1. HbA: 96-98%, Composed of two alpha(α) and two beta(β) globin chains.
2. HbA2: 1.5-3.5%, Composed of two alpha(α) and two delta(δ) globin chains.
3. HbF: < 1.0%, Composed of two alpha(α) and two gamma(γ) globin chains.

Hemoglobin can combine with other substances, some normally and some abnormally and can also occur as:

1. Oxyhemoglobin: Oxygen combined with hemoglobin.
2. Carboxyhemoglobin: Carbon monoxide (CO) combined with hemoglobin.
3. Carbaminohemoglobin: Carbon dioxide (CO₂) combined with hemoglobin.
4. Methemoglobin: Iron oxidized from its ferrous state to ferric state.
5. Sulfhemoglobin: Sulfur combined with the hemoglobin.
6. Cyanmethemoglobin: Methemoglobin bonded to cyanide ions.

Methods for Hemoglobin Estimation:

The different methods used for the estimation of hemoglobin can be divided as follows:

1. Visual methods:
   • Sahli’s method
   • Dare method
   • Haden method
   • Wintrobe method
   • Tallqvist method
2. Spectrophotometric methods:
   • Oxyhemoglobin method
   • Cyanmethemoglobin method
3. Gasometric method
4. Automated hemoglobinometry
5. Other methods:
   • Alkaline-hematin method
   • Specific gravity method
   • Lovibond Comparator method

Sahli’s method for hemoglobin estimation

Sahli’s method, also called as acid hematin method is the visual comparator method for the estimation of hemoglobin. As visual comparison may lead to unacceptable imprecision and accuracy, this method is not recommended nowadays and the use of spectrophotometric methods like Cyanmethemoglobin method is preferred to it.

Principle:

When the blood is added to dilute hydrochloric acid (HCl), hemoglobin present in the RBCs is converted into brown-colored acid hematin. The acid hematin solution is further diluted until it’s color matches exactly with the permanent standard brown glass compared by direct vision.

Requirements:

Specimen:

Capillary or venous blood. Venous blood should be anticoagulated with 1.5-1.8 mg EDTA per mL of blood and mixed immediately.

Instruments:

1. Sahli’s hemoglobinometer
   It is a set of devices that includes a comparator, hemoglobin tube, hemoglobin pipette, and stirrer.
   
   • Comparator: It is a rectangular plastic box with a slot in the middle which accommodates a hemoglobin tube. Brown standard glasses are provided on either side of the slot for color matching. White opaque glass is present at the back to provide uniform illumination.
   • Hemoglobin tube: Sahli’s graduated hemoglobin tube is graduated in one side in gram percentage (g%) from 2 to 24, and on the other side in percentage (%) from 20 to 140. The tube is also called Sahli-Adams tube.
   • Sahli’s pipette or hemoglobin pipette: It contains only one mark at 20μl or 0.02ml. Unlike WBC and RBC diluting pipettes, it contains no bulb.
   • Stirrer: It is a thin glass rod used for stirring the mixture inside the hemoglobin tube.

Reagents:

1. N/10 Hydrochloric acid (HCl): Mixing 36 grams HCl in distilled water to 1 liter gives 1 N HCl. Diluting it 10 times will give N/10 HCl.
2. Distilled water

Procedure:

1. Ensure that the hemoglobinometer tubes and pipette are clean and dry.
2. Fill the hemoglobinometer tube with N/10 HCl up to its lowest mark i.e. 2 g% or 10% mark with the help of a dropper.
3. Take blood up to mark in the Sahli’s pipette (20 μl). Wipe the extra blood outside the pipette and deliver it to N/10 HCl in the hemoglobin tube.
4. Mix and leave it for 10 minutes in order for a complete conversion of hemoglobin to hematin.
5. Add distilled water drop by drop and stir till color matches with the standard glass of the comparator.
6. Take the reading at lower meniscus, which directly gives the hemoglobin concentration in 100 ml of blood.

Advantages and Disadvantages of Sahli’s method

Advantages:

1. Easy to perform and convenient.
2. Not very time consuming. Can be performed within maximum 15 minutes.
3. Reagents and apparatus are cheap and easily available. Reagents are less harmful.
4. Can be used in mass surveys. Doesn’t require electricity.

Disadvantages:

1. Acid hematin is a suspension, not a true solution. So, some turbidity may result.
2. This method can’t measure all hemoglobins. It estimates only oxyhemoglobin and reduced hemoglobins. But carboxyhemoglobin, methemoglobin and sulfhemoglobin (which all constitute about 2-12% of total hemoglobin) are not converted to acid hematin.
3. HbF is not converted to acid hematin. Therefore, Shali’s method is not suitable for measuring hemoglobin in infants upto 3 months.
4. WBC counts >100,000/cumm will produce turbid solution of acid hematin, which will increase the hemoglobin report by 5-10%.
5. Chances of visual error are high. Color of acid hematin also fades gradually.
6. The color of glass standard may fade over time.

