[F] Week 13: Enzyme Histochemistry and Diagnostic Application Flashcards
(101 cards)
1
Q
Detect early metabolic changes in biopsy and autopsy tissue before manifestation on H&E staining or immunohistochemistry
A
Enzyme histochemistry
2
Q
- Essential not to inactivate an appreciable proportion of the enzyme
- Precision varies inversely with the length of time required in the incubation medium
A
Enzyme histochemistry
3
Q
TOF
Enzyme histochemistry is commonly requested
A
False
4
Q
- May be bound to specific cell components or may be free and soluble in the cytoplasm and body fluids
- Speeds up a biochemical process
A
Enzyme
5
Q
generally required for histochemical demonstration of enzymes
A
Frozen Sections
Tissues frozen to -70°C or below are usually well preserved, with little loss of enzyme activity
6
Q
Several destructive factors
causeing false negative, and disadvantages of unfixed frozen sections:
A
- Mechanical disruption by freezing and thawing
- Uneven section thickness
- Diffusion of soluble enzymes
- Co-factors leading to loss of reproducibility
- False localization
7
Q
The best fixative for all enzymes is?
`
A
chilled acetone
8
Q
What are the disadvantage of using chilled acetone as the fixative for enzyme
A
Even the cytological details are not so good, you can still preserve a lot of enzymes
9
Q
Chilled acetone as fixative is especially recommended when staining for?
A
Acid Phosphatase
10
Q
For other enzymes (other than acid phosphatase) this is the prefarable fixative because it gives a better cytological fixation and the tissue is easier to handle.
A
COLD 90-100% ETHYL ALCOHOL
11
Q
Most hydrolytic enzymes are reasonably resistant to formalin and can be fixed in?
A
10% Formalin
(preferably adjusted to pH 6-6.5 with a small amount of phosphate buffer)
12
Q
entirely unsuitable as a fixative, because it destroys most enzymes
A
Methyl alcohol
13
Q
most common technique
for histochemical demonstration of enzymes
A
METAL PRECIPITATION
14
Q
Enzymes for which histochemical techniques are known belong in one of the two groups:
A
- Oxidative enzymes
- Hydrolytic enzymes
15
Q
catalyze the reaction
between substrate and atmospheric oxygen
A
Oxidative Enzymes
16
Q
Transferring of electron from one
substrate to another substrate - the hydrogen
A
Oxidative Enzymes
17
Q
Oxidative enzyme can be demonstrated by?
A
simultaneous coupling
method
18
Q
simultaneous coupling method, which involves oxidation of the substrate and subsequent reduction of a tetrazolium salt, resulting in
the formation of a?
A
relatively insoluble formazan deposit at the site of enzyme activity
19
Q
The two tetrazolium salts commonly used as hydrogen acceptors:
A
monotetrazolium (MTT) and ditetrazolium chloride-nitro (NBT)
20
Q
The oxidative enzymes fall into three groups:
A
- dehydrogenases
- oxidases
- peroxidases
21
Q
Catalyze the transfer of hydrogen to immediate acceptors other than oxygen and peroxides
A
Dehydrogenases
other elements can be used such as peroxides.
22
Q
What are the required coenzymes for dehydrogenases?
A
linked to the diaphorase or cytochrome system
seen in respiratory/krebs cycle
23
Q
Delicate enzymes which are largely destroyed by any sort of fixation and
destroyed by embedding
A
Dehydrogenases
One disadvantage of dehydrogenases, making the procedures HARD
24
Q
Dehydrogenase
Demonstrates mitochondria and the fine detail of the sarcoplasmic reticulum of the fiber
A
Nicotinamide Adenine Dinucleotide (NADH) diaphorase
25
# Dehydrogenase
used to detect very minor or early
structural abnormality in the **sarcoplasmic reticulum network of the fiber**, as well as **mitochondrial abnormalities**
NADH diaphorase
26
# Dehydrogenases
The three main types of compounds used are
* Methylene blue (not recommended)
* Tetrazolium method (most sensitive; reduced to bright red)
* Tellurite( reduced to black; less sensitive)
27
What are the guidlines for the specimen when using dehydrogenase?
- tissue **need not be absolutely fresh**
- Refrigeration for **4 hours at 4°C** does not cause any noticeable loss of activity
- Fixation in chilled acetone for 4 hours causes only **40% inactivation of the enzyme**
28
Catalyzing the oxidation of various substrates, mainly **phenols
and amines**
Oxidase
29
What are the enzymes under oxidase
1. Polyphenol oxidases (PPOs)
2. Indophenol Oxidase (Cytochrome Oxidase)
3. Tyrosinase
4. Dopa oxidase
30
# Oxidase
- belong to **copper-containing metalloproteins.**
* They also belong to **oxidoreductases** that catalyze the oxidation of a wide range of phenolic compounds by utilizing molecular **oxygen**.
