Monday, October 29, 2007

SIP blogging

Hello to all, this week I will be blogging on body fluids for examination. Body cavity fluids other than blood or urine are referred as extravascular fluids and they are:
1.) Cerebrospinal (around the brain and spinal cords)
2.) Synovial (around the joints)
3.) Pleural (around the lungs)
4.) Peritoneal (around the abdominal and pelvis cavities)

This blog i going to talk about synovial fluid.

Normal synovial fluid is clear straw-coloured or pale yellow fluid found in small amount in joints and tendons sheaths. It is an ultrafiltrate of plasma plus a mucopolysaccharide, produced by the lining synovial cells which make the fluid thick and viscous. Normal volume of fluid in knee joints is about 3.5ml.

Function of synovial fluid is to lubricate the joint space, transport nutrients to the articular cartilage, remove waste and debris from joint and medium for leucocytes to circulate and phagocytize debris.

Clinical significance for synovial fluid analysis is in the differential diagnosis of swollen joint: distinguish crystal-inducing disease from septic joints.

Sample Collection/Preparation
Synovial fluid is collected by aspiration of fluid from the joint space with a needle. Due to cells are easily destroyed and glucose may also undergo glycolysis on standing, thus the analysis of fluid must be done immediately once received.

Laboratory Analysis of Synovial Fluid
1. Appearance

First, state the appearance and clarity of the fluid received. Synovial fluid can be described as:
- Straw colour or pale yellow and clear is the normal appearance of synovial fluid
- Turbid or purulent sample is seen in bacterial infection or high leucocytes count.
- Blood-stained
- Chylous fluid refer to having a characteristics milky, opaque appearance which remains in the supernatant after centrifugation, which is caused by lymphatic leakage or obstruction.
If the fluid is blood stained or turbid, spun it down and state the appearance of the supernatant.

2. Cell Count and WBC Differential

A cell-count with differential would aid in making the diagnosis as bacterial infections will have a predominance of neutrophils while viral, fungues and mycobacterial infections may have a predominance of lymphocytes or show a mixed inflammatory response.

If the effusion is predominantly neutrophils, an acute inflammatory process is the cause and differential count showing essentially all lymphocytes suggests a chronic process.

3. Glucose

Fluid glucose is reduced due to bacterial, increased WBCs or infiltration with malignant cells the reduction is the result of the metabolic requirement of the infecting organisma and of the inflammatory cells as well as the tumour cells.

4. Total Protein

Total protein is abnormally raised in: infection, malignant infiltration, chronic inflammatory conditions or traumatic tap (false positive)

Differentiation of exudate and transudate fluid

5. Crystals

The 3 most common types of crystals present in the joint fluid are:
- Monosodium urate
- Calcium pyrophosohate
- Cholesterol

Monosodium Urate Crystals(MSU):
These crystals are needle shaped, double refractile of 8-10um long, negatively birefringent and soluble in water. They are associated with gout and may be intracellular. MSU crystals are negatively birefringent and when aligned parallel to the compensator will show a yellow colour and when turned perpendicular to the compensator the colour change to blue.

Calcium Pyrophosphate Dihydrate Crystals(CPPD):
These are associated with pseudogout, which most frequently involves the wrists and knees. These crystals have various shapes but usually rhomboid in shape and are positively birefringent. They are broader than uric acid crystals and up to 25um long, have a line running through them. The crystals are blue in colour when parallel to the compensator and yellow when perpendicular to it.

Cholesterol Crystal:
Cholesterol crystals have a characteristics notched-plate and birefringent. They are present in chronic inflamed joints such as rheumatoid arrthritis. In pseudogout, serum uric acid is normal while serum uric acid is high in gout. Cholesterol crystals may be found in any chronic effusion.

6. Summary:

Normal Synovial Fluid
- Clear , yellow fluid which does not clot spontaneously
- UP to 200 WBC/uL of which less than 25% are neutrophils
- No Crystals
- Total protein: 18g/L
- Glucose level is similar to serum glucose level


Friday, October 19, 2007

SIP- Histopathology

Hey everyone! I shall touch on the 2 special stains that I did when I was attached to Special Staining. They are namely the Liver Orcein stain and the Victoria Blue stain.

Liver Orcein Stain

Function: Demonstrates Hep B surface antigen and elastic tissues

Principle: The disulphide bridges in the elastic tissue are broken down into its anionic derivatives during oxidation. These derivatives can then be stain by Orcein stain. Virus inclusion bodies can be stain by Orcein after oxidation.