References:

1. Pal, G.K., 2006. Textbook Of Practical Physiology-2Nd Edn. Orient Blackswan.
2. Ghai, C.L., 2012. A textbook of practical physiology. JP Medical Ltd.
3. A. V. Naigaonkar. A Manual Of Medical Laboratory Technology.
4. Cheesebrough, M., 1998. District laboratory practice in tropical countries, part II. Cambridgeshire Tropical Health Technology, Cambridge, UK, 231.
5. Laboratory Procedures in Clinical Hematology By United States. Department of the Army
6. Mohan, H. and Mohan, S., 2011. Practical Pathology for Dental Students. JP Medical Ltd.

The post Sahli’s Method For The Estimation Of Hemoglobin appeared first on LaboratoryTests.org.

1. HbA: 96-98%, Composed of two alpha(α) and two beta(β) globin chains.
2. HbA2: 1.5-3.5%, Composed of two alpha(α) and two delta(δ) globin chains.
3. HbF: < 1.0%, Composed of two alpha(α) and two gamma(γ) globin chains.

Hemoglobin can combine with other substances, some normally and some abnormally and can also occur as:

1. Oxyhemoglobin: Oxygen combined with hemoglobin.
2. Carboxyhemoglobin: Carbon monoxide (CO) combined with hemoglobin.
3. Carbaminohemoglobin: Carbon dioxide (CO₂) combined with hemoglobin.
4. Methemoglobin: Iron oxidized from its ferrous state to ferric state.
5. Sulfhemoglobin: Sulfur combined with the hemoglobin.
6. Cyanmethemoglobin: Methemoglobin bonded to cyanide ions.

Methods for Hemoglobin Estimation:

The different methods used for the estimation of hemoglobin can be divided as follows:

1. Visual methods:
   • Sahli’s method
   • Dare method
   • Haden method
   • Wintrobe method
   • Tallqvist method
2. Spectrophotometric methods:
   • Oxyhemoglobin method
   • Cyanmethemoglobin method
3. Gasometric method
4. Automated hemoglobinometry
5. Other methods:
   • Alkaline-hematin method
   • Specific gravity method
   • Lovibond Comparator method

Cyanmethemoglobin method for hemoglobin estimation

The cyanmethemoglobin method or hemoglobincyanide method is the most accurate method of choice for the measurement of hemoglobin. It is a type of colorimetric/spectrophotometric method which has the following three major advantages over other methods:

1. Measures all forms of hemoglobin except sulfhemoglobin, which is normally not present in the blood.
2. Can be easily standardized, and
3. Cyanmethemoglobin reagent is very stable.

Principle:

Blood is diluted 1:201 in a solution containing potassium ferricyanide (C₆N₆FeK₃) and potassium cyanide (KCN). Potassium ferricyanide oxidizes hemoglobin in the sample to methemoglobin. The methemoglobin further reacts with potassium cyanide to form a stable-colored cyanmethemoglobin (hemiglobincyanide- HiCN) complex. The intensity of the colored complex is measured at 540 nm which is directly proportional to the amount of hemoglobin present in the specimen.

Requirements:

Specimen:

Capillary or venous blood. Venous blood should be anticoagulated with 1.5-1.8 mg EDTA per mL of blood and mixed immediately.

Reagents:

1. Drabkin’s reagent, pH 7.0–7.4
   The original cyanmethemoglobin technique was proposed by Stadie in 1920. This method used separated alkaline ferricyanide and cyanide reagents. A single reagent was introduced by Drabkin and Austin in 1935.

   This fluid contains:

   • Potassium ferricyanide = 200 mg
   • Potassium cyanide = 50 mg
   • Potassium dihydrogen phosphate = 140 mg
   • Non-ionic detergent = 1 ml
   • Distilled or deionized water = to 1 liter
2. Hemoglobin standard

Procedure:

1. Label three clean, dry test tubes as Blank (B), Standard (S), and Test (T).
2. Pipette as follows:

	Blank	Standard	Test
Drabkin’s Reagent	5 ml	5 ml	5 ml
Hemoglobin standard	–	20 µl	–
Sample	–	–	20 µl

3. Mix well and allow to stand at room temperature (25⁰C) for 5 minutes.
4. Measure the absorbance of the standard and test sample at 540 nm (green filter) against blank in a colorimeter. The color will be stable for up to several hours.

Calculation:

Calculate the concentration of hemoglobin in the specimen using the following formula:

Alternatively, a calibration graph can be prepared using multiple standards of varying concentration and the result can be obtained quickly by checking hemoglobin concentration which corresponds to obtained absorbance. This is markedly acceptable when a large number of specimens are daily processed on the same instrument.

References:

1. Pal, G.K., 2006. Textbook Of Practical Physiology-2Nd Edn. Orient Blackswan.
2. DR .Sara Fadhil Bunea, Medical laboratory techniqes, Human physiology practical.
3. Haemoglobin Reagent Product Insert – Tulip Diagnostics

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Principle of Colorimeter :

When a beam of incident light of intensity I₀ passes through a solution, following events occur:

• A part of incident light is reflected. It is denoted by I_r
• A part of incident light is absorbed. It is denoted by I_a
• Remaining incident light is transmitted. It is denoted by I_t

As I_r is kept constant by using cells with identical properties, The light that is not absorbed is transmitted through the solution and gives the solution its color. Note that color of the incident light should be complementary to that of color of the solution.

