As the name suggest, this test is used to detect cold antibody. Not only do we detect whether there is cold antibody, we also detect the cold antibody titre.
Firstly, we serial dilute the antibody of the patient. Then we test the different concentrations of antibody against 3-5% patient cells and control cells. The titre value is determined by the highest dilution that can still cause a positive reaction. Titration value can help quantify the relative antibody concentration in the plasma.
The most common type of cold auto-antibody is IgM. This antibody is capable to cause cold haemagglutin disease (CHD). The most common specific cold antibody seen is auto anti-I.
CHD can be acute or chronic. Acute condition is usually caused by mycoplasma pneumoniae infection. For chronic condition, it usually occurs in elderly suffering from chronic hemolytic anaemia.
As a control, a segment group O cells will be taken from a random blood unit that has no blood antigen listed on the unit of blood. (The pints of blood have a sticky label on top of it. Should there be any antigen, it'll be listed on the pint of blood) For example, some units are (K-). Since the units are selected at random, we must also note that should this test be repeated with a segment from another group O cells, we expect similar results, but never the same results.
Procedure:
1. Label 2 sets of tubes, first set with "own cells followed by the dilution", e.g. OC 1, second set with "control cells followed by the dilution, e.g. CC 1024.
2. We pipette 6 drops of saline into tubes from: OC 2 to OC 1024.
3. Then, 3 drops of patient's serum into OC 1 and CC 1, 6 drops into OC 2.
4. From OC 2, we mix the saline and serum well, then pipette 6 drops into OC 4, and 3 drops into CC 2. This ensures that the OC and CC concentration are the same.
5.From OC 4, we pipette 6 drops into OC 8 and 3 drops into CC 4.
6.From OC 8, we pipette 6 drops into OC 16 and 3 drops into CC 8.
7. This process is repeated until we reach OC 1024. 3 drops are pipetted into CC 1024 and 6 drops are pipetted out into the waste container.
8. We should obtain 3 drops in every tube, and we must be careful as to not introduce any air bubbles in the process of pipetting as it may influence the readings.
9. Add 1 drop of 3-5% patient's own washed cells to each of the OC tubes
10. Add 1 drop of 3-5% control own cells into each of the CC tubes
11. Incubate the tubes at 4 degrees for at least 2 hours.
12. Read the tubes and rate them from 4+ to +w(where the w is in superscript), +w represents plus weak, i.e a very very weak agglutination reaction where you see numerous agglutinates in a background of free cells.
13. To check for microscopic agglutination reaction, cold glass slides are are in the refrigerator is used.
Agglutination reaction at 1:32 dilution or lower is regarded as clinically insignificant while agglutination reaction at 1:64 or higher is regarded as clinically significant.
Results are expressed as the reciprocal of the highest serum dilution able to cause a macroscopic reaction with group O cells. Reactions in both sets of tubes should appear very similar unless there is a mistake somewhere. It is also important that we read the results inside the fridge, that way, the positive results will not become negative reactions
Yanhong
0703979E
Saturday, August 29, 2009
Saturday, August 22, 2009
Tissue Typing (HLA) Laboratory
I was attached to the tissue typing (HLA) laboratory for a week and had fun learning new things. It was a bit stressful at times, as the procedures performed in this laboratory requires critical attention with much understanding of the HLA system.
Human leukocyte antigen (HLA) is a specialised category of immunology that deals with human histocompatibility testing. This laboratory discipline supports clinical specialities in transplantation, transfusion and immunogenetics. Hence, a laboratory officer must be specialised in this field to handle the heavy responsibilities. All of the preliminary result is double-checked by senior laboratory officers for confirmation. The HLA laboratory in BSG handles samples that are mainly for organ and bone marrow transplantation. We also receive samples from regional countries such as Vietnam and Malaysia.
It is necessary to determine the HLA antigen composition in an organ or bone marrow recipient to match with the prospective donor, who may be living family-related or unrelated deceased. For transfusion cases, patients that underwent platelet therapy may develop platelet refactoriness that can result in poor platelet survival following random donor platelet infusion. Hence, it is important to identify HLA alloantibodies in the recipient serum before transplantation or transfusion. HLA alloantibodies are majority IgG.
