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Confusing Carbs!! – Carbohydrate staining simplified

Carbohydrate staining is confusing isn’t it? Lets try and simplify it!

(Staining procedures and methods will not be discussed, only the interpretation)

PAS and Alcian blue staining will be discussed

TABLE OF CONTENTS
1. Types of carbohydrates
2. PAS( Periodic acid Schiff) staining
3. Alcian blue staining
4. Combined PAS + Alcian blue staining
5. Combined Alcian blue + High iron diamine staining
6. Summary
TABLE OF CONTENTS

1.TYPES OF CARBOHYDRATES

Group 1: Neural Polysaccharides or non-ionic homoglycans: This group has glucose containing glycogen, starch and cellulose as well as N-acetyl glucosamine containing chitin.

Group 2: Acid mucopolysaccharides or anionic heteroglycans: Hyaluronic acid and Chondroitin sulfate belong to this group.

Group 3: Neutral mucins, Carboxylated mucins (sialomucins) as well as sulfated mucins (sulfomucins) are present in this category.

NEUTRAL MUCINS CARBOXYLATED MUCINS SULFATED MUCINS
  • Surface epithelia of the stomach

  • Brunner glands of the duodenum

  • Prostatic epithelium
  • Bronchial submucous glands

  • Goblet cells

  • Salivary glands
  • Bronchial mucus glands
    Group 3

    Remember: Carboxylated mucins or sialomucins (also called simple mucins) are weak acids whereas sulfated mucins (also called complex mucins) are strong acids

    Group 4: Glycolipids and Phosphatides belong to this group.

    WHICH OF THESE GROUPS IS PAS (PERIODIC ACID SCHIFF) POSITIVE?

    Group 1 is always PAS positive

    Group 2 is always PAS negative.

    Group 3 and 4 is also PAS positive.

    Remember: Mesenchymal polysaccharides such as Hyaluronic acid and Connective tissues polysaccharides such as Chondroitin sulfate are always negative for PAS.

    carbohydrate staining- PAS stained gastric foveolar cells
    Carbohydrate staining – Periodic acid-Schiff (PAS) stain -Positive gastric foveolar cells6. SUMMARY OF CARBOHYDRATE STAINING

    PAS Positive cell and tissue components

    1.Glycogen
    2.Starch
    3.Mucins (Neutral mucins>sialomucins>sulfomucins)
    4. Basement membranes
    5. α-antitrypsin
    6.Reticulin
    7.Fungi (capsules)
    8.Pancreatic zymogen granules
    9.Thyroid colloid
    10.Corpora amylacea
    11. Russell bodies

    Carbohydrate staining- Periodic acid-Schiff (PAS) stain highlights the microvillous along the apical surface of the absorptive cells.
    Carbohydrate staining- Periodic acid-Schiff (PAS) stain highlights the microvillous along the apical surface of the absorptive cells.

    Diseases in which PAS staining can be used for diagnosis

    1. Glycogen storage disease
    2. Adenocarcinoma
    3. Paget’s disease of the breast
    4. Staining macrophages in Whipple’s disease
    5. Fungal infection
    6. Alveolar soft part sarcoma
    7. Erythroleukemia
    8. α1-antitrypsin deficiency
    9. Ewing sarcoma
    10. Pulmonary alveolar proteinosis

    3. LET’S MOVE ON TO ALCIAN BLUE STAINING

    Group 1 is always Alcian blue negative

    Group 2 is always Alcian blue positive. Hyaluronic acid is positive at a pH of 2.5 and Chondroitin sulfate is positive at a pH of 0.5.

    Group 3 is the confusing part, we need to divide it into three parts for better understanding

    1.Neutral mucins– Always alcian blue negative.

    2. Sialomucins or carboxylated mucins– Alcian blue positive at a pH of 2.5

    3. Sulfated mucins– Alcian blue positive at a pH of 1.

    Group 4 is Alcian blue negative.

    Alcian blue staining of barrett mucosa
    Alcian blue staining of Barrett mucosa at a pH of 2.5- Goblet cells are stained blue and gastric mucous cells are clear.

    Alcian blue stain is frequently combined with PAS and High iron diamine for diagnostic purposes (discussed below).

    Alcian blue staining of cartilage at pH 0.5
    Alcian blue staining of cartilage at pH 0.5

    4. COMBINED PAS AND ALCIAN BLUE STAINING

    Primary use of PAS+Alcian blue combined staining is to distinguish neutral and acidic especially sialomucns.