The ratio of the intensity of transmitted light (I_t) to the intensity of incident light (I₀) is called transmittance (T). Photometric instruments measure transmittance. In mathematical terms,
T = I_t÷I₀

The absorbance (A) of the solution (at a given wavelength) is defined as equal to the logarithm (base 10) of 1÷T. That is,
A = log (1÷T)

These measurements are dependent on two important laws:

1. Beer’s law:

   When monochromatic light passes through a colored solution, the amount of light absorbed is directly proportional to the concentration (C) of solute in the solution.

2. Lambert’s law:

   When monochromatic light passes through a colored solution, the amount of light absorbed is directly proportional to the length (L) or thickness of the solution.

When combining Beer-Lambert’s law,
Absorbance (A) α CL
Or, A= KCL
where K is a constant known as absorption coefficient.

As the path length is same (as same cuvette is used), Concentration of an unknown solution can be determined by using equation:

Instrumentation of Colorimeter :

1. Light Source:

The light source should produce energy at sufficient intensity throughout the whole visible spectrum (380-780nm). Tungsten lamp is frequently used.

2. Slit:

It allows a beam of light to path and minimize unwanted light.

3. Condensing lens:

Give parallel beam of light.

4. Monochromator:

It is used to produce monochromatic radiation (one wavelength band) from polychromatic radiation (white light) produced from light source. It allows required wavelength to pass through it. Prism, gelatin fibers, grating monochromators or interference filters can be used.

5. Sample Holder (Cuvette):

Must be transparent. Glass or clear plastic cuvettes are preferred.

6. Photo detectors:

Detector of colorimeter basically receives the resultant light beam once it has passed through the sample and converts it into electrical signal. Selenium photocell, silicon photocell, phototube, photomultiplier tube etc are used.

7. Display:

It detects and measures the electric signal and makes visible output.

Uses and Applications

In clinical laboratory, colorimeter is used for the estimation of various biochemical compounds in variety of biological samples like blood, plasma, serum, CSF, urine and other body fluids. All those methods which involve the formation of colored product with specific analyte, the analyte can be estimated quantitatively. Colorimeters are also widely used for monitoring the growth of bacterial or yeast cells in liquid cultures.

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Different methods based on different properties of glucose are described for blood glucose estimation. They are:

1. Reduction Methods:

• Ferric reduction methods
1. Hagedorn-Jensen ferric reduction method
2. Hoffman’s method
• Cupric reduction methods
1. Somogyi-Nelsen method
2. Neocuproine method
3. Shaffer-Hartmann method
4. Folin-Wu method
5. Benedict’s method

2. Aromatic amine condensation methods:

1. O-toluidine method

3. Enzymatic Methods:

1. Glucose-oxidase Peroxidase (GOD POD) method (Trinder method)
2. Hexokinase method
3. Glucose dehydrogenase (GDH) method
4. Kinetic method
5. Polarographic method

4. Electrochemical methods:

1. Glucometer

GOD-POD Method for Glucose Estimation

Being more specific, easier, and more accurate, enzymatic methods are preferred these days. Among them, the GOD-POD method is the most common method of glucose estimation.

Principle:

In the presence of atmospheric oxygen, glucose present in the specimen is oxidized by the enzyme glucose oxidase (GOD) to gluconic acid and hydrogen peroxide (H₂O₂).

Thus formed H₂O₂ oxidatively couples with 4-aminoantipyrine and phenol in presence of peroxidase (POD) to form red-colored quinoneimine dye, which is measured colorimetrically at 540nm. The intensity of the color is directly proportional to the concentration of glucose present in the specimen.

Requirements:

Specimen:

Serum, or plasma free of hemolysis. Sodium fluoride is preferred as an anticoagulant due to its antiglycolytic activity.

Reagents:

1. Glucose standard (100 mg/dl)
2. GOD-POD reagent: Enzyme reagent mixture containing glucose oxidase (GOD), peroxidase (POD), 4-aminoantipyrine, phenol, and phosphate buffer (pH≈7.0), some stabilizers and activators.

Instruments:

1. Test tubes
2. Pipettes, disposable tips, rack
3. Water bath
4. Colorimeter

Procedure:

1. Label three clean, dry test tubes as Blank (B), Standard (S), and Test (T).
2. Pipette as follows:

	Blank	Standard	Test
GOD-POD Reagent	1 ml	1 ml	1 ml
Distilled water	10 µl	–	–
Glucose standard	–	10 µl	–
Sample	–	–	10 µl

3. Mix well and incubate at 37⁰C for 10 minutes. Or, at room temperature (25⁰C) for 30 minutes.
4. Measure the absorbance of the standard and test sample at 540nm (green filter) against blank within 60 minutes.

