Seminal Analysis

Section: Hematology
Task: Seminal Analysis


When is the test requested?
Seminal analysis is usually performed on 2 occasions:
1) To investigate whether a man might be infertile.
It is only a ‘screening test’, not a confirmatory test for fertility.
Studies shown that 30% of all patients with normal semen analysis have abnormal sperm function.
(It is a common request in couples planning for marriage!)

2) After a vasectomy to determine whether the procedure was successful.

How to collect?
A colleague joking told me that in olden days, hospitals have a special room playing RA-ted movies in order for males to collect the semen.

That I am not sure whether it is true, but now semen can be collected through:
1) Masturbation
2) If intercourse is the only way to collect sample, special nonreactive condoms are available. They are made up of silicone or polyurethane, instead of latex, which is non-harmful to sperm.

Semen specimen are collected into a sterile and transparent container and required to be analyzed within 1 hour of collection. Therefore, the patients should be strongly recommended to collect samples within clinic area.

Procedures
Upon receiving the semen, we will put it into the incubator for an hour before performing seminal analysis.

Why do we need to incubate for an hour?
- It is estimated that the sperms after being ejaculated, take an hour to pass through the vagina to the uterus to the fallopian tubes to reach the ovaries. Hence 1 hour is the standardized time to measure the amount of sperm that can reach the eggs of females.
- Putting in the incubator is to maintain the viability of the sperms.

Tests done:
1) Appearance
How? Observing the color of semen
Normal result: White or pale yellow
Comments: Reddish semen is abnormal

2)Viscosity
How? Observing the thickness of semen
Normal result: Liquid-like
Comments: Glue-like(very thick) is abnormal

3) pH
How? Use pH dip strip
Normal result: 7.2-7.8
Comments: Semen is alkaline by nature; Acidic ejaculate can be associated with blockage of seminal vesicles.

4) Volume
How? Using 1ml dropper
Normal result: 2-6 ml
Comments: Low volume may be due to absence of seminal vesicle component or decrease of retrograde ejaculation

5) Density
How? Count 10 squares of neuber chamber under microscope
Normal result: >20x106/ml
Comments: The result = (no. of sperm counted) ×10^6/ml

6) 1st hr and 2nd hr motility
How?
1) Drip a drop of semen onto the glass slide and place a cover slide on top
2) Count the motile and non-motile sperms under microscope
3) Motility can be calculated by dividing the motile sperms with total sperm count.
Normal result: >60% for 1st hr
Comments: 2nd count is usually lower as more sperms die.

7) Morphology
How? RBCs , WBCs , Abnormal sperm
Comments: Abnormal sperm includes those with short tail, either head that are too big or small, sperm with 2 heads etc

Factors leading to low sperm count:
Smoking:
Toxins in cigarettes affects the lifespan of sperm and it was also proven that smokers had far less sex drive than their non-smoking peers.

Alcohol:
Drinking in excess damages developing sperm and decreases healthy sperm.

Testicular overheating:
Putting laptop on your laps or wearing tight fitting underwear can raise scrotal temperatures, which slow down the rate of sperm production. Laptop heat insulators and loose fitting garments are recommended to allow air to circulate and keep the testicles cool

Vigorous exercises:
Exercises that cause repeated trauma or impounding damage to the testes and scrotum result in semen alterations. Examples of such exercises are cycling or mountain biking.

There are many more interesting factors! You can read more on www.spermtest.com/causes_of_abnormal_low_sperm_counts.php

Other sites visited:
www.umc.sunysb.edu/urology/male_infertility/SEMEN_ANALYSIS.html
www.labtestsonline.org/understanding/analytes/semen/test.html

Cheers,
Kum Hui Min =)
tg01

Hi people, hope you guys enjoy your SIP. I’ve been posted to the same lab as Maya, Lloyd, Azeimah and Ivan so what we learned are the same. But I realized even those who are not in our lab, they learned similar things as us too.

For the first 2 weeks I’ve been posted to Immunology with Maya and the 3rd and 4th week we are posted to Biochemistry. For these two sections they are closely link as they used the same automated track that helps them to automatically load samples into their machine. As mentioned in Maya’s and Ivan’s blog, for Immunology section they have this machine called Centaur and for Biochemistry, their machine is called Bayer Advia 1650 Chemistry System. Since both of them had already posted on the machines, I guess I’ll post on the manual test that biochemistry section does then.