* The concept involves the transfer of energy from **one side to another.** They are useful for various biotechnological applications.
Polyphenol oxidases (PPOs)
31
* A copper-yielding cytochrome complex that catalyzes the oxidation of **ferrocytochrome C to produce ferricytochrome C and 2H2O**
Indophenol Oxidase (Cytochrome Oxidase)
32
# Indophenol Oxidase (Cytochrome Oxidase)
To stain for cytochrome oxidase, a mixture of
solutions of a
**phenol** or **naphthol** and an **aromatic diamine** is slowly oxidized on exposure to air
| intensely blue indophenol dyes
33
# Oxidase
Copper containing monooxygenases that catalyze the production of **melanin and other pigments** from tyrosine by oxidation.
Tyrosinase
34
- Increased tyrosinase activity:
- Decreased tyrosinase activity:
* Increased tyrosinase activity: increased melanin
synthesis thereby causing melanoma
* Decreased tyrosinase activity: hyperpigmentation (albinism)
35
Melanin pigment is formed from the amino acid, dihydroxy-phenylalanine (DOPA), by the action of a specific oxidative enzyme that is responsible for the oxidation of tyrosine to **dopa and dopa quinone**
Dopa oxidase
36
what are the samples for DOPA OXIDASE?
Adrenal Glands
37
To demonstrate dopa oxidase, use **frozen** sections of **fresh** material or of tissue fixed for only a few hours in?
5% formalin
38
What are the optimal temerature used to when using Dopa oxidase
20o and 37oC
39
# TOF
Longer fixation may cause partial inactivation of the enzyme dopa oxidase
True
40
The sections are rinsed very briefly in distilled water and transferred a 0.1 per cent solution of **dihydroxyphenylalanine**, buffered with a phosphate buffer to pH ____ in an open dish for 4-5 hours
7.3-7.5
41
# Dopa oxidase
It is advisable to change the incubating solution
once or twice
42
Color reaction for dopa oxidase
dark brown -gray or color black
| increased pH = more melanin precipitate
43
Heme-containing enzymes that use **hydrogen peroxide** as the electron acceptor to catalyze a few oxidative reactions
Peroxidases
44
Peroxidase is seen in ____ or ____ wherein positive reaction produces blue color. In this test, it uses
PSEUDOPEROXIDASE.
Guaiac Test or FOBT
45
Quite resistant to various chemical and physical agents, especially to acids and heat
Peroxidases
46
a method for demonstrating peroxidase granules in some neutrophils and in eosinophils
Peroxidase stain
47
What are the dyes used for peroxidase stain
- **Benzidine**: if oxidized, it will produce *blue or brown dye*
- **Naphthol**: if oxidized, it will produce *purple-black color*
- **Leuco-dyes** are *re-colorized to their original shades* (If it does not turn back to its original color, it means it did not react)
48
Perosxidase smears are fixed with
acetone, alcohol, or formalin alcohol (1:10)
49
# Peroxidases
For tissues, the same fixatives or
formalin-saline
50
what is the best specimen for the use of peroxidase
Frozen Section
| can either use paraffin or celloidin embedding material
51
* **Complex catalytic proteins** that use **water** to break down protein, carbohydrate, nucleic acids, starch, fats, phosphate esters and other molecules
* Group of **esterase**
* Reactions have been devised for the demonstration of either the
**acid or the alcoholic moiety**.
Hydrolytic Enzyme
52
# 2 HYDROLYTIC ENZYMES
Acids and alcohol combined with?
Acids: metal ions: cobalt, iron, and copper
Alcoholic: (thio?)alcohol or naphtol
53
What are the hydrolytic enzymes
1. Phosphatase (Alkaline phosphatase, Acid phosphatase)
2. 5- nucleotidase
3. Adenosine Triphosphatase (ATPase)
4. Nonspecific esterase
5. Chloroacetate esterase
6. Acetylcholinesterase
7. Phosphorylase
8. Aldolase
9. Sulfatase
54
- *Complex catalytic proteins* that use **water** to break down protein, carbohydrate, nucleic acids, starch, fats, phosphate esters and other molecules
- Most of this enzymes belong to the group of **esterase**
- Reactions have been devised for the demonstration of either the **acid or the alcoholic moiety**
Hydrolytic Enzyme
55
# Hydrolytic Enzyme
What are the phosphatase enzymes?
- Alkaline Phosphatase
- Acid Phosphatase
56
# Hydrolytic Enzyme
Reactions have been devised for the demonstration of either the acid or the alcoholic moiety, what chemicals are they combined with?
Acids: with metal ions (cobalt, iron, and copper)
Alcoholic: Thioalcohol or naphtol
57
# Hydrolytic Enzymes
* These are enzymes that are capable of hydrolyzing organic **phosphate esters**.