Calculation:

Calculate the concentration of blood glucose in the specimen using the following formula:

References:

1. Burrin, J. M., & Price, C. P. (1985). Measurement of blood glucose. Annals of clinical biochemistry.
2. Dandekar, S. P., Rane, S. A. (2004) Practical and Viva in Medical Biochemistry, New Delhi, Elsevier/Reed Elsevier. India PVT LTD.




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]]> https://laboratorytests.org/god-pod-method/feed/ 0 https://laboratorytests.org/folin-wu-method/ https://laboratorytests.org/folin-wu-method/#respond Sun, 20 Feb 2022 09:26:04 +0000 http://laboratorytests.org/?p=778 Folin-Wu method is one of the oldest methods for the estimation of blood sugar. However, it is almost obsolete for now but is in use in countries where enzyme preparations are not easy to obtain. [...]

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]]>

Folin-Wu method is one of the oldest methods for the estimation of blood sugar. However, it is almost obsolete for now but is in use in countries where enzyme preparations are not easy to obtain. This method is old and not specific for glucose determination since other substances (e.g. fructose, lactose, and glutathione) also bring about a reduction. The blood glucose level when estimated by the Folin-Wu method is higher than true glucose.

Principle

Proteins from the blood are removed by 10% sodium tungstate and 2/3N sulphuric acid. The glucose present in the protein-free filtrate on boiling in an alkaline medium will be changed to enediol form. This enediol will reduce cupric ions to the precipitate of cuprous oxide. This oxide is dissolved and reacted by phosphomolybdic acid to form phosphomolybdenum blue which is blue in color. Constricted tubes (Folin-Wu tubes) are used to avoid reoxidation of cuprous oxide by atmospheric oxygen. The final blue color is measured at 680 nm which is proportional to the amount of glucose present in the specimen.

Requirements

1. Folin Wu tubes
2. Colorimeter
3. Reagents:
   1. 2/3 N H2SO4: Add 2ml H2SO4 to about 50ml of D/W and dilute up to 100ml.
   2. 10% Sodium Tungstate: Dissolve 10 gm in 100ml of D/W.
   3. Alkaline Copper tartarate:
      A) Dissolve 40gm sodium carbonate and 7.5 gm tartaric acid in about 400ml of D/W.
      B) Dissolve 4.5 gm copper sulphate in about 100ml D/W.
      Mix A and B and make volume up to 1000ml with D/W.
   4. Phosphomolybdic acid: Dissolve 35 gm molybdic acid and 5 gm sodium tungstate in 200 ml 10% NaOH. Add it to 200ml D/W and boil for 45 minutes to remove ammonia. Cool and add slowly 125 ml of 89% phosphoric acid. Make up the volume to 500ml with D/W.
   5. Distilled water
   6. Glucose standard
      Stock (1g/dl): Dissolve 1 gm of glucose in 100 ml saturated benzoic acid (0.3%).
      Working standard (10mg/dl): Dilute stock 1:100 with saturated benzoic acid.

Procedure



Step 1: Preparation of protein-free filtrate:

1. Add 1 ml of blood to 7 ml of distilled water and mix.
2. Add 1 ml of 10% sodium tungstate.
3. Add 1 ml of 2/3N H2SO4 and mix. Allow standing for 5 minutes.
4. Centrifuge or filter using Whatmann number 1 filter paper.

Step 2: Testing

1. Set up 3 Folin-Wu tubes as follows:
   

   	Blank	Standard	Test
   Distilled water	1 ml	–	–
   Working glucose standard	–	1 ml	–
   Protein-free filtrate	–	–	1 ml
   Alkaline copper tartarate	1 ml	1 ml	1 ml

2. Place the tubes in a boiling water bath for 10 minutes.
3. Cool and add 1ml phosphomolybdic acid reagent to each tube.
4. Shake the tubes to get rid of air bubbles. Add distilled water up to 12.5 ml mark.
5. Mix and read the absorbance at 680 or red filter. Set the zero using the blank.

Calculations

Calculate the concentration of glucose in the blood specimen using the following
formula:

Note: 1 ml of blood was diluted 1:10 for protein precipitation. 1 ml of the diluted blood was then used for test. Therefore, the actual volume of blood used for the test is 0.1 ml.
Also, 1 ml of the working standard (10 mg/dl) contains 0.1 mg of glucose.

References

1. Kolhatkar, A., Ochei, J., & McGraw, T. (2008). Medical Laboratory Science: Theory and Practice.
2. Naigaonkar, A.V., (2007). A Manual Of Medical Laboratory Technology.
3. Geetha, D. K. (2011). Practical Biochemistry. Jaypee Brothers Medical Publishers (P) Ltd, UK.

