Finger Prick

I’ve been really busy working overtime for weeks due to the shift I’m assigned to and since I’m having half day on my second last day of official work, I shall clear all my overdue posts. Once again, I won’t be sharing about the routine work I do in the lab since everybody is doing so. I will be sharing about the things I do but not in the lab and within the usual job scope. For the place that I’m attached to, we’ve outreach program. For the one that I was asked to go, a finger prick to test for cholesterol and glucose level was required to be done by me.

Things needed:

1. Lancet

2. Cholesterol meters

3. Glucose meters

4. Cholesterol test strips

5. Glucose test strips

6. Alcohol swabs

7. Cotton wool

Steps involved:

1. Turn on the glucose meter then the cholesterol meter.

2. Insert the respective test strips into the respective meters.

3. Swab the patient’s finger (to be pricked) with alcohol swab

4. Feel the thickness of the patient’s skin and adjust the depth of the lancet accordingly

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Taken from: http://www.poc.roche.com/poc/rewrite/generalContent/en_US/article/POC_general_article_37.htm

5. Prick the finger and clean off the first drop of blood.

6. Cover the test area completely with blood (first cholesterol then glucose) and wait for results.

I will only be sharing in details about the Cobas Accutrend Cholesterol test. It is used to determine cholesterol in fresh capillary blood in diabetics and non diabetics, for self monitoring, for early detection of a risk of astherosclerosis, for monitoring treatment with lipid-lowering drugs and for screening purposes. Cholesterol esters are cleaved by enzymes into fatty acids and cholesterol, oxidation of cholesterol to cholestenone with the simultaneous formation of hydrogen peroxide, which oxidizes an indicator to its blue radical cation.

Whenever a new pack of Accutrend Cholesterol strips is opened, the meter must be coded. This is done by inserting the code strip into the meter. After which, a QC is done using the Accutrend Control CH1 control solution to check if the meter and test strips are fit for use. When blood is applied, a chemical reaction takes place and the test area changes colour. The meter records this change in colour and converts the measurement signal to the displayed result using the data previously entered via the code strip. Normal cholesterol values are less than 5.2 mmol/L. The measuring range for this meter that I’m using is 3.88 – 7.76 mmol/L. Cholesterol determination may be affected by intravenous infusion of ascorbic acid, bilirubin values greater than 10mg/dL, hematocrit values exceeding 55%, methylaminoantipyrine and gentisic acid.

Enjoy!

Michelle

TG02

0703478H

ABO Blood Grouping

ABO Blood Group

ABO Blood Grouping is a common test that we have done in our school. However, it is slightly different in the hospital.

ABO Blood Grouping is done to determine the Blood Group of a person using Antibody-Antigen reaction. It has two different parts, one Forward grouping and the other Reverse Grouping. Forward grouping is the reaction between Commercial Serum/Antibody and Patient’s Diluted Blood Antigen. Reverse Grouping is the reaction between Commercial Red Blood Antigen and Patient’s Serum/Antibody.

The tubes are then centrifuged and are checked for agglutination. If there’s agglutination, it shows that there is Ag-Ab reaction. For example, under forward grouping for α tube, only have agglutination; it means that the person has A antigens from the red blood cell. Thus there might be a possibility that the person is Blood Group A. Reverse Grouping is then used to check, thus if there is agglutination in tube B, it shows that the patient has anti-B. This can confirm that the patient is Blood group A since the patient cannot have anti-A if not it will cause transfusion reaction.

Adult ABO Blood Group Procedure

· Print out ABO Blood Group worksheet from the Laboratory Information System using the test code 420500

· Collect the EDTA blood tube and ensure the name and sample number matches with the worksheet printed before proceeding to the test

· Prepare 9 glass/plastic tubes

· Label 4 tubes α, β, αβ and anti D for forward grouping

· Label 3 tubes A, B and O for reverse grouping

· Pour about 1-2ml of Blood into a new tube

· Centrifuge the tube for 5 minutes to collect serum

· Pour about 1-2ml of saline solution into a new tube, prepare blood suspension by pipetting
blood into the saline, and ensure that the blood suspension has about the same colour as the
commercial Red Cell Antigen.

