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HISTOPATH LEC > Fixation + Addtl. Discussion > Flashcards

Fixation + Addtl. Discussion Flashcards

1
Q

This step includes entering the details of the specimen in a log book

A

NUMBERING

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2
Q

Gross description is always done by?

A

PATHOLOGIST

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3
Q

Most important and most crucial step in tissue processing. It has to be carried out adequately.

A

FIXATION

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4
Q

Step in tissue processing that is define as the killing, penetration and hardening of tissues

A

FIXATION

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5
Q

May also be defined as the alteration of tissues by stabilizing protein so that tissues become resistant to further changes

A

FIXATION

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6
Q

Primary goal of Fixation:

A

To preserve the morphological and chemical integrity of the cells and tissues as close to the original as possible

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7
Q

Secondary goal of Fixation:

A

To harden tissues;
To facilitate easy cutting;
To protect the tissue from trauma of further handling

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8
Q

2 Fixation Methods:

A

PHYSICAL and CHEMICAL

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9
Q

Types of Physical Method:

A

Heat Fixation and Microwave Technique
Cryo-preservation (freeze drying)/ freeze substitution

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10
Q
  • Mostly done in microbiology to fix bacterial
    smears
  • Not usually carried out in Histopath (rarely used)
  • For rapid diagnosis
  • For frozen tissue section
A

Heat Fixation

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11
Q

Fixatives under Chromate Fixatives: (CROP)

A

Chromic acid (1-2%)
Regaud’s/ Moller’s fluid
ORTH’S FLUID
Potassium Dichromate (3%)

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12
Q
  • physical technique that is widely used but not a common method.
  • it increases movement of molecules thereby accelerating fixation, staining, decalcification, immunohistochemistry and EM
A

Microwave Technique

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13
Q

Neurochemical substances in brain that can be used by microwave techniue

A

Acetylcholine

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14
Q

A method of fixation where the specimen is immersed in chemical fixative

A

CHEMICAL METHOD

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15
Q

Reagent used to fix tissue is called?

A

Fixative

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16
Q

Factors involved in fixation/ practical considerations

A
  1. Temperature
  2. Thickness/ Size
  3. pH
  4. Osmolality
  5. Concentration
  6. Time and Duration of Fixation
  7. Volume
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17
Q

Temp. required for EM and histochemistry

A

0-4 degree Celsius with Autotechnicon

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18
Q

Fixation is traditionally carried out at what temperature?

A

Room temperature

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19
Q

Required temp. in Autotechnicon?

A

40 degree Celsius

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20
Q

TRUE or FALSE. Autotechnicon is done usually in laboratory and it uses constant agitation?

A

TRUE

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21
Q

TRUE or FALSE. Formalin @ 60 deg C may be use to fix tissues with TB

A

FALSE

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22
Q

TRUE or FALSE. Formalin at 60 deg C is for rapid fixation for urgent biopsy

A

TRUE

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23
Q

Temperature for formalin required in order to fix tissues with tuberculosis

A

Formalin @ 100 deg C

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24
Q

3 Fixatives for Smear (SME)

A
  1. Methanol
  2. Ether/ Ethanol
  3. Schaudinns Fluid
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25
Q

Required thickness of tissue spx for electron microscopy (EM)

A

1-2 mm^2

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26
Q

Required thickness of tissue spx for light microscopy (LM)

A

2 cm^2 wide and not more than 4mm thick

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27
Q

TRUE or FALSE. The thickness of tissues spx for LM should be more than 0.5 cm

A

FALSE; should be no more than 0.4 cm - 0.5 cm (4-5 mm) for LM

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28
Q

TRUE or FALSE. Many laboratories use tissue processors that work at 45 deg C for regular tissue processing?

A

FALSE; 40 deg C

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29
Q

TRUE or FALSE. Refrigeration is used to speed up decomposition if the tissue needs to be photographed and cannot be fixed immediately

A

FALSE; refrigeration is used to SLOW DOWN

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30
Q

TRUE or FALSE. Brain cells can deteriorate very quickly

A

TRUE

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31
Q

TRUE or FALSE. Nucleic acids react with fixatives to any extent at room temperatures and chemical reactions including those involved in fixation are more slower at higher temperatures

A

FALSE; nucleic acids does not react; rapid at higher temperatures

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32
Q

TRUE or FALSE. An increase in temperature can increase the rate of fixation but can also increase the rate of autolysis

A

TRUE

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33
Q

Required thickness for processing lung specimen

A

1-2 cm

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34
Q

TRUE or FALSE. Tissues should not be more than 4-5mm except in processing lung specimen

A

TRUE

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35
Q

TRUE or FALSE. Uterus and other large solid tissues must be opened/ slice thinly to improve penetration of fixatives

A

TRUE

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36
Q

TRUE or FALSE. Fecal matter and stomach contents can inhibit the penetration of fixatives and must NOT removed before the fixation process

A

FALSE; it should be remove before fixation process

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37
Q

Brain is usually suspended whole in what fixative for how many weeks to ensure fixation and some hardening prior to sectioning

A

10% buffered formalin for 2-3 weeks

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38
Q

TRUE or FALSE. Most tissues can be cut and trimmed without prior fixation such as the brain

A

FALSE; most tissues EXCEPT brain is generally soft when unfixed so it must be fixed before sectioning

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39
Q

TRUE or FALSE. An incompletely fixed tissue may lead to improper and incomplete clearing and impregnation, and may later prove to be a hindrance to normal sectioning and staining of specimen

A

TRUE; FIXATION IS MOST IMPORTANT STEP IN TISSUE PROCESSING

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40
Q

TRUE or FALSE. Penetration into a thin section will occur more rapidly than for a thick section

A

TRUE

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41
Q

TRUE or FALSE. Formalin penetrates tissues slowly so specimens may need to be opened, incised, or sliced and left to fix for an adequate period of time prior to processing

A

TRUE

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42
Q

TRUE or FALSE. Larger tissues require more fixatives and longer fixation time

A

TRUE

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43
Q

TRUE or FALSE. To maintain an adequate fixation time of 4- hours, the recommended size of the tissue is 2 cm^2 and no more than 4mm thick

A

TRUE

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44
Q

Fixation is best carried out close to neutral pH, in what range?