    Diagnostic utility: 1. To distinguish eccentric gastro-esophageal junction from Barret’s mucosa

    2. Gastric intestinal metaplasia

    Remember: Key finding an diagnostic feature intestinal metaplasia is the presence of goblet cells. Goblet cells and surface epithelial cells of stomach may be difficult to distinguish in routine Hematoxylin and Eosin stained sections.

    Goblet cells are positive with both PAS and Alcian blue but gastric epithelial cells are positive with PAS alone. When combined PAS+Alcian blue staining is performed, Goblet cells stain PURPLE and Gastric foveolar cells stain MAGENTA.

    Batter esophagus showing goblet cells
    Barrets esophagus showing goblet cells
    PAS + Alcian blue stain showing purple colored goblet cells and magenta colored gastric foveolar cells
    PAS + Alcian blue stain showing purple colored goblet cells and magenta colored gastric foveolar cells at pH 2.5.

    5. COMBINED ALCIAN BLUE AND HIGH IRON DIAMINE

    Combined PAS + High iron diamine is used to distinguish between sulfomucins and sialomucins.

    Diagnostic utility: Large bowel metaplasia can be identified, since it contains both sulfomucins and sialomucins, in contrast to small bowel metaplasia (sialomucins only)

    With Alcian blue + High iron diamine staining- Sulfomucins: BROWN COLOR and Sialomucins: BLUE COLOR

    Carbohydrate staining- Sulfomucins stained brown with High iron diamine and few sialomucins stained blue.
    Sulfomucins stained brown with High iron diamine and few sialomucins stained blue.

    6. SUMMARY OF CARBOHYDRATE STAINING

    1. PAS is always negative in mesenchymal and connective tissue mucopolysaccharides
    2. Alcian blue is always negative in neutral mucins
    3. PAS+Alcian blue is key to distinguish ectopic gastric mucosa or eccentric gastroesophageal junction from Barret’s mucosa in the esophagus.

    Comment your queries and suggestions!! Thank you!

    Check this link for Key Points on Hematoxylin and eosin staining

    Breast Pathology MCQ 1

    MCQ neuroendocrine differentiation in invasive ductile carcinoma of breast

    MCQ breast pathology

    Image courtesy

    CLICK HERE FOR ANSWER WITH COMPLETE EXPLANATION

    CORRECT ANSWER IS C

    Diagnosis is Solid papillary breast carcinoma

    BREAST CARCINOMAS SHOWING NEUROENDOCRINE DIFFERENTIATION ARE

    1. Solid papillary carcinoma in 50% cases
    2. Mucinous carcinoma

    Solid papillary breast carcinoma are Luminal type and show ER/PR positivity in most cases

    Myoepithelial cells are not present

    Solid papillary breast cancer has a favorable prognosis with rare lymph nodal metastasis.

    CNS Pathology case based MCQs-2

    CASE : The spinal MRI of a 22-year old male with neurofibromatosis type-2 showed a hyperintense lesion. The lesion was resected and histopathologic section is shown in image.

    Question 1: What is the diagnosis?

    A. Medulloblastoma
    B. Ependymoma
    C. Retinoblastoma
    D. Central neurocytoma

    Question 2: Which of the following statements is true regarding this tumor?

    A. They are predominantly supratentorial
    B. Posterior fossa tumors in adults have a good prognosis
    C. Spinal tumors have a bad prognosis
    D. IDH 1 and 2 mutations are present in 100% cases.

    Question 3: Identify the variant of the tumor shown in the image below.

    A. Tancytic
    B. Myxopapillary
    C. Subependymoma
    D. Anaplastic

    Question 4: Which of the following statements is true regarding tancytic ependymoma?