Control: A known Hep positive liver tissue

Fixative: 10% buffered neutral formalin

Chemicals required: Orcein solution
0.5% potassium permanganate
1% oxalic acid
3% sulphuric acid

1) Dewax (removal of wax) and bring tissue section to water
2) Place in acidified potassium permanganate solution for 5 mintues (section will appear brown in colour)
3) Wash in water
4) Bleach section till colourless in 1% oxalic acid
5) Wash in water
6) Stain in Orcein solution for 4 hours at room temperature or overnight in fridge
7) Wash quickly in 2 changes of absolute alcohol
8) Dehydrate, clear then mount

Results: Coarse elastic fibers- reddish brown
Fine elastic fibers- dark brown
Hep B surface antigen- dark brown

Victoria Blue Nuclear Fast Red

Function: Stain elastic fibers

Principle: Elastic fibers are highly cross-linked by disulphide bridges. Following oxidative treatment, these bridges may convert into anionic sulphuric acid derivatives. These derivatives are strongly basophilic and capable of selective reactions with the basic dyes.

Control: Skin and artery for elastic fibers demonstration. Inflamed skin for mast cells demonstration.

Fixative: 10% buffered neutral formalin

Chemicals required: 0.5% potassium permanganate
3% sulphuric acid
4% sodium bisulphate
VB solution
Nuclear Fast Red

1) Dewax and bring section to water
2) Stain with acidified potassium permanganate solution for 5 minutes (section will appear brown in colour)
3) Bleach with 1% sodium bisulphite until colourless
4) Wash in running water
5) Dry on hotplate
6) Leave in VB solution for 24 hours
7) Differentiate in 70% alcohol
8) Wash in running water
9) Stain with Nuclear Fast Red for 5 minutes
10) Rinse in running water
11) Dehydrate, clear then mount

Results: Elastic fibers, lipofuchsin and mast cells- blue
Cytoplasm and nuclei- red

This is the end of my posting. Thanks for reading! Enjoy the rest of your SIP! =)

*I want to make an important note. According to my colleague, the Liver Orcein Stain will be used to stain the HepB antigen while the VB Stain is only used for elastic fibers demonstration.

Tham Wan Jin June

Sunday, October 14, 2007

SIP Week 16 Blog Posting - Special Stains in Histopathology

Hey poly peeps, Desmond here. It's week 16 and just another 4 more weeks before we go back to campus. Having covered Cytopathology and Histopathology routine (embedding and microtomy) in my 2 previous posts, I've decided to touch on some of the special stains performed in Histopathology.


This stain differentiates Gram positive and Gram negative bacteria

Both bacteria’s, positive and negative cell wall is composed of peptidoglycan, (the gram positive has a thicker wall) and both will take up the crystal violet. The gram-negative has a layer of lipopolysaccharide external to the peptidoglycan wall, which is disrupted in the acetone rinse, allowing the crystal violet to be differentiated out. This allows the gram-negative bacteria to take up the fuchsin stain.

An infected appendix, or any tissue containing both negative and positive gram rods.

1. 10% Buffered Neutral Formalin

Reagents required:
1% aqueous crystal violet solution
1% Basic fuchsin
Potassium iodide
Formalin 37-40%
Glacial acetic acid
Picric acid

Reagent Preparation:
1. Gram iodine solution
Iodine 1g
Potassium iodide 2g
Type II water 300ml
Allow iodine and potassium iodide to dissolve completely. Mix before use. Solution is stable for 6 months at room temperature.

2. Gallego’s differentiating solution
Type II water 100ml
Formalin 37-40% 2ml
Glacial acetic acid 1ml
Mix before use. Solution is stable for 6 months at room temperature

3. Picric acid-acetone solution
Picric acid 1g
Acetone 100ml
Solution is stable at room temperature for 6 months.

4. Acetone-Xylene solution
Equal parts of acetone and xylene.
Solution is stable at room temperature for 6 months.

5. 1% Crystal violet
Crystal violet 1g
Type II water 100ml
Filter into bottle. Solution is stable for 1 year at room temperature.

6. 1% Basic Fuchsin
Basic Fuchsin 1g
Type II water 100ml
Filter into bottle. Solution is available for 6 months at room temperature.

