WEEK NUMBER EIGHTEEN

Hello everyone! A few weeks more and we're all back at school. This will be my last post for our SIP so, here it goes...


Before a person is allowed to donate blood, he or she has to undergo certain physical examinations. The donor centre representative evaluates the prospective donor with regard to general appearance, weight, temperature, pulse, blood pressure, pulse, blood pressure, haemoglobin, and presence of skin lesions.

In this post, I will be talking about donor haemoglobin testing.

One of the methods used at the blood bank to estimate the level of haemoglobin in a donor is by the copper sulphate method.

Principle

i. The method of haemoglobin estimation using copper sulphate method is based on the principle of specific gravity. If a drop of blood is dropped into copper sulphate solution, it becomes encased in a sac of copper proteinate which will prevent the specific gravity from changing for at least 15 seconds. If the specific gravity of the blood is higher than that of the solution, the drop will sink within 15 seconds. If not, the drop will hesitate, remain suspended, or rise to the top of the solution.

ii. A specific gravity of 1.053 corresponds to a haemoglobin concentration of approximately 12.5g/dL

iii. False positive results are rare and donors whose drop of blood sinks nearly always have an acceptable haemoglobin level. However, false negative reactions occur fairly commonly and can cause inappropriate deferral unless the haemoglobin level is checked using another method.


Specimen
Sample of blood obtained by finger prick.


Materials
i. Copper Sulphate Solution (specific gravity: 1.053)
ii. Glass beaker
iii. Cotton swabs, with 70% alcohol or spirit
iv. Sterile disposable lancets
v. Capillary tube
vi. Biohazard sharps box
vii. Disposable gloves
viii. Alcohol swab (individual pack)


Procedures

i. Make sure that the glass beaker (250ml) is clean and free of debris. Fill the beaker with copper sulphate solution until it is slightly above 200ml marking.

ii. Put on a pair of gloves.

iii. The donor’s middle or ring finger is normally chosen for the finger prick procedure. Avoid fingers with rings on. Clean the chosen finger using a piece of alcohol swab. Allow the alcohol to dry or wipe dry with a new piece of dry cotton swab. Discard the swabs in the biohazard waste bin. Do not re-use the swab.

iv. Prepare a disposable sterile lancet by pressing and turning the knob into the lancet until there is a ‘click’ sound.

v. Use your thumb to slightly press the donor’s finger from the top of the knuckle towards the tip. Puncture the finger firmly, near the end but slightly to the side, with the sterile disposable lancet. Ensure that there is a good free flow of blood. DO NOT SQUEEZE the puncture site repeatedly as this will dilute the drop of blood with excess tissue fluid and lower the specific gravity. Dispose off the lancet in the sharps box.

vi. Wipe away the first drop of blood from the punctured site with a new piece of cotton swab. Collect the second drop of blood in a capillary tube until it is at least three quarters full, without allowing air to enter the tube.

vii. Hold the capillary tube about 1cm above the surface of the copper sulphate solution in the glass beaker, and let one drop of blood fall gently by unassisted gravity from the tube into the solution. Dispose off the capillary tube in the sharps box. DO NOT FLICK the drop of blood out of the tube as this will lead to in accurate haemoglobin measurement.

viii. Observe the drop for 15seconds. If the blood drop has a higher specific gravity than the copper sulphate solution, it will sink within 15 seconds. If not, the sinking drop will hesitate, remain suspended or rise to the top of the solution.

ix. Discard the copper sulphate solution after every 20 tests or every 2-3 hour intervals or when the solution is turbid. If there is any floating blood residue on the surface, it should be removed with an applicator stick.


Interpretation

i. If the drop of blood sinks within 15 seconds, the haemoglobin is greater than 12.5g/dL which is acceptable for blood donation unless there are other conditions which require a higher pre-donation haemoglobin level.

ii. If the drop of blood does not sink or sinks very slowly, the haemoglobin is most likely less than 12.5g/dL, and the result must be rechecked using quantative haemoglobin estimation by the HemoCue haemoglobin test.

iii. Because the copper sulphate method is not a quantitative test, the quantitative method by the HemoCue haemoglobin test is necessary to check the exact level in donors who fail the test as it suggests that the haemoglobin is low and the donor may need medical advice


Limitations

i. This is not a quantitative test and it shows only whether the potential donor’s haemoglobin is below or above 12.5g/dL

ii. The copper sulphate solution must be stored in tightly capped containers to prevent evaporation. The solution should be kept at room temperature to brought to room temperature before it is used.


Rusydiana binte Kusni
0608485I
TG 02

WeeK 17!!!

Oh my its Week 17 already!!!