· Pipette 1 drop of Commercial Serum into tubes α, β, αβ and anti D

· Pipette 1 drop of Commercial Red Cell Antigen into tubes, A, B and O

· Pipette 1 drop of Blood Suspension into tubes α, β, αβ and anti D

· Pipette 2 drops of Serum into tubes A, B and O

· Place all 7 tubes into the centrifuge machine and centrifuge for 15 s

· Take all the tubes out and check for agglutination

· Record the Blood Group of Patient

· Ask another Medical Technologist to verify the results you recorded


Baby ABO Blood Group Procedure

Baby ABO Blood Group Procedure is about the same as the adult one. Only that, forward grouping is done only. Reverse grouping is not done, this is because baby do not produce antibodies at young age. So even serum is retrieved by centrifuging, there are no antibodies.

· Print out ABO Blood Group worksheet from the Laboratory Information System using the test code 420500C

· Collect the EDTA blood tube and ensure the name and sample number matches with the worksheet printed before proceeding to the test

· Prepare 5 glass/plastic tubes

· Label 4 tubes α, β, αβ and anti D for forward grouping

· Pour 1-2ml of blood into a new plastic/glass tube

· Wash 3 times with saline solution with each sending for centrifugation

· Each centrifugation is to be 1 minute at 1006g

· Prepare blood suspension and ensure it is of the same tone/colour as the commercial Red Cell Antigen

· Pipette 1 drop of Commercial Serum into tubes α, β, αβ and anti D

· Pipette 1 drop of Blood Suspension into tubes α, β, αβ and anti D

· Place all 4 tubes into the centrifuge machine and centrifuge for 15 s

· Take all the tubes out and check for agglutination

· Record the Blood Group of Patient

· Ask another Medical Technologist to verify the results you recorded

Take note of the blood group, if the results are either AB blood group or Rhesus negative for any blood groups. There is a need to request for baby’s own blood to determine and reconfirm its blood group. As the test is done by using Baby’s Cord Blood, which may have a possibility that the blood of the mother got mixed with Baby’s cord blood, thus giving a discrepancy in the result.

Thats all for me!

Jordan Wong Wei Jie
TG02 0703992H
Grp 09

Bone Marrow Aspiration

Hi all! I won't be sharing about the usual routine things that I do in the laboratory. Instead, I'll be sharing about how to prepare the bone marrow slides as I had the chance to witness a bone marrow aspirate procedure!

First of all, after the lab has been informed about the appointment made, one of the medical technologists (usually the haematologist) will be sent over to the ward to prepare the bone marrow slides. The physician would perform the bone marrow puncture and biopsy using aseptic technique and this is the kit they use:

As for the procedures for bone marrow aspiration, I’ll let the pictures do most of the talking. If you’re interested to know more about it, click on this link: http://www.pathology.vcu.edu/education/lymph/How%20to%20Marrow.pdf

Preparation of bone marrow on slides

Bone Marrow examination is important in the study of haemopoietic disorders and may be the only way to make a correct diagnosis. Comprehensive bone marrow morphology requires the examination of the bone marrow particles, push preparations, trephine biopsy imprints, trephine biopsy and correlation with peripheral blood picture. Valid interpretation requires consistent preparation of these samples.

The reagents and equipments needed are 20 microscope slides, at least 2 good spreaders, gauze, plastic plates for isolation of bone marrow particles, glass marker pen, disposable transfer pipette, EDTA tubes and slides holder.