Staining Procedure:
1. Dewax and bring section to water.
2. Place in 1% crystal violet solution for 1 minute.
3. Rinse in tap water.
4. Place in Gram iodine solution for 1 minute
5. Rinse in tap water.
6. Decolourize in acetone until background is clear.
7. Immediately wash in tap water.
8. Place in 1% Basic Fuchsin solution for 5 minutes.
9. Rinse in tap water.
10. Place in Gallego’s differentiating solution, 2 changes, 1 minute each.
11. Rinse in tap water.
12. Transfer to a staining dish.
13. Treat with acetone for 30 seconds.
14. lace in picric acid-acetone solution for 2-3 minutes.
15. Place in acetone-xylene solution for 2 changes.
16. Clear in xylene, 2 changes.
17. Mount in DPX.

Gram positive - Blue
Gram negative - Red
Background - Yellow

Ziehl Neelsen Stain

To demonstrate acid fast bacteria belonging to the genus mucobacterium

The lipid capsule of the acid-fast organism takes up carbol-fuchsin and resists decolourization with a dilute acid rinse. The lipoid capsule of the mycobacteria is of such high molecule weight that it is waxy at room temperature and is not stained by bluing solutions such as methylene blue.

Any tissue containing acid-fast organisms

1. 10% Buffered Neutral Formalin

Reagents required:
Commercial TB stains (Merck)
Loeffler’s Methylene Blue
1% Potassium Hydroxide
1% acid alcohol

Reagent Preparation:
1. Stock Loeffler’s Methylene Blue solution
Methylene Blue 1gm
95% Alcohol 100ml
2. 1% Potassium Hydroxide
Potassium Hydroxide 1gm
Type II water 100ml
3. Working solution
1% Loeffler’s Methylene Blue 30ml
Type II water 99ml
1% Potassium Hydroxide 1ml
4. 1% Acid Alcohol
Concentrated Hydrochloric Acid 20ml
70% Alcohol 1980ml

Staining Procedure:
1. Dewax and bring sections to water.
2. Commercial TB colour – 5 minutes (or hot carbol fuchsin solution for 30 mins)
3. Differentiate with 1% alcohol
4. Wash in water
5. Couterstain with 1% Loeffler’s methylene blue.
6. Wash in water and go straight to 95% alcohol to control blue colour.
7. Dehydrate clear and mount in DPX.

Tubercle bacilli - Red
Background - Blue


Stains Bone marrow lymph node. Differentiates cells present in hematopoietic (lymph nodes).

The neutral dyes combining the basic dyes methylene blue and dye eosin, give a wide colour range when staining. The more acid pH levels give more selective chromatin staining and less cytoplasmic basophilla. Less acid pH levels give denser nuclei and increased cytoplasmic basophilla.


1. 10% Buffered Neutral Formalin

Reagents required:
Commercial Giemsa Stain

Reagent Preparation:
Working Giemsa solution (for 5 slides or less)
Giemsa Solution 3ml
Type II water 12ml
Mix and use in plastic slides mailer. Discard after use.

Staining Procedure:
1. Dewax and bring section to water.
2. Rinse section in Type II water
3. Place sections in working Giemsa solution for 1 hour at room temperature.
4. The sections are removed from the Giemsa solution and put into 100ml Type II water, to which 3-4 drops of undiluted glacial acetic acid have been added. The sections are agitated gently in this solution for a few seconds, slightly differentiated and then immediately put into 96% ethyl alcohol, in which they are differentiated further until the desired staining is achieved (microscopic control).
5. Differentiation is stopped and, at the same time, dehydration is achieved by dipping in 3 changes of isopropanol for 2 minutes each.
6. Dehydrate, clear and mount with DPX.

RNA, DNA - Blue (Basophillic)
Acidophilic substances - Pink or reddish orange
Acid mucopolysaccharides - Reddish violet


Stains Bone marrow and gastric biopsies. To demonstrate helicobacter. To differentiate cells present in hematopoeietic tissue (lymph nodes). Use in blood smears.

The neutral dyes combining the basic dyes methylene blue and dye eosin, give a wide colour range when staining. The more acid pH levels give more selective chromatin staining and less cytoplasmic basophilla. Less acid pH levels give denser nuclei and increased cytoplasmic basophilla. Methylene blue in an alkaline pH solution stains metachromatic.

Stomach or colon tissue containing helicobacter. Skin for mast cells.

1. 10% Buffered Neutral Formalin

Reagents required:
Commercial Jenner solution
Commercial Giemsa solution
1% acetic acid

Reagent Preparation:
1. Jenner working solution
Stock Jenner Solution 2ml
Type II water 2ml
Mix and use. Discard after use.
2. Giemsa working solution
Stock Giemsa solution 2ml
Type II water 40ml
Mix and use. Discard after use.

