Monday, September 24, 2007
Principle of Test: The Anti-HCV assay is a two-step immunoassay, using Chemiluminescent Microparticle Immunoassay (CMIA) technology for the qualitative detection of anti-HCV in human serum and plasma. In the first step, patient sample, HCV coated microparticles and assay diluent are combined. Anti-HCV that is present in the sample binds to the HCV coated microparticles. After washing, anti-human acridinium-labeled conjugate is added in the second step. After washing again, Pre-trigger and trigger solutions are added to the mixture. The resulting chemiluminescent reaction is measured in relative light unit. The amount of anti-HCV in the sample is proportional to the relative light unit measured.
Test Result with reference range: Specimens with cut-off values <1.00 are considered nonreactive by the Anti-HCV assay and need not be tested further.
Specimens with cut-off value >/= 1.00 are considered reactive by the Anti-HCV assay.
Clinical interpretation: A positive anti-HCV test provides evidence of exposure to HCV. Specific antibodies against HCV may not always be detectable in early acute disease because antibody production is usually not detected until 7 to 31 weeks after infection. However, antibody detection doesn't differentiate between acute, chronic or past HSV infection. For diagnostic purposes, anti-HCV result should be used together with patient history. If Anti-HCV results are inconsistent with clincial evidence, further test would have to performed. Additional information can be obtained by testing for viral RNA in serum by PCR. A positive HCV RNA test is suggestive of viral replication in the liver and can confirm a diagnosis of either acute or chronic infection.
Yong Yang (TG02)
Monday, September 17, 2007
G6PD is the most common enzyme deficiency in the world, affecting approximately 400 million people. It is an X-linked inherited disorder which affects Africans, Asians, Mediterraneans and people of Middle-Eastern descent.
G6PD catalyzes NADP to its reduced form, NADPH, through the pentose phosphate pathway, which prevents oxidative damage to cells.
Diagnosis of G6PD deficiency is made through 2 ways, a rapid fluorescence spot test (screening), or a quantitative spectrophotometric analysis.
I will elaborate more on the screening test in the lab I am working at. In Singapore, all newborns are screened for G6PD deficiency.
Glucose-6 Phosphate Dehydrogenase (G6PD) screening test
Procedure for G6PD screening
1. Pipette 100µL of lysing agent into the wells of a titration plate. The first 4 wells are for the 3 levels of controls (Deficient, Intermediate and Normal) and for a blank. Patient samples are placed in rows of 4s below the controls.
2. Blood in the EDTA tube is mixed by gently inverting the tube several times.
3. Pipette 5µL of whole blood/controls into the wells and shake the plate to mix the solution.
4. Incubate at room temperature for 10 minutes for complete lysis of the RBCs to release the enzyme.
5. Using a stick, take 2 drops of reaction mix and spot on to the absorbent filter paper.
6. Dry the spots completely for about 3 minutes with the aid of a hair-dryer.
7. View the spots under long-wave (365nm) UV light. Deficient samples will not show any fluorescence. Intermediate samples will show little or some fluorescence. Normal samples will fluoresce brightly.
8. The screening test for G6PD is based on the following principle:
Glucose-6-Phosphate + NADP+ --(G6PD)--> Gluconate-6-phosphate + NADPH + H+
The NADPH produced in the reaction fluoresces under long-wave UV light (365nm). No fluorescence will be observed if there is marked deficiency or lack of the enzyme G6PD. This screening test might be tricky at times, especially when there is little fluorescence. Some people may think it is deficient, while others think it is normal (i.e very subjective to which level of enzyme activity). Thus, if a technician is unsure of the result, he/she will consult another technician to confirm the result.
Ng Wei Shen Martin
Sunday, September 9, 2007
WBC Impedance Count (WIC)
WBC Optical Count (WOC)
Mean Platelet Volume (MPV) : measurement of the average size of your platelets. New platelets are larger, and an increased MPV occurs when increased numbers of platelets are being produced. MPV gives information about platelet production in your bone marrow.
WBC Optical Count (WOC) and WBC Impedance Count (WIC) are just 2 different methods used by the analyzer to assure correct leukocyte count.
The Reagents involved:
Dilute samples so the analyzer can measure the various components. It also rinses aspiration probes to reduce carry-over from the previous test.
-Rapidly lyse RBCs
-Strip white cells cytoplasm leaving the nucleus membrane intact so the white cells’ nuclei can be enumerated
-converts haemaglobin to a single chromogen that is measurable at 540nm
-osmotically lyse RBCs
-maintain light scattering properties of WOC during the measuring period
- prevent accumulation of air bubble in WOC flow system.
-removes protein build-up within the analyzer
Blood can be collected via venipuncture or finger-prick and sent to the lab in EDTA tubes (big purple tube and small pink tubes). After the blood has been checked for clots and is well-mixed, it is loaded into the analyzer. Clotted blood tubes are rejected as they cause low platelet count.
CD 3500 is able to aspirate blood in 2 modes- OPEN and CLOSE. Open mode involves manual positioning of blood tube to the aspirating probe. In the case of Close mode, the blood tubes just need to be place in the cartridge and the probe will automatically mix and aspirate blood.
After blood is aspirated into the Shear Valve (inside the analyzer), the valve will cut the blood into different segment for various testing.
Blood will be diluted and mixed with lysis solution. The diluted sample passes through a thin aperture where electrical impedance is used to count the WBC as they transverse through the aperture.
Blood is diluted and transferred to the WOC flow cell by the peristaltic pump. A laser beam is focused on the flow cell, as the sample stream (one cell at a time) intersects the laser beam, the light scattered by the cells is measured.
RBCs and Platelets
Blood is diluted 10,812 times. Electrical impedance is used to count RBCs and platelets as they transverse through the aperture.