The steps involve are:
1) Obtaining Specimen
- Check patient's name ID
- Ask for EDTA blood for peripheral blood smears ( at least 2) before the bone marrow aspiration procedure
- Transfer aspirate (0.5 ml) into EDTA tube as quickly as possible. Mix gently but thoroughly. Recollect the specimen if there are no fragments.
- If trephine biopsy specimen is also taken, make imprints by gently rolling specimen (at least 1 cm) between 2 microscope slides. Make at least 3-4 imprint smears.

- Transfer biopsy specimen into a bottle of formalin.
- Label all smears and EDTA tubes wit patient's ID, accession number and date.

2) On return to laboratory
- Expel the aspirate onto a plastic plate. Hold the plate at an angle so that fluid will run down the plate leaving bone marrow particles isolated.
- Transfer suitable materials onto microscopic slides and make smears as quickly as possible. At least 12 slides are required. Make extra smears if the patient is a suspected case of leukemia.

- Label all specimens and smears
- Patient's original request form, slides holder containing 10 marrow slides and 2 blood smears, EDTA tube of excess marrow and EDTA blood are all placed into a dispatched box and acknowledgement slip to be sent to NUH haematology laboratory and dispatch trephine biopsy specimen to NUH histology department.

Bone marrow aspiration is a really painful process. Hence, the physician doing the procedure must be trained and skilful in it so that the patient does not need to go through unnecessary amount of puncture before the physician succeed. Furthermore, the medical technologist preparing the bone marrow slides must know the procedures and criteria well so as to be able to produce satisfactory slides for proper evaluation. This is to prevent the slides being rejected by NUH and patient undergoing a second bone marrow aspiration.

All pictures are taken from: Riley, R. S. (2009, October). An Illustrated Guide to Performing the Bone Marrow Aspiration and Biopsy. Richmond, Virginia, United States of America.

That’s all folks!

Michelle

TG02

0703478H

Laboratory Techniques - In vitro organ bath experiment

HELLO PEOPLE! THIS IS ADDITIONAL POST FROM RENEE!!

hahaha! just feel like sharing another skill that i have learnt during my attachment - that is ORGAN BATH!

In-vitro organ bath is actually a set-up for us to conduct experiments on muscle strips. The muscle strips are either obtained from humans or laboratory animals and placed into the organ baths for isometric or isotonic measurement.

Isotonic measurement would estimate the variation in length of the muscle tissue without any change in tension, while isometric measurement would study the tension that is accumulated within muscle tissues without any visible change in the length of the tissue.

In my experiment, we take the isometric measurement of the corpus cavernosum smooth muscle under the influence of my test drug. We do this because a relaxation in the corpus cavernosum smooth muscle would mean erection, and therefore, we want to see if the drug actually causes decreased tension in the corpus cavernosum.

What i did was to pre-contract the corpus cavernosum strips with noradrenaline, which is a drug that would cause increase tension in the corpus cavernosum. After that, i add in cumulative concentrations of the drug that i am testing to see if there is a decrease in tension.

What i hypothesize to get would be concentration dependent decrease in corpus cavernosum tension, so that i would be able to get a dose response curve.



Picture retrieved from : http://www.currentprotocols.com/protocol/ph0403

This is the schematic diagram of an organ bath. The tissue is tied with a thread and mounted onto the organ bath. Tissue is bathed in tyrode solution and bubbled with oxygen and carbon dioxide. Drugs are added into the solution.

Thats all folks.
feel free to ask me questions: ))

Love
Renee
TG02
0703634F

Colony Counting Assay

Colony counting assay also determines the toxicity of the anti-cancer therapy, but in this case it determines if the cells will grow after the cells have been treated. So the cancer cells will first be seeded on 24 well plates and incubated overnight. The next day, the cells are treated with the respective treatments and incubated. After treatment, the cells are trypsinised and replated on a new 6 well plate at a seeding density of 2000 cells per well. The cells are then maintained for 2-3 weeks with the media replaced every three to four days. At the end of the assay, the cells are stained with crystal violet for 1 hour. Lastly, the colonies are counted and imaged using a stereomicroscope (Nikon, U.S.A.) at 10× magnification, in five microscopic fields per well (one central, four peripheral).