* Can either be an acid (lower pH) or alkaline (higher pH)
* This uses **methyl** precipitate.
* The higher the end-number of enzymes, the **darker** or more intense the staining is.
Phosphatases
58
# Hydrolytic Enzymes
* Higher **pH 9**
* It is the largest group among all and it will **hydrolyze any monoesters of phosphoric acid** in addition to nucleic acid.
* The tissue that can be used here is the **frozen section.**
Alkaline phosphatase
59
# Hydrolytic Enzymes
Alkaline Phosphatase are partnered with to be activated
magnesium
60
# Hydrolytic Enzymes
- **not too sensitive** to minor variations in temperature; incubation at any temperature between **30° and 45°C** will do.
- The cheapest and easiest available substrate for this is glycerophosphate.
- **Blackens** the cell
Alkaline phosphatase
61
- what is the most common metal used for alkaline phosphatase?
- What are the method used?
- Calcium
- Gomori Calcium (color reaction --> BLACK)
62
# Hydrolytic Enzyme
* Lower** pH 5**
* Used to recognize **lysosomes**.
* Stain is used to identify **macrophages in necrotic fibers** and **abnormal lysosomal activity** in muscle fibers / tissues and anything with this phosphatase content
Acid phosphatase
63
# Hydrolytic Enzyme
What is the method used for acid phosphatase?
Gomori Lead
64
# Hydrolytic Enzyme
* If there is an **increased number** of enzymes, it will *darken* or lead to *black deposit* reaction in tissues.
* It can be inhibited by **fluoride**.
Acid phosphatase
65
# Hydrolytic Enzyme
fixative and embedding material used for acid phosphatase
Fixatice: Cold Acetone
Embedding: Paraffin
66
# Hydrolytic Enzyme
* Optimum pH around **7.8** and quite active at pH **9**
o The problem here is that if the pH decreases, there will be artifactual changes or deposit reactions, which may result in a **false positive result.**
* METHOD: **Lead method**
5- nucleotidase
67
# Hydrolytic Enzyme
* To **differentiate** Type 1 and Type 2 muscle fibers (Also have subtypes)
* This is commonly used in **muscle diseases** or when there is atrophy (loss, weakening, or decrease in size of muscle tissue)
Adenosine Triphosphatase (ATPase)
68
# Adenosine Triphosphatase (ATPase)
* It involves metal using ____ ____ to form phosphate as a primary reaction product.
* It can also be precipitated either by **LEAD** to form lead phosphate, resulting in a ____ (The alternative for this is calcium cobalt. The end color
will be the same)
- What technique is used?
- adenosine triphosphate
- dark brownish-black color
- Metal Precipitation
69
# Hydrolytic Enzyme
* Used to identify cell types containing **esterases** that have a characteristic ability to split esters on their particular conditions.
* Increased staining can be seen in people with problems in **macrophages** or **lysosomal activities**.
* The color is **dark brown-red** or **black** colored deposits
* The enzyme is primarily detected in the **monocytes**, **macrophages**, and **histiocytes**.
* It is NOT found in the granulocytes.
Nonspecific esterase
70
# Hydrolytic Enzyme
Nonspecific esterase are paired with?
diazonium compound (para labasan ng esterase hehe)
71
# Nonspecific esterase
- Used during enzyme hydrolysis
- This method uses bromo-indoxyl acetate as subtrate
- α- Napththyl acetate method
- lndoxyl acetate method
72
# Hydrolytic Enzyme
* Also called **specific esterase**
* A useful stain for identification of **neutrophil polymorphs** in both frozen and paraffin section
- Enymatically hydrolyzed (to free napthol compound)
Chloroacetate esterase
73
# Hydrolytic Enzyme
An enzyme that is present in nervous tissue, muscle, and red blood cells
Acetylcholinesterase
74
# Hydrolytic Enzyme
- catalyzes the hydrolysis of acetylcholine to?
- seen in neuromuscular unctions, issues with nerve synapses
acetic acid and choline
75
# Hydrolytic Enzyme
* Enzyme that in the presence of inorganic phosphate, catalyzes the conversion of **glycogen** into **glucose-1 phosphate** (Gkycogenolysis)
* It is seen in **liver** and **muscles**.