A

Between pH of 6-8

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45
Q

TRUE or FALSE. Acidity favors formation of formalin-heme pigment that appears black, polarizable deposits in tissue

A

TRUE

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46
Q

Common buffers includes…

A

Phosphate
Bicarbonate
Cacodylate
Veronal

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47
Q

Commercial formalin is buffered with phosphate at a pH of…?

A

pH of 7

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48
Q

What will happen if the cells are fixed in a hypertonic solution?

A

Cell Shrinkage

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49
Q

Effect of hypotonic solution?

A

Cells may swell

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50
Q

Preferred osmolality for fixation

A

Slightly hypertonic (400-450 mOsm)

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51
Q

What component is commonly added to osmium tetroxide fixatives for electron microscopy?

A

Sucrose

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52
Q

Osmolality classification used in practice and may be use as holding solutions for tissues to be transported to frozen sections or kidney biopsies for special processing

A

Isotonic solution

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53
Q

TRUE or FALSE. Concentration of fixative should be adjusted down to the lowest level possible

A

TRUE

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54
Q

Concentration of fixative used for formaldehyde?

A

10% solution

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55
Q

Concentration of fixative used for glutaraldehyde and used for EM

A

3% solution

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56
Q

Ideal concentration of fixative for immuno-electron microscopy

A

0.25% glutaraldehyde

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57
Q

Ideal time to perform fixation

A

20-30 minutes following interruption blood supply

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58
Q

TRUE or FALSE. In order to maintain tissue morphology, samples SHOULD BE FIXED IMMEDIATELY after removal or death to prevent autolysis or putrefaction.

A

TRUE

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59
Q

TRUE or FALSE. More cellular organelles will be lost, more nuclear shrinkage and artefactual clumping will occur and tissue spx can be irreversibly damaged when the fixation is delayed

A

TRUE

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60
Q

It refers to the period the tissue is exposed to formalin

A

Fixation time

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61
Q

Fixation is retarded by:

A

Large and thick tissues
Presence of mucus
Presence of blood
Presence of fat
Cold temperature
(can make the fixation time LONGER/ PROLONG)

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62
Q

TRUE or FALSE. Cold temperature can inactivate enzymes

A

TRUE

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63
Q

TRUE or FALSE. Cold temperature can inactivate enzymes

A

TRUE

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64
Q

Fixation is accelerated by:

A

Smaller and thin tissues
Agitation — continuous stirring
Heat (37-56 degrees C)
(can make fixation time SHORTER/FASTER)

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65
Q

Required temperature for heat in tissues

A

37-56 deg C only

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66
Q

TRUE or FALSE. Beyond 56 deg C heat is acceptable for tissues spx

A

FALSE; 37-56 deg C heat only, beyond 56 deg C can be damaging to tissues

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67
Q

Fixative volume for maximum effective fixation

A

20x the volume of the specimen

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68
Q

Ratio of fixative to tissue

A

20:1

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69
Q

Formalin diffuses into the tissue at the rate of:

A

1mm per hour

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70
Q

Fixatives used for EM: (KAPOG)

A

Karnovsky’s
Acrolein
Paraformaldehyde
Osmium tetroxide
Glutaraldehyde

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71
Q

Volume required if osmium tetroxide is used for EM

A

5-10 times the volume of specimen

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72
Q

Volume required for museum preparations

A

Should not be less than 50-100 times the volume of the specimen

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73
Q

TRUE or FALSE. Agitation will also enhance fixation of the specimen

A

TRUE

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74
Q

Most common error in histotechnology is…

A

Insufficient ratio of tissue volume to fixative

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75
Q

Human brain must undergo in what method in fixation process

A

INTRAVASCULAR PERFUSION

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76
Q

This is used to wash out blood in human brain during intravascular perfusion

A

RINGER’S LACTATE

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77
Q

TRUE or FALSE. Eyes should be dissected before they are fixed

A

FALSE; it SHOULD NOT be dissected; tissues may wrinkle; inject formol alcohol before immersing the organ to fixative

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78
Q

If the autopsy materials is not possible to fix immediately after death, the body must be placed where and at what temperature?

A

Mortuary ref (4 deg C)

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79
Q

Hard tissues (cervix, uterine, fibroid, etc.) must undergo in what method?

A

LENDRUM’S METHOD

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80
Q

What method washes with running water overnight then immerse specimen in 4% aqueous phenol for 1-3 days.

A

Method of Lendrum’s Method

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81
Q

Lendrum’s method is done before, during or after fixation?

A

After fixation

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82
Q

Hollow organs (stomach, intestine) should be…

A

Packed with cotton soak in fixative;
The spx can be sliced and it must be completely open before immersing it in adequate fixing solution

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83
Q

What specific organ may float on fixative?

A

Air filled lungs

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84
Q

How to prevent air filled lungs floating on a fixative?

A

May cover it on several layers of gauze to keep it at the bottom of the container

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85
Q

Effects of fixatives in general:

A

Harden tissues
Makes cells resistant to damage
Increase optical differentiation of cells
Acts as mordants or accentuators to facilitate easy staining

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86
Q

(Problems in Fixation) What artefacts that may appear in tissue spx when the washing was incomplete?

A

Presence of artefacts —> white paraformaldehyde, precipitate artefacts

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87
Q

PROBLEMS IN FIXATION

A
  1. Incomplete washing may lead to: presence of artefacts on tissues
  2. Overfixation will render the tissue brittle and hard, shrinkage and swelling
  3. Loss of substance soluble in fixing agent, loss or inactivation of enzyme may result from wrong choice of fixative.
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88
Q

Cause of failure to arrest early autolysis of cells

A

Failure to fix immediately (the tissue was probably allowed to dry before fixing); Insufficient fixative

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89
Q

Cause of removal of substances soluble in fixing agent

A

Wrong choice of fixative

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90
Q

Presence of artefact pigments on tissue sections

A

Incomplete washing of fixative

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91
Q

Cause of issues are soft and feather-like in consistency

A

Incomplete fixation

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92
Q

Cause of loss or inactivation of enzymes needed for study

A

Wrong choice of fixative

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93
Q

Cause of shrinkage and swelling of cells and tissue structure

A

Overfixation

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94
Q

Cause of tissues block are brittle and hard

A

Prolonged fixation

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95
Q

Well-known artifact that may be produced under acid conditions

A

Formalin pigment

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96
Q

What substance should use to eliminate the pigment or may be used to reduce the fixation of the specimen?