Basically, the manual test procedures are not difficult or as many steps as we thought. The types of manual test that biochemistry does in out lab are OGTT, G6PD Qualitative test, and Drug Analysis (Drug Screen Test). I’ll share a little about the Drug screen test since no one has really talked about it.

Drug analysis (Drug Screen Test)

Samples to collect: Urine

The Drug Screen Test is a fast and easy test to determine drug abuse. The company purchases these cassettes (test device) that can be used to test for different drugs such as Amphetamine (AMP), Barbiturates (BAR), Benzodiazepines (BZO), Cocaine (COC), Marijuana (THC), Methadone (MTD), Methamphetamine (MET), Methylenedioxymethamphetamine (MDMA), Opiate (OPI), Phencyclidine (PCP) and Tricyclic Antidepressants (TCA). Simply just drop 3 drops of urine to the cassette well and the results will be out in a few seconds.

Signs and symptoms of patient with drug abuse:

Eg. Benzodiazepines— this drug is usually prescribed for treatment foe anxiety and sleep disorder. When this drug is taken more frequently, it will cause dependence and stopping abruptly can bring about symptoms such as trouble sleeping, gastrointestinal upset, feeling unwell, loss of appetite, sweating, trembling, weakness, anxiety and changes in perception.

Principle: Competitive binding.

During testing, the urine migrates upwards through capillary action. The drug present in the urine competes against their respective drug conjugate for binding sites on their specific Ab. If the presence of drug in the urine is below the cut-off concentration, it will not saturate the binding sites of its specific Ab. The Ab will then react with the drug-protein conjugate and a visible coloured line will appear at the test region. If the drug present in the urine is above the cut off concentration, it will saturate all the binding sites of the Ab, thus the coloured line will not appear due to drug competition.

Interpretation of result:

Coloured line appears—negative
No coloured line appears—positive—drug abuse


Note: The assay only provides a preliminary analytical test result. A more specific alternate method should be used to confirm this analytical result. The preferred confirmatory test is Gas chromatography/mass spectrometry (GC/MS). However these test are not done in our lab so I’m not sure how is it done. The lab would usually report positive cases to which ever clinic or hospital that requested for the test. When this preliminary test shows positive result, clinical consideration and judgment from professional are needed in order to make sure that the patient is really having drug abuse.


Sharon
TG01

Week 4 SIP...

Hi all, enjoying your SIP?? I hope you do enjoy it.. Left with 4 months!!!

Alright here to share with you my experiences during my attachment in school..

For my SIP, what I will be doing is mostly on data entry. Well seem very unrelated to what we doing right.. haha.. but it plays a major role in operating the whole lab smoothly. For examples what I dealing with is the purchasing or maintaining of equipments, chemicals products etc. So what I do is key in the amount and what is the amount use for then sending the invoices to the finance department. I will also be updating the actual prices for chemicals products for the next semester use. When lecturer wanted to buy chemical products, they will refer to the price stated to estimate the amount they going to spend on the items.

For my MP, I will be doing a project on 'Evaluation of anti-microbial activity in different tea extracts'. Tea is one of the most commonly consumed beverage(other than water) and reports have shown the benefits of tea to our health. So I will be setting up a whole cell-based assays which is(in my case) S.aureus to evaluate different tea extracts for anti-microbial activity. For now I will be focusing on what I have done in the past 4 weeks.

During the past 4 weeks I had done a series of experiments to determine the log phase of S.aureus. This is just the first part of my whole experiment.

Aims: To determine the log(exponential) phase of S.aureus.

Methods:

1. Preparing of 1L of Trypic-soy agar and 1L Trypic-soy broth.

2. Inoculate S.aureus into TSB and incubate at 37oC for 18 hours, shake with 150-200rpm, as

S.aureus is in beads form this is to dissolve the beads and allow the S.aureus to be release.

3. Streaking of S.aureus from TSB broth to TSA agar plate so as to obtain isolated colonies. The

streak plate is incubated at 37oC for 18 hours.

4. Gram stain of S.aureus, this is to check for the purity of the culture. For S.aureus, it will

appear purple and cocci in cluster.

5. Inoculation of one colony from the TSA agar plate to TSB broth and incubate at 37oC for

18 hours, shake with 150-200rpm.

