Hello everyone!! It’s my turn to blog for the week. I am currently at coagulation lab. The test which I am going to share with you guys is 50%PT/APTT. I suppose you should be familiar with PT and APTT which we had done before in during haematology lab session. Does it ring a bell? To refresh your memory…

PT is used to screen for abnormalities of those factors that are involved in the extrinsic pathway (factor 5,7,10) and prothrombin and fibrinogen.

APTT is sensitive to deficiencies of factor II,V,VIII,IX,X,XI,XII( Factor 2,5,8,9,10,11,12) involved in the intrinsic pathway.


Test: PT

1) PT reagent is incubated in water bath at 37 degrees for 3mins
2) 0.1ml of test plasma is incubated in 37 degrees water bath for 3mins
3) 0.2ml of PT reagent was pipette into the 0.1ml of test plasma after the incubation period
4) start the stopwatch simultaneously and shake the test tube to mix the contents well and observed for clot

Test:APTT

1) 0.1ml of APTT reagent and 0.1ml of calcium chloride was incubated in water bath at 37 degrees for 5mins
2) 0.1ml of test plasma was added to 0.1ml of APTT reagent. Remember to shake the tube to mix the contents well
3) the tube was placed at 37 degree water bath for 4mins
4) 0.1ml of calcium chloride was pipette into the plasma and APTT reagent
5) start the stop watch simultaneously and observed for clot





Test: 50%PT and APTT correction test

Intended use:
It is used to screen for the presence of inhibitors in patient’s plasma.

Principle:

50% addition of normal plasma is sufficient to restore the deficient factor. Complete or partial correction time suggests a clotting factor deficiency, whereas failure to correct the clotting time suggest the presence of inhibitors. Normal plasma contains all coagulation factors and is mixed with patient’s plasma to detect the presence of circulating inhibitors in the patient’s plasma. If a factor deficiency is responsible for the abnormal clotting time addition of normal plasma should provide the deficient factors and correct the PT and APTT time. However if abnormal clotting time is caused by a circulating inhibitors in the patient’s plasma, the clotting time will remain prolonged even when normal plasma is added. The inhibitor inhibits factors in normal plasma as well as in the patient’s plasma.

Example: if the patient’s PT is 18secs
Normal plasma is 12secs
Average is (18+12)/2=15secs
PT is corrected if 50%PT <15secs>

that's all! feel free to ask if in doubt..

kai lin

0503211E

SIP posting

For this past few weeks I was attached to Haematology

At haematology section, I have learn various principle of the tests such as retic count, Dengue testing.

RETIC COUNT
For retic count, the analyzer used is CELL-DYN Ruby. Reticulocytes are defined as transitional red cells between nucleated red cells and the mature erythrocytes. The reticulocyte assay enables determination of the percentage of reticulocytes using a whole blood specimen. The method used is based on light scatter measurement of stained cells.

Staining Procedure:
1. Label one tube of CELL-DYN Reticulocyte Reagent(Phosphate buffered saline solution contain New Methylene Blue N) for one specimen.
2. Verify the whole blood specimen have no clotting and mixed well.
3. Pipette 20 micro-litre of whole blood specimen into the tube of reticulocyte reagent.
4. Incubate the stained reticulocyte specimen for 15 mins at room temperature.
5. Ready for sampling in CELL-DYN Ruby

Principle of Procedure:
Reticulocytes contain ribosomal RNA, this RNA can be seen by certain dyes that simultaneously stain and precipitate the polyanion to form a reticulum. The CELL-DYN Ruby reticulocyte method uses the thiazine dye New Methylene Blue N. Sample preparation is done by diluting a small volume of blood into pre-measured staining solution and incubate for at room temperature for 15 mins for the staining of reticulum to complete. The stained sample could be tested using CELL-DYN Ruby. The stained sample will be aspirated in the analyzer and diluted with the Reagent(WBC Lyse). Once diluted, the RBCs sphere due to the influence of the nonionic detergent incorporated into the staining solution. Sphering is necessary to eliminate optical orientational noise that would otherwise be introduced into the scatter measurement. The usual lytic action of the Reagent is prevented by electrolytes contained in the staining solution and the lack of the usual incubation period used in this channel during WBC analysis. Also the high New Methylene Blue concentration in the staining reagent exerts a stabilizing effects on RBCs.

During data acquisition, 0,10 and 90 degrees scatter is collected. The 0 degrees threshold is set high enough to exclude most platelets. Histogram data is used to differentiate reticulocytes, mature RBCs, platelet clumps and nucleated cells. Reticulocytes have similar 10 degree scatter to mature RBCs but differ them by exhibiting greater 90 degree scatter.

