly incubated at 35 degree Celsius and are agitated throughout the 5 days.
For aerobic and anaerobic bacteria, the vials are read every 10minutes by the detector and the bottles are discarded after 5 days if no positive are flag on the bottles.
In some cases such as patients with Endocarditis or Brucella, the bottles are tied with red or blue ribbons before incubated and cannot be thrown away even if flagged negative.
For positive vials, immediately being removed from incubator and sent to for blood culture.
Blood Culture
The BACTEC Fluorescent Series
If bacteria or fungi grow in a medium that provides the essential nutrients which enhances their growth, they may cause haemolysis of red blood cells if the medium is blood. They may also cause the medium to be turbid or producing gaseous by product such as carbon dioxide production.
The BACTEC Fluorescent Series detects the production of carbon dioxide by the increasing numbers of bacteria in the blood.
If bacteria or fungi are present in the blood or other body fluids, they will break down the nutrients in these medium and release carbon dioxide. The dye in the sensor will react with the carbon dioxide produced. This reaction affects the amount of light that is absorbed by a fluorescent material in the sensor. The instrument’s photo detector measures the amount of fluorescence which responds proportionally to the amount of carbon dioxide produced by bacteria or fungi.
The instrument begins continuous automated testing and the vials’ fluorescent sensors are activated by the LED (Light Emitting Diodes) behind the vials that laminated the racks. The instrument’s detector would start taking the readings after a warm up period. The vials will be read by the detector every ten minutes and the cycle for all the racks inside the incubator will be finished after every ten minutes. If there is a positive culture, the indicator light will light up in front of the instrument and also shown on the monitor and the alarm will sound. After the positive vial was detected, the medical technologist in charge will remove the vial from the incubator and send them for blood culture for the isolation and detect the identity of the organisms present in the blood or medium.
Steps for the positive culture to be lighted up or identified:
1. Carbon dioxide was released by bacteria
2. Carbon dioxide reacts with dye in the sensor
3. LED, modulated by dye, activates fluorescent material in sensor
4. Increasing amount of fluorescent is read by photo detector
5. Raw data from detector is sent to rack microprocessor
6. To where positivity analysis is performed
7. Positive vial lamp illuminates, audible alarm sounds, positive stations are displayed
Usually, when blood specimens or other body fluids are received such as stem cells, peritoneum fluid from renal, and dialysis fluid, it comes in two bottles where one is to detect for present of aerobic bacteria while another is for anaerobic bacteria. But in some cases if there is a need to detect for the present of fungi, a third bottle will be sent together with the two bottles.
For aerobic bacteria, the cap of the bottle is in blue colour while gold colour for anaerobic bacteria.
For fungi or mycobacterium, it is in red colour.
Below is a picture that shows the BACTEC blood culture bottles with different cap colours for different types of purposes:
Procedures
The BACTEC bottles or vials are inserted into the rackets of the incubator and the racks constantly incubated at 35 degree Celsius and are agitated throughout the 5 days.
For aerobic and anaerobic bacteria, the vials are read every 10minutes by the detector and the bottles are discarded after 5 days if no positive are flag on the bottles.
In some cases such as patients with Endocarditis or Brucella, the bottles are tied with red or blue ribbons before incubated and cannot be thrown away even if flagged negative.
For positive vials, immediately being removed from incubator and sent to for blood culture.
Saturday, August 1, 2009
medical mircobiology [serology] (part I)
Hello all~ yanling here.. its my turn to blog this wk~!
I'm going to talk about serology as im in that department for 3 wks (again). This week is my last week. xD
I will talk about one test in a post. So there might be quite a few posts at a go. But at least questions can be directed to each test~! xDDecided to write about some of the manual tests done in serology, because I think they require more skills, especially in pipetting. xD
Venereal Disease Research Laboratory Test
Its is to check if the person has syphilis.
The first test done is toscreen for presence of syphilis/ Treponoma pallidum Ab using RPR reagent which consist of carbon particles coated with syphilis Ag. [I had made a mistake here, SO SORRY~!] screen for presence of Ab that are specific to the substance that were released by the cells that are attacked by Treponoma pallidum which causes syphilis. Since its about Ag-Ab, so the principle of this test is agglutination.