Above is an example on how the plate will look like after the colonies have been stained. The first one would most likely be the control , hence many colonies can be seen. The last one would be the drug polymer and TRAIL, a ligand that binds to the
TRAIL receptors found on cancer cells, to induce apoptosis.

Gwendolynn Tan
TG02
0703953J

Occult Blood (OB) Test

Hi!Terribly Sorry for the delay

I'm Jordan Wong from TG02 and will be posting on a post on "Occult Blood Test"

OB Test

OB Test refers to Occult Blood Test. Occult Blood Test is done usually to determine if there is any hidden (occult) blood in the stool/faeces. It helps us to find out whether if a particular person is suffering from any colorectal cancer or gastrointestinal bleeding (from mouth all the way to colon).

In order to perform the OB Test, stool samples from patients are collected as specimen and stored in an OB cassette, which will then be sent for processing using the Occult Blood Test Analyzer, Diana OC Sensor by Eiken.

1. Quality Control

As usual, for all analyzers or machines that are used for running and processing samples, there is a need to run/perform Quality Control before the samples are sent for running. This is to ensure that the results/values the machine or analyzer is producing is precise and accurate. If not the results of samples may not be accurate, which may caused an analytical variation. Therefore Quality Control is an important procedure for all machines and analyzers in the laboratory.

Performing of Quality Control

• Prepare 4 Hitachi Cups for QC High and 4 Hitachi Cups for QC low

• Label the date (for example 24/07) and QC High/Low on the Hitachi Cups

• Take out the QC reagent from the fridge and let it cool to room temperature for 15 – 30 minutes

• Ensure and check that the Lot number of the QC reagent and the Lot number noted on the machine is the same before continuing

• Using the blue pipette, calibrate it to exactly 1000ul (1ml)

• Using the blue pipette, pipette exactly 1ml of deionised water into each QC reagent bottle

• To ensure well-mixed, Spin the QC reagent bottle for 15 – 30 minutes using the Nutating Mixer by GyroMini

• Pipette 200ul of QC reagent into each Hitachi cup (Total 4 cups)

• Remaining 200ul, pipette into each Hitachi cup drop by drop, ensuring that all 4 Hitachi cups has the same amount of well mixed QC reagent

• Place the Hitachi Cups for QC High and QC Low into the dark blue rack and send it for machine to run. [Dark Blue racks are meant for Quality Control]

• Wait for results to come out and ensure that the values produced for QC High and QC Low is with-in the reference range given by a standard/company

2. Preparation of Samples

For OB Test in the laboratory, samples sent come in two types. Some come in OB cassettes while some come in stool bottles. As usual, samples that are sent were being signed, opened and registered. The samples will then be labelled, for OB cassettes; the label sticker must be labelled with the barcode facing the middle so that the sensor in the OB machine is able to read/scan the barcode into the system. OB cassettes has stool specimen in it, so the cassettes can be sent for processing right away.
However, some patients will send in Stool Bottle, where there might be other stool tests to be done other than Occult Blood (OB). Other stool tests may include Stool Culture and Stool FEME, thus a stool bottle containing the stool will be sent. Once we receive stool bottles from the admin department, we will go into LIS Edit Lab Request to check for the pending tests the patient enquired, if there is a pending test for Occult Blood, then there is a need to prepare OB cassette from the stool bottle.
As I said, in order to run Occult Blood for the ones with stool bottle, we will have to place the stool specimen from the bottle into the OB cassettes. This procedure will usually be performed in the safety cabinet at microbiology department.

• Label the OB cassette with the patient’s lab number

• Remove the cover of the OB cassette

• Place it into/onto the Stool Bottle, to retrieve some stool specimen

• Cover back the cover of the OB cassette

• Proceed to the Admin to print the correct label before the cassette is sent for processing

• During printing of label, enter the patient’s lab number, the code of the specimen, in this it will be 30 and click print

• Label the cassette properly, ensuring that the barcode is labelled on the middle of the OB cassette.

