From:Rainbow Ron

Dear Roxanne,

I have copied and pasted our method which I developed back in 1993 and
published in an Australian Histology Bulletin called Tissue Talk. The
reference is: Rainbow, R.D., (1994). A Rapid Method For Routine Processing
of Iliac Crest Bone Marrow Trephine Biopsies. Tissue Talk. May Ed. 3-4

Not included in the article is the fact that you section H&Es at 0.5-1
microns, reticulin stains at 5 microns and immunohistochemistry sections at
3 microns. Light and heavy chain immunoglobulin IHC sections are done at 1-2
microns. You must include celestine blue for 3-5 minutes prior to Harris
Haematoxylin in your H&E to provide a mordanting effect which will give you
excellent nuclear detail.

This is the article and the method we still use today.

Good luck.


R D RAINBOW   Scientist in Charge, Department of Anatomical Pathology, Royal
Hobart Hospital, Hobart, Tasmania, Australia.

Until recently, the method for processing iliac crest bone marrow trephine
biopsies in our laboratory involved overnight fixation of 1.0 - 1.5 x 0.2 cm
cores in neutral buffered formalin followed by mild decalcification for 8
hours in formic acid / sodium citrate (1) ,
neutralization in saturated lithium carbonate (2)for 30 minutes, 15 minutes
washing in running tap water, then overnight processing using a routine
standard 16 hour alcohol, xylene, paraffin processing schedule. 
Previous methods  involved mercury based fixatives which yielded slightly
improved cytological detail. For reasons of safety and concern for the
environment the routine fixative was  changed to neutral neutral buffered
formalin. The use of disposable microtome blades such as the Feather S35
produced excellent quality thin 1.0 micron sections which provided the
necessary cytological detail .
In an effort to provide a faster turn around time processing systems taking
less time were examined. 
25 iliac crest bone marrow trephine cores were taken at post mortem and were
fixed by microwave fixation for different times and temperatures until
optimal results were obtained (3). Decalcification fluids and times were
also examined to determine the minimum decalcification time that would
decalcify a trephine core with an above average bone content, allow easy
sectioning at 1 micron, and have no deleterious effect on the tissue.
The method described is the method which gave the most optimum results in
terms of time, fixation, decalcification and which would easily fit into our
routine tissue processing schedule. Consideration was also given to the
routine of the clinician responsible for taking the trephine biopsies.

1.	On receipt of the bone marrow trephine biopsy, which will be either
fresh or in 	neutral buffered formalin, immediately transfer the biopsy
into a plastic microwave 	coplin jar and 3/4 fill with neutral
buffered formalin.
2.	Ensure that the biopsy core is positioned near the side of the jar
so that it will not be 	in contact with the temperature probe when
3.	Insert Sharp R9570 Microwave Oven temperature probe into the coplin
jar through a  	central hole in the coplin jar lid. Place coplin jar in 700W
Sharp R9570 Microwave 	Oven in centre of carousel and insert temperature
probe in the receptacle in the top 	centre of the oven cavity.  NOTE: An
additional dedicated temperature probe to that 	supplied with the microwave
oven should be used for this purpose and cleaned 	thoroughly after
each use. This prevents the possibility of contamination during other
microwave procedures.
4.	Microwave at 68 0C for 5 minutes at medium high power which is about
490 W.	
5.	On completion of microwave fixation, remove temperature probe and
thoroughly 	clean with distilled water to prevent corrosion.
6.	Transfer the biopsy back into its original specimen container
containing neutral 	buffered formalin. Place a microwave fixed label on
the lid of the container and 	transfer to the decalcification site in the
7.	Specimens remain in neutral buffered formalin until 2.30PM when
decalcification 	commences. Specimens arriving after 2.30PM are
microwave fixed as above but are 	not decalcified until the following

8.	Rinse biopsy thoroughly in distilled water then place in a labelled
25ml specimen 	container filled with HISTOLABS FASTCAL decalcifier
(available from Fronine 	Pty. Ltd. NSW). 
9.	Place on roller mixer and set timer for 2 hours.
10. Wash thoroughly in distilled water for 10 minutes.
11. Place in a coloured tissue cassette which will identify the tissue as
being decalcified 	and process overnight with the tissue from the
routine surgical cut up. 

This method has less than halved the previous time in which a trephine
biopsy report would have been available.This time, however, could  be
further improved by shortening the processing cycle from a 16 hour schedule
to possibly 9 hours, this would  advantage laboratories operating a shift
system. Shorter cycles are available for urgent specimens requiring results
on the same day (3).
The schedule described fits into our routine biopsy schedule and allows for
any bone marrow trephine biopsies arriving no later than 2.15PM to be fixed,
decalcified, processed and reported on by 11.00AM on the following day.
The use of Histolabs Fastcal in preference to formic acid / sodium citrate
allows for rapid decalcifying and has no deleterious effects on cytological
detail. The only disadvantage in using Fastcal, or similar commercial rapid
decalcifying fluids such as RDO, is the loss of  haemosiderin, thus
preventing Perls Prussian Blue staining. This is not a problem however,
since Perls stains are routinely performed on the bone marrow aspirate in
the haematology laboratory.
Immunohistochemical staining is actually enhanced by the effects of
microwave fixation as opposed to the cross linking properties of formalin
which tend to mask some epitopes.
The only problem with this method is that lipid droplets in the bone marrow
appear to show signs of minor dislodgement presumably occurring during
microwave fixation. This has had no effect on cellular morphology but does
cause slight displacement of reticulin fibres.
The method has now been in routine use in our department for all bone marrow
trephine biopsies, except those for metabolic bone disease assessment, since
May 1993 without adverse comment from pathologists or haematologists.

1.	Preece, A. (1972). A Manual for Histologic Technicians. Third
Edition. Little and 	Brown. pp 143.
2.	Villaneuva, A. R. (1982). Post Decalcification Treatment of Bone
Specimen. 	Histologic Bulletin. XII. 3 :181.
3.	Leong AS-Y. (1991). Microwave Fixation and Rapid Processing in a
Large 	Throughput Histopathology Laboratory. Pathology. 23: 271-273. 

Ron Rainbow
Scientist in Charge
Department of Anatomical Pathology
Royal Hobart Hospital
GPO Box 1061L
Australia 7001
Ph 0362228771
Facsimile 0362228191
E-mail: ron.rainbow@dchs.tas.gov.au

-----Original Message-----
From: Soto, Roxanne [mailto:RSoto@covhealth.org] 
Sent: Friday, February 22, 2002 5:18 AM
To: 'Histonet@pathology.swmed.edu'

Hello all,
I have a question about bone marrow's.  We are having a big problem with
bone marrow's, as far as decal and processing consistently.  Does anyone
have a good protocol for a microwave decal procedure and a processing
procedure for bone marrow's?
Thanks in advance

Roxanne Soto
AP Supervisor
Franciscan Shared Laboratories
Covenant Healthcare
Milwaukee, WI

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