Dengue Testing using DENGUE DUO CASSETTE
The Dengue Duo Cassette is for the qualitative presumptive detection fo IgM and IgG Ab to dengue virus in human serum, plasma and whole blood. The assay can be used for the presumptive differentiation between primary and secondary infection. Positive result are presumptive and must be confirmed by virus isolation, paired serum analysis, Ag detection by immunochemistry or viral nucleic acid detection for confirmation of dengue virus infection.


Dengue, a flavivirus is found in large areas of the tropics and subtropics. Transmission is by mosquito, principally Aedes aegypto and Aedes albopictus. Dengue virus infection causes a spectrum of clinical manifestation ranging from asymptomatic to fatal haemorrhagic disease.


In the Dengue Duo Cassette, IgM and IgG are determined simultaneously using a single addition of serum, plasma or whole blood. Thus, a differentiation between primary and secondary infection can be made by a single application of serum, plasma or whole blood. In primary infections, serum IgM Ab can be detected from dengue patients as early as 3-5 days after onset of fever, generally persisting for 30-90 days, although detectable levels may be present for 8 months post-infection.

Secondary infection is characterised by high IgG levels that may or may not be accompanied by elevated IgM levels. The sensitivity of this assay has been set so that in patients with primary dengue, IgM is positive while IgG is negative. In contrast, patient with secondary infections will have a positive IgM result.

Assay Procedure
1. Add 10 micro-litre of whole blood, serum or plasma to the cirular well and allow the sample to be absorb into the specimen pad within the circular well.



2. Hold the buffer bottle vertically and 1 cm above the square well and add 2 drops of the buffer to the square well at the base of the cassette.


3. Read the result exactly after 15 mins after adding the buffer.


Princple
When present in the patient sample, dengue-specific IgM or IgG Ab bind to the anti-human IgM or IgG Ab immobilized in two lines across the cassette membrane. Colloidal gold complexes containing recombinant dengue 1-4 Ag are captured by the bound patient's IgM or IgG to give visible pink line. A procedural control is included to indicate that the assay has been performed correctly.

Interpretation of Result

Primary infection

Pink bands appear in the IgM and Control regions

The test is positive for IgM Ab and is suggestive of primary dengue infection.






Secondary infection


Pink bands appear in the IgM, IgG and Control regions.

The test is positive for the IgM and IgG Ab and is suggestive of a secondary dengue infection.

Secondary infection

Pink bands appear in the IgG and Control region.

The test is positive for IgG Ab and is suggestive of secondary infection.















Negative

A pink band appears in the control region only.

No detectable IgG and IgM Ab to dengue. The result does not exclude dengue infection. Retest in 3-4 days if dengue infection is suspected.

Invalid

No pink band appear in the control region.


The test is invalid and should be repeated.

This are the 2 tests I have learn. Feel free to ask question.
Juexiu TG02

SIP- Cytology

This month, i'm scheduled to the Cytology department in which the bulk of the specimens it deals with are mainly PAP smears, urine and body fluids (such as peritoneal, pleural and pericardial fluids, CSF).

Scope of Cytology:

• Diagnosis of malignancy
• Identify premalignant conditions
• Identify infections and associated organisms
• Evaluation of hormonal effects of estrogen and progesterone

PAP (Papanicolaou) Smearing and Staining

Purpose: Diagnosis of cervix cancer (causative agent: Human Papillomavirus).

Procedures:

1. After smearing on slides, specimems will be sent to the Cytology lab.
2. Check to make sure the patients' data on the request log tallies with that on the slides.
3. Load them into a rack then into the automated staining machine which has been programmed to perform PAP staining.
4. After staining, the slides are mounted for microscopic examination.

PAP Staining (The Regressive Method- Nuclei Overstained)

Principle: Exhibit differences in cellular morphology, maturity and metabolic activity. As intact cells in a cytologic smear tend to appear in 3-D configurations and overlap, PAP allows resultant transparency and clear definition of nuclear details.