• OB cassette is ready for processing.

There are times where the stool cannot be scooped by the spoon of the stool bottle into the OB cassette, therefore we were taught to use ice cream sticks to retrieve some stool specimen and place it into the OB cassette.

3. Merge Stool and OB cassettes

There are times where stool samples or OB cassettes do not come together on the day itself with other samples in the package. Thus these samples will arrive to the lab the next day, a few days later or even months later; we call these samples as merge samples. These merge samples come with a merge form. On the merge form itself, they will tick the type of sample. For example, merge urine is sent to the lab; the merge form will record and tick that urine is being sent. So upon receiving such merge samples, we will help merge the samples into the LIS system.

I. Ensure the samples received matches with the record on the merge form (A tick on the urine means only urine sample is sent)

II. In the LIS system, go to edit Lab Results, a screen will open; enter either the name or the IC number of the patient into the LIS.

III. A new window will open, now search for the recent package the patient has recently registered, record down the lab number of the patient.

IV. Now go to edit lab request, enter the lab number of the patient. Next, under cancel test and test info, ensure that the package has the test where the respective sample was sent. (Let’s say Urine Bottle was sent, ensure that the package includes urine test in the system)

V. If it correct, record the lab number on the merge form (0909090123) and also record down the day the sample was received.

VI. Under specimen information in edit lab request, enter the code for the specimen to register the samples into the system. (Urine Bottle = 23, Stool Bottle = 30, OB cassette = 31 & 32, Plain tube = 01, EDTA tube = 02 and Pap smear = 09)

VII. Ensuring what you merged was correct in the system, click saved. Next, go to print the label by entering the lab number and the respective code for the specimen.

VIII. Paste the label on the sample, highlight the lab number on the label and sent it to the respective department to run/process.

4. Running of Samples

All OB cassettes and Stool Bottles will be sent to the Haematology Department and be placed into a yellow rack. In order to process/run the cassettes, we will place all OB cassettes into a light blue rack. Each light blue rack consists of 10 slots, which enable to hold 10 OB cassettes and a rack number is provided. [Light Blue racks are meant for processing/running of OB samples]

While slotting the OB cassettes into the test rack, we have to ensure that the barcode label of the OB cassette is in the middle. We also need to ensure that all papers or sticker must be removed from the OB such that it will not cause a hindrance when the OB is being processed. After ensuring that the OB cassettes are properly placed into the test rack, placed the rack onto the machine, aligning with the machine tray. Click set sample for the machine to run the OB cassettes.

5. Verifying of Results

Once all the samples are processed/run, the light blue racks will be shifted to the left hand side of the machine, result slip will be printed out from the top. Then, a screen will appear saying analysis is done. The result slip consists of several information such as rack number and position, results of the OB sample, lab number of the patient and the number of OB cassette being processed. Upon completion of the analysis by the OB machine, we can then verify the results of the patient.

For verifying of OB results, we can verify in two ways, the first one is by Lab number and the other way is by Test Code. By verifying through Lab Number, enter the lab number of the patient, click under the logical column, under the Occult Blood row, and enter the results of the patient. 1 represent for Negative and 2 represent for Positive. In addition, for all positive cases, we will have to enter the value of the results in ng/mL under the memo corner. Lastly, click verify changes for whatever changes that was done and click save. After all those procedures as described, the patient’s results for OB were verified.

To verify using the Test Code method, we will open 2 LIS window, and enter two test codes, as Occult Blood has two test codes, 475700 and 475702. 475700 refer to Occult Blood while 475702 represent Immunological Faecal Occult Blood. Some samples are registered under 475700 test code while some are registered under the 476702 test code. Thus we open both to ensure that all registered OB samples appear in the system so as to verify them. However, using this method will hang the LIS system in the whole lab, therefore it is not often used unless necessary like when checking pending only. Verifying is usually done through lab number. After opening both test codes, once again enter 1 which represent for Negative and 2 which represent for Positive. In addition, for all positive cases, we will have to enter the value of the results in ng/mL under the memo corner. Clicks verify changes for whatever changes that was done and click save.