 

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]]> https://laboratorytests.org/folin-wu-method/feed/ 0 https://laboratorytests.org/may-grunwald-giemsa-stain-principle-preparation-and-procedure/ https://laboratorytests.org/may-grunwald-giemsa-stain-principle-preparation-and-procedure/#respond Sat, 19 Feb 2022 15:40:22 +0000 http://laboratorytests.org/?p=761 May Grunwald-Giemsa stain(MGG) is a type of Romanowsky stain, which is used routinely for staining of air-dried cytological smears, blood, and bone marrow smears. Cytological preparations made from FNAC and serous fluids are normally processed [...]

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]]> May Grunwald-Giemsa stain(MGG) is a type of Romanowsky stain, which is used routinely for staining of air-dried cytological smears, blood, and bone marrow smears. Cytological preparations made from FNAC and serous fluids are normally processed by May Grunwald-Giemsa stain. It is useful for studying cellular morphology and is superior to PAP stain to study cytoplasm, granules, vacuoles, and basement membrane.

Principle

May Grunwald-Giemsa stain is a combination of two stains: May Grunwald stain and Giemsa stain.

• May Grunwald stain is alcohol-based stain composed of methylene blue and eosin.
• Giemsa stain is alcohol-based stain composed of methylene blue, eosin and azure B.

The working principle of MGG stain is the same as that of other Romanowksy stains. Polychromatic Romanowksy dyes contain different ratios of methylene blue (and the reagent-related thiazine dyes, such as azure B), as the cation (+vely charged, basic) component, and eosin Y as the anion (-vely charged, acidic) component. Cation and anion components in combination produce the well-known Romanowsky effect or metachromasia.

The solvent methanol initially fixes the cells. The basic dyes carry net positive charges; consequently, they stain nuclei (because of the negative charges of phosphate groups of DNA and RNA molecules), granules of basophil granulocytes, and RNA molecules of the cytoplasm. The eosin carries a net negative charge and stains red blood cells and granules of eosinophil granulocytes. Buffer solution of pH 6.5-6.8 is used to enable the dye to precipitate and bind well with the cellular material.

Reagents

May Grunwald Stain

• Stock solution:
  May Grunwald dye= 0.3 gm
  Methanol = 100 ml
• Working Solution:
  Stock solution= 20 part
  Phosphate Buffer (pH 6.8)= 30 part

Giemsa Stain

• Stock Solution:
  Giemsa Powder= 1 gm
  Glycerine= 66ml
  Absolute ethanol= 66ml
  Mix giemsa and glycerine, place in 60C oven for 30 minutes 2 hr. Add 66ml methanol
• Working Solution:
  Stock Giemsa= 50 drops
  Distilled Water=50ml

Procedure

• Prepare a thin smear and air dry.
• Fix smears for 5-10 minutes with methanol.
• Stain the smear in May Grunwald working solution for 10 minutes.
• Rinse in pH 6.8 buffer.
• Stain the slides with diluted Giemsa stain for 30 minutes.
• Wash the smears with distilled water and let them dry.
• Mount the slide with DPX and examine under microscope.

Results


• Erythrocytes: Light pink to light purple
• Platelets: Granules – Reddish purple
• Lymphocytes/monocytes: Nuclei – Dark purple, Cytoplasm – Sky blue
• Neutrophils: Nuclei – Dark blue, Granules – Reddish purple, Cytoplasm – Pale pink
• Eosinophils: Nuclei – Blue, Granules – Red/orange red, Cytoplasm – Blue
• Basophils: Nuclei – Dark blue, Granules – Purple

References

1. Dey, P. (2018). Basic and advanced laboratory techniques in histopathology and cytology. Springer Singapore.
2. Matutes, E., Pickl, W. F., Van’t Veer, M., Morilla, R., Swansbury, J., Strobl, H., … & Ludwig, W. D. (2011). Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification. Blood, The Journal of the American Society of Hematology, 117(11), 3163-3171.
3. Product Information May-Grünwald Giemsa, Avantor Performance Materials.

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]]> https://laboratorytests.org/may-grunwald-giemsa-stain-principle-preparation-and-procedure/feed/ 0 https://laboratorytests.org/periodic-acid-schiff-pas-stain/ https://laboratorytests.org/periodic-acid-schiff-pas-stain/#respond Tue, 14 Sep 2021 14:55:03 +0000 http://laboratorytests.org/?p=736 The Periodic Acid-Schiff (PAS) Stain is widely used technique in histopathology for the demonstration of carbohydrates and carbohydrate rich compounds in tissues. PAS stain demonstrates polysaccharides, mucin, glycogen, certain glycoproteins and glycolipids, basement membrane and [...]

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]]> The Periodic Acid-Schiff (PAS) Stain is widely used technique in histopathology for the demonstration of carbohydrates and carbohydrate rich compounds in tissues. PAS stain demonstrates polysaccharides, mucin, glycogen, certain glycoproteins and glycolipids, basement membrane and certain fungus in tissues.