Staining Procedure:
1. Dewax and bring sections to water.
2. Place slides in 2 changes of methyl alcohol for 3 minutes,
3. Dip direct into working Jenner solution for 5 to 6 minutes.
4. Transfer direct to working Giemsa solution for at least 45 minutes.
5. Rinse in distilled water. Check under microcope and monitor differentiation
6. Differentiate in 1% acetic acid.
7. Dip in 95% alcohol for 5 to 6 times.
8. Dehydrate clear and mount in DPX.

Cytoplasm - Pink
Nuclei - Blue
Erythrocytes - Red
Mast Cell granules - Purple
Bacteria - Blue
Malaria Parasite - Blue

Desmond Heng

Saturday, October 6, 2007

Student Internship Programme (SIP) HAEM

Neutrophil Alkaline Phosphatase (NAP) stain
Intended use
NAP score is used to distinguish Chronic Granulocytic Leukaemia from other myeloproliferative disorders and Polycythaemia Rubra Vera from secondary polycythaemia.

At an alkaline pH, NAP hydrolyses substrate naphthol AS-BI phosphate to liberate napthol-AS. The liberated naphthol couples with a diazonium salt, Fast Blue BB to produce an insoluble complex which precipitates at the site of enzyme activity. The sites of alkaline phosphatase activity will appear as blue granules. The NAP score is the sum of the rating of 100 consecutive segmented and band neutrophils using a scale of 0 to 4+ according to the appearance and intensity of the precipitated dye.

Specimen collection and handling conditions
1. Fresh capillary/venous blood preparation, not anticoagulated is required.
2. EDTA blood is not recommended as enzyme activity is inhibited.

Preparation of stock substrate solution
a) Dissolve 30mg naphthol AS phosphate in 0.5ml N,N-dimethylformamide. CAUTION: N,N-dimethylformamide is toxic by inhalation. To be prepared in fume hood.
b) Add 0.2M tris buffer, pH 9.1 to bring volume to 100ml.
c) Store at 4-10 deg celcius. Stable for 2 months.

Preparation of working substrate
a) Add 10mg Fast Blue BB to 10ml stock substrate solution. Mix well. CAUTION: Fast Blue BB is carcinogenic and a possible mutagen. To be prepared in fume hood.
b) Prepare fresh when needed.

Quality control
A normal control smear must be included with every batch of stain and results should fall within tolerance limits.

1. Fix air-dried smear in buffered 60% Acetone in Citrate brought to room temperature for at least 5 mins.
2. Rinse with tap water and dry slide.
3. Using filter paper No.41, filter the freshly prepared working substrate directly onto slides. Stain for 15 mins at room temperature. Discard working solution into the toxic waste container.
4. Wash in tap water.
5. Counterstain with 0.5% neutral red for 1 min.
6. Wash and dry.
7. Count under oil immersion 100 neutrophils. The slides are examined and counted by 2 technologists and an average of 2 results is reported.

Sites of enzyme activity are represented by discrete bright blue granules of varying sizes. Nucleus is stained red. The enzyme is present predominantly in segmented neutrophils. Cytoplasmic granules of eosinophils do not stain while basophils cannot be differentiated from other granulocytes.

One hundred segmented or band forms of neutrophils are rated. The sum of 100 cell ratings give a score with a possible range from 0-400.

Normal range: 30-100

Clinical significance
Chronic Granulocytic Leukaemia and Paroxysmal Nocturnal Haemoglobinuria are associated with abnormally low or absent staining. Conversely, leukaemoid reactions, polycythaemia vera and myelofibrosis are associated with markedly elevated NAP scores. High scores can also be obtained from neonates, pregnant women or women taking oral contraceptives.

1. Do not make the smear too thick to avoid inadequate fixation.
2. Fixed smears should be stored at -20 deg celsius if staining delayed for >5-6 hours.
3. Scoring of enzyme activity should be made in areas of slide with optimal cell morphology.

Answers to possible questions

Why must the working solution be prepared fresh before every staining?
Precipitation will occur over time once the Fast Blue BB is added to the stock substrate solution that will be indicated by a colour change from bright yellow to dull brown. Therefore it is important that the working solution be mixed and applied fast.

What are the various illnesses stated?
Please visit Wikipedia or other search engines to administer your enquiries. I'm sure you will learn more than what I'll be able to explain this way. :)

I've also taken some pictures and will seek to post them as soon as possible once I get relevant approval. Hope you've learnt something!
Alex Tan 0503222B TG02