6. Closing of OB machine

Occult Blood Machine only run OB cassettes from morning till afternoon about 4pm only. After which, the machine will be closed. Before closing OB machine, we will usually check OB pending first where it serves to ensure/ to make sure that all OB cassettes that were registered are processed and verified or if it wasn’t processed then must ensure that the OB cassette is present for processing the following day. After checking OB pending, if there are no problems, then the OB machine can be closed.

• From the analysis screen, Press/Click close

• Wait till screen appears as Main Screen

• Press/Click Maintenance

• Press/Click Close Mode

• On the next screen, ensure that that it is Yes No Yes.

7. Checking Occult Blood Pending

Occult Blood Pending is done three times daily. Once in the morning, after running a batch of samples that was left during the previous day after the OB machine was closed. And another once more, in the afternoon, before the machine is closed at 4pm. Lastly, one more time of pending after all OB cassettes / Stool samples are merged and registered.

To check Occult Blood Pending, 2 LIS windows must be logged in. Then, open the two test codes for OB, 475700 (Occult Blood) and 475702 (Immunological Faecal Occult Blood). Wait till both test codes are completely opened. Then, look at both the screens; ensure that there are no samples listed in the screen. If there is, it is either not verified yet or the samples are just registered. Therefore, make sure that all OB results are verified. If samples are not process yet, make sure there are available for processing the following day.

As stated, after all OB cassettes / Stool samples are merged and registered. Open the OB pending for the third time. Using Microsoft Excel, print the OB pending (475700 and 475702) and ensure that the list of samples found in the pending matches with the number and the correct OB cassette(s) that were registered.



Transferring of Stool Specimen from Stool Bottle into OB cassette




Transferring of Stool Specimen from Bottle to OB cassette



This is a Stool Bottle : Used for Preparation of OB cassette specimen and other Stool Tests



These are all the require solutiones for Occult Blood Machine.

They are mainly Purified Water, Wash Solution and Drain (Waste)



This is our lab's Occult Blood Machine



10 OB cassettes slotting into 10 slots on the Light Blue Racks



This is a OB cassette



This is the Light Blue Racks for Patient's OB cassettes to be processed



Placing Stool from Stool Bottle into OB cassette



This is the Dark Blue Racks for running Quality Control in the OB machine before processing patients samples.



This is the Orange Racks used for running samples that requires Dilution in the OB machine.

Do you have any questions, do post it on our blog

This is all i have to share for today!

2 MORE DAYS!

Enjoy your final moments!

With Regards
Jordan Wong Wei Jie
TG02 0703992H
Group 9

Cell Cycle Analysis

Cell cycle analysis involves using flow cytometry so as to analyse the toxicity of the anti-cancer treatment through the number of cells in each cell cycle. The process is done by first seeding the cells overnight and treated with various kinds of treatments such as anticancer drug, doxorubicin. The cells are then trypsinised from the wells and harvested by centrifugation. Resuspension of the cells is conducted and the cells are fixed with fixing solution, then stored at -20 ºC overnight. The next day, the fixing solution is removed by centrifugation and then decanting of the solution. The cells are then stained with the staining solution for 1 hour, so that will be detected by the flow cytometer. The samples are then measured at low flow rate at an excitation wavelength of 488nm and emission of orange/red fluorescent. Cells at sub G1 phase indicate death. So the higher the number of cells at the sub G1 phase, the more toxic is the anticancer treatments.


This above shows how the flow cytometer presents the results after reading the various samples treated by the various treatments such as the anticancer drug, doxorubicin.

Gwendolynn Tan
TG02
0703953J