Re: Hirschprung's - Urgent rqst ACHE. (Long!)

From:"J. A. Kiernan" <jkiernan@uwo.ca>

On Sun, 8 Apr 2001, David & Marjorie Cardwell wrote:

> A clinician wants us to carry out the ACHE procedure for Hirschprung's
> Disease. One of our pathologists has experience in reading them at a
> paediatric hospital, and I have done the lab procedure many times before,
> but I don't have a copy of the procedure.

Here is a procedure. This is published (copyright) material, so please
use it but don't disseminate it without prior permission and due
acknowledgement of the printed source. I'm sending it to you verbatim
because you say this is a matter of clinical urgency. You should urge
the clinician to buy any one of dozens of textbooks that contain
instructions for simple histochemical procedures. There are several
techniques for acetylcholinesterase activity, and this may not be the
one you have used before. The only book that gives an authoritative
account (IMHO) is Vol 3 of Pearse's Histochemistry (ed. Stoward &
Pearse, 1991). This covers the whole field of enzyme histochemistry
and it costs about US$180. How does this compare with a pathologist's
fee for making a diagnosis of Hirschsprung's in one child?

Here's the method. It doesn't include bibliographic details for the
references. These are in the book, but for an urgent case you won't
have time to go to the library. This is just an informal internet
message, of no academic, clinical or scientific value. If you use
the technique and a wrong diagnosis is made, the pathologist who
requested the procedure and examines the slides is answerable for
being unable to give you proper instructions. 
------------------

This is the direct-colouring method of Karnovsky & Roots (1964), with
modifications based on the work of Hanker et al. (1973) and Tago et
al. (1986).

Frozen sections are cut from specimens fixed at 4C for 12-24 hours in
formal-saline or neutral, buffered formaldehyde. Glutaraldehyde (1.0-3.0%,
pH 7.2-7.6) is also a suitable fixative. Formal-sucrose-ammonia (formalin
(40% HCHO), 10 ml; sucrose, 15 g; strong ammonia (NH4OH, S.G. 0.880), 1.0
ml; water to 100 ml) is superior to neutral buffered formaldehyde for the
preservation of these enzymes (Pearson, 1963; Pearse, 1972)***. 

Two incubation media, C.(1) and C.(2), are prescribed, each for a
different purpose.

Reagents required

A. Substrate and inhibitors

     (i) Acetylthiocholine iodide (substrate for AChE)
    (ii) Butyrylthiocholine iodide (substrate for ChE)
   (iii) Inhibitors, according to the requirements of the investigation.
The solid substrates are stored desiccated at -20C. Instructiuons for
using inhibitors are given in Section 15.2.6.

B. Stock solution for incubating medium

0.1 M acetate buffer, pH 6.0 (see Chapter 20):   65 ml
Sodium citrate (trisodium salt; dihydrate):     147 mg 
Cupric sulphate (anhydrous):                     48 mg 
Water:                                 to make  100 ml 
  Dissolve, then add: 
Potassium ferricyanide (K3Fe(CN)6):      17 mg

This pale green solution keeps for about 1 week at room temperature. It
should not be used if it contains a brown precipitate.

C.(1). Incubation medium (working solution, full strength)

Dissolve 5.0 mg (approximately) of either acetylthiocholine iodide or
butyrylthiocholine iodide in a drop of water and add 10 ml of solution
B. This medium is stable for several hours and may be used repeatedly if
not cloudy, but should be discarded at the end of the day. This medium is
used for tissues containing high enzymatic activity, including skeletal
muscle (motor end-plates).

C.(2). Incubation medium (working solution, 10 x dilution)

Mix 10 ml of Solution C(1) above with 90 ml of 0.1 M acetate buffer, pH
6.0. This medium is used for tissues containing thin AChE-positive nerve
fibres, or cell-bodies with low enzymatic activity.