The testing services offered by HLA laboratory in BSG are HLA antigen typing, HLA antibody identification (i.e. determining the specificity of the anti-HLA class I and II antibodies) and lymphocyte crossmatching between donor cells and recipient serum for compatibility. I got the opportunity to view and do both serological and molecular techniques in a HLA typing test. Since it is very lengthy to explain both, I will focus on explaining the serology part.
For serological procedures, the very commonly requested test is the HLA-B27 typing. This tissue antigen is associated with ankylosing spondylitis. Samples are received in acid-citrate-dextrose (ACD) tube or heparin tube (green cap). We will do layering of the blood sample with Ficoll-Paque in the ratio 3:1 and centrifuged the tube at 2000 rpm for 20 minutes. This condition is ideal to create a density gradient so that we can separate out the lymphocytes. 4 layers are obtained: bottom – red cells, Ficoll-Paque, lympocytes and monocytes, and plasma – top. Discard the plasma and carefully transfer the lymphocytes and monocytes layer into a clean tube without taking so much Ficoll-Paque using Pasteur pipette. Wash 3X this layer with cold PBS for purification.
Next, we want to harvest T cells. Why T cells? This is because HLA-B27 is categorised as HLA Class I molecule and we don’t want B cells that have both Class I and II molecules. We will use the magnetic particle concentrator (MPC), where we add the Class I beads (they are actually monoclonal antibodies specific against Class I antigens). This will positively select the T cells. We will dilute the cells to the optimal concentration with prepared solution that contains Hank’s balance salt solution (HBSS), 2% fetal calf serum (FCS) and acridine orange (AO).
Next, we will perform the complement-dependent cytotoxicity (CDC) assay. Before doing so, we will check if we got the optimum cell concentration and cell viability (graded with score of 1 i.e. 0-10% dead cell) by adding 2μl of cells to an empty well of a microtiter plate and 2μl of Propodium Iodine (PI). This will stain the cells under the fluorescent microscope. Green means cells are alive while red means cells are dead.
We use a 60-well commercial microtiter plate. The plate has 1μl of known antisera with mineral oil layer on top to protect the antisera from evaporation. We will add 2μl of the patient T cells and incubate at room temperature for 40-50 minutes. Then we add 5-6μl of rabbit complement to each well and incubate for 60 minutes in the dark (we just put the plate in a metal plate with a lid). After that we add 2μl of PI in 5% EDTA solution to each well for 15 minutes at room temperature. This is for staining and to also stop the cytotoxic reaction. Lastly, we will remove excess fluid and add paraffin oil to prevent evaporation when viewing under the fluorescent microscope (10X objective). If the 81-100% of the cells are dead (i.e. graded with a score of 8), the cells are strongly positive for the HLA-B27 antigen.
Indah.
0705361D
Human leukocyte antigen (HLA) is a specialised category of immunology that deals with human histocompatibility testing. This laboratory discipline supports clinical specialities in transplantation, transfusion and immunogenetics. Hence, a laboratory officer must be specialised in this field to handle the heavy responsibilities. All of the preliminary result is double-checked by senior laboratory officers for confirmation. The HLA laboratory in BSG handles samples that are mainly for organ and bone marrow transplantation. We also receive samples from regional countries such as Vietnam and Malaysia.
It is necessary to determine the HLA antigen composition in an organ or bone marrow recipient to match with the prospective donor, who may be living family-related or unrelated deceased. For transfusion cases, patients that underwent platelet therapy may develop platelet refactoriness that can result in poor platelet survival following random donor platelet infusion. Hence, it is important to identify HLA alloantibodies in the recipient serum before transplantation or transfusion. HLA alloantibodies are majority IgG.
The testing services offered by HLA laboratory in BSG are HLA antigen typing, HLA antibody identification (i.e. determining the specificity of the anti-HLA class I and II antibodies) and lymphocyte crossmatching between donor cells and recipient serum for compatibility. I got the opportunity to view and do both serological and molecular techniques in a HLA typing test. Since it is very lengthy to explain both, I will focus on explaining the serology part.