So how the screening test is done? First, 50ul of serum is dropped onto a circle of RPR card (will upload picture soon). Then a drop of RPR reagent will be added to each circle then rotate for 8 mins at 100rpm.
Then 8 minutes later, one has to check for agglutination. Here comes the difficult part as for weak positive, the agglutination will not be very obvious so there are chances that one may mistaken with the RPR carbon. So everytime in doubt, got to ask the staff for help.
So for the reactive samples, they will be keep aside for further tests. Then for the non-reactive samples, they will be scan and reported as "non-reactive".
Now I'm going to talk abt the further tests for reactive samples. The next test is venereal disease titre, which to find how strong the syphilis Ab is. There will be 6 circles required, 50ul of saline is pipetted onto second to sixth circles. Then 50ul of reactive serum is added to 1st circle (neat). Then another 50ul of the same serum will be pipette to 2nd circle. Then a 50ul serial dilution is done from 2nd circle to 6th circle. Then the serum is spread to the entire circle from the highest dilution to the neat. Then 1 drop of RPR reagent is added to each circle and rotate for 8 mins at 100 rpm. Then titre of the serum will then be recorded b4 proceeding to the next test. The titre from neat to the 6th circle is as follows: 1:1 1:2 1:4 1:8 1: 16 and 1: 32. And if there are still strong agglutination at the 6th circle it will be recorded as >1:32.
The next test done is Treponoma pallidum Haemagglutination (TPHA), is a more specific test for syphilis as VDRL test can be reactive if there is present of malaria parasite.
The principle for TPHA is haemagglutination, which uses cells with the Ag to check for agglutination whereas for VDRL, it uses particle coated with the Ag for agglutination, and for this case is carbon particle.
The steps are a little complicated, but i will try to explain to the best i can. The test uses 96 u-shaped well. For each sample, 6 wells will be used. So the max number that can be done in a plate is 15 as one row will be used for control. xD
So before adding anything to the plate, the side of each row of 6 wells must be labelled with the last 3 number of the barcode no. or for control, it will be labelled as C. Then 190ul of sample diluent will be added to the 1st well of each sample or control. Next, leaving the 2nd well empty, add 25ul of sample diluent into 3rd to 6th wells. As for control, the sample diluent is added till the 4th well. This because the control will show agglutination from 3rd well onwards, so in order to save the diluent, it is done till the 4th well. After that, 10ul of control/serum is added to the 1st well and mix well. Then take 25ul from the 1st well to then 2nd well and the 3rd well. Then a 25ul serial dilution is done from 3rd well onwards.
After the serial dilution, one drop of unsensitised cells will be added to 2nd wells of each sample and control. And 1 drop of sensitised cells will be added to 3rd wells onwards. The unsensitised cells contains cells without any T. pallidum Ag, so the results will always be negative. Then as for sensitised cells, it contains the T. pallidum Ag, so if there is any presence of T. pallidum Ab in the patient's serum, there will be agglutination. Then the sides of the plate is tapped to ensure the contents are mixed. Its like how we do our ELISA during AIMM i think? Then it will be incubate for minimum of 45 mins at room temp.
So how the TPHA negative and positive results will look like? For negative results, it will appear as a compact button or small ring at the center of the well as the cells will settle. Then for positive results, there will be agglutination and so there will not be any button or small ring in the center of the well. The titre of the test will be then recorded and reported. The titre is from 3rd well onwards, 1:80, 1:160 and 1:320. If there is still no button or small ring, it will report as >1:320.
yaNLing xD
I'm going to talk about serology as im in that department for 3 wks (again). This week is my last week. xD
Venereal Disease Research Laboratory Test
Its is to check if the person has syphilis.
The first test done is to
So how the screening test is done? First, 50ul of serum is dropped onto a circle of RPR card (will upload picture soon). Then a drop of RPR reagent will be added to each circle then rotate for 8 mins at 100rpm.