Importance and Uses of Periodic Acid-Schiff (PAS) Stain

• PAS helps to demonstrate glycogen, cellulose and starch. This is useful to detect glycogen deposits in liver when glycogen storage disease is suspected.
• Basement membrane of various tissues may also be visualized through the PAS stain. The PAS is most commonly used to demonstrate the thickness of glomerular basement membrane when renal disease is being assessed.
• Certain fungi in tissue samples such as Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumiagtus, Blastomyces etc can be demonstrated by PAS stain because of high carbohydrate content in their cell wall/capsule.
• Mucin, particularly acid mucin is demonstrated by PAS. It is important un endocervical glands, intestinal glands and bronchial glands.
• PAS helps to demonstrate cerebrosides and gangliosides. It helps in diagnosis of Gaucher’s disease, Krabbe’s disease and lysosomal storage diseases etc.
• Certain pigments such as lipofuchsin and pigments of Dubin-Johnson syndrome are demonstrated by PAS stain.
• Russell bodies of plasma cells are stained by PAS.

Principle of PAS Stain

Glycol group of carbohydrates are oxidized by periodic acid to release dialdehydes. These dialdehydes on subsequent combination with Schiff’s reagent result in the formation of magenta colored complex, localized at the site of aldehyde formation.

Preparation of Reagents for PAS Stain

Periodic Acid Solution :

Periodic acid solution, used for oxidation is used in any strength between 0.5% and 2.5%. 1% solution is preferred.
Periodic acid= 1gm
Distilled water= 100ml

Schiff’s Reagent

Fuchsin Basic : 1 gm
Distilled water : 100 ml
Sodium metabisulphite : 2 gm
Conc. HCl : 2 ml
Charcoal activated : 0.3 gm

Dissolve basic fuchsin in boiling water, cool at 50°C and filter. Add sodium metabisulphite and HCl. Store at dark room at room temperature overnight. Add charcoal, shake for one minute and filter.

Procedure of PAS Stain

1. Deparaffinization: flame the slide on burner and place in the xylene. Repeat the treatment to remove the wax.
2. Hydration: Drain xylene and hydrate the tissue section by passing through decreasing concentration of alcohol baths (100%, 90%, 80%, 70%) and water.
3. Oxidation: Place the sections in periodic acid solution (1%) for 5-10 minutes.
4. Rinse: Wash in at least two changes of distilled water.
5. Treatment with Schiff’s reagent: Cover with Schiff’s reagent for 20-30 minutes.
6. Rinse: Rinse in running tap water for 5-10 minutes.
7. Counterstain: Cover with hematoxylin for 3-5 minutes. Differentiate and blue for the sections.
8. Dehydration: Dehydrate in increasing concentration of alcohols.
9. Clearing: Put slides in two xylene baths for clearing.
10. Mounting: Mount in DPX or other mounting media.
11. Observe under microscope.

 

Results and Intrepretation

Substance	Resulting color
PAS positive material	Magenta Pink to Red
Nuclei	Blue








The substances positive for PAS are:

1. Polysaccharides: Glycogen, cellulose and starch. Many leucocytes contain glycogen, capsule of fungi (Candida albicans, Histoplasma capsualtum, Cryptococcus and Blastomycosis), actinomycosis and bacteria.
2. Glycoproteins: Mucins, mucoid secrection of intestinal tracts, uterine glands, ducts, tracheobronchial tress, hormones (TSH), megakaryocytes etc.
3. Glycolipids: Gangliosides, mainly gray matter composed of fatty acids.
4. Non-carbohydrate containing substances: Unsaturated lipids, phospholipids and phosphoinositides.
5. Certain pigments and substances: Ceroid, lipofucsin, pigment in melanosis coli and Dubin-Johnson pigment.
6. Plasmogens: They are acetyl phospholipids, eg: Russell bodies.
7. Miscellaneous: Amyloid, cartilage matrix, colloid and ocular lens material.

References

1. Shariff, S., & Kaler, A. K. (2016). Principles & Interpretation of Laboratory Practices in Surgical Pathology. JP Medical Ltd.
2. Dey, P. (2018). Basic and advanced laboratory techniques in histopathology and cytology. Springer Singapore.
3. Bancroft, J. D., & Gamble, M. (Eds.). (2008). Theory and practice of histological techniques. Elsevier health sciences.
4. Löffler, H., & Rastetter, J. (2012). Atlas of clinical hematology. Springer Science & Business Media.
5. https://www.pathologyoutlines.com/topic/stainspas.html
6. https://www.labce.com/spg949466_periodic_acid_schiff_pas_diagnostic_applications.aspx

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]]> https://laboratorytests.org/periodic-acid-schiff-pas-stain/feed/ 0 https://laboratorytests.org/convert-g-to-rpm/ https://laboratorytests.org/convert-g-to-rpm/#comments Sat, 11 Sep 2021 16:37:08 +0000 http://laboratorytests.org/?p=725 Centrifuagtion is a technique that helps to separate mixtures by applying centrifugal force. A centrifuge machine works by using a principle of sedimentation. Under the influence of gravitational force, substances separate according to their density. [...]

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]]> Centrifuagtion is a technique that helps to separate mixtures by applying centrifugal force. A centrifuge machine works by using a principle of sedimentation. Under the influence of gravitational force, substances separate according to their density.