A

Phenol-formalin

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97
Q

May be found in surgical specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue stained in H&E stained sections

A

Crush artifact

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98
Q

Can be used to vapor-fix freeze-dried tissues

A

Paraformaldehyde and Osmium tetroxide

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99
Q

TRUE or FALSE. Fixation does not prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body

A

FALSE; fixation PREVENTS

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100
Q

TRUE or FALSE. Fixation in 10% buffered formalin will initially cause slight swelling of tissue specimens

A

TRUE

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101
Q

TRUE or FALSE. During processing, the spx may shrink and lose 20%-30% of its volume

A

TRUE

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102
Q

TRUE or FALSE. The particular fixative employed will also influence the degree to which individual elements will stain with various histochemical and immuno-histochemical reagents

A

TRUE

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103
Q

TRUE or FALSE. Leaving a tissue spx in air for a prolonged period of time will cause it to dry out, and will result in distortion of its morphological appearance

A

TRUE

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104
Q

BENEFITS OF FIXATION

A
  1. Allows thin sectioning of tissue by hardening it
  2. Prevents autolysis and inactivates infectious agents (except prion diseases)
  3. Improves cell avidity for special stains
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105
Q

Factors to be considered when choosing the right fixative

A
  1. The need for immediate examination: urgency of the case (urgent biopsy = use a rapid fixative in action)
  2. Type of specimen to be processed
  3. The tissue structure to be studied.
  4. Structure technique to be applied (the fixative must be compatible with the stain to be used)
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106
Q

Types of fixatives as to MECHANISM OF ACTION

A

Additive
Non-additive

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107
Q

Fixatives that when used is ABSORBED BY THE TISSUE; stabilizes tissue proteins

A

Additive Fixatives

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108
Q

Examples of Additive Fixation

A

Formaldehyde
Mercuric Chloride
Chromium Trioxide
Picric Acid
Glutaraldehyde
Osmium Tetroxide
Zinc Sulfate or Chloride

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109
Q

Fixing agent that is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attach to H-bonds of certain groups within the protein molecule.

A

Non-additive Fixation

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110
Q

Fixative is not absorbed by the tissue; alteration of tissue components

A

Non-additive Fixation

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111
Q

Example of Non-additive Fixatives

A

Alcohol
Acetone

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112
Q

Acts by cross-linking proteins

A

Aldehyde and Oxidizing Agents

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113
Q

Protein-denaturing agents

A

Alcohol based fixatives

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114
Q

Acts by forming insoluble metallic precipitates

A

Metallic fixatives

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115
Q
  • Cheap
  • Stable
  • Safe to handle
  • Must kill the cell quickly
  • Must inhibit bacterial decomposition and autolysis
  • Produce minimum shrinkage of tissue
  • Permit rapid and even penetration of tissues
  • Must harden tissues
  • Must be isotonic (some are hypotonic solutions)
  • Must make cellular component insoluble to hypotonic solutions and render them insensitive to subsequent processing
  • Must permit subsequent application of many staining procedures to facilitate easier and more profitable examination
A

Characteristics of a good fixative

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116
Q

Non-additive fixative that alters the tissue component

A

Acetone

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117
Q

Types of Fixative According to Action: (MCH)

A

Cytological
Microanatomical
Histochemical

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118
Q

Use to preserve PART OF THE CELLS (nucleus or cytoplasm)

A

Cytological

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119
Q

Preserves parts of cytoplasm;
pH greater than 4.6;
DO NOT CONTAIN GLACIAL ACETIC ACID because HAc destroys the mitochondria and golgi bodies of the cytoplasm

A

Cytoplasmic Fixatives

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120
Q

Fixatives that (should) contain glacial acetic acid;
pH of < 4.6;
Preserves nuclear structures like nuclear chromatin and chromosomes

A

Nuclear Fixatives

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121
Q

Examples of Nuclear Fixatives: (BF(e)NCH)

A

Bouin’s Fluid
Flemming’s Fluid
Newcomer’s Fluid
Carnoy’s Fluid
Heidenhain’s susa

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122
Q

Fixatives for Cytoplasmic: (HORFF)

A

Helly’s/ Kelly’s/ Zenker Formol
Orth’s Fluid
Regaud’s Fluid (Moller’s)
Flemming’s without HAc
Formalin with post-chroming

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123
Q

Fixes Rickettsia org. and other bacteria

A

Orth’s fluid

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124
Q

Fixative that reacts with viruses, and causes the loss of their infective power

A

Mercuric Chloride

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125
Q

TRUE or FALSE. Glacial acetic acid destroys mitochondria and golgi bodies of the cytoplasm

A

TRUE

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126
Q

Gives the best quantitative results using frozen tissues as the standard

A

Ethanol and Acetone

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127
Q

Allows the GENERAL MICROSCOPIC STUDY of tissue structures WITHOUT ALTERING THE STRUCTURAL PATTERN normal intercellular relationship of tissues

A

Microanatomical Fixatives

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128
Q

Fixatives for Microanatomical:

A

10% Formol saline
10% Neutral buffered formalin
Heidenhain’s susa
Formol sublimate/ formol corrosive
Zenker’s formol (helly’s)
Zenker’s solution
Bouin’s
Brasil’s

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129
Q

Used to preserve chemical components of tissues like enzymes

A

Histochemical

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130
Q

Fixatives for Histochemical: (FAcNAe)

A

10% Formol saline
Acetone
Newcomer’s fluid
Absolute ethyl alcohol

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131
Q

Fixative used for the detection of rabies

A

Acetone

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132
Q

Types of Aldehyde Fixatives: (FGG)

A

Formaldehyde/ Formalin
Glutaraldehyde
Glyoxal

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133
Q

Types of Metallic Fixatives: (MCL)

A

Mercuric Chloride
Chromate Fixatives
Lead Fixatives

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134
Q

Types of Picric Acid:

A

Bouin’s solution
Brasil’s alcoholic picformol
Holllande’s solution

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135
Q

Types of Alcohol Fixatives (MINCER)

A

Methyl alcohol
Isopropyl
Newcomer’s
Carnoy’s
Ethyl alcohol
Rossmann’ solution
Others: Clarke’s solution, Methacarn

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136
Q

Used for routine; m
Most common fixative reagent
Soluble in water to the extent of 37-40% solution volume
AKA 100% formalin

A

Formaldehyde/ Formalin

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137
Q

Fixative recommended for mailing specimens (tolerant fixative) and for colored tissue photography

A

Formalin

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138
Q

Diluted form of the concentrated solution;
Routine tissue fixative;
Most commonly used fixative

A

10% formalin

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139
Q

Advantages of Formalin:

A

Cheap
Easy to prepare
Readily available
Can preserve fats
Stable

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140
Q

Disadvantages of Formalin:

A

Fumes are irritating
May cause dermatitis on prolonged contact
May form brown pigment on blood containing tissues like spleen

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141
Q

Methods in removal of Formalin pigments:

A

Kardasewitsch method
Lillie’s method
Picric Acid method
1% KOH in 80% alcohol

142
Q

Component of Kardasewitsch method:

A

70% ethanol
28% ammonia water

143
Q

Components of Lillie’s method:

A

Hydrogen Peroxide
28% ammonia water
Acetone

144
Q

Components of Picric Acid method:

A

Saturated Alcoholic Picric Acid

145
Q

Most widely used fixative for routine histology buffered to pH 7 with phosphate buffer

A

10% neutral buffered formalin (phosphate buffer)

146
Q

It is the considered the fixative of choice for many other procedures that require paraffin embedding, including immunohistochemistry and interphase Fluorescent In-Situ Hybridization (FISH)

A

10% neutral buffered formalin

147
Q

Fixative which is for post-mortem tissue and CNS tissues
Classified as histochemical fixative

A

10% formol saline

148
Q

Remedy for precipitation of white paraformaldehyde

A

May add 10% methanol or filter it

149
Q

Prolonged storage of formaldehyde = ?

A

Precipitation of white paraformaldehyde

150
Q

TRUE or FALSE. Overnight fixation is generally indicated for 10mm thick slices of tissues

A

TRUE

151
Q

TRUE or FALSE. Variations in time and conditions of fixation cause the majority of problems in histochemistry

A

TRUE

152
Q

TRUE or FALSE. Formalin cannot preserve fats, mucin, and glycogen

A

FALSE; Formalin can preserve

153
Q

TRUE or FALSE. Formalin can preserve proteins but it does not precipitate proteins

A

TRUE

154
Q

TRUE or FALSE. Formalin does not make tissues brittle and therefore, it is the recommended for nervous tissue preservation

A

TRUE

155
Q

TRUE or FALSE. Formalin is a soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding

A

TRUE

156
Q

Prolonged fixation may produce:

A
  1. Bleaching of the specimen and loss of natural tissue colors
  2. Dispersal of fat grom the tissue into the fluid
  3. Dissolution or loss of glycogen and urate crystals
157
Q

Recommended for fixing tissues with iron pigments and elastic fibers

A

10% Neutral Buffered Formalin or Phosphate Buffered Formalin

158
Q

AKA FORMOL SUBLIMATE

A

Formol Corrosive

159
Q

Mercuric chloride + formaldehyde; recommended for lipids, neutral fats, and phospholipids

A

Formol corrosive/ Formol sublimate

160
Q

AKA Gendre’s solution

A

Alcoholic Formalin

161
Q

Composition of Alcoholic Formalin:

A

95% ETOH
Picric Acid
Glacial Acetic Acid

162
Q

Fixative to use to fix sputum specimen and for microincineration techniques

A

Alcoholic Formalin

163
Q

When small tissues are burn into ashes to identify mineral elements from the ashes

A

Microincineration Techniques

164
Q

Fixative used for enzyme histochemistry and electron microscopy

A

Glutaraldehyde

165
Q

Formula of 10% formal-saline:

A

40% formaldehyde: 100ml
Distilled water: 900ml
Sodium dihydrogen phosphate monohydrate: 4gm
Disodium hydrogen phosphate anhydrous: 6.5gm

166
Q

Formula of 10% neutral -buffered formalin

A

Distilled water
40% formaldehyde
Sodium dihydrogen phosphate, anhydrous
Sodium dihydrogen phosphate

167
Q

Fixative that considered as alternatives to mercuric chloride formulations;
Can give improves results with immunohistochemistry

A

Zinc Formalin

168
Q

Fixatives under Glutaraldehyde that are also for EM

A

Karnovsky’s paraformaldehyde-glutaraldehyde and ACROLEIN

169
Q

Required solution of glutaraldehyde for small tissue fragments

A

2.5% solution

170
Q

Required solution of glutaraldehyde for large tissues less than 4mm thick

A

4% solution

171
Q

TRUE or FALSE. Glutaraldehyde can cause rapid and irreversible changes but it can fixes quickly and well suited for EM, it fixes well at 4 deg C and gives best overall cytoplasmic and nuclear detail

A

TRUE

172
Q

TRUE or FALSE. Glutaraldehyde does not cause dermititis

A

TRUE

173
Q

TRUE or FALSE. Glutaraldehyde is more expensive and less stable

A

TRUE

174
Q

Supplied as 40% aqueous solution;
Commercially available;
Suited for urgent biopsies

A

Glyoxal

175
Q

Fast acting fixative;
Can fix tissues rapidly;
Smallest aldehyde fixative;
Suited in urgent biopsies

A

Glyoxal

176
Q

Surgical specimens are fixed within how many hours using glyoxal fixativesurgical specimens are fixed within how many hours using glyoxal fixative

A

4-6 hours

177
Q

Small biopsy specimens are fixed within how many minutes using glyoxal fixative

A

45 minutes

178
Q

Most common metallic fixative and may form black mercury deposits

A

Mercuric Chloride

179
Q

Remedy for black mercury deposits formed by Mercuric Chloride

A

Wash tissue with alcoholic iodine (treating the section with 0.5% iodine solution in 70% ethanol for 5-10 minutes)

180
Q

Mercuric Chloride is excellent for:

A

Trichrome staining

181
Q

TRUE or FALSE. Mercuric chloride precipitates all proteins; has greater affinity to acid dyes and preferred in lieu of formaldehyde for cytoplasmic staining.