For serological procedures, the very commonly requested test is the HLA-B27 typing. This tissue antigen is associated with ankylosing spondylitis. Samples are received in acid-citrate-dextrose (ACD) tube or heparin tube (green cap). We will do layering of the blood sample with Ficoll-Paque in the ratio 3:1 and centrifuged the tube at 2000 rpm for 20 minutes. This condition is ideal to create a density gradient so that we can separate out the lymphocytes. 4 layers are obtained: bottom – red cells, Ficoll-Paque, lympocytes and monocytes, and plasma – top. Discard the plasma and carefully transfer the lymphocytes and monocytes layer into a clean tube without taking so much Ficoll-Paque using Pasteur pipette. Wash 3X this layer with cold PBS for purification.
Next, we want to harvest T cells. Why T cells? This is because HLA-B27 is categorised as HLA Class I molecule and we don’t want B cells that have both Class I and II molecules. We will use the magnetic particle concentrator (MPC), where we add the Class I beads (they are actually monoclonal antibodies specific against Class I antigens). This will positively select the T cells. We will dilute the cells to the optimal concentration with prepared solution that contains Hank’s balance salt solution (HBSS), 2% fetal calf serum (FCS) and acridine orange (AO).
Next, we will perform the complement-dependent cytotoxicity (CDC) assay. Before doing so, we will check if we got the optimum cell concentration and cell viability (graded with score of 1 i.e. 0-10% dead cell) by adding 2μl of cells to an empty well of a microtiter plate and 2μl of Propodium Iodine (PI). This will stain the cells under the fluorescent microscope. Green means cells are alive while red means cells are dead.
We use a 60-well commercial microtiter plate. The plate has 1μl of known antisera with mineral oil layer on top to protect the antisera from evaporation. We will add 2μl of the patient T cells and incubate at room temperature for 40-50 minutes. Then we add 5-6μl of rabbit complement to each well and incubate for 60 minutes in the dark (we just put the plate in a metal plate with a lid). After that we add 2μl of PI in 5% EDTA solution to each well for 15 minutes at room temperature. This is for staining and to also stop the cytotoxic reaction. Lastly, we will remove excess fluid and add paraffin oil to prevent evaporation when viewing under the fluorescent microscope (10X objective). If the 81-100% of the cells are dead (i.e. graded with a score of 8), the cells are strongly positive for the HLA-B27 antigen.
Indah.
0705361D
Friday, August 14, 2009
Bone Marrow Harvest
Hello everyone ! I’m Justin here again. Sorry for my super late posting. Hope all of you are enjoying your SIP so far =) Today I’ll talk about the harvesting process in more detail, covering the protocol, as well as some stuff to note.
As mentioned before in my previous post, harvesting is the processing step to obtain metaphases for analysis. The 3steps involved are mitotic arrest, hypotonic treatment, and fixation.
The cell cycle is arrested at metaphase by colcemid, which depolymerises tubulin, thus preventing spindle fiber formation. Although an increase in duration of exposure to colcemid causes more metaphases to be collected, a prolonged exposure to colcemid also causes chromosome contraction as an effect. This would result in shortened chromosomes, which are not suitable for analysis. Hence, the duration of colcemid exposure should be limited.
Hypotonic treatment causes water to enter the cell. 0.075M of potassium chloride is used. Cells would swell and increase in volume, thus causing chromosomes to spread out. The incubation at 37ºC will speed up the process. The duration of hypotonic treatment has to be controlled, as over-treatment will cause excess spreading of chromosomes and cell lysis.
The final step is fixation, using modified Carnoy’s fixative, which consists of methanol to acetic acid in the ratio of 3:1. The fixative has to be prepared fresh and refrigerated. This is because over time the fixative would absorb water from the surroundings, thus causing the fixative to lose its properties. Fixation hardens cells and makes them resistant to changes. Changing of the fixative (through centrifugation and removing the supernatant) also lyses red blood cells, providing a clear suspension.
In this protocol, the colcemid solution and potassium chloride are incorporated into 1 solution, known as the harvest media. 0.08µg/ml of colcemid is used. Cells are exposed to the harvest media for a duration of 30 minutes.