Then 8 minutes later, one has to check for agglutination. Here comes the difficult part as for weak positive, the agglutination will not be very obvious so there are chances that one may mistaken with the RPR carbon. So everytime in doubt, got to ask the staff for help.
So for the reactive samples, they will be keep aside for further tests. Then for the non-reactive samples, they will be scan and reported as "non-reactive".
Now I'm going to talk abt the further tests for reactive samples. The next test is venereal disease titre, which to find how strong the syphilis Ab is. There will be 6 circles required, 50ul of saline is pipetted onto second to sixth circles. Then 50ul of reactive serum is added to 1st circle (neat). Then another 50ul of the same serum will be pipette to 2nd circle. Then a 50ul serial dilution is done from 2nd circle to 6th circle. Then the serum is spread to the entire circle from the highest dilution to the neat. Then 1 drop of RPR reagent is added to each circle and rotate for 8 mins at 100 rpm. Then titre of the serum will then be recorded b4 proceeding to the next test. The titre from neat to the 6th circle is as follows: 1:1 1:2 1:4 1:8 1: 16 and 1: 32. And if there are still strong agglutination at the 6th circle it will be recorded as >1:32.
The next test done is Treponoma pallidum Haemagglutination (TPHA), is a more specific test for syphilis as VDRL test can be reactive if there is present of malaria parasite.
The principle for TPHA is haemagglutination, which uses cells with the Ag to check for agglutination whereas for VDRL, it uses particle coated with the Ag for agglutination, and for this case is carbon particle.
The steps are a little complicated, but i will try to explain to the best i can. The test uses 96 u-shaped well. For each sample, 6 wells will be used. So the max number that can be done in a plate is 15 as one row will be used for control. xD
So before adding anything to the plate, the side of each row of 6 wells must be labelled with the last 3 number of the barcode no. or for control, it will be labelled as C. Then 190ul of sample diluent will be added to the 1st well of each sample or control. Next, leaving the 2nd well empty, add 25ul of sample diluent into 3rd to 6th wells. As for control, the sample diluent is added till the 4th well. This because the control will show agglutination from 3rd well onwards, so in order to save the diluent, it is done till the 4th well. After that, 10ul of control/serum is added to the 1st well and mix well. Then take 25ul from the 1st well to then 2nd well and the 3rd well. Then a 25ul serial dilution is done from 3rd well onwards.
After the serial dilution, one drop of unsensitised cells will be added to 2nd wells of each sample and control. And 1 drop of sensitised cells will be added to 3rd wells onwards. The unsensitised cells contains cells without any T. pallidum Ag, so the results will always be negative. Then as for sensitised cells, it contains the T. pallidum Ag, so if there is any presence of T. pallidum Ab in the patient's serum, there will be agglutination. Then the sides of the plate is tapped to ensure the contents are mixed. Its like how we do our ELISA during AIMM i think? Then it will be incubate for minimum of 45 mins at room temp.
So how the TPHA negative and positive results will look like? For negative results, it will appear as a compact button or small ring at the center of the well as the cells will settle. Then for positive results, there will be agglutination and so there will not be any button or small ring in the center of the well. The titre of the test will be then recorded and reported. The titre is from 3rd well onwards, 1:80, 1:160 and 1:320. If there is still no button or small ring, it will report as >1:320.
yaNLing xD
medical mircobiology [serology] (part II)
Mycoplasma pneumoniae antibodies
if one is infected by M. pneumoniae, one will produce Ab against it, thus in this test, it is to see if the patient had been infected with the bacteria.