A

TRUE

182
Q

It is recommended for renal tissue, fibrin, connective tissue and muscle

A

MERCURIC CHLORIDE

183
Q

Contains mercuric chloride and glacial HAc;
Recommended for fixing liver, spleen, connective tissue fibers, and nuclei

A

Zenker’s Fluid

184
Q

AKA Helly’s Fluid

A

Zenker’s formol

185
Q

Contains potassium dichromate and 40% formaldehyde;
Preserves pituitary glands, bone marrow, and other blood containing organs

A

Zenker’s formol

186
Q

Mercuric deposits may be removed by immersing tissues in ALCOHOLIC IODINE prior to staining, through a process known as …

A

De-zenkerization

187
Q

Done by oxidation with iodine to form mercuric iodide

A

De-zenkerization (iodine - sodium thiosulfate - water)

188
Q

Excellent fixative for bone marrow, extramedullary hematopoiesis and intercalated discs of cardiac muscle

A

Zenker’s Formol or HELLEY’S

189
Q

Fixative combined with anhydrous acetate;
For preserving bone marrow

A

Lillie’s B5 fixative

190
Q

Composition of B-5 Fixative

A

4% aqueous formaldehyde with 0.22M chloride and 0.22M acetic acid

191
Q

TRUE or FALSE. Mercuric chloride ensures rapid structural stabilization and also facilitates bright staining by many of the dyes used in microtechnique

A

TRUE

192
Q

Estimate fixation time for Lillie’s B5 fixative

A

4-8 hours

193
Q

Fixatives under Mercuric Chloride: (BHZZ)

A

B5
Heidenhain’s susa
Zenker’s Fluid
Zenker’s Formol
Others: Schaudinn’s Ohlmacher’s, Carnoy-Lebrun solution

194
Q

Used for acid mucopolysaccharides and tissue mucin

A

Lead Fixatives

195
Q

Chromate fixative that preserves carbohydrates and precipitates all proteins

A

Chromic acid (1-2%)

196
Q

Chromate fixative that preserve lipids and mitochondria

A

Potassium dichromate (3%)

197
Q

AKA Muller’s fluid

A

Regaud’s

198
Q

Chromic fixative for mitochondria, mitotic figures, golgi bodies, RBC, and containing colloid tissues

A

Regaud’s (Moller’s)

199
Q
  • chromate fixative for rickettsia and other bacteria
  • also for tissue necrosis, and the early degenerative process.
A

Orth’s fluid

200
Q

pH value used in potassium dichromate to fix mitochondria

A

pH 4.5-5.2

201
Q

Agent can act as a fixative, stain, and decalcifying agent

A

PICRIC ACID

202
Q

Excellent for glycogen demonstration and Brilliant staining with trichome method

A

PICRIC ACID

203
Q

Major drawback of Picric Acid

A

Imparts a YELLOW color when used as a fixative

204
Q

Remedy for drawback (yellow impart) of Picric Acid:

A

Saturated solution of Lithium carbonate in 70% alcohol then wash with water
The tissue is then placed in 70% ethanol followed by sodium thiosulfate and washed with water

205
Q

TRUE or FALSE. Orth’s fluid preserves myelin better than buttered formalin

A

TRUE

206
Q

TRUE or FALSE. Regaud’s and Orth’s fluid can preserve fats

A

FALSE; Regaud’s and Orth’s fluid does not preserve fats

207
Q

Good fixative for connective tissue, preserves glycogen well, and extracts lipids to give superior results in immunostaining of biogenic and polypeptide hormones

A

Picric Acid Fixatives

208
Q

TRUE or FALSE. Picric Acid is explosive in dry form

A

TRUE; should be stored in moist distilled water or saturated alcohol

209
Q

This stain use combinations of anionic dyes with phosphotungstic or phosphomolybdic acid to impart contrasting colors to cytoplasm, collagen fibers and other components of tissues

A

Trichrome stains

210
Q
  • Fixative used for the fixation of embryo, pituitary biopsies, and endometrial curetting
  • Usually an excellent fixative for preserving soft and delicate structures
A

Bouin’s solution

211
Q
  • This fixative is NOT for kidneys, lipids and mucus
  • It abolishes Feulgen’s reaction
A

Bouin’s Fluid

212
Q

Demonstrates RNAs and DNAs

A

Feulgen reaction

213
Q

Excess picric should be washed from tissues prior to staining with…?

A

70% ethanol

214
Q

A fixative known to be excellent for glycogen

A

Brasil’s Alcoholic Picformol

215
Q

Fixative for GIT biopsies and endocrine tissues

A

Hollande’s solution

216
Q

TRUE or FALSE. One disadvantage of using Bouin’s solution is that it causes RBC hemolysis and reduces the amount the demonstrable ferric iron in tissue

A

TRUE

217
Q

TRUE or FALSE. Hollande’s solution produces LESS lysis than Bouin’s solution

A

TRUE

218
Q

Fixative that is considered as Picric Acid and Alcohol

A

Gender’s fluid

219
Q

It is the preferred fixative for tissues to be stained by Masson’s trichrome for collagen, elastic or connective tissue;
It does not need “washing out”

A

Gender’s fluid

220
Q

Fixative that is better and less messy than Bouin’s solution

A

Brasil’s Alcoholic Picroformol Fixative

221
Q

The major effects of Acetic Acid are…

A

Precipitate DNA and for preservation of nuclei

222
Q

A compound fixative, recommended for nucleoproteins

A

Glacial Acetic Acid

223
Q

Glacial acetic acid solidifies at what temperature?