Method:
1.Obtain the cultures from the incubator (after their respective durations of incubation- recall first post on culture setup) and transfer the culture contents into a centrifuge tube. (This is done because the culture flasks cannot fit into the centrifuge) Centrifuge at 1500rpm for 10 minutes.
2.Remove the supernatant and add 10 ml of harvest medium to the cell pellet. (The harvest medium contains the colcemid and the hypotonic solution)
3.Incubate in a 37ºC incubator for 30 minutes.
4.After which, remove from the incubator and perform a pre-fix step by adding 2ml of fresh cold fixative. (The purpose of the pre-fix step is because cells are fragile after the 30 minutes exposure to the hypotonic solution. The fixative will harden the cells and prevent them from being destroyed by the force of centrifugation) Ensure that cell clumps are well dislodged by mixing well.
5.Centrifuge at 1200 rpm for 10 minutes.
6.Remove the supernatant and resuspend the cell pellet in 6 ml of cold fixative. (This is the fixation step) Ensure that cell clumps are well dislodged by mixing well.
7.Centrifuge at 1200 rpm for 10 minutes.
8.Remove the supernatant and resuspend the pellet in 4ml of cold fixative. (this is to change the fixative) If supernatant is still coloured, change the fixative (through centrifugation, removing the supernatant and re-suspending the pellet in 4ml of cold fixative) until it becomes clear.
9.Place the tubes in the refrigerator. (the next process would be slide making)
Per culture- 10 ml of harvest media
12 ml of fixative (2ml pre-fixation+6ml fixation+ 4ml changing of fixative)
That’s all for now. Take care everyone and enjoy SIP !
Ng Tze Yang Justin
0703747F
As mentioned before in my previous post, harvesting is the processing step to obtain metaphases for analysis. The 3steps involved are mitotic arrest, hypotonic treatment, and fixation.
The cell cycle is arrested at metaphase by colcemid, which depolymerises tubulin, thus preventing spindle fiber formation. Although an increase in duration of exposure to colcemid causes more metaphases to be collected, a prolonged exposure to colcemid also causes chromosome contraction as an effect. This would result in shortened chromosomes, which are not suitable for analysis. Hence, the duration of colcemid exposure should be limited.
Hypotonic treatment causes water to enter the cell. 0.075M of potassium chloride is used. Cells would swell and increase in volume, thus causing chromosomes to spread out. The incubation at 37ºC will speed up the process. The duration of hypotonic treatment has to be controlled, as over-treatment will cause excess spreading of chromosomes and cell lysis.
The final step is fixation, using modified Carnoy’s fixative, which consists of methanol to acetic acid in the ratio of 3:1. The fixative has to be prepared fresh and refrigerated. This is because over time the fixative would absorb water from the surroundings, thus causing the fixative to lose its properties. Fixation hardens cells and makes them resistant to changes. Changing of the fixative (through centrifugation and removing the supernatant) also lyses red blood cells, providing a clear suspension.
In this protocol, the colcemid solution and potassium chloride are incorporated into 1 solution, known as the harvest media. 0.08µg/ml of colcemid is used. Cells are exposed to the harvest media for a duration of 30 minutes.
Method:
1.Obtain the cultures from the incubator (after their respective durations of incubation- recall first post on culture setup) and transfer the culture contents into a centrifuge tube. (This is done because the culture flasks cannot fit into the centrifuge) Centrifuge at 1500rpm for 10 minutes.
2.Remove the supernatant and add 10 ml of harvest medium to the cell pellet. (The harvest medium contains the colcemid and the hypotonic solution)
3.Incubate in a 37ºC incubator for 30 minutes.
4.After which, remove from the incubator and perform a pre-fix step by adding 2ml of fresh cold fixative. (The purpose of the pre-fix step is because cells are fragile after the 30 minutes exposure to the hypotonic solution. The fixative will harden the cells and prevent them from being destroyed by the force of centrifugation) Ensure that cell clumps are well dislodged by mixing well.
5.Centrifuge at 1200 rpm for 10 minutes.