The principle is this test is also agglutination, where the results are similar to the results of TPHA i mentioned earlier.. if the result is negative, the sensitised particles will settle to the bottom of the well to form a compact ring or button.
the steps is more simpler here. 1st, add 100ul of sample diluent into 1st well, and add 25ul of sample diluent into 2nd to 4th well for control and 2nd to 6th well for each patient's serum. Then add 25ul of serum into 1st well and do a 25ul serial dilution down the row. Next, add i drop of unsensitised particle into 2nd well and add 1 drop of sensitised particles into the 3rd and 4th well for control and 3rd to 6th well for specimens. Pat the sides of the plate and incubate for 3 hours or overnight.
it is important that the 2nd well must be negative results, as the unsensitised particles are not coated with the bacteria Ag, thus there will not be any agglutination. If there is positive results for 2nd well, it might be due to cross contamination or added wrong reagent.
the results of each specimen are then recorded and report in terms of the titre as follow: 1:40(3rd well), 1:80(4th well), 1:160(5th well), 1:320(6th well) and >1:320(when there is still agglutination at 6th well).
yaNLing xD
*will add more tests soon when im free~!*
if one is infected by M. pneumoniae, one will produce Ab against it, thus in this test, it is to see if the patient had been infected with the bacteria.
The principle is this test is also agglutination, where the results are similar to the results of TPHA i mentioned earlier.. if the result is negative, the sensitised particles will settle to the bottom of the well to form a compact ring or button.
the steps is more simpler here. 1st, add 100ul of sample diluent into 1st well, and add 25ul of sample diluent into 2nd to 4th well for control and 2nd to 6th well for each patient's serum. Then add 25ul of serum into 1st well and do a 25ul serial dilution down the row. Next, add i drop of unsensitised particle into 2nd well and add 1 drop of sensitised particles into the 3rd and 4th well for control and 3rd to 6th well for specimens. Pat the sides of the plate and incubate for 3 hours or overnight.
it is important that the 2nd well must be negative results, as the unsensitised particles are not coated with the bacteria Ag, thus there will not be any agglutination. If there is positive results for 2nd well, it might be due to cross contamination or added wrong reagent.
the results of each specimen are then recorded and report in terms of the titre as follow: 1:40(3rd well), 1:80(4th well), 1:160(5th well), 1:320(6th well) and >1:320(when there is still agglutination at 6th well).
yaNLing xD
*will add more tests soon when im free~!*
medical microbiology [serology] (part III)
Thyroglobulin & Thyroid Microsomal Antibodies (THYMA)
It is to test for presence of thyroidic autoantibodies, where the Ab attacks the thyroid. 2 tests are usually done, thyroglobulin antibodies test (ATG) and thyroid microsomal antiboides test (AMC). these 2 tests are usually ordered together. Presence of either Ab means that the person has autoAb to his/her thyroid.
The principle is agglutination and the steps are almost the same with MPA, only the volume of sample diluent and serum are different.
For both tests, the first and second well is added with 50ul of sample diluent and 75ul for 3-4th well for control and 3-6th well for each sample. Then, 10ul of control is added to the 1st well of control. Same goes to each specimen. After that, a 25 serial dilution is done down the row till the 4th well for control and 6th well for each specimen.
Same with MPA, 1 drop of unsensitised particles is added to second well and 1 drop of sensitised particles to 3 wells onwards. The sides of the plates are then patted and incubate overnight at room temperature.
The results is same with MPA, where unsensitised cells and negative results will show button in the center of the well, while positive results will show agglutination and absence of button in the center of well.
yaNLing xD
It is to test for presence of thyroidic autoantibodies, where the Ab attacks the thyroid. 2 tests are usually done, thyroglobulin antibodies test (ATG) and thyroid microsomal antiboides test (AMC). these 2 tests are usually ordered together. Presence of either Ab means that the person has autoAb to his/her thyroid.
The principle is agglutination and the steps are almost the same with MPA, only the volume of sample diluent and serum are different.
For both tests, the first and second well is added with 50ul of sample diluent and 75ul for 3-4th well for control and 3-6th well for each sample. Then, 10ul of control is added to the 1st well of control. Same goes to each specimen. After that, a 25 serial dilution is done down the row till the 4th well for control and 6th well for each specimen.
Same with MPA, 1 drop of unsensitised particles is added to second well and 1 drop of sensitised particles to 3 wells onwards. The sides of the plates are then patted and incubate overnight at room temperature.
The results is same with MPA, where unsensitised cells and negative results will show button in the center of the well, while positive results will show agglutination and absence of button in the center of well.
yaNLing xD
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