week 14th.

I am going to explain a test which I had the oppotunity to view for myself during my placement at Haematology Laboratory.


Bleeding Time using Surgicutt

This test is to estimate the integrity of the hemostatic plug. It is a screening test for
qualitative and quantitative platelet functions and generally helps to diagnose hemostatic defects and platelet dysfunctions.

Principle

When a minor standardized incision is made on the forearm, time between the infliction of the incision and the moment the bleeding stops is taken. A hemostatic plug is formed after the incision to stop the bleeding. This plug formation depends on adequate platelets to adhere to the subendothelium to form aggregates.
For this test is a screening test, an increased bleeding time would not qualitatively or quantatively diagnose an underlying platelet disorders. However, it is indicative of the need for more quantifiable testing such as platelet counts, APPT, TT testing etc.

Image of Surgicutt Bleeding device taken from http://www.medcompare.com/

Procedure

1. First, a sphygmomanometer is used to cuff the upper hand of the patient. The pressure is raised to 40 mm Hg.This is the standard pressure for children and adults. For infants, the pressure would be dependant on the weight. Patient’s blood pressure was to remain at such throughout the duration of the test.
2. The volar surface of the forearm, where the incision is to be made, is rubbed with alcohol swab.
3. The incision is made using Surgicutt bleeding time device and the timer is started simultaneously. There are three different devices to suit different age group, mainly infants, children and adults. This is shown below.

Age group :1 - 4 months
Device:Surgicutt Newborn
Depth of blade (mm) :0.50

Age Group:5 months to 15 yrs
Device: Surgicutt Junior
Depth of blade (mm): 1.00

Age Group: 16 years and above
Device: Surgicutt
Depth of blade(mm): 1.00

4. Every 30 seconds, a blotting paper was used to wick the blood close to the incision without touching the incision itself.
5. The timer is stopped when the bleeding stopped.
6. Reading on timer is recorded down.

The following are the reference values for normal bleeding time:

Infants (1-4 months) :
0.85- 1.65 mins

Children (5 months to 15 years) :
1.3- 8.99 mins


Adults (16 years and above) :
2-8 mins

For neonates whose bleeding does not stop after 5 minutes, time is reported as >5 mins. For adults whose bleeding does not stop for more than 20 minutes, time is reported as >20 mins.

Raihana~

WEEK NUMBER THIRTEEN

HELLO EVERYONE!!!!
Hope you guys are doing well.

This week, I will be touching on a topic which may be foreign to some of you. It will be on glycerolization/freezing and deglycerolization/thawing of red blood cells. This process is done in the cryopreservation lab of the blood bank.

Glycerolization/Freezing
Freezing RBCs with glycerol dates back to the 1950s. Frozen RBCs can be stored for up to 10 years for:
1. Patients with rare phenotypes
2. Autologous use
3. In case of national emergencies in which blood cannot be dispositioned out to hospitals quickly enough to prevent expiration.

The resulting deglycerolized product is free of leucocytes, platelets, and plasma due to washing process. Cryoprotective agents can be categorized as penetrating and nonpenetrating. A penetrating agent involves small molecules that cross the cell membrane into the cytoplasm. The osmotic force of the agent prevents water from migrating outward as extracellular ice is formed, preventing intracellular dehydration. An example of a penetrating agent is glycerol. An example of a non-penetrating agent is hydroxyethyl starch (HES). This comprises large molecules that do not enter the cell but instead form a shell around the cell, preventing loss of water and subsequent dehydration.

Two procedures used for glycerolizing RBCs are high glycerol and low glycerol methods. The methods differ in the equipment used, the temperature storage and the rate of freezing. Most blood centres practice the high glycerol method.

High Glycerol (40% w/v)
This method increases the cryoprotective power of the glycerol, thus allowing a slow, uncontrolled freezing process. The freezer is generally a mechanical freezer that provides storage at -80oC. This particular procedure is probably the most widely used because the equipment is fairly simple and the products require less delicate handling. It does however require a large volume of wash solution for deglycerolization. RBCs are frozen within 6 days of collection when the preservative is CPD or CPDA-1 and up to 42 days when preserved in AS-1, AS-3 and AS-5. AABB Standards states RBCs must be placed in the freezer within 4 hours of opening the system. It is advisable to freeze a sample of donor serum in the event additional testing is required for donor screening.

Low Glycerol (20% w/v)
In this method, the cryoprotection of the glycerol is minimal, and very rapid, more controlled freezing procedure is required. Liquid nitrogen (N2) is routinely used for this method. The frozen units must be stored at about -120oC, which is the temperature of liquid N2 vapour. Because of the minimal amount of protection by the glycerol, temperature fluctuations during storage can cause RBC destruction.

Deglycerolization/Thawing
Red cells that have been frozen require thawing and deglycerolization before they can be safely transfused. Glycerol will be removed from RBC to avoid in vivo and/or in vitro heamolysis.