6.Remove the supernatant and resuspend the cell pellet in 6 ml of cold fixative. (This is the fixation step) Ensure that cell clumps are well dislodged by mixing well.
7.Centrifuge at 1200 rpm for 10 minutes.
8.Remove the supernatant and resuspend the pellet in 4ml of cold fixative. (this is to change the fixative) If supernatant is still coloured, change the fixative (through centrifugation, removing the supernatant and re-suspending the pellet in 4ml of cold fixative) until it becomes clear.
9.Place the tubes in the refrigerator. (the next process would be slide making)
Per culture- 10 ml of harvest media
12 ml of fixative (2ml pre-fixation+6ml fixation+ 4ml changing of fixative)
That’s all for now. Take care everyone and enjoy SIP !
Ng Tze Yang Justin
0703747F
Sunday, August 9, 2009
Immunology
Architect is the analyzer that is used to detect the presence of antigens, Abs and analytes in the sample. The principle that it uses is the chemiluminescent microparticle immunoassay (CMIA) technology.
When the sample is loaded into the analyzer, the barcode on the plain tube will be scanned for the analyzer to conduct the respective test that was requested.
When the sample is processed, microparticles (paramagnetic microparticles coated with capture molecules) will be dispensed into the reaction vessel (RV) containing the sample (i.e.: serum). It will then be vortexed and incubated to allow the reagents and the sample to interact. If the analyte is present in the sample, it will bind to the corresponding capture molecule on the microparticles forming immune complexes. After incubation and vortexing, a magnet will attract the bound paramagnetic microparticles to one side of the surface of the RV. It will then undergo a first wash to remove unbound materials.
Next, a chemiluminescent acridinium labelled conjugate will be added into the RV. Similarly, the mixture will be vortexed and incubated. This conjugate will bind to the immune complexes. It will then undergo a second wash to remove unbound materials.
A pre-trigger solution (hydrogen peroxide) is added. It is used to acidify the environment to prevent early release of energy (i.e.: light emission) by the acridinium, it also prevents clumping of the microparticles and it will split the acridinium dye from the conjugate that is bound to the microparticle complex.
A trigger solution is added. It will cause the acridinium to be oxidized due to the presence of peroxide and alkaline solution. Oxidation of acridinium will produce N-methylacridone and release of energy (i.e.: light emission). The CMIA optical system will then measure the chemiluminescent emission to quantitate the concentration of the analyte present in the sample.
An example of a test conducted using the above principle is the Syphillis TP (Treponema pallidum). Syphillis is caused by an infection by the TP bacteria. It can be transmitted congenitally or through sexual contact. The analyzer will determine the amount (if any) of anti-TP in the patient’s serum. This will determine the diagnosis of syphilis.
Once the sample is loaded onto the analyzer, the patient’s serum, microparticles coated with recombinant TP Ag and assay diluents are aspirated and dispensed into the RV. Anti-TP present in the patient’s sample will bind to the TP Ag coated microparticle. After vortexing, incubating and washing the mixture, acridinium-labelled anti-human IgG and IgM conjugate is added. After a second vortexing, incubating and washing the mixture, the pre-trigger and trigger solutions are added to the RV. The resulting chemiluminescent reaction is measured using relative light units (RLUs). The higher the RLUs, the higher the amount of anti-TP measured.
Liyana
(0703827F)
When the sample is loaded into the analyzer, the barcode on the plain tube will be scanned for the analyzer to conduct the respective test that was requested.
When the sample is processed, microparticles (paramagnetic microparticles coated with capture molecules) will be dispensed into the reaction vessel (RV) containing the sample (i.e.: serum). It will then be vortexed and incubated to allow the reagents and the sample to interact. If the analyte is present in the sample, it will bind to the corresponding capture molecule on the microparticles forming immune complexes. After incubation and vortexing, a magnet will attract the bound paramagnetic microparticles to one side of the surface of the RV. It will then undergo a first wash to remove unbound materials.
Next, a chemiluminescent acridinium labelled conjugate will be added into the RV. Similarly, the mixture will be vortexed and incubated. This conjugate will bind to the immune complexes. It will then undergo a second wash to remove unbound materials.