6. Take OD reading at 600nm to find out the log phase of S.aureus at a specfic time(for me I

take the reading once every half an hour). The reading is to be continue until the culture

reached stationary phase.

7. Confirm OD reading and do a serial dilution. Serial dilution is necessary so that when doing

spread plate it will not be overcrowded.

8. After serial dilution do spread plate and incubate at 37oC for 18 hours. For a good plate/ good

dilution, the cells number in the plate should be in the range of 30-300 colonies.

9. Count plate to get the CFU/ml. This result will be use for reference for further experiments.

For the experiment above, I have repeated a few times so as to ensure the result is reproducible. Alright that is all I had done so far.

Cheers;)
Justina
0605950E
TG01

Biochemistry Lab

Hi All,

On the second and third week of my attachment in a Clinical Biochemistry lab, I was stationed at the TDX station to observe how blood levels of therapeutic drugs are tested.

For this week's post, I'm zooming in to one of the most commonly tested drug - Cyclosprine A.

Cyclosporine A is an immunosuppressive drug which is used to combat tissue rejection following organ transplantation. For example, after an organ (liver, kidney, skin etc) transplant, the immune system of the recipient may reject the introduction of a foreign tissue. T cells and other immune cells may be induced to destroy the foreign cells, leading to graft rejection. Cyclosporine A is thus administered to enable graft survival by reducing the activity of the recipient’s immune system.

Cyclosporine A has a narrow TI (Therapeutic Index) and is also associated with nephrontoxicity and hepatotoxcity, it is therefore recommended to have regular drug monitoring for the patients on this medication. (This explains why I notice a handful of repeated names everyday.)

Procedure

The specimens, which are whole blood specimens received in EDTA tubes, are labeled with a specific bar code that gives each specimen a code number.

1. Microcentrifuge tubes are labeled with patient’s code number. Control tubes are labeled with L (low), M (medium) and H (high).
   
2. Control reagents are provided and treated in the same way as a patient’s sample.
   
3. The samples are mixed by gentle inversion and 150ul of the sample are accurately pipetted into the labeled mircocentrifuge tubes accordingly. (During pipetting, it is important to make sure that blood do not stick to outside of the pipette tips as this would cause additional volume to be added to microcentrifuge tube)
   
4. 50ul of solubilization reagent and 300ul of whole blood precipitation reagent are pipetted into each mircocentrifuge tube.
   
5. After pipetting all the samples, each tube are capped securely and vortex for a full 10 seconds to ensure thorough mixing. After which, samples are centrifuged for 5 minutes at 10900rpm. A clear supernatant and a hard, compact pellet of denatured proteins would be obtained after centrifugation.
   
6. The supernatant of each specimen is decanted completely into the sample well of a sample cartridge. The cartridges are then positioned on a carousel and are ready to be placed into the TDX machine.


7. Other than the carousel, a reagent pack is also placed in the TDX machine. This reagent pack should be mixed by gentle inversion and bubbles should be removed prior to placing the pack on the reagent platform in the TDX.


8. When both carousel and reagent pack are placed stably on their respective platforms, the cover is closed and the analyzer would begin upon pressing RUN, ASSAY 50.


9. Allow time for the machine to operate and once the results are produced, the control values are matched to ensure it falls within the acceptable range. If it does, patient’s results are accepted. If it does not, a rerun of the test is required.

Tan Zhao Rong

TG01

Histopatology!

Fig1.1 Hotplate.
Added to explain in my reply to one of the comments.


Subject: Histology

I am posted to Histopathology/Cytology department, together with Ernest and Tira. ;) (Hope they can stand my craziness at times as the formalin is making us real high. LOl)
This week we are at the Histopathology department, and I heard that we will be in cytology for a month! We been there before, during our tour and I saw the Biosafety Hood (remember what Ms. Chew mentioned before? Yes. The expensive one. )
_____________________________________________________________________________________
The overview of the Histopathology was explained to us by one of our friendly colleagues.
Surgeon cuts specimen of interest-->sends to Histopathology department receiving room-->request forms are checked thoroughly and biopsy no is issued to each patient/case-->sends specimen to trimming room(2 steps away only. Haha)-->pathologists trim big/large specimen while medical technologists trims smaller ones-->cut specimen placed in cassettes--> send for tissue processing--> embedding-->microtomy-->heat slides before staining (H&E as the most common staining)-->sorting slides according to the different doctors and the request forms--> send out for pathologists to read the slides