Procedures:

1. Fix the cells in 95% ethanol for 15 minutes to allow the cells to absorb the dyes in the subsequent staining procedures which are fully automated.
2. Unacidified haematoxylin
3. Blue in water (nuclear staining)
4. Dilute hydrochloric acid (remove background staining caused by excess haematoxyin)
5. Rinse in water(3 rounds to set the haematoxylin)
6. 95% alcohol (3 rounds to remove all water as subsequent staining reagents are alcohol-based and water cannot mix with alcohol)
7. OG (Orange-G) stain (stains keratin orange)
8. 95% alcohol (2 rounds)
9. EA Polychrome stain (stains mature cells- mostly cancerous cells red and immature cells green)
10. 95% alcohol (2 rounds to provide clearer view of overlapping cells)
11. 100% alcohol (2 rounds for final dehydration)
12. Xylene (2 rounds for clearing to allow microscopic examination)

Results: Nuclei- blue or black

Cytoplasm (non-keratinised)- green or blue

Cytoplasm (keratinised)- pink or orange

Red blood cells- orange

Quality Control of PAP Staining

1. Checking of nuclear staining

• Staining is crisp and sharp
• Parachromatin is clear (not muddy)

2. Cytoplasmic counterstains

• Sharp and clean
• 3 distinct colours: red (Eosin-Y); orange (OG); green (EA)

Troubleshooting Guidelines for PAP Staining

1. Problems with absorption of haematoxylin due to inadequate removal of carbowax resulting in irregular and spotty staining (poor quality).

• Soak in 95% ethanol for complete fixation to enable the cells to have better absorption of haematoxylin

2. Lack of contrast in nuclear staining due to poor quality of haematoxylin.

• Replace water rinses used for blueing purposes
• Tap water should be slightly alkaline. Acidic tap water will result in nuclear fading while heavily chlorinated tap water will bleach out the haematoxylin.

3. Problems with cytoplasmic stains due to improper rinsing of stains after OG and EA

• Do not allow the slides to sit in alcohol solution following OG and EA stains as it can wash the stains away
• Rotate ethanol rinses as ethanol nearest the dye becomes discoloured

4. Water droplets seen on slides due to water in xylene (xylene will appear milky)

• Change the xylene
• Add Silica-Gel pellets to absolute alcohol to minimize the possibility of water contamination of xylene

That's all! Enjoy your SIP! Take care! ^_^

June Tham

TG02

0505073G

Embedding and Microtomy

Embedding

Introduction
After the completion of the processing cycle, the tissues are removed from the tissue processing machines to the blocking room for embedding. Each batch of blocks is tallied against the listing of the blocks for the corresponding batch to ensure that no blocks are missing before embedding.

Principles
The tissue is placed in molten paraffin (56* melting point) such that after the paraffin cools, the tissue and paraffin will form a block of sufficient consistency to cut thin sections

Materials
Embedding machine
Embedding moulds
Forceps

Embedding technique
1. Open the processing cassette carefully and view the tissue.
2. Select a mould that best corresponds to the size of the tissue.
3. Partially fill the mould with paraffin.
4. With warm forceps remove the tissue from the cassette and place it at the bottom of the mould – refer to specimen orientation below.
5. Gently press the surface of the tissue against the solidifying wax to hold it in the desired position.
6.Ensure no tissue is stuck on the lid then discard the lid of the cassette and place the bottom of the cassette firmly on the top of the mould.
7. Fill the combined mould and cassette with paraffin.
8. Place the mould on ice to solidify the wax, thus separating embedding tissue-cassette from the mould.
9. The tissue and cassette forms a paraffin block ready for sectioning.

Specimen orientation
1. Tissue sections are embedded flat to ensure that complete section is obtained.
2. Orientation should be such that the resistance the tissue offers the knife proceeds from the lesser amount towards the greater amount as the block is sectioned. This prevents the harder tissue from compressing the softer tissues and produces a smoother section.
3. There should be an adequate margin of embedding medium surrounding all sides of the tissue for maximum cutting support.
4. Tubular structures such as vas deferens, veins, arteries and fallopian tubes must be embedded such that the knife cuts across the lumen. These should be placed vertically in the mould.
5. Tissues with epithelial surfaces such as skin, intestine, gallbladder, urinary bladder and uterus must be positioned such that the plane of the section is across all tissue layers. The epithelial surface should be placed such that the plane of section is perpendicular.
6. Multiple specimens should be placed side by side close together so that call pieces can be sectioned.
7. Rectangular tissues should be orientated parallel to each other and with their long axis perpendicular to the plane of section.
8. Small bisected cysts should be embedded with the cut surface down and ensure that no air bubbles are trapped in the paraffin.
9. Muscle biopsy in 2 pieces should be embedded with one piece in a longitudinal and the other in a vertical position.