When dealing with centrifuge machines, we come across two different units of measurement: The Revolutions per minute (RPM) and Relative centrifugal force (RCF) or g-force. In fact these units are not same. This article discusses about what they actually are and how they are realated to each other.

Revolutions per minute (RPM)

RPM (Revolutions per minute) basically describes how fast the centrifuge goes. It is a measurement of how fast the centrifuge rotor does a full rotation in one minute. The force applied to the contents varies by the size of the centrifuge rotor.

Relative centrifugal force (RCF)

RCF (Relative centrifugal force) is the amount of force exerted on the contents on the rotor, resulting from revolutions of the rotor. It depends on the rotation speed and radius of the rotor. It is relative to the force of earth’s gravity and is measured in force*gravity (or g-force).

Which one to use?

The bigger the radius the more acceleration is applied to the samples for the same RPM. Since different centrifuge machines may have different rotor sizes, using RCF or g-force will give very accurate setting for experiments. RCF remains constant irrelevant of the centrifuge you are using

Converting RPM to RCF and vice versa

To calculate RCF from RPM, use following equation:

To calculate RPM from RCF, use following equation:

Where,

• RCF(g) = Relative Centrifugal Force
• r = Radius of the rotor (cm). It is the distance from the rotor axis to the bottom of the tube.
• RPM(n) = Revolutions Per Minute

References

1. https://handling-solutions.eppendorf.com/sample-handling/centrifugation/safe-use-of-centrifuges/basics-in-centrifugation/
2. https://www.westlab.com/blog/2019/01/29/difference-between-rcf-and-rpm-in-centrifugation
3. https://blog.btlabsystems.com/blog/rpm-rcf-g-force
4. http://www.fao.org/3/ac802e/ac802e0u.htm

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]]> https://laboratorytests.org/convert-g-to-rpm/feed/ 1 https://laboratorytests.org/diacetyl-monoxime-dam-method-for-estimation-of-urea/ https://laboratorytests.org/diacetyl-monoxime-dam-method-for-estimation-of-urea/#respond Sun, 28 Mar 2021 15:26:32 +0000 http://laboratorytests.org/?p=695 Urea is a waste product formed in liver following the breakdown of proteins. It passes into the blood, is filtered out of the kidneys and excreted in urine. Thus determination of blood urea is the [...]

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]]> Urea is a waste product formed in liver following the breakdown of proteins. It passes into the blood, is filtered out of the kidneys and excreted in urine. Thus determination of blood urea is the most widely used screening test for the evaluation of kidney function. There are various methods used for the estimation of urea in a laboratory. Diacetyl monoxime (DAM) method is an older method.

Principle

Proteins are first precipitated by trichloroacetic acid. The urea present in the protein-free filtrate reacts with diacetyl monoxime in a hot acidic medium in presence of ferric/cadmium ions and thiosemicarbazide to form pink or red colored complex- diazine. The intensity of the color developed is measured photometrically at 530nm, which is directly proportional to the concentration of the urea present in the fluid.



Requirements

• Apparatus:
  Colorimeter
  Conical flasks and test tubes to hold 20ml
  Pipettes: 50ul, 0.1ml, 0.5ml, 5 ml
  Measuring cylinder, 50 ml
  Water bath at 100°C
• Reagents:
  Benzoic acid
  Ferric Chloride
  Diacetyl monoxime
  orthophosphoric acid
  Thiosemicarbazide
  Tricholoroacetic acid
  Urea
• Specimen:
  Serum, heparinized plasma or fluoride plasma.

Preparation of Regaents

1. Reagent 1: Trichloroacetic acid, 50g/l (5%) solution
   Trichloroacetic acid = 10g
   Distilled water = upto 200ml
2. Reagent 2: Diacetyl monoxime (2,3-butanedione monoxime) solution
   Diacetyl Monoxime = 2g
   Distilled water = upto 500ml
3. Reagent 3: Acid reagent
   Concentrated sulfuric acid = 44ml
   Orthophosphoric acid (H3PO4), 85% = 66ml
   Cadmium sulfate = 1.6 g
   Thiosemicarbazide = 50mg
   Distilled water = upto 500ml
4. Reagent 4: Colour reagent
   Acid reagent (Reagent 3) = 50ml
   Diacetyl monoxime reagent = 50ml
5. Reagent 5: Benzoic acid solution 1 g/l
   Benzoic acid = 1 g
   Distilled Water = 1000ml.
6. Reagent 6: Urea stock reference solution, 125mmol/l
   Urea = 750mg
   Benzoic acid, 1 g/l (0.1%) solution = upto 100ml
7. Reagent 7: Urea working reference solution, 10mmol/l
   Urea stock reference solution = 8ml
   Benzoic acid (C7H6O2), 1 g/l (0.1%) solution = upto 100ml

Preparation of Sample

To obtain protein free filtrate, take 50 ul of whole blood/serum/plasma in a centrifuge tube. Add 1 ml of TCA solution and mix. Centrifuge at high speed (3000 g) for 5 minutes to sediment the precipitated proteins and obtain a clear supenatent fluid. Do same for standard/control sample.

