New Technology in Laboratory Medicine
Continuous-Specimen-Flow, High-Throughput, 1-Hour
Tissue Processing
A System for Rapid Diagnostic Tissue Preparation
Azorides R. Morales, MD; Harold Essenfeld, MD; Ervin Essenfeld, MD; Maria Carmen Duboue, HTL(ASCP);
Vladimir Vincek, MD, PhD; Mehrdad Nadji, MD
Context. Current conventional tissue-processing
methods employ fixation of tissues with neutral buffered
formalin, dehydration with alcohol, and clearing with
xylene before paraffin impregnation. Because the time
required for this procedure is usually 8 hours or longer,
it is customary to process tissues in automated instru-
ments throughout the night. Although this time-honored
method continues to serve histology laboratories well, it
has a number of shortcomings, such as a 1-day delay of
diagnosis, the need to batch specimens, the relatively
large volumes and toxicity of reagents used, and the ex-
tent of RNA degradation.
Objective.To describe a rapid new method of tissue
processing using a continuous-throughput technique.
Design.We used a combination of common histologic
reagents, excluding formalin and xylene, as well as micro-
wave energy, to develop a rapid processing method. The
effect of this method on the quality of histomorphology,
histochemistry, immunohistochemistry, and RNA content
of processed tissue was compared with that of adjacent
tissue sections processed by the conventional processing
technique. We also assessed the impact of this rapid pro-
cessing system on our practice by comparing the turn-
around times of surgical pathology reports before and after
its implementation.
A waiting period of 1 day or longer for a pathologic di-
agnosis following any surgical procedure is custom-
ary.1,2 Patient anxiety, the delay to plan or institute treat-
Accepted for publication January 2, 2002.
From the Department of Pathology, University of Miami/Jackson
Memorial Medical Center, Miami, Fla (Drs Morales, Vincek, and Nad-
ji and Ms Duboue); and the Essenfeld Pathology Laboratory, Policlin-
ica Metropolitana, Caracas, Venezuela (Drs H. Essenfeld and E. Es-
senfeld).
Dr Morales, Dr E. Essenfeld, and Dr H. Essenfeld participate in a
Sponsored Research Program with Sakura Finetek USA through the
University of Miami and are listed as inventors of US patent 6 207 408.
The University of Miami has licensed this patent to Sakura Finetek USA,
and the inventors will receive a percentage of the proceeds gained by
the University of Miami.
Reprints: Azorides R. Morales, MD, Department of Pathology (D-33),
University of Miami School of Medicine, PO Box 016960, Miami, FL
33101 (e-mail: amorale@med.miami.edu).
Arch Pathol Lab MedVol 126, May 2002
Results.The new processing method permitted prepa-
ration of paraffin blocks from fresh or prefixed tissue in
about 1 hour. The procedure allowed continuous flow of
specimens at 15-minute intervals. It eliminated the use of
formalin and xylene in the processing and used consider-
ably lower volumes of other chemical reagents. Histomor-
phologic, histochemical, and immunohistochemical results
were comparable to the parallel sections prepared by the
conventional method. The new technique, however, pre-
served higher quality RNA. Use of the new methodology
led to the diagnosis and reporting of more than one third
of surgical pathology specimens on the same day that they
were received, as compared to 1% of same-day reporting
before the implementation of the rapid processing system.
Conclusion.The quality of hematoxylin-eosin, histo-
chemical, and immunohistochemical tissue sections provid-
ed by the new system is comparable to that obtained fol-
lowing the conventional processing method. The new system
preserves RNA better than the conventional method. It also
shortens the processing time to about 1 hour from the re-
ceipt of fresh or prefixed tissue, eliminates the need for for-
malin and xylene, and reduces the volume of other chemi-
cals. Most importantly, it impacts overall patient manage-
ment by allowing for considerably shorter turnaround times
for completion of surgical pathology reports.
(Arch Pathol Lab Med. 2002;126:583590)
ment, and other adverse aspects related to the delay be-
tween surgery and diagnosis are consequences of con-
straints imposed by the time required to prepare tissue
for microscopy. In particular, the tissue-processing stage
of diagnostic tissue preparation takes 8 hours or longer.
Another concern associated with the conventional pro-
cessing method (CPM) is the toxicity of reagents used,
especially formaldehyde and xylene. Formaldehyde, for in-
stance, is known to irritate conjunctiva and the respiratory
mucosa. It can also cause allergic dermatitis and asthma,
and has been implicated in the etiology of cancer of the
oropharynx and the respiratory tract.3,4 Furthermore, be-
cause the study of diseases at the molecular level in ar-
chival tissue is being greatly expanded, severe degradation
of RNA by the conventional method constitutes a major
disadvantage for practitioners.5
During the last 2 decades, as a consequence of the in-
troduction of microwave energy into histology laborato-
Rapid Diagnostic Tissue PreparationMorales et al 583
Figure 1. Dissecting board. Inset, A closer
view of 1 of the cutouts of the metallic plate.
Arrows point to the slit that permits inserting
the scalpel blade in the guiding assembly. Us-
ing this assembly assures that the cuts through
the tissue placed in the slots result in uniform
slices of desired thickness.

