Sudan Black B Stain – Purpose, Principle, Procedure, Preparation, Protocol and Clinical importance

Many staining methods are used in the laboratory setting and one of which is Sudan Black B Stain. It is used to visualize a wide array of lipids such as sterols, phospholipids, and neutral triglycerides.

Unlike other Sudan dyes, Sudan Black B Stain is not lipid specific, which means you can use it for other staining purposes such as leukocyte granules, Golgi apparatus, and chromosome staining. (1, 2)

What is the purpose of Sudan Black B Stain?

The main purpose of Black Sudan B is for the study of hematologic pathologies as it is effective in staining myeloblasts. Sudan Black B Stain has a high affinity for lipids and neutral fats, which effectively differentiates acute myeloid leukemia from people who have acute lymphoid leukemia.

It is similar to myeloperoxidase staining pattern of both monocytes and leukocytes, but offer more advantage when compared to myeloperoxidase. In addition, it has the ability to stain two weeks’ old smears. More so, it can also stain azurophilic and specific granules found in neutrophils. (1, 2, and 3)

A microscopic image of a specimen that has tested positive for Sudan Black B Stain pictures

Picture1 : A microscopic image of a specimen tests positive for Sudan Black B Stain.

What are the principles and protocols (leukemia)?

Procedures like Sudan Black B Stain makes use of frozen tissues with the aid of formalin solution. It also uses paraffinized sections. Of all dyes from the Sudan dye group, Sudan Black B dye is the most used dye.

It combines acidic groups in the lipid compounds, therefore, effective in staining lipoproteins, phospholipids, and triglycerides found in staining specimen. During the staining process, the dye leaves the solvent due to lipids’ high solubility.

The solubility is higher than the solvent. Under microscopic exam, various degrees of black-colored pigments are noticed in the positive reaction. (3, 4)

What is the clinical importance?

Sudan Black B Stain is used in a variety of clinical applications. It has the ability to react against lipofuscin, which is an aggregate of lipids, metals, and oxidized proteins. It is a substance linked to many aging processes, which over time will accumulate in senescent cells.

Lipofuscin can be easily and thoroughly detected when Sudan Black B Staining is applied. Sudan Black B Stain helps understand physiological responses as well as pathophysiologic details of many diseases, especially in coming up with the best interventions for a specific disease condition. (4, 5)

The image above shows the actual Sudan Black B Stain used in special hematological process pictures

Picture 2 : The image above shows the actual Sudan Black B Stain used in special hematological procedures.

Requirements

  • Sample: bone marrow smear or fresh anticoagulated whole blood.
  • Fixative agent: formaldehyde solution vapor (40%)
  • Staining agent: The staining agent used is Sudan Black B Stain 0/3 g in 100 ml ethanol.
  • Phenol Buffer: 16 grams of crystalline phenol is dissolved in 30 ml absolute ethanol to create a phenol buffer. It is then added to 100 ml distilled water, and 0.3 grams of Na2HPO4.12H20 is dissolved. (4, 5)
  • Working Sudan Black B Stain: You need to add at least 49 ml buffer to 60 ml Sudan Black B Stain solution.
  • Counterstaining: For counterstaining, two substances can be used, such as Leishman stain and May-Grunwald-Giemsa stain. (6)

Procedures

  1. The frozen sections are placed on the sterile glass slide using 10% fresh formalin.
  2. The fixed section is washed with distilled water. Make sure to tap off the excess water.
  3. The next step is to add propylene glycol twice at five minutes interval.
  4. Get the Sudan Black Solution and let it stay there for seven minutes; enough time to agitate the solution. (7)
  5. Get 85% propylene glycol and let it stay there for three minutes.
  6. A distilled water is used to rinse the stain.
  7. Add nuclear fast red and let is stay there for at least three minutes.
  8. Rinse the solution using tap water twice, but use distilled water for the final rinse.
  9. The final step is to mount the solution using aqueous mounting jelly (glycerin jelly).

Results and Interpretation

  • The result is positive if you notice the presence of black and granular piments.
  • If you notice the presence of lipids in Azurophilic and secondary granules of myelocytic cells, they indicate a positive Sudan Black B stain result. As the cells mature, the stain becomes more intense. (8)
  • It does not necessarily mean a positive reaction if you notice a bright red to purple metachromatic staining of granules when testing basophils.
  • If lipids are present in monocytic cells’ lysosomal granules, you will notice a negative to a weakly positive result.
  • The result is negative is lymphoid cells are present. However, in people with acute lymphoblastic leukemia (ALL), less than 3% of blast cells will indicate a positive reaction. (9)

A Sudan Black B stain result for patient diagnosed with leukemia image

Picture 3: A Sudan Black B stain result for patient diagnosed with leukemia.

Frequently Asked Questions (FAQs)

Q1. What is Sudan Black B Stain Test?

 
It is a non-lipid-specific staining procedure used for visualizing lipids. It has the ability to stain a wide array of lipids like neutral triglycerides, sterols, and phospholipids. Aside from staining lipids, it can also be used to stain chromosomes, leukocyte granules, and Golgi apparatus. (9)

Q2. What does Sudan Black B Stain detect?

It effectively detects lipofuscin, an aggregate of lipids, metals, and oxidized proteins. It is a substance linked to various aging processes, which later on accumulate in senescent cells. (10)

Q3. How does Sudan Black B stain lipids?

It stains lipids using frozen tissue sections fixed to a glass slide using formalin solution or paraffinized sections. It is a basic dye that combines acidic groups in lipid compounds. With the use of the Sudan Black B stain, it can thoroughly detect specific substances in a staining specimen, specifically lipoproteins, phospholipids, and triglycerides. (10)
 

Q4. What is the positive staining reaction of Sudan Black B?

A positive Sudan Black B reaction is noticed when there’s black and granular pigment on the slide. You will also notice the presence of lipids in azurophilic and secondary granules of myelocytic cells. When you notice these things, they all indicate a positive Sudan Black B Stain result. As the cells mature, the staining gets more intense. (8)

Q5. How do you make Sudan Black B?

To make a Sudan Black B Solution, you need to prepare a staining solution of Sudan Black B (500 mg) in acetone (20 ml). The solution will be added to acetic acid (15ml) and water (85ml). Centrifuge the mixture by stirring it for at least 30 minutes. The purpose of stirring the mixture continuously is to get rid of the precipitate. (8)

Q6. What are Sudan stains used for?

Sudan stains are primarily used for lipids and phospholipids staining, such as Sudan IV and Sudan Black B stain. (6)

Q7. What does Sudan Black B bind to?

It is a lipophilic dye that irreversibly binds to granulocytes’ undefined granule components. It also binds to eosinophils and monocytes. (10)

References
  1. https://www.clinisciences.com/en/buy/cat-sudan-black-b-stain-3954.html
  2. https://webpath.med.utah.edu/HISTHTML/MANUALS/SUDANF.PDF
  3. http://laboratorytests.org/sudan-black-b-stain/
  4. https://www.urmc.rochester.edu/urmc-labs/pathology/stainsmanual/index.html
  5. https://www.sciencedirect.com/topics/medicine-and-dentistry/sudan-black-b
  6. https://www.bio-diagnostic.com/images/sudan_black__b__stain.pdf
  7. https://pubmed.ncbi.nlm.nih.gov/27812872/
  8. https://www.tandfonline.com/doi/abs/10.3109/10520294009110328?journalCode=ibih19
  9. https://www.scbt.com/p/sudan-black-b-4197-25-5
  10. http://www.baso.com.cn/en/view.asp?id=155

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