Dear Carmen,

I have included  below a method I developed a number of years ago and
published in an Australian Histo journal which no longer exists. The
method has also been presented at a National Scientific Meeting here
in Australia and has also been cited for use by the Royal College of
Australasia Quality Assurance Program. We have used this method
routinely since it was developed and have had consistent excellent
results. H&E sections are routinely cut at 1micron with ease but
because of the thinness you should use  celestine blue as a mordant
before Harris haematoxylin in your H&E. Cytological detail is
excellent. We have had excellent immuno results as well.

Hope this helps






ROYAL HOBART HOSPITAL

BONE MARROW TREPHINE BIOPSIES

FIXATION AND DECALCIFICATION

MICROWAVE FIXATION
1.	On receipt of the bone marrow trephine biopsy (which will either be
in neutral buffered formalin or fresh), IMMEDIATELY transfer the
biopsy into the 	dedicated microwave coplin jar located on the
decalcification bench. 
 Note: Those specimens for undecalcified bone processing as described
 in the the 	notice on the previous page must be stamped DO NOT
 DECALCIFY  and given to Ron Rainbow for special resin embedding or
 despatch.    
2.	3/4 fill the microwave coplin jar with neutral buffered formalin
and ensure that the trephine biopsy is positioned near the side of
the jar so that it will not be in contact with the temperature probe
when microwaving. 3.	Insert temperature probe into the coplin jar
through the hole in the coplin jar lid. 	NOTE: Use only the dedicated
temp. probe from box on decalcification bench. 4.	Place in Sharp R9570
Microwave Oven and insert temperature probe plug
 in the receptacle in the top centre of the oven cavity.
5.	Position coplin jar in centre of dish on carousel and arrange cord
so that 
 the coplin jar will not fall over during microwaving.
6.	Set temperature at 68  C, touch the TEMP pad, select MED HIGH,
enter 5 min., 
 then touch the START pad. Monitor coplin jar so that the coplin jar
 doesn't fall over during microwaving.
7.	On completion of microwave fixation, remove temperature probe,
 thoroughly wash in distilled water to prevent corrosion and return
 with the coplin 	jar, to the box on the decalcification bench.
8.	Transfer the biopsy back into its original specimen container
containing
 neutral buffered formalin and write MICROWAVE FIXED on the lid.
9.	Specimens remain in neutral buffered formalin until 2.30PM when
decalcification 	commences. Specimens arriving after 2.30PM are
microfixed as above but are not 	decalcified until the following day.

DECALCIFICATION (2.30-4.30PM)
10.Rinse biopsy in distilled water then place in a labelled 25ml
specimen container filled with       HISTOLABS FASTCAL
decalcifier.CAUTION:Fastcal is corrosive, handle with care.

11.Place on roller mixer and set timer for 2.0 hours.
12.Wash thoroughly in distilled water for 10 minutes.
13.Place in labelled tan coloured unicassette and process overnight.

Reference:
Rainbow, R.D., (1994). A Rapid Method For Routine Processing of Iliac
Crest Bone Marrow Trephine Biopsies. Tissue Talk. May Ed. 3-4.

ISSUE DATE :             6/9/99	AMENDED DATE 
..............................	REVIEW DATE .........................

AUTHORISED BY:    RD Rainbow	AMENDED BY 
....................................	REVIEWED BY
........................
  Page 1 of 1




A RAPID METHOD FOR ROUTINE PROCESSING OF ILIAC CREST BONE MARROW
TREPHINE BIOPSIES


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




INTRODUCTION
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.

METHOD
MICROWAVE FIXATION
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 microwaving. 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 laboratory. 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 day.

DECALCIFICATION (2.30 - 4.30PM)
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. 

DISCUSSION
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.

REFERENCES
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
Hobart
Tasmania
Australia 7001
Ph 0362228771
Facsimile 0362228191
eMAIL:  ron.rainbow@dchs.tas.gov.au