The thawing process takes approximately 30 minutes and involves immersion of units into a 37oC water bath and washing the RBCs with solutions of decreasing osmolarity (eg. 12% NaCl, 1.6% NaCl, 0.9% NaCl, + 0.2% dextrose). An exception to this rule is a donor with sickle cell trait in which RBCs would haemolyze upon suspension in hypertonic solutions; in this case the cells would be washed in 12% NaCl and then 0.9% NaCl with 0.2% dextrose, omitting the 1.6% solution. Automated continuous-flow instruments can be utilized for washing. In our blood bank, a machine called the Haemonetic 215 is used. Once the RBCs have been deglycerolized, the unit is considered an open system with an expiration date of 24hrs and is stored at 1oC to 6oC.

Hope my post this week has enlightened you on some of the processes that occurs in a blood bank. Till next time..... Adios!!!

Rusydiana binte Kusni
TG02
0608485I

AMir's back...and its week 12!!!

How is everyone doing?.. wah 3 months of attachment already..thats fast.. 2 more months and we can meet up already..ok what shall i say about this week.. hmmmmmm


PARASITOLOGY!!


For last week I was posted at parasite lab.. In this lab, they mainly deal with protozoans and helminths(i hope you guys remember what we learn in BMic).. if not nvm i shall give a brief intro...


Parasite are organisms that live on or in a host organism, usually causing it some harm. They are relatively smaller in size and are dependent on their host for nourishment. There are many forms of parasites like, arthropod parasites (ticks), plant parasites (mistletoe), single-celled protozoan (amoebas) and even viruses. Numerous parasites live in the gastrointestinal tract, known as intestinal parasites which are common human pathogens. Parasite are subdivided into protozoans (unicellular) and helminths ova/larva (multicellular)


Eg of protozoans include: Amoeba histolytica, Giardia lamblia, Cryptosporidium
Eg of helminths include: Roundworms (Ascaris lumbricoides), tapeworm, flatworms

Parasite infections in Singapore is not as common as bacterial infections. So, we do not recieve much specimens. There are many different tests carried out: Ova and Parasite Exam, Silver stain for Pneumocystis jirovecii (PCP), leukocytes examination and occult blood. For this blog i will focus more on Ova and Parasite exam


Ova & Parasite exam

Ova and parasite exam is basically analysis of stool to check for the presence of a parasite or worm-like infection of the intestine. Most of the specimens, or i shall say ALL of the specimens are stool specimens, as intestinal parasites there will be released in the stool of infected humans. There will be a large population of parasites in the stool, there it will be easier to detect the presence of this parasites.

Principle

• Ova and parasite exam is the most common performed procedure in diagnostic parasitology
• For identification of intestinal protozoan parasites, it is based on the recognition of their cyst or trophozoites stages or even both
• Cysts are spherical with smooth uniform walls formed during the dormant resting stage
• Trophozoites have thin limiting membrane with variations in size and shape, they are formed during active feeding stage
  
  
image taken from: http://www.stanford.edu/group/parasites/ParaSites2006/Giardiasis/images/Giardia_LifeCycle%20cdc.gif



• For Helminthic infections, it is based on identification of eggs, larvae or proglottids in faeces
• Eggs or ova are usually found in stool specimen as it is passed out in faeces
• Ova and parasite exam consists of 3 separate protocols: Direct wet mount, Concentration and Permanent stained smear
• Direct wet mount allows detection of motile protozoans tryphozoites and helminth worms
• Concentration is designed to facilitate recovery of protozoan cysts, coccidian oocysts, microsporidial spores, and helminth eggs and larvae.
• Formalin-ethyl acetate sedimentation method is used to prepare concentrated specimen for direct wet mount.
• Permanent stained smear is used to identify intestinal protozoa, using the common Trichrome stain method
• It is the most important procedure as it aids in the confirmation of any suspicious objects seen in the direct wet mount

Materials

• Para-Pak® ULTRA Zn-PVA fixative


• Para-Pak® Macro-Con® Kit
  - Surfactant bottle (30ml)
  - Conical 50ml centrifuge tubes with filtration units and screw caps


• Disposable ice-cream stick and applicator sticks


• Disposable Pasteur’s pipette


• Paper towel


• Microscopic slides


• Timer


• 10% Formalin


• Ethyl acetate (Clearance)
  
• Disposable cotton swabs
  
• Normal saline


• Trichrome stain
  · For destaining: 10% acetic acid in water, 50% and 70% ethyl alcohol

