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Histotechnology > Week 12 > Flashcards

Week 12 Flashcards

1
Q

What is used for nuclear staining

A

-Mordant dyes for routine
-Hemotoxylin -colorless but turns red brown, not a true dye
-Hematein-synthetic, oxidized product of hematoxylin. Weak anionic negatively charged dye

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2
Q

Oxidation of Hematoxylin what is natural and chemical Ripening

A

Natural
-exposed to O2 like Delafield and Ehrlich Solutions

Chemical
Mercuric oxide, sodium iodate and KMG like Harris, Mayer and Gill

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3
Q

What is the oxidation of hematoxylin

A

when hematoxylin is converted to hematein by the removal of 2 hydrogen atoms during oxidization of hematoxylin
-oxidized hematoxylin is called hematein and it has little affinity for tissue but becomes a strong dye with particular affinity for nuclear chromatin when it combines with metallic mordant
-oxidizer in Hematoxylin solutions can be a mordant
-solutions should always have unoxidized hematoxylin because oxidation continues with O2
-incomplete oxidation or over ox can cause solution breakdown
-oxidation rate in influenced by pH- faster in alkaline and slower in acidic

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4
Q

Why is the use of mordant in Hematein (hematoxylin) more favoured than using Hematein?

A
  • Inferior quality of hematein dye
  • Short shelf life of hematein dye
  • Hematein dye is expensive

DOES HEMATEIN BECOME POSITIVE

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5
Q

Different types of Mordants

A

Mordants should not oxidize hematoxylin

-Non oxidizing metallic salts

Aluminum salts
– Ammonium aluminum sulfate
– Potassium aluminum sulfate

Iron salts

Weak acids
– Phosphotungstic acid
– Phosphomolybdic acid

Dye + Mordant = Lake

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6
Q

How to achieve selective staining

A

Add

Excess acid: H combines with acidophilic tissue components so they dont take up nuclear staining

Excess aluminum - couteracts over oxidation

Too much aluminum can cause precipitation

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7
Q

Ingredients of a Nuclear staining

A
  • Mordant - aluminum or iron sulfate
  • Oxidizer - sodium iodate, mercuric oxide
  • Stabilizer - glycerol
  • pH adjuster -Citric acid
  • Prevents scum formation - Chloral hydrate
  • Solvent - Alcohol, water, ethylene glycol
  • Usage - progressively or regressively
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8
Q

How will ripened Hematoxylin appear

A

Metallic sheen with aluminum hematoxylin

-Filter before use - blue/black precipitate

-Acetic acid prevents scum by slowing oxidation

Color indicates freshness
– Blue - fresh mordant
– Red - aged but usable
– Brown - over-oxidized

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9
Q

Why is it necessary to add acid to the Hematoxylin solutions?

A

-extends hematoxylin solution shelf life
-reduces number of available anionic group by suppressing ionization of COOH and other weak groups
-hematein selectivity is increased for phosphate groups of nucleic acids - ACCENTUATOR

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10
Q

What is background staining?

A

-non specific staining where non basophilic tissue takes up HX like RBCs, muscle, collagen

Caused by
– Presence of COO- group
– H-bonding
– Presence of amino acid

Can be removed by mild acid treatment

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11
Q

Progressive VS Regressive

A

-depends on the ratio between dye and mordant (D/m) and on the stain application time

– Hx is PROGRESSIVE- high D/M (1:50) with a less than 2 min application time
– Hx is REGRESSIVE- low D/M (1:10) with a greater than 5 minute application time

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12
Q

Differentiation

A
  • removal of excess stain
    -1% Hcl in 70% ethanol (acid alcohol)
    -hydrogen will compete with mordant for tissue

Result
-cytoplasm and CT are colorless
-Nuclei is red for nuclear detail
-alkali reagent is used to reverse blue to red discoloration because the next stain is eosin which is also red. With two red dyes there will be no differentiation between acidophilic tissue and nuclei

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13
Q

What is bluing

A

-reversal of color change during differentiation

-Weakly alkaline solutions are:
– Alkaline running tap water (RTW)
– Lithium carbonate
– Dilute ammonium hydroxide
– Scott’s Tap Water Substitute (TWS)

Bluing reagent changes solubility of the dye lake
– Aluminum-hematoxylin is red
– Soluble in pH <5; blue lake is insoluble in pH >5.