Hb concentration is measured spectrophotometrically. Lysis solution converts Hb to hemiglobinhydrozylamine complex. Light-emitting didode (LED) with a wavelength of 540nm determines the amount of Hb, by capturing the light emitted, by a photodetector.
The rest of the parameters are reported based on the calculation automatically done by the analyzer.
Sunday, September 2, 2007
Receiving of Specimen
Check that the name, identification number of the form matches that of the bottles or the bags
Once every item received tallies with the request form, sign your name, stamp the time and write the date.
Sort the specimens out according to type, some specimens such as placenta require trimming, and must be put into a special box.
Scribing of gross description
Samples such as product of conception (POC), Breast biopsies and endometrial samplings have to go through this.
For POC, the information to be written down on the form are:
-The amount of sample used in milliliters
-If there is no more sample left after putting into a cassette, NR or No Reserve is written on the -form.
-The number of cassettes used is also written down
For breast biopsy, the information to be written down on the form are:
-The number of strips received
-The length, Breadth and width of strips
-The number of cassettes used is also written down
-The number of strips in each cassette is written down
Since the histopathology laboratory has to generate many blocks of tissue a day,
In order to differentiate them, different specimens have different colours.
-For paediatrics specimens, a yellow cassette is used
-For adult specimens, a green cassette is used
-For urgent specimens, a purple cassette is used
-For Post Mortem cases, a pink casstte is used.
The cassettes containing the tissues are put into the Shandon Excelsior tissue processor,
It is an automated processor that removes the job of having someone to manually do the tissue processing process which takes about 18 hours. With the Shandon Excelsior tissue processor, the histopathology laboratory does not need to be a 24-hour laboratory, all the medical technologists have to do is to put in the cassettes at the end of the day and allow it to run overnight
Processes that the samples undergo during tissue processing:
-A process that fixes the tissue samples in their current state so that the tissues can be studied
-Fixation is done with Formalin
-A process done for the removal of water
-Dehydration is done usually with alcohol, although other laboratories have been known to use alcoholic variants
-Clearing agents used in the process must be miscible with both the dehydrating agent and the infiltration medium
-Clearing is done, to remove the dehydrating agents from the tissue and to prepare the tissue for addition of infiltrating medium
-Clearing is done with Xylene
-done with paraffin wax
-done to remove the clearing medium from the tissue and to prepare the tissue sample for embedding
-The embedding process is done with the aid of the machine Sakura Tissue-Tek
-Prepare 3 different kinds of forceps for aiding of the tissue embedding process
-Soak them in in melted paraffin wax
-On the left panel of the tissue embedder, molds of different sizes are kept there and ready for use for tissue samples of different sizes
-The right panel is used to put the cassettes after they have been removed from the tissue processing machine
-Once the tissue has been removed from the cassette, it is placed into the the mold of an appropriate size
-Fill the mold with wax and use the forcep to press down the tissue, to ensure that the tissue sample is level. This ensures that shaving and sectioning would be easier
-Once the tissue is leveled, attach the base of the cassette to the top of the mold and fill up with more wax
-A piece of colour coded paper is embedded into the wax before it cools, this is done for tracebility purposes.
-The molds are then placed in ice to cool down
-Do not force the cassettes out of the mold, but instead wait for the paraffin wax to contract and become loose on its own
-In the cases where the cassette is still stuck on the mold after a period of time use abit of force but remember to place the tissue block back on the ice
-The tissue blocks are shaved at 10 microns, to expose the full surface of the tissue to make sectioning easier
-It makes sectioning easier, as the person doing sectioning requires less shaving of the tissue block
-When shaving the tissue block, always check how much of the tissue has been exposed, as if too much is shaved off some parts of the tissue may be lost
-Some tissue samples such as fibroid are hard to cut as they contain calcium deposits, therefore they must be placed in softerner for 5 mins to soften the tissue again to make tissue sectioning easier
-After the tissue blocks have been softened, wash them under running water until the soapy feel is removed
-Dry the blocks before placing them on a cooler set at between –10 to –20 degrees, this makes sectioning easier
-Sectioning is done with either the leica or sakura manufactured rotary microtomes
-After the tissue blocks have been placed on the cooler for a period of time, they can be ready for cutting
-Set the microtome to cut at 4 microns
-After the appropriate section has been obtained, transfer it from the microtome to a water bath
-Fish the section onto a clean glass slide, before transferring it to another water bath
-The section is then fished back onto the same slide and left to dry off
-After the slides have been dried, they are put into racks to prepare for staining by baking in the oven at around 80°c for about 15 minutes
Routine H & E staining
-In the laboratory, routine Haematoxylin & Eosin ( H&E) staining is done on an automated machine the leica ST5010
-All we have to do is insert the rack of prepared slides (each rack can accommodate 30 slides) into the machine, ensure that the correct program is selected and then press load.
The machine has 16 different stations, and starts of with:
Xylene (removes wax)
Absolute alcohol (hydration)
95% alcohol (hydration)
Haematoxylin (staining of the nucleus)
Once the process has been completed, the machine will sound alerting the user
Attaching of coverslip
Once the slides have been removed from the autostainer, they will be transferred into the Leica CV 5030, a fully automated glass coverslipper.
The machine individually applies Depex onto each glass slide before covering it with a cover slip.
Once the process is done, the machine will also alert the user that the process has been completed.
The slides are then sorted according to case number, and compared to the original tissue blocks. If overlapping or folding of the tissue is observed, the tissue block would have to be recut to obtain a new slide.
Once the slides have been double checked and confirmed as okay, the appropriate labels are stuck on to them before the cases are dispatched to their respective pathologists to read the slides.
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