A

17 deg C

224
Q

TRUE or FALSE. Acetic acid is always incorporated into other fixatives to form a compound solution, most commonly at a concentration of approximately 5%

A

TRUE

225
Q

Can be a fixative or decalcifying agent;
Also used for precipitation of proteins and nucleic acids

A

Trichloroacetic Acid (TCA)

226
Q

TRUE or FALSE. TCA is a poor penetrating agent, and suitable only for small pieces of tissues or bones

A

TRUE

227
Q
  • Fixative used at ice cold temperature (-5-4 deg C);
  • Can also fix and dehydrate tissue at the same time
A

ACETONE

228
Q

Fixative recommended for preservation of water-diffusable enzymes (lipases, phosphatases) and for to fix brain tissue for diagnosis of rabies

A

ACETONE

229
Q

Type of fixatives that rapidly denatures and precipitates proteins;
Can also act both as fixative and dehydrating agents;
Ideal for small tissue fragments

A

Alcohol Fixatives

230
Q

TRUE or FALSE. Alcohols are protein denaturant and can cause too much brittleness and hardness, hence, not used routinely for tissues

A

TRUE

231
Q

TRUE or FALSE. Solid specimens taken from patients with gout are usually fixed with 70% methanol for subsequent histochemical detection of sodium urate crystals

A

FALSE; 95% ETHANOL

232
Q

Alcohol fixative that appears to give the most usable DNA fragments for PCR

A

ETHANOL

233
Q

TRUE or FALSE. Alcohol can cause tissue shrinkage and not good for EM

A

TRUE

234
Q

TRUE or FALSE. Absolute alcohol can be used to fix and preserve glycogen, pigments, blood, tissue films and smears

A

TRUE

235
Q

Alcohol fixative for fixing wet and dry smears, blood smears and BM tissues;
Fixes and dehydrates at the same time

A

METHANOL (100%) or Methyl Alcohol

236
Q

Alcohol fixative that is recommended for touch preparations, for special staining (Wright-Giemsa staining)

A

ISOPROPYL (95%)

237
Q

For blood, tissue films and smears, and DNA (alcohol fixative);
Preserves nucleoproteins and nucleic acids, used for histochemistry especially for enzyme studies

A

ETHYL ALCOHOL (70-100%)

238
Q

Most rapid fixative;
Contains alcohol, glacial HAc, and chloroform;
Can also used as fixative and can dehydrate at the same time

A

Carnoy’s fluid

239
Q

Most rapid alcohol fixative;
For urgent biopsies (Chromosomes, Lymph glands), brain for diagnosis of Rabies

A

Carnoy’s fluid

240
Q
  • Fixative that is classified both as nuclear and histochemical fixative
  • Can also preserve mucopolysaccharide and nuclear proteins
A

NEWCOMER’S FLUID

241
Q

Alcohol fixative for CT mucins and umbilical cord

A

ROSSMANN’S

242
Q

TRUE or FALSE. Lower concentration of ethanol (70-80%) will cause TBC hemolysis and inadequately preserve leukocytes

A

TRUE

243
Q

Alcohol fixative that preserves Nissl granules and cytoplasmic granules;
Also permits good nuclear staining and differentiation

A

CARNOY’S

244
Q

TRUE or FALSE. Carnoy’s fluid causes considerable tissue shrinkage and is suitable only for small pieces of tissues due to slow penetration. It also dissolves fat, lipids, and myelin

A

TRUE

245
Q
  • Alcoholic fixative
  • Recommended for frozen sections and smears
  • Can produce fair results after conventional processing if fixation time is kept very short
  • Preserves nucleic acids but extracts lipids.
A

Clarke’s solution

246
Q

Fixative for EM that is not commonly used and quite expensive;
It is also slow-acting fixative;
Excellent stain for lipids in membranous structures and vesicles

A

OSMIUM TETROXIDE

247
Q

Tissues fspx of OSMIUM TETROXIDE

A

Myeline
Peripheral nerves
Processing neurological tissues

248
Q

Most common chrome osmium acetic acid fixative, and excellent for nuclear structures

A

Flemming’s

249
Q
  • Known for cytoplasmic fixatives
  • Preserves cytoplasmic structures particularly the mitochondria
  • Composed of osmic acid and chromic acid
A

Flemming’s without HAc

250
Q

Fixation time for Flemming’s solution

A

24-48 hours

251
Q

Flemming’s solution has a tendency to form artifact pigments, what is the remedy of it?

A

Washing the fixed tissue in running tap water for 24 hours before dehydration

252
Q

Fixatives for Enzyme Histochemistry:

A

4% formaldehyde
Formal saline

253
Q

Fixative for electron histochemistry and electron immunocytochemistry

A

Karnovsky’s paraformaldehyde-glutaraldehyde

254
Q

What substance is used in post chromatization during secondary fixation occurs

A

2.5-3% potassium dichromate (chromate-containing)
- Acts as a mordant for better staining

255
Q

Placing an already fixed tissue to a second fixative to:
- Improve demonstration of particular substance
- Ensure complete hardening and
- For special staining
(not a mandatory step in tissue processing)

A

Secondary fixation

256
Q

What fluids or solution that can be used during washing out process in order to remove excess fixative in tissue?

A
  1. Tap Water (often used)
    - Excess chromates in tissues fixed and in Helly’s, Zenker’s and Flemming’s
    - To remove excess
  2. Alcoholic Iodine
    50-70% alcohol
257
Q

First step of tissue processing wherein the spx will be placed in 10% formalin for the first time?

A

Primary fixation

258
Q

Microscopic study of NORMAL tissues

A

HISTOLOGY

259
Q

4 Types of Tissues:

A

Connective tissues
Epithelial tissue
Muscle tissues
Nervous tissue

260
Q

Microscopic study of ABNORMAL tissues

A

HISTOPATH

261
Q

3 Types of Specimen that is processed in Histopathology:

A

Specimen of cytology
Autopsy specimen
Biopsy specimen

262
Q

Tissues specimen obtained from a patient (ALIVE) for the examination in the lab

A

Biopsy specimen

263
Q

Most common type of biopsy:

A

Incisional
Excisional
Needle biopsy/ Aspiration/ Fine needle aspiration biopsy (FNAB)

264
Q

Type of biopsy that are not usually carried out:

A

Core biopsy and Shave biopsy

265
Q

Type of biopsy that involves the removal of a part of a mass or organ

A

Incisional biopsy

266
Q

Type of biopsy that involves removal of ENTIRE mass or organ;
Known as the most reliable and will give a greater chance to detect cancer