A pre-trigger solution (hydrogen peroxide) is added. It is used to acidify the environment to prevent early release of energy (i.e.: light emission) by the acridinium, it also prevents clumping of the microparticles and it will split the acridinium dye from the conjugate that is bound to the microparticle complex.
A trigger solution is added. It will cause the acridinium to be oxidized due to the presence of peroxide and alkaline solution. Oxidation of acridinium will produce N-methylacridone and release of energy (i.e.: light emission). The CMIA optical system will then measure the chemiluminescent emission to quantitate the concentration of the analyte present in the sample.
An example of a test conducted using the above principle is the Syphillis TP (Treponema pallidum). Syphillis is caused by an infection by the TP bacteria. It can be transmitted congenitally or through sexual contact. The analyzer will determine the amount (if any) of anti-TP in the patient’s serum. This will determine the diagnosis of syphilis.
Once the sample is loaded onto the analyzer, the patient’s serum, microparticles coated with recombinant TP Ag and assay diluents are aspirated and dispensed into the RV. Anti-TP present in the patient’s sample will bind to the TP Ag coated microparticle. After vortexing, incubating and washing the mixture, acridinium-labelled anti-human IgG and IgM conjugate is added. After a second vortexing, incubating and washing the mixture, the pre-trigger and trigger solutions are added to the RV. The resulting chemiluminescent reaction is measured using relative light units (RLUs). The higher the RLUs, the higher the amount of anti-TP measured.
Liyana
(0703827F)
Sunday, August 2, 2009
Sorry for the late posting... Starting end of July to end of August, I will be attach to the microbiology department of the laboratory. In the microbiology lab, the specimens that we handled are usually stools, blood tubes and blood culture bottles. Reading the agar plates, antimicrobial sensitivity testing and identification of the pathogenic organisms are done by my supervisor, as he is much more experience and is able to determine which of the colonies on the agar plates are pathogenic almost immediately.
So for me, I'm doing the serology testing in the micro laboratory such as Venereal Disease Research Laboratory (VDRL) test and Treponema Pallidum Haemagglutination (TPHA) test which are syphilis tests that test for the presence of the antibody that is produced in the presence of Treponema Pallidum. Although both of the tests are syphilis test, VDRL is non-specific as the antibody produced with Treponema Pallidum infection that it tests for can also be found when the patient is not suffering for syphilis but other diseases such as hepatitis A and malaria. TPHA is used as a confirmation tests (unless the doctor ordered the test) for syphilis. TPHA uses cells sensitized with Treponema Pallidum specific antigen, hence any agglutination observed would mean a positive that patient is tested positive for syphilis.
Steps involved for VDRL:
1. A drop of serum is taken and spread evenly on the test card.
2. A drop of reagin is added to the serum. (Remove the first few drops to remove the bubbles trapped to prevent uneven volume of reagin added)
3. Test card is placed on an electronic rotator for 8 minutes with approximately two rotations per minute.
4. Test result is observed and recorded.
If agglutination or reaction is observed, proceed to serial dilution to determine the titre:
1. 50µl of saline is placed on 4 different test areas on the test card.
2. 50µl of serum is added to the first test area labelled 1:2. Mix well by pipetting up and down.
3. 50µl of serum+saline mixture is transfered from the first test area to the next labelled 1:4. Mix well by pipetting up and down. Continue till the fourth well and discard the remaining 50µl of mixture.
4. A drop of reagin is added to the different test areas.
5. Test card is placed on an electronic rotator for 8 minutes with approximately two rotations per minute.
6. Test result is observed and recorded.
The titre is taken as the lowest dilution with reaction (agglutination). Once the titre is obtained. TPHA will be performed to confirm the result if the patient is doing a package (a series of test). If the patient is not doing a package, the confirmation will only be done if the doctor order for the test.
Since VDRL is testing for the non-specific antibody for Treponema Pallidum, it is not specific and cannot be used clinically to determine whether patient is suffering form syphilis without a confirmation test.
Hui Juan
TG01
0702012F
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