Hello friends!! How are you guys doing?? I hope everything is fine especially with the projects. Well, this week Im doing on Quality Assurance. Theres not that much work to be done actually. We carry out QA tests on media and agars used in the laboratory. Here are 2 of the tests that I did for the week,

A. Citrate Test

Objective
· To determine the ability of an organism to utilize citrate as a sole source of carbon for metabolism and growth

Principle
· Certain organisms are able to utilise citrate, an intermediate metabolite in TCA (Kreb’s) cycle, as the sole source of carbon.
· In bacteria, cleavage of citrate involves an enzyme system without coenzyme A involvement
· Bacteria break the conjugate base salt of citrate into organic acids (formic acid and acetic acid) and carbon dioxide.
· An organism that can use citrate as its sole carbon source also uses ammonium salts as its sole nitrogen source.
· Simmon's citrate media contains ammonium salts and a pH indicator, bromthymol blue
· Bacteria extract nitrogen from ammonium salts with production of ammonia leading to alkalinisation of medium which causes the indicator, bromthymol blue to change to an intense blue or a Prussian blue colour when pH is above 7.6 showing a positive result

Materials
· Simmon’s citrate media
· Disposable inoculating loops
· Enterobacter aerogenes W 3/3/90 (positive control)
· Escherichia coli (E.coli) ATCC 25922 (negative control)

Procedure
1. Streak the surface of the Simmon’s citrate slant
2. Incubate at 35⁰C overnight (usually 18hrs)

Left: Negative, Right: Positive Picture taken dddddddddddddddddddddddddddddddfrom: http://www.ams.cmu.ac.th/mt/clinmcrb/CMBwebsite/Price%20list_media.htm

Results and Conclusion
· Positive result (Enterobacter aerogenes): Deep blue colour of agar
· Negative result (E.coli): Absence of growth or no colour change
· Citrate test can be use to differentiate members of the Enterobacteriaceae family and other Gram negative organisms

Possible errors
· Too heavy inoculums may give a false-positive result
· Preformed organic compounds from dying bacteria may release carbon and nitrogen which may result in false positive results

Urease Test

Objective
· To test the ability of an organism to break down urea by the action of the enzyme urease

Principle
· Urea which is often referred to as carbamide, a diamide of carbonic acid
· Due to the presence of amides, urea is readily hydrolyzed by a specific enzyme, urease
· Urease is an important microbial enzyme which is used in the decomposition of organic compounds
· Urease breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia, and water
· Urease is detected by plating bacteria onto an amide containing medium, specifically urea
· Urea will be hydrolyzed into ammonium carbonate as the end product,
· Formation of ammonia alkalinises the medium causing a change in the phenol red indicator
· Urea agar is slanted with an adequate butt portion to allow gradation of positive reactions
· Proteus, Providencia and Morganella species produce urease in large quantities

Materials
· Christensen’s urea agar slant
· Disposable Inoculating loops

Procedure
1. Streak the surface of urea agar slant
2. Incubate aerobically at 35⁰C overnight (usually 18hrs)
Left:Negative, Right: Positive Picture taken from: www.nhlmmcgym.com/culture.htm
Conclusion
· Positive result: urea slant change to an intense pink-red to red-violet colourchange Positive result
· Negative result: no colour change; urea slant remain yellow
· Some organisms like Pseudomonas aeruginosa may produce ammonia by breaking down peptones present in the agar slant à False positive results
· Presence of glucose in the Christensen’s medium prevent organisms from using ammonia produced as its sole source of nitrogen which will lead to inaccurate results
· Organism like Helicobacter pylori are able to hydrolyze urea rapidly, producing a positive reaction within 1 to 2 hours

So thats all! Any questions just ask me aites!! Bye! Take care!!

week 16...!!!!

Well..... For the past 2 weeks i was attached to the Specimen Reception (SR)department. It is the pre-analytical part of the whole laboratory, where the specimens are received and labelled.

It is here, in this department where I found out that mistakes are simply not tolerated. The personnels must be constantly on their toes, fast and at the same time, do their work accurately. A simple mistake of not tallying up the names on the specimens to the name on the form, can spell disaster.

There's no tests involved, but I am to include the workflow of the SR department.

• The dispatch crew will arrive with the samples from the various clinics. The samples are received by the personnels there, where the number of samples and specimens are counted and tallied with those counted by the dispatch crew.

• The samples are then passed over to another group of personnels who will open the samples. Here, they will check that the names on the specimens tally with those in the request form, and write down the number and type of specimens that they receive for that request form only.

For example: 2 long plain tubes, 1 EDTA tube and 1 urine will be written as 'LP x 2, E, U' in a vertical fashion.