    A. GFAP negative
    B. Abundant rosettes
    C. Can be confused with pilocytic astrocytoma
    D. WHO Grade 1

    Question 5: Identify the FALSE statement regarding the variant of ependymoma shown below

    Image credit- http://www.twitter.com/DrAldehyde

    A. WHO Grade 1
    B. Arises from filum terminale
    C. Aggressive clinical course
    D. Mucin stains positive

    Question 6: TRUE statement regarding recent molecular classification (WHO CNS 2021 5th edition ) of ependymoma is

    A. Spinal ependymomas with MYCN have a good prognosis
    B. ZFTA Fusion- favorable prognosis
    C. YAP Fusion- poor prognosis
    D. Spinal ependymomas with NF 2 mutation- favorable prognosis

    ANSWER- Question 1

    Correct answer is B- Ependymoma

    Ependymomas are characterized by perivascular rosettes and ependymal rosettes which have an empty central lumen

    Types of rosettes
    ANSWER- Question 2

    CORRECT ANSWER IS B

    🐱 Majority of of Ependymomas are infratentorial.

    🐱Posterior fossa tumors in children have a poor prognosis (PF-A)- associated with loss of H3K27me, whereas Posterior fossa tumors in adults have a god prognosis (PF-B)- H3K27me is retained.

    🐱 Spinal ependymomas have a good prognosis, whereas a small number of ependymomas associated with MYCN have a poor outcome.

    🐱IDH 1 and 2 are associated with astroctyomas and oligodendroglomas but nor ependymomas.

    ANSWER-Question 3

    CORRECT ANSWER IS A

    Diagnosis is TANCYTIC EPENDYMOMA, 🐱 Tancytes are cells with fibrillary processes which line ventricles along with ependymal cells.

    Types of tancytes
    ANSWER – Question 4

    Correct They are GFAP positive 🐱Confused with pilocytic astrocytoma because of the prominent fibrillary projections 🐱It is a GRADE II Ependymoma 🐱 Ependymal rosettes and pseudorosettes are scanty in tanytic ependymoma, which makes diagnosis challenging.

    ANSWER- Question 5

    Correct answer is C
    💀Diagnosis is myxopapillary ependymoma
    💀It is a Grade II Tumor according to latest WHO
    💀It is common in adults and arises from the filum terminale
    💀It is clinically indolent ( nor aggressive)
    💀Stains for Mucin is positive.

    Mucin stains positive in myxopapillary ependymoma
    ANSWER – Question 6

    Correct answer is D

    Spinal ependymomas with NF2 mutation has a good prognosis

    Summary of latest molecular updates in ependymoma

    CNS Pathology case based MCQs-1

    Case: A 12- year old boy presents with headache , projectile vomiting and blurry vision. CT scan shows a posterior fossa mass lesion. Histopathologic image of the resected section is seen below.

    CNS Pathology case based  MCQs-1

    Answer the questions based on this case

    Question 1: Which of the following genetic alterations has the BEST prognosis in this tumor?

    A. WNT-activation
    B. SHH- activation, TP53 wildtype
    C. NMYC-amplification
    D. SHH- amplification, TP53 mutated

    Question 2: Identify the false statement regarding this tumor.

    A. Associated with turcot syndrome
    B. Extremely radiosensitive
    C. WHO Grade 3
    D. TP53 mutation is associated with a bad prognosis

    Question 3: Most common cytogenetic abnormality associated with this tumor is?

    A. Isochromosome 17q
    B. Monosomy 6
    C. Isochromosome 12p
    D. Balanced translocations

    DIAGNOSIS

    Diagnosis is medulloblastoma

    ANSWERQuestion 1

    Correct answer is A: WNT-activated medulloblastomas have the best prognosis among all.

    SHH- amplification, TP53 mutated medulloblasyomas have a bad prognosis.

    ANSWER Question 2

    Correct answer is C
    🍉Medulloblastoma is WHO Grade 4
    🍉Syndromes associated with medulloblastoma- Gorlin syndrome, Turcot syndrome
    🍉It is a radiosensitive tumor.
    🍉Isochromosome 17q is association with a bad prognosis.

    ANSWER Question 3

    Correct answer is A

    Most common cytogenetic abnormality in medulloblastoma is isochromosome 17 q

    WNT-activated medulloblastomas are associated with monosomy 6 and have a better prognosis.

    Central nervous system pathology MCQ 1

    Try to answer this CNS pathology MCQ

    Click here to view the answer and complete explanation

    Correct answer is D
    🥮The most common BRAF mutation in pilocytic astrocytoma is
    KIAA1549-BRAF fusions, however BRAF V600E mutations are also seen.
    🍕Pilocytic astrocytoma is of glial origin and hence is GFAP positive
    🍪Pilocytic astrocytoma is associated with mutations in NF-1 but not TSC- 2 gene.