* distinguishes type one to type two muscels
Phosphorylase
76
# Hydrolytic Enzyme
hydrolyzing sulfates of phenols and of thioglucosides are quite resistant to dehydration by acetone
Sulfatase
76
# Hydrolytic Enzyme
* Demonstration of this enzyme is based on the fact that the enzyme splits **hexose diphosphate** into two molecules of **triose phosphate.**
* Uses **frozen** section
Aldolase
77
# Hydrolytic Enzyme
since sulfatase have low concentration in tissue, theres a need to precipitate it using?
benzedene
78
- the **most common staining technique** that we use in laboratory and is mostly used for diagnosing of **prognosis** and **treatment** of patients
* Identification of specific or highly selective **cellular epitopes** or **antigens** in frozen or paraffin-embedded tissues
* Detect organisms in **cytologic preparations**
* Utilizes **antigen - antibody interaction**s but this is more
specific wherein we detect the presence of antigens
* **IgG** is the most used antibody since this is the easiest antibody to develop
IMMUNOHISTOCHEMISTRY (IHC)
79
# IMMUNOHISTOCHEMISTRY (IHC)
* The best tissue to be used is ____
* To visualize the distribution and localization of specific ____ within cells and in the proper tissue context
* Disease ____, drug development and biological research
* Immunofluorescence is often, but not always performed on frozen tissue due to the high background ____ seen in formalin fixed paraffin embedded tissue
- routine tissue processing
- cellular components
- Diagnosis
- auto-fluorescence
80
TOF
stain in immunostaining
only **brown** in color
eurt
81
What is the first satge of immunohistochemistry?
application of a **primary antibody** that binds specifically to the target antigen
82
Poly or Mono -clonal antibody
- have an affinity to only **one epitope** and tend to produce, cleaner, more specific staining but are less sensitive or intense
- have the affinity with, and bind to, **multiple** epitopes (or parts) or the target antigen, and as such are more prone to cross-react to non-target antigens
- Monoclonal
- Polyclonal
83
# IHC
- bind to the **primary antibody**
* This is known as **INDIRECT IHC**
* **Most commonly** used in the laboratory
Secondary Antibody
84
# IHC - TOF
It is now commonly used as multiple secondaries can bind to a single primary to further amplify the staining intensity
True
| Horseradish peroxidase most common
85
Best tissue specimen for Immunohistochemistry
Routine tissue processing
86
# IHC
The best is those tissues who undergo a routine tissue processing. But for some cases, cryostat or frozen section are received or those tissue that are fixed in ____
Methanol
87
Tissue processed from cryostat or frozen section, can cause what problem?
antigen sites are being **masked** because of the **frozen artifacts** or the **cryomatrix gels**.
88
When receiving tissue, it is important to know where the tissue came from (frozen or routine tissue processing). If from frozen section, there is an additional step to do
which is the?
antigen retrieval
(longer fixation, less Ag binding stie)
89
Masked antigens can now be retrieved in routinely processed tissue by these steps:
1. Proteolytic enzyme digestion (for *formalin fixed* section)
2. Microwave antigen retrieval (for *frozen* tissues)
3. Microwave and trypsin antigen retrieval
4. Pressure cooker antigen retrieval
90
# Proteolytic enzyme digestion
Formalin fixed paraffin sections are usually pre-treated with
proteolytic enzymes
| to breakdown crosslinking, allow their exposures
91
Proteolytic Enzyme Digestion are especially useful for demonstrating in formalin-fixed paraffin-embedded biopsies
- heavy chain immunoglobulins,
- complement
- specific antigens (such as cytokeratin)
92
What are the most common enzymes for Proteolytic Enzyme Digestion
**trypsin and protease**
useful for CK cases; not recommended for frozen sections not embedded in parafffin
93
* **Most common**, very simple laboratory technique that is used
* **Heat**, coupled with specific **buffered solutions**, is utilized to recover antigen reactivity in formalin fixed paraffin embedded tissue
Heat-Induced Epitope Retrieval (HIER)
94
What are the action of Heat-Induced Epitope Retrieval (HIER)
1. heat would break the **cross-link** in the antigen site or epitope
2. It removes **bound calcium** (e.g bone marrows samples in EDTA or citrate)
95
HIER heating sources include
- microwave (most common)
- vegetable steamer
- pressure cooker
- water bath
96
In general, the ____ the temperature of the HIER solutions, the more effective the recovery of the epitope is
Higher
97
# Antigen Retrieval
Boiling of **formalin-fixed deparaffinized sections** in certain solutions with buffer, such as **0.01 M-citrate buffer (pH 6.0)**, **EDTA at pH 8.0 or Tris EDTA**
Microwave Antigen Retrieval
98
# Microwave Antigen Retrieval
* Temperatures near the boiling point of water for ____
mins.
* Boiling of poorly fixed material often damages ____ details.
- Fibrous and fatty tissues tend to ____ from the slide
* Amplification of nucleic acids from paraffin-embedded material by the ____ is increasingly being used to detect **viral genomes and oncogene** mutations, although not common.
* ____ samples can be done in batches.
- 10 to 60 mins
- nuclear
- detach
- polymerase chain reaction (PCR)
- Multiple
99
99
# Antigen retrieval
**Less time-consuming** and allows for more consistent recovery of many antigens, compared to large batch microwave oven technique.
Heating is not equal meaning, the unmasking of antigen is also not equal
Pressure Cooking Antigen Retrieval