_____________________________________________________________________________________
Assisting during trimming:
These few days, we were allowed to observe how our colleagues and help out what we could. I assisted the pathologist during trimmer by preparing and labeling cassettes (using a special tissue and cassette marker which cannot be dissolved by any solvent known to the histo-department) for them according to the specimen received and jot down the number of cassettes used for each case before putting them into formalin. The most common specimens received are breast, uterus, colon and kidney and the others are lungs, bile, prostate, fetus, and placenta. I even found out from our colleagues that they had received eyeball and an entire leg before! During trimming, the pathologist will also dictate what they observed, for instance, a cyst in found __ cm near the right margin. These dictations will be sent to the admin staff who will type it out as a report to be attached to the respective request form. Sometimes, the pathologists will also ‘colour’ the specimen using special dyes of different colours as to denote the different parts of the specimen.

Fishing:
Other than assisting during trimming, we also did fishing, heating of slides and sorting slides. After cutting the tissues using the microtomy, we will put the tissues into cold water first before putting them into the water bath. This is to increase the surface tension of the tissues, so that there will be no folds on the tissues which can hinder reading of slides. The water bath has an alarm system so that once the temperature is above 550C, the alarm will beep. The optimum temperature is between 500C to 540C. We will fish the tissues before labeling them according to what is stated on the cassettes. Everyday, we will fish from around 8am to 10 plus am, depending on the workload. Tissues are to be fished in the middle of the slide with the same orientation as the specimen embedded in the paraffin wax. If tissues are fished too high on the slide, it may be properly stained by the auto-stainer (machine for staining). If too low, tissue may be damaged during heating when not properly handled. The correct orientation facilitates checking of slides with cassettes before sending them out to the doctors and pathologists. Smaller tissues (app. 1cm by 1cm) are fished 6 in a slide, 2 by 2 by 2 slices, while normal size tissues are 1 per slide.

Heating:
After fishing, the slides will be tap dry before placing them slanted(partially) on the hot plate to remove all the water on the slide first. This is to prevent ‘smearing’ or moving of tissues out of their positions (on the slide) when they are heated on the hot plate for 3 mins at the highest temperature on the machine (the knob is pointing at 10, the maximum).

Staining:
After heating the slides, we will load the slides for H&E staining into this awesome machine called an auto-stainer. There is a total of 18 buckets of reagents, with 0.5% acid alcohol and lithium carbonate (not for H&E but for other types of staining). This machine also incorporates a part where it mounts the coverslip for you! So fast and with no air bubbles in it! Not like that time where our fingers trembled when placing coverslip. Some slides are requested for special staining like GMS and PAS where they will be placed in another machine. Some types of staining even need up to 1.5 hours!

Sorting:
After the slides are mounted, we will sort them according to their batch no and biopsy no before sorting them again according to respective request forms and the different doctors. These will be checked by our colleagues before sending them out.

The manpower in histopathology department is rotated throughout the different sections, for instance, receiving, trimming, embedding, microtomy, and staining. The hospital we are in receives the most no of specimens and requests everyday, which explains the large no of slides we do everyday. The staff there are also very experience, many of whom with at least 8 years of experience in histopathology department!

Posted by:
Ting Ying Chee =D
0606530D

My experience in bio/immuno lab :)

Subject: Chemistry and Immunology

Hi guys!

I'm posted to Biology and Immunology for this week:)

Basically in the bio/immuno section, we handle samples that requires chemistry and immunology testing. Upon receiving the samples, we are to sort them into different catergories and send them to respective machines for testing.

The categories are:
1) Chemistry
2) Immunology
3) Mixed

Chemistry
Types of tests:
Tchol, HDL, Tg, Chol, CO2, Glu, K, Na, Cl, TP, ALT, AST, GGT, alb, glob, ratio(A/G), T-Bil, Ca, Phos, CPR Quant/latex, HS CRP, Amylase, UA, APO-A, APO-B, D-Bil, Fe, D-Dimer, Mg

Those highligted in green are normal tests requested.
Here we test the sample under a machine called ModP. These samples are easy to handle as we only need to load them in the machine, press START and wait for the results that will be shown on the computer:)

Those highlighted in purple, they are samples we have to take note of.
ModP will not be able to detect them hence we use a more specialized machine Cobas501.