Microtomy

Introduction
Paraffin blocks from the blocking room are brought to the cutting room for sectioning

Principles
Microtomy is the use of a microtome to make thin sections for microscopy. Rotary microtomes are used in the laboratory

Materials
Room Temperature floatation bath
Warm floatation bath – 48 plus-minus 4 degrees Celsius thermostatically controlled
Cryoplate
Disposable microtome blade
Microscope glass slides
Rotatry microtome
Soft pencil

Solution preparation
1. Type II Water 2000ml

Allow the water in the floatation bath to reach 48 plus-minus 4 degrees Celsius on a thermostat before sectioning. Technologist must record this temperature on the temperature chart before sectioning

2. 1% alcohol floatation bath
95% alcohol 5ml
Type II Water 500ml

This is used to float the tissue section prior to transferring on the heated floatation bath above. It is an alternative when folds on the tissue are difficult to get rid, as alcohol having low vapour pressure will increase the surface tension when transferring the tissue onto the heated floatation bath.

Microtomy Technique
1. Rough cutting – Secure the paraffin block in the block holder of the microtome. Adjust it to ensure that it clears the knife. Readjust the block holder screws to place the block parallel to the knife if necessary. During the rough-cut, while being manually advanced, the block is repeatedly sectioned at 20 microns thickness per slice. Sectioning stops when the entire surface of the tissue is exposed. The block is then removed from the holder.
2. All blocks should be rough cut before sectioning because:

a.Dense and hard tissue that may cause nicks and blunt the knife more rapidly can be identified e.g. bone, cervix and thyroid. Such blocks could be separated from the rest and appropriately pre-treated before sectioning. These blocks should be sectioned last as they may still cause nicks and score lines to the blade.
b. Sutures and staples attached to the surgical specimens may not have been removed and can be detected during rough cutting.
c. Hard bone is identified and additional surface decalcification can be done, by placing the block face down in decalcifying agent e.g. RDO for further decalcification prior to sectioning.
d. Fibrous tissue e.g. fibroids can be softened in mollifex or 10% fabric softener for 5 minutes. The paraffin blocks are washed in water after the appropriate treatment porior to sectioning.
3. Chilling – the blocks are then placed face down on the cryoplate to chill the block to facilitate fast sectioning. This renders the block sufficiently hard for thin sections.
4. Sectioning – secure and adjust the block in a similar manner as rough cutting. Using the handwheel, section the block at 3-4 microns, by allowing the block to advance automatically. Some tissue biopsies are sectioned at different thickness.
5. Flotation- gradually lower the section or sections if a ribbon is cut, onto the water bath. If difficulty is encountered in spreading of the tissue, float the section on the alcohol bath first so as to increase the surface tension before transferring onto the water bath. Allow the section to remain on the water bath until it has spread sufficiently. Transfer the section onto a glass slide. The corresponding biopsy number is written on the frosted end of the glass slide.
6. Separation of slides – Slides for routine HE stain, special stains and unstained sections should be separated on different racks.

Serial Sections
First submission
Tissues less than 0.5cm in size
Gastric biopsies
i. 1 HE slide with 6 serial sections
Liver biopsies
i. Slide 1 – 2 consecutive complete sections for HE
ii. Slide 2 – 1 section for MT
iii. Slide 3 – 1 section for Ret
iv. Slide 4 – 2 consecutive complete sections for HE
Bone marrow
i. Section at 2um thick
ii. 1 HE slide with 6 serial sections for small specimen or 3 HE sections on 3 slides labelled VL1, VL2, VL3 for larger specimens and 1 slide for Retic
Renal biopsies
i. Section at 2um thick
ii. 10 slides with 3 sections each as follows:
1. HE on slide 2, 5 and 8
2. Pas on slide 1m 6 and 9
3. PaAg on slide 4
4. PgMT on slide 3, 7 and 10
TBLB – Trans bronchial lung bx
i. Section at 5um thick
ii. 10 slides with 2 sections each as follows:
1. HE on slide 2, 5 and 8
2. TB stain on slide 10 for TB cases only
Endomyocardial bx
i. Section at 5um thick.
ii. 10 slides with 2 sections each as follows:
1. HE on slide 4, 6, 10
2. MT on slide 7
Prostatic needle bx
i. 1 HE slide with 6 serial sections
Tissue biopsies less than 0.5cm in size not specificed above
i. 1 HE slide with 6 serial sections
Larger tissue biopsies
i. 1 HE slide
Request for variable levels
For small tissue approximately less than 0.5cm in size
i. Repeat 6 serial sections on 1 slide labelled VL 1 for HE
For larger tissue approximately more than 0.5cm in size
i. Repeat 3 serial sections on 3 slides labelled VL1, VL2 and VL3 respectively for HE
Request for special stains
Addition sections will be cut and stained as requested
For additional levels of routine biopsy specimens or irregular number of sections required, serial sections will be cut according to the request of the pathologist.
All personnel receiving verbal or phone orders must read back the entire order to verify accuracy and transcription.

Desmond Heng
TG02
0503179D