Inhibitors should be added to the medium C.(1) or C.(2) as needed. See
Section 15.2.6 for instructions on use of inhibitors.

D. TRlS buffer, pH 7.2 (see Chapter 20)

E. DAB solution

3,3'-diaminobenzidine tetrahydrochloride:        10 mg
Water:                                            1 ml
  Dissolve, then add to
TRIS buffer (see Chapter 20; pH 7.6):            20 ml 
Nickel ammonium sulphate (Ni(NH4)2(SO4)2.6H2O): 150 mg,

Use within one hour of dissolving the DAB.

F. Hydrogen peroxide (0.03%)

A stock solution of 30% H202 (= "100 volumes available oxygen": handle
carefully) is diluted to 0.03% with water, less than 15 minutes before it
is needed (see Procedure below). 2 ml of the diluted solution will be
needed.

Alternatively use urea hydrogen peroxide (UHP), which is a solid compound
containing approximately 35% H2O2 by weight, which is released by
dissolving in water. For 0.03% H2O2 dissolve 0.1 g of UHP in 115 ml of
water. 

Procedure

1. Collect frozen sections into water in which they may remain for up to 1
hour. Pre-incubate any control sections in appropriate inhibitors. Wash
irreversibly inhibited sections in four changes of water. Do not wash
after pre-incubation with competitive inhibitors.
2. Transfer sections to the incubation medium, C.(1) or C.(2), for 10-30
minutes at room temperature. When regions of enzymatic activity go reddish
brown, incubation is adequate. Check under a microscope for isolated sites
of activity such as motor endplates.
3. Wash in 3 changes of TRIS buffer, each about 2 minutes. The sections
may be left in the last change, but total time in TRIS buffer should not
exceed 30 minutes.
4. Transfer sections to the 20 ml of DAB solution (E), and wait for 5
minutes.
5. Add 2 ml freshly diluted 0.03% hydrogen peroxide (Solution F) to the
DAB solution containing the sections. Mix well by stirring with a glass
rod. Make sure the sections are not collapsed into little balls or knots.
6. Wait for 10 minutes, then wash in 3 changes of water, each at least 1
minute.
7. Mount the sections onto slides and allow to dry.
8. Either mount in an aqueous medium, or dehydrate through graded
alcohols, clear in xylene, and mount in a resinous medium.

Result

Sites of enzymatic activity black (brown if steps 4 and 5 are omitted).

Notes

1. Stages 4 and 5 may be omitted when only sites of strong activity, such
as AChE at motor end plates or ChE in cerebral capillaries and some
central neurons, are of interest. Stages 4 and 5 are necessary when medium
C.(2) is used.
2. Although the substrates (especially butyrylthiocholine) show partial
specificity, the use of inhibitors is imperative for certain
identification of the two enzymes. A control with eserine should also be
provided, though non-enzymatic precipitation of the end product is unusual
with this technique. If the DAB-nickel-H2O2 intensification is carried
out, there may be a non-specific grey background, and sites of endogenous
peroxidase activity (erythrocytes, granular leukocytes, occasional groups
of neurons in the brain) will be stained.
3. Endogenous peroxidase activity can be blocked by treating the sections
with 0.1% hydrogen peroxide for 30 minutes, before Step 1 of the method.
4. A counterstain may be applied if desired between Steps 7 and 8. Most
dye-based stains may be used. Silver reduction methods (Chapter 18) are
unsatisfactory if Steps 4 and 4 are omitted, because the copper
ferrocyanide deposit is slowly decolorized or removed when exposed to
silver nitrate.
---------------------------------- 
Text between  --------------  lines above is Copyright (c) 1999 and 2000
J.A.Kiernan, Butterworth-Heinemann Ltd and Arnold Publishing. Unauthorized
reproduction is illegal without permission of the author(s) and
publisher(s).

----------------------------------------
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1
   kiernan@uwo.ca
   http://publish.uwo.ca/~jkiernan




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