For Cobas501, it is able to detect all type of chemistry tests.
So why don't we just use Cobas for all chemistry tests?
It is because Cobas is a much smaller machine than ModP... Hence ModP is usually used as it is able to process more speicmens.

Immunology
Type of tests:
TSH, FTy, FT3, CA, CEA, PSA, Testro, HIV, FSH, LH, Prolactin, Progesterone, Ferretom, Abs, Ag, HAV, HBeAg, Afp, Trop T, CK-MB, B-hCG, anti-HBe, anti-HBC, anti-HBCIgM, anti-HCV, anti-EBV, anti-dsDNA, Rubella-IgG

Similarly to chemistry, there are samples that we run normally and others that we must take note of.
For normal tests requested, we use modEAO
For specialized tests, we use Eles2010. These samples we have to run maunally, where we have to key into the machine what specific tests to perform.

Mixed
The tests requested consist of both type! So samples are run in respective machines.

All samples are run in plain tubes except D-dimer where it is run in sodum citrate tube.


The procedures above are the general tests we perform. However we also handle other cases like:
-Oral Glucose Tolerance Test(OGTT)
test for: diabetes
sample: fasting glucose, 1st he glucose and 2nd hr glucose
machine used: modP
tube used: fluoride tube

-HbAIC (more specific test for diabetes)
machine used: D10
tube used: EDTA tube

-Micoalbumin(Quanti)
test for: urine(normal/abonormal)
uses: Clinitex and urine strips

-G6PD
test: manually perform on test strip and looked under long wave
results: If baby is normal, sample will fluoresce)

-Microsomal Ab
test: performed on microtitre plate


ok so heres a recap!
chemistry testing---- modP or Cobas
immunology testing-- modEAO or Eles2010
diabetes------------- OGTT or HbAIC in D10
urine(microalbumin)- Clinitex
G6PD---------------- Manual testing on test strip
Microsomal Ab------- Manual testing on microtitre plate

Additional info~
Test tubes:
Yellow- plain tube with gel
Red --- plain without gel
Blue--- sodium citrate (D-dimer)
Purple- EDTA
Grey--- fluoride (glucose)
Green-- lithium haprine

Posted by=)
Kum Hui Min
0605989C

LMQA: Ways to reduce waste

Generation of waste is inevitable, especially in clinical labs where routine tests or research programs[1] are being conducted. However, minimization of waste should be in place[2], whenever possible to minimize the pollution of the environment[2],[3], exposure of personnel to toxic and hazardous materials[2],[3] and to help in conservation of the world resources[3]. By cutting down the generation of waste, clinical labs can also cut down on the cost of purchasing raw materials[3].

How can we minimize waste production?

Management

1. Set goals in the department to limit the generation of waste[2]
2. Create an inventory of chemicals and other raw materials and equipments[2]
   a) With the inventory, unused materials or materials that are close to the expiry date can be sent to other department to be used, if possible[2].
   b) Using updated inventory, purchase only the materials needed as to minimize having expired or deteriorated chemicals or materials that add to the generation of waste[2].

Lab Technician

1. Perform small-scaled experiments whenever possible[2]
2. Limit the use of highly hazardous materials[2],[4]
   Examples of hazardous chemicals are: mercury, ethylene oxide, formaldehyde[3]
   For instance, use Syber Green instead of ethidium bromide[2]
3. Recycle spent solvents by reusing[2]
4. In catalyst reaction, retrieve the metal used[2]
5. Recycle solid materials such as packaging, papers, computer monitors[1],[2]

References

1. University of Minnesota. Minnesota Technical Assistance Program. Laboratories. Retrieve on 15 June, 2008 from http://mntap.umn.edu/labs/index.htm
2. The Geroge Washington University. The Office of Risk Management. Retrieve on 15 June , 2008 from http://www.gwu.edu/~riskmgnt/wasteminimize.cfm
3. Basal Action Network. Basal Convention Meetings- Clinical Waste in Developing Countries. Retrieve on 15 June, 2008 fromhttp://www.ban.org/subsidiary/clinical.html
4. University of Minnesota. Minnesota Technical Assistance Program. Source Newsletter. (2006) Retrieve on 15 June, 2008 from http://mntap.umn.edu/source/2006-2/procedures.htm
   
   

Done by: Ting Ying Chee TG01
Topic: Safety aspects in clinical laboratories and ISO14K