Then, they would sign their initials before seprating the samples into single profile (includes only HIV, VDRL and tests for malaria parasites) and profile (which is all the other tests other than those previously named).

• The seperated samples are then subjected to primary barcoding, where the request forms and specimens are labelled with their own individual barcode, and allocated into baskets according to the tests that are requested (such as HIV samples are placed into the HIV basket, and so forth). Plain tubes, fluoride tubes, and certain EDTA tubes and urine (which are already poured into 5ml test tubes) are centrifuged. The rest are collected by the respective departments, such as Microbiology collecting the urine, stool and swab samples.

• The centrifuged samples then goes through secondary barcoding, where certain specimens' serum are required to be aliquoted out due to the type of tests involved. The specimens are then loaded onto a sample tray, where they will be placed in the Sample Manager (explained in my earlier posts). The sample manager will then sort the specimens according to the type of tests to be run, and send them to the various machines via the transportation track.

• At the end of the day, the specimens will undergo allocation and be placed in the cold room, where the will be kept for a week to 10days. This is so that if there is any additional tests that is to be run, we can still use the specimen and not bother the patient to go for another blood-taking session.

Mayafirhana

TG02

Hello everyone!! Before i start talking about my attachment, i would like to wish all my Muslim frenz Selamat Hari Raya!! We shall go visiting one day alryte??

So this week, i am going to talk about the stool occult blood test and OC Light test.

Stool occult blood test

Introduction – To detect occult blood in stool

Principle of analysis - When stool specimens containing occult blood are applied to Hemoccult SENSA test paper, which is impregnated with guaiac, the hemoglobin portion of the occult blood comes in contact with the guaiac. When the Hemoccult SENSA peroxide developing solution is added, a guaiac peroxidase like reaction occurs. Oxidation of guaiac becomes visible by the appearance of a blue color within 30 seconds. A food that contains substances with peroxidase activity may cause false positive results.

Specimen – Stool

Reagents –

Hemoccult SENSA test slides
Hemoccult developing reagent

Retrieved from : http://www.beckman.com/products/rapidtestkits/hemoccultsensa.asp

Method –

1) Apply a very thin smear of stool inside section 1 and 2 with an applicator stick.

2) After 3-5 minutes, open the perforated section on the back of the slide.

3) Apply 1 drop of Hemoccult SCREEN developing reagent to the positive and negative well each and 2 drops to the reaction wells.

4) Read results after 30 seconds and before 1 minute.

Interpretation –

Read the results only when the controls give the correct color after the addition of the developing reagent.

• Positive – Traces of blue colors in either one or both of the reaction wells

• Negative – No indication of blue color in either one or both of the reaction wells

Positive stool samples are confirmed using the OC Light Occult Blood Method.

Limitations of procedure –

Hemoglobin is not usually found in the stool of healthy individuals. Its presence indicates bleeding in the gastrointestinal tract. Causes include tumors. Tumors are common in the elderly and this test is used as a screen to select patients for further invasive investigation.

Stool OC Light

Introduction – The OC Light method is used to confirm the positive cases by the Hemoccult SENSA method.

Principle of analysis – The OC Light is designed to measure human hemoglobin in fecal samples. The OC Light immunochromatographic test has a test detection range from 10µg to 100mg/g of feces. The test strip reacts only with human hemoglobin and does not react with hemoglobin of other animal origin.

Specimen - Stool

Reagents -

OC Light Sampling Bottles

OC Light Strips

Retrieved from : http://www.eiken.co.jp/en/product/index.html

Method -

1) Remove the green cap from the sampling bottle and dilute the stool inside the bottle.

2) Remove the white cone nozzle.

3) Insert the strip into the sampling bottle from the dip slide.

4) Read test result, presence of a blue line at the lower and upper center of the strip after 5 minutes.

Interpretation –

Read the result only when the controls give the correct color after the addition of the developing reagent.

Positive result – Presence of blue line at the lower and upper center of the strip may be considered positive.

Negative result – Presence of blue line only at the upper center of the strip is negative.

Equivocal result – No blue in line in both the lower and upper center of the strip or at the lower center of the strip only should be considered equivocal. Such samples should be retested by changing OC Light test strip.

That's all for now peepz!!

Before i sign off, i would like to say sorry if i have not answered any of your questions regarding my last two posts.. It's been hard for me to get access to the internet. I will try my best to reply to all the questions ASAP.. Really sorry especially to Ms Chew. I hope i have not been penalized by my lack of participation. I will try to participate more often once i got my laptop soon.. Sorry once again..

Toodles~~

** Sofie **