Procedures

A. Preparation of material for Trichrome staining

1. Using a disposable ice-cream stick, place fresh faeces into the PVA fixative in the
Slides ready to be stained ratio of 3 parts PVA fixatives to 1 part faeces.
2. Mix thoroughly so that it appears homogenous
3. Allow the faeces to fix for a minimum of 30mins; overnight fixation is acceptable
4. Pipette out some of the well-mixed PVA-faecal mixture onto a paper towel using a disposable Pasteur’s pipette and allow to stand for 3mins to absorb out the excess PVA (This step is critical to obtain the best possible stain)
5. With an applicator stick, apply some of the faeces from the paper towel onto a clean slide (Spread the material onto the edge of the slide for better adherence)
6. Dry the slides for 3 hours or overnight at room temperature (Slide must be dried thoroughly to prevent material from being washed off during staining)

B. Concentration procedure
1. Add 10 drops of surfactant into the ParaPak specimen vial
2. Insert the filtration tightly into the specimen vial
3. Invert assembled unit, tapping sharply to force the solution into the conical tube
4. Unscrew the filtration unit and discard appropriately
5. Add 10% Formalin to the dotted line, plus 5 ml of Ethyl Acetate (Clearance)
6. Tightly screw the cap and shake vigorously for 1min
7. Centrifuge at 1500xg for 10mins
8. After centrifugation the specimen is clearly separated into four layers
9. Using an applicator stick, rim the debris layer. Pour off the debris and supernatant fluid, leaving the sediment
10. With the tube still inverted, use a cotton swab to clean and remove the remaining debris
11. Return the tube to upright position and add a few drops of saline to mix the sediment

C. Wet Mount
1. Using an applicator stick, prepare a coverslip preparation from the sediment
2. Examine microscopically

D. Trichrome Staining
1. Place slide in Trichrome stain solution for 10mins
2. Destain by placing the slide in acidified alcohol for 1-3sec (Do not allow slides to remain in this solution)
3. Dip slide several times in 100% ethyl alcohol
4. Place slide in 2 additional changes of 100% ethyl alcohol for 5mins each
5. Place slide in 2 changes of xylene substitute for 5mins each
6. Mount in immersion oil, using a No.1 thickness cover glass
7. Examine microscopically

Conclusion



In Trichrome stain, cytoplasm of cysts and trophozoites will stain blue-green tinged with purple. Parasite eggs and larvae usually stain red. All ova, pathogenic protozoan trophozoites or cysts seen must be reported. The reports will be sent to the doctors, and they will decide whether to do any further confirmatory tests depending on the patient's clinical symptoms. image taken from: http://www.med-chem.com/Para/prob%20of%20month/IMAGES/Giardia,%20trophs%20(2)%20in%20mucus%20string,fixed%20great.jpg

So, thats about all!!!.. I shall be back sooon!!.. take care peeps!!

AmiR


TG02

week 11 already people!!

Know what? I'm really having a hard time finding what to blog about, considering most of the tests have already been posted. Haha.


Well anyways, I've finally decided to write about a test to detect anti-Mycoplasma pneumoniae antibodies, using SERODIA-MYCO II (also known as MPA), since now I'm currently in serology department. Alot of manual tests, if I may say.

First of all, Mycoplasma pneumoniae is a very small kind of bacterium that is usually associated with upper respiratory tract infections, together with fever, headaches, cough and malaise. It may lead to tracheobronchitis, which is a common respiratory infection characterized by inflammation of the trachea and bronchi. Its mode of transmission is person-to-person transmission by direct contact with the infected respiratory secretions, and it will usually take about 1-4 weeks before the symptoms will develop.

Principle
SERODIA-MYCO II is used in the detection of antibodies to Mycoplasma pneumoniae. It uses gelatin particles which are sensitized with extracted cell membrane components of Mycoplasma pneumoniae. Serum containing specific antibodies will react with the sensitized couloured gekatin particles to form a smooth mat of agglutinated particles in the microtitration tray.


Reagents
SERODIA-MYCO II kit, which includes:

• Sensitized particles
• Unsensitized partiles
• Positive control
• Sample diluent

Procedures

6 wells are required for a patient's sample

4 wells are required for control

1. Label the wells (ID number or Control)
2. Add 100ul of sample diluent to the first wells
3. Add 25ul of sample diluent to the rest of the wells
4. Add 25ul of positive control to the first well and mix well
5. Bring 25ul of the mixed solution to the next well and mix well
6. Repeat Step 5 until the last well, where 25ul will be discarded after mixing
7. Repeat Steps 4-6 with patient's sample
8. Add one drop of unsensitized particles into the second wells
9. Add one drop of sensitized psricles into the rest of the wells starting from well 3
10. Tap at the sides of the microtitration plate gentle to ensure proper mixing
11. Cover the plate and incubate at room temperature
12. Results to be read 3 hours later

Interpretation

Negative (-):

Definite compact button in the center of well with smooth round outer margin

Positive (+):

Definite large ring with firmly agglutinated particles spread within the circle

Positive (++):

Agglutinated particles spread out to cover th bottom of the well entirely

MAYAFIRHANA

TG02