Procedure

1. Take three (or more if needed) large test-tubes and label as follows:
   Blank tube (B)
   Standard tube (S)
   Test tube (T)
2. Pipette into each tube as follows:
   

   	Test	Standard	Blank
   Color Reagent (Reagent 4)	3 ml	3 ml	3 ml
   Protein Free Filtrate	0.1 ml	–	–
   Urea Standard 10 mmol/l	–	0.1 ml	–
   Distilled water	–	–	0.1 ml

3. Mix the contents of each tube. Place all the tubes in the water-bath at 100°C for exactly 15 minutes to allow the red color to develop.
4. Remove the tubes and allow them to cool in a beaker of cold water for 5 minutes.
5. Measure the colour produced in a colorimeter at a wavelength of 530nm.

Calculations

Calculate the concentration of urea in the blood specimen using the following formula:


References

1. World Health Organization, 2003. Manual of basic techniques for a health laboratory. World Health Organization.
2. World Health Organization, 1986. Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies. In Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies.
3. Godkar, P.B. and Godkar, D.P., 2003. Textbook of medical laboratory technology. Bhalani.
4. Mukherjee, K.L., 2013. Medical Laboratory Technology Volume 3 (Vol. 3). Tata McGraw-Hill Education.




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]]> https://laboratorytests.org/diacetyl-monoxime-dam-method-for-estimation-of-urea/feed/ 0 https://laboratorytests.org/o-toluidine-method/ https://laboratorytests.org/o-toluidine-method/#respond Sat, 27 Mar 2021 17:16:22 +0000 http://laboratorytests.org/?p=687 The O-toluidine method is an older method of blood glucose estimation. This method is no longer used today because O-toluidine is believed to be a carcinogen and is replaced by enzymatic methods. This method is [...]

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]]> The O-toluidine method is an older method of blood glucose estimation. This method is no longer used today because O-toluidine is believed to be a carcinogen and is replaced by enzymatic methods. This method is still popular because of its simplicity, sensitivity and accuracy.

Principle

The proteins are first precipitated by tricholoroacetic acid. The glucose present in a protein free filtrate react with O-toluidine (primary aromatic amine) in a hot acidic medium to form a stable green colored complex, namely N-glycosamine. The presence of thiourea stabilizes the o-toluidine reaction. The intensity of the color developed is measured photometrically at 630nm, which is directly proportional to the concentration of the glucose present in the fluid.



Requirements

• Apparatus:
  Graduated pipettes
  Test tubes
  Micropipettes
  Heating Bath, 100C
  Spectrophotometer, wavelength 630nm
• Reagents:
  Benzoic acid
  D-glucose
  Glacial acetic acid
  O-toluidine
  Thiourea
  Trichloroacetic acid
• Specimen:
  Collect 2-3 ml of blood in a fluoride tube. Use plasma for testing. Serum/Whole blood can also be used. Prepare protein-free filtrate if the sample is grossly hemolyzed or icteric. Add 100 ul specimen to 900 ul of 5gm/dl TCA, mix and centrifuge to get the filtrate.

Preparation of Regaents

1. Benzoic acid solution 1 g/l:
   Dissolve 1 gm of benzoic acid in water and make upto 1 liter. Prepare the benzoic acid at least 24 hours before use.
2. Stock glucose solution 100 mg/dl:
   Dissolve 1 gm of glucose in 1 liter of benzoic acid solution (1 g/l).
3. O-toluidine reagent:
   Dissolve 1.5 gm thiourea in 940 ml of glacial acetic acid. When completely dissolved, add 60ml of O-toluidine. Mix well and store in amber bottle.

Procedure

1. Take three (or more if needed) large test-tubes and label as follows:
   Blank tube (B)
   Standard tube (S)
   Test tube (T)
2. Pipette into each tube as follows:
   

   	Test (T)	Standard (S)	Blank (B)
   O-toluidine reagent	3 ml	3 ml	3 ml
   Serum/Plasma	50 μl	–	–
   Glucose Standard 100 mg/dl	–	50 μl	–
   Distilled water	–	–	50 μl

   Note: If protein free filtrate is used instead of serum/plasma, pipette 500 ul of protein free filtrate instead of 50 μl serum/plasma.

3. Mix the contents of each tube. Place all the tubes in the water-bath at 100°C for exactly 12 minutes.
4. Remove the tubes and allow them to cool in a beaker of cold water for 5 minutes.
5. Measure the colour produced in a colorimeter at a wavelength of 630nm.

Calculations

Calculate the concentration of glucose in the blood specimen using the following
formula:


References

1. World Health Organization, 2003. Manual of basic techniques for a health laboratory. World Health Organization.
2. World Health Organization, 1986. Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies. In Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies.
3. Mukherjee, K.L., 2013. Medical Laboratory Technology Volume 3 (Vol. 3). Tata McGraw-Hill Education.




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