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14
Q

Decolorization VS Differentiation

A
  • Decolorization
    – removal of excess stain macroscopically
  • Differentiation
    – controlled removal of excess stain
    microscopically
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15
Q

Substitute for hematoxylin in H&E procedure

A
  • Celestine blue
  • Mordant - iron
  • Used progressively
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16
Q

Cytoplasmic staining

A

consists of eosin Y and Eosin B
-sodium salt of colored acid
-has BR in it and 4 rings
-para quinoid ring

  • Nuclei - blue
  • RBC - dark pink
  • Muscles - pink
  • Collagen - light pink
17
Q

What is Eosin

A

Chromophore - in the anionic part of the molecule

– @ pH 7 - negatively charged
– @ pH < 6 [4.6 - 5] - can stain proteins (IEP pH 6)
– @ pH < 4 - decreased negative charges on the
dye because the molecule converts to a free
acid.

Free acid binds to tissues by hydrogen binding called Muddy tissues

18
Q

Factors affecting cytoplasmic staining

A
  • Fixative
  • Duration of fixation
  • Heat during processing
  • Stain formulation (concentration)
  • Differentiation step
  • Nature of the collagen
19
Q

Substitute for eosin in H&E procedure

A
  • Eosin - Phloxine B
    – Vivid pink shades but easy to overstain
  • Phloxine B – Saffron
    – Phloxine and saffron used separately
    – Counterstaining takes longest
    – Saffron more expensive

HPS (Hematoxylin, Phloxine, Saffron)
* Nuclei - blue
* Collagen – yellow
* Muscle and cytoplasm - pink

20
Q

How to to achieve good H&E Staining

A

1- Microscopically check slides from each basket to make sure proper staining occurs
-nuclei must be well defined with proper nuclear membrane and crisp chromatin
-eosin shades must be present
-intensity and contrast balanced between 2 dyes

2-stain control slide first

3- dont let sections dry out during staining

4-solutions must be covered to prevent evaporation, if there is precipitation then you need to filter

5 after bluing rinse in tap water, if too much alkaline solution is carried into eosin cytoplasmic staining will be inadequate pH of eosin is critical

6- dont pass through alcohol too quick as it helps to diff

7- some tap water cant be used as rinse because Iron, sulfur and chlorine will produce weak nuclear staining.

21
Q

Why would Tissue Basophilia be lost

A

-if wet tissue is left too long in Bouins solution or unbuffered formalin
-tissues have residual water from improper dehydration
-over decalcification of bones because over decaled bones cant be restored

22
Q

Restoring Tissue Basophilia in three ways

A

1- put deparaffinized slides from tissue over-exposed to Bouin solution in 5% aqueous lithium carbonate for 1hr. Wash in RTW for 10 mins and stain

2- put deparaffinized slides in 5% aqueous sodium
bicarbonate for 3 hrs. Wash in RTW for 5 mins and stain

3- put deparaffinized slides in 5% aqueous periodic acid for 30 mins. Rinse in 3 changes of dh20 and stain

23
Q

What is frozen sectioning

A

Needs to be done right after cryotomy. 2 mins to do and its permenant.
-Hx is filtered and changed daily
-progressive

-Alcohol, acetone, alcoholic formalin or
formaldehyde are used as fixative for frozen
sections.

-fix slides immediately dont let them air dry or the macroscopic preservation will be poor

24
Q

H&E Technique with Steps and Reagent manual and progressive

A
  • Nuclear staining- Harris hematoxylin
  • Differentiation- Acid alcohol (only during regressive)
  • Blueing- Scott’s tap water substitute (STW)
  • Cytoplasmic staining- Eosin Y

in regressive check differentiation microscopically before you counter stain

Histotechnology (13 decks)

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