A

Excisional biopsy

267
Q

Type of biopsy that uses needle and syringe to collect tissue spx

A

FNAB or Needle biopsy

268
Q

Type of specimen that is obtained from a dead patient;
Also called as necropsy

A

Autopsy specimen

269
Q

Specimens obtained for studying cell biology;
Example is PAP’s smear

A

Cytology specimens

270
Q

Small fragments are shave from a surface usually skin

A

Shave biopsy

271
Q
  • Kind of specimen that will not undergo tissue processing
  • It allows examination of protoplasmic activity (phagocytosis, motility)
  • Tissues are not permanent it cannot be kept for future purposes
A

Fresh specimen

272
Q
  • Kind of specimen that usually facilitate or examine in histopath lab
  • Specimen that are better and more effective
  • Can keep the specimen on slides for future references
  • Better and more effective
A

Processed tissues

273
Q
  • Type of cytology spx
  • Example is PAPANICOLAU STAIN
  • Used as a screening procedure for cervical cancer
A

Gynecologic specimens

274
Q
  • Cytology specimens such as sputum, CSF, gastric lavage, urine
  • Can be used to detect urothelial malignancies
A

Non-gynecological specimen collected

275
Q

4 method of examination are:

A

Teasing (Dissociation,
Squash Preparation (Crushing)
Smear Preparation
Frozen Section

276
Q

Steps in Teasing/ dissociation:

A
  1. Get the watch glass, place small pieces of tissues, then add NSS
  2. Using a loop or needle aspirator, dissociate or separate.
  3. Place the separated tissues on a slide and examine it under the microscope
277
Q

Stains that can be used in fresh tissue specimen

A

Supravital dyes/stains

278
Q

Type of microscope that can be used to detect movement and mitotic division

A

Phase Contrast Microscope

279
Q

Steps in squash preparation:

A
  1. On a slide, place the specimen
  2. Get another slide
  3. Compress the tissue in between two slides
    (slide-to-slide then compress)
280
Q

4 types of smear preparation

A
  1. Streaking
  2. Spreading
  3. Pull-apart
  4. T ouch preparation
281
Q

Most recommended method of examination if dealing with cells

A

Smear preparation

282
Q

Manner used in Streaking preparation

A

Zigzag manner

283
Q

Advantage of Spreading preparation

A

Preserve intercellular relationship

284
Q

Types of smear preparation that is suited for viscous specimens

A

Pull-apart and Streaking

285
Q

Smear preparation that also called Impression smear and Abraded cytology

A

Touch preparation

286
Q

Recommended glass slides for Touch Preparation
A. Frosted Glass Slide
B. Polished Ends Glass Slide

A

B. Polished Ends Glass Slide

287
Q

Method of examination for rapid diagnosis and done intraoperatively;
Method used to demonstrate heat sensitive structures

A

Frozen section

288
Q

Smear technique that allows slide to come in contact with freshly cut tissues (lymph node)

A

Touch Preparation/ Impression Smear/ Abdraded Cytology

289
Q

Smear technique recommended for fresh sputum and bronchial aspirate, and thick mucoid secretions;
It also maintains intercellular components

A

Spreading

290
Q

Method used for demonstration of fats/ lipids, nervous tissues elemets, and enzymes

A

Frozen section

291
Q

Apparatus used in Frozen Section:

A

Freezing Microtome and Cryostat/ Cold Microtome

292
Q
  • Rapid diagnosis during surgery
  • Diagnostic and research enzyme histochemistry
  • Demonstration of soluble substances like lipids and carbohydrates
  • Immunofluorescent and immunohistochemical staining
  • Special staining in neuropathology
A

Applications of Frozen Sections:

293
Q

FREEZING AGENTS:

A
  1. Liquid nitrogen
  2. Isopentane cooled by liquid nitrogen
  3. Carbon dioxide gas
  4. Aerosol sprays
294
Q

BENEFITS OF FIXATION/ EFFECTS OF FIXATION:

A
  • Prevents autolysis, reduce risk of infection
  • Allows thin sectioning
  • Acts as mordant thus facilitating staining
295
Q

Results from frozen section must be released by how many minutes? (TAT)

A

5-15 minutes

296
Q

Most common freezing agent used

A

Carbon dioxide

297
Q

Average temp of cryostat or comb microtome

A

-20 deg C

298
Q

First and most important step;
This is where you enter the details of patient in LOGBOOK

A

Numbering/ Accessioning

299
Q

TRUE or FALSE. BRAIN tissues must be FIXED BEFORE GROSSING

A

TRUE

300
Q

Before Fixation, large solid tissues should be…?

A

Opened or sliced thinly

301
Q

Factors that can accelerate fixation:

A
  1. Heat application = 37-56 deg C only
  2. Size and thickness of tissue
  3. Agitation - continuous mixing —> allows rapid entry of fixative to tissue
302
Q

Request form must include…

A
  • Name of the patient
  • Age
  • Attending doctor
  • Initial diagnosis
  • Type of specimen
303
Q

Not computerized

A

NUMBERING

304
Q

Computerized

A

ACCESSIONING

305
Q

Size of a tissue cassette

A

2.5 x 4 cm

306
Q

Depth of a tissue cassette

A

5mm

307
Q

An automatic tissue processor that can do fixation, dehydration, clearing, and infiltration;
Consists of 10 1L beakers that arranged in circular position

A

Autotech

308
Q

Highest concertation of formalin

A

37 - 40% Formalin

309
Q

Factors that can DECREASE the fixation time:

A

Increased heat
Agitation
Vacuum
Microwave
Small and thinner tissues specimen

310
Q

Factors that ENHANCE fixation process:

A

Size and thickness
Agitation
Moderate heat (37 deg C)

311
Q

TRUE or FALSE. Presence of mucus and blood in a specimen can be flush with NSS if there’s too many

A

TRUE

312
Q
  1. Spx should be transferred to fixative immediately after surgery (< 1 hr.)
  2. Fixative to specimen: 20:1 or 10:1
  3. Anatomical barriers to fixation (should be removed)
    - fascia (covering of organ)
    - bones
    - feces
    - thick tissue
  4. Large specimens must be sectioned or inflated with fixative or opened and cleaned to allow penetration
    - Lungs —> inflate/ sectioned
    - Intestines/gastrointestinal tract —> opened
  5. Fixatives diluted and contaminated fixative - should be replaced to ensure effectiveness
  6. Pinning of spx to corkboard or inserting a paper or gauze “wick” into tubular structures can improve fixation and reduce tissue distortion
A

Practical Considerations to Optimize Fixation of Tissue

313
Q

May be more difficult to reverse and may also result in loss of immunohistochemical antigenicity

A

Prolonged fixation
Increased fixation –> decreased immunohistochemical antigenicity

314
Q

2 types of COMPOSITION for fixative:

A

Simple fixative
Compound fixative

315
Q

Composition of Saline:

A

Distilled water
NaCl

316
Q

Formaldehyde linked by 3C’

A

Glutaraldehyde

317
Q

TRUE or FALSE. Methyl alcohol can cause BLINDNESS

A

TRUE

318
Q

Microanatomical fixative should never contain ________ because it inhibits hematoxylin (for staining nucleus)

A

Osmium tetroxide

319
Q

Preserving or demonstrating chemical substances in the tissue

A

Histochemical fixatives

320
Q

Formaldehyde waste ways:

A
  1. Recycle by distillation
  2. Drain disposal
  3. Disposal by a licensed waste howler
  4. Detoxification by a commercial product
321
Q

Mercurial reagents/ used to dezenkerize

A
  • Releases mercury and must not go through drain
322
Q

Disposal of mercury is expensive but it can replaced by ____

A

Zinc formalin or Glyoxal solutions

323
Q

Fixative agent cannot be used in lipid fixation

A

ALCOHOL

324
Q

Agents used in Lipid Fixation: (FB MAP D)

A
  • Formaldehydes
  • Baker’s formol-calcium -> for phospholipids
  • Mercuric chloride
  • Aldehydes
  • Potassium dichromate - lipid for cryostat
  • Digitonin -> cholesterol for ultra-structural demonstration
325
Q

Fats (Frozen Section)

A
  • Frozen section
  • Formalin
  • Potassium dichromate
  • Osmic acid
  • Formol calcium
326
Q

Agents for Protein Fixation:

A
  • Neutral Buffered Formalin
  • Formaldehyde Vapor
327
Q

Agents for Carbohydrate Fixation:

A
  • Alcoholic fixatives (glycogen fixation)
  • Rossman’s fluid
  • Cold absolute alcohol
328
Q

BRAIN must suspend WHOLE in ____ for 2-3 weeks to ensure hardening

A

10% buffered formalin

329
Q

TRUE or FALSE. Formaldehyde should never be neutralized because it may cause violent explosion

A

TRUE

330
Q

REMOVAL OF PARAFORMALDEHYDE

A
  • Use methanol to prevent its decomposition to formic acid
  • Can filter paraformaldehyde
331
Q

Color of formalin pigments

A

Brown or Black

332
Q

Formalin pigments occur because of…?

A

Reaction of formic acid and hemoglobin (blood)

333
Q

Formaldehyde (Formalin) recommended for ___

A

CNS

334
Q

It fixes sputum since it coagulates mucus

A

Alcoholic formalin (Gendre’s)

335
Q

All mercuric chloride fixatives cause black precipitate except…?
A. Zenker’s fluid
B. Zenker’s formol
C. Heidenhain’s Susa
D. B5

A

C. Heidenhain’s Susa

336
Q

TRUE or FALSE. Mercuric chloride is used for tissue photography

A

TRUE

337
Q

What to use for removal of black deposits from Mercuric chloride?

A

Use saturated iodine in 96% alcohol

338
Q
  • Contains glacial acetic acid, mercuric chloride, potassium dichromate, sodium sulfate, and distilled water
  • Recommended for small pieces of liver, spleen, connective tissue fibers, nucleic components
A

Zenker’s fluid

339
Q
  • Zenker’s formol
  • Contains formaldehyde, mercuric chloride, potassium dichromate
  • Good for microanatomical fixative
  • Pituitary gland, bone marrow, blood-containing organs (spleen and liver)
A

Helly’s fluid

340
Q
  • Recommended for tumor biopsies of the skin
A

Heidenhain’s Susa

341
Q

Fixative used for PRESERVATION OF BONE MARROW BIOPSIES

A

B5 fixative

342
Q

Composition of Bouin’s solution:

A

Picric acid saturated aqueous solution
40% Formaldehyde
Glacial acetic acid

343
Q
  • Process of removing the excess fixative after fixation
  • To improve staining
  • To remove artifacts from the tissue
A

Washing out

344
Q
  • Less concentrated alcohol fixatives = less lysis
  • Used in small tissue fragments
A

Alcoholic fixatives used in concentration of 70-100%

345
Q

In WASHING OUT:
- Excess chromates in tissues fixed and in Helly’s, Zenker’s and Flemming’s
- To remove excess formalin and cosmic acid

A

Tap water (washing out)

346
Q

In WASHING OUT:
- To remove excess picric acid fixatives (Bouin’s solution)

A

50-70% alcohol (washing out)

347
Q

In WASHING OUT:
- To remove excess mercuric fixatives

A

Alcoholic iodine (washing out)

348
Q
  • Tissue in a fixated
  • 450 watts at 55 deg C for 1.5 minutes to 4 minutes
  • May create fumes
A

Microwave assisted fixation

349
Q

ADVANTAGE OF FIXING TISSUES IN MICROWAVE:

A
  • Tissues is heated through the block for a very short time; potentially allowing rapid study of cellular processes.
  • Can used in neurochemical substances (brain, acetylcholine)
  • For rapid fixation: routine surgical specimens
  • Immunohistochemistry and In-Situ hybridization
350
Q

DISADVANTAGE OF FIXING TISSUES IN MICROWAVE:

A
  • Only penetrates tissues to thickness of 10-15mm
  • No significant cross-linking of protein molecules, and subsequent chemical fixation may be needed
  • Viable spores and pathogens may remain in tissues processed with alcohol-based fixatives or microwave alone
351
Q

Physical method that preserves tissue by rapid exposure of the spx to cold temperatures (-160 to -180 dec C)

A

Freeze drying/ Freeze substitution

352
Q

3 types of Specimen

A

Biopsy
Autopsy
Cytology

HISTOPATH LEC (4 decks)

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