Table 1. Conventional Processing Method

Concentra- Time, Heat, 8C Pressure/
Step Solution tion, % min (Convective) Vacuum Agitation Volume, L
1 Formalin 10 120 40 Yes Yes 4
2 Formalin 10 120 40 Yes Yes 4
3 Alcohol 80 30 40 Yes Yes 4
4 Alcohol 95 30 40 Yes Yes 4
5 Alcohol 95 45 40 Yes Yes 4
6 Alcohol 100 45 40 Yes Yes 4
7 Alcohol 100 45 40 Yes Yes 4
8 Alcohol 100 45 40 Yes Yes 4
9 Xylene 100 45 40 Yes Yes 4
10 Xylene 100 45 40 Yes Yes 4
11 Paraffin ... 30 60 Yes Yes 4
12 Paraffin ... 30 60 Yes Yes 4
13 Paraffin ... 30 60 Yes Yes 4
14 Paraffin ... 30 60 Yes Yes 4

ries, progress has been made in the development of rapid
processing methods and the elimination of toxic re-
agents.610 Methods reported so far, however, are neither
sufficiently practical nor shorten tissue-processing time to
the extent required for their widespread acceptance.
We report a simple, practical, and fully automated meth-
od that reduces processing time to about 1 hour, and
therefore enables pathologists to render a diagnosis in 2
to 3 hours. This method does not require prior fixation of
tissue and obviates the need for formaldehyde and xylene
during processing, leading to improved preservation of
RNA.
MATERIALS AND METHODS
Specimens
Approximately 32 000 surgical specimens are submitted an-
nually to the Pathology Laboratory at the University of Miami/
Jackson Memorial Medical Center, Miami, Fla. About 27 000 of
these specimens originate from surgical procedures at Jackson
Memorial Hospital, and the remaining are from the University
of Miami Hospital and Clinics, including the Sylvester Compre-
hensive Cancer Center. About 110 000 paraffin blocks are pre-
584 Arch Pathol Lab MedVol 126, May 2002
pared every year from these tissues. Small biopsy specimens are
received in the laboratory in containers partially filled with 10%
sodium phosphatebuffered formalin (4% formaldehyde). Large
specimens, such as uteri, breasts, intestines, and placentas, are
usually received fresh or in various-sized containers with for-
malin. Until September 1997, the tissues were processed over-
night following the steps outlined in Table 1. A few emergency
biopsies were processed within 2 hours using the short cycle of
the automated processor (Tissue-Tek VIP, Sakura Finetek, Tor-
rance, Calif).
During the developmental stages of our continuous-throughput
processing method (CTPM), we used samples from residual
large surgical specimens for evaluation. As soon as the efficacy
of the new methodology was established, we began routine pro-
cessing of specimens with CTPM. The system uses either fresh
or prefixed specimens. The tissue slices, however, should be no
thicker than 1.5 mm. Consequently, small biopsy specimens re-
quire no trimming. Large specimens are trimmed on a dissecting
board specifically developed to prepare uniform slices of desired
thickness (Figure 1). This board was designed by inserting a 26.0
3 5.0-cm metallic plate on a 45.0 3 30.0 3 2.5-cm flat plastic
slab. The metallic plate has two 1.5-mm-deep cutouts, measuring
3.0 3 2.5 cm and 2.5 3 1.8 cm. Tissues are placed over either of
the cutouts and slid horizontally along the slicing plate. During
Rapid Diagnostic Tissue PreparationMorales et al
 Table 2. Continuous-Throughput Processing Method
Heat, 8C
Time,
Step Liquids min Convective
1 Solution I 15 ...
2 Solution II 15 ...
3 Solution II 5 ...
4 Mineral oil/
paraffin 5 65
5 Mineral oil/
paraffin 5 65
6 Paraffin 5 65
7 Paraffin 5 65
8 Paraffin 5 65
9 Paraffin 5 65
Solution of isopropyl alcohol, acetone, polyethylene glycol, glacial acetic acid, and dimethyl sulfoxide.
Solution containing same reagents as step 1, with the addition of low-viscosity mineral oil.
slicing, specimens are held in place by the index finger or with
a plastic holder applied to the exposed surface of the tissues. The
use of this cutting board produces uniform, 1.5-mm-thick slices
of tissue. To make this board equally useful for conventional pro-
cessing, an additional 3.0-mm-deep slot was cut in the metallic
plate, which can procure thicker sections if needed.
Processing
The CTPM as presently practiced in the Department of Pa-
thology at the University of Miami/Jackson Memorial Medical
Center is outlined in Table 2 and schematized in Figure 2. It is
fully described elsewhere.11 Briefly, we use mixtures of common
histology reagents, such as isopropyl alcohol, acetone, polyeth-
ylene glycol, mineral oil, paraffin, and minute amounts of glacial
acetic acid and dimethyl sulfoxide. Formaldehyde and xylene are
not used during processing. During gross dissection, the sections
are placed in regular plastic cassettes and immersed in a holding
solution identical to that used in the first step (see Step 1), but
without acetic acid. The sections are kept in this solution for about
5 to 15 minutes, depending on the time required for dissecting
the surgical specimen. Because the first step of processing takes
15 minutes, this holding stage does not cause any delay in adding
new samples to the system. Up to 30 cassettes are loaded into a
plastic basket and subjected to the following steps:
Step 1. The samples are microwaved at 628C for 15 minutes
in 1000 mL of a solution of acetone, isopropyl alcohol, polyeth-
ylene glycol, glacial acetic acid, and dimethyl sulfoxide. Air bub-
bling produces agitation.
Step 2. The samples are heated for 15 minutes at 628C in a
second microwave in 1000 mL of a solution similar to that used
in step 1, to which low-viscosity mineral oil has been added. The
solution is agitated by bubbling.
Step 3. The samples are heated for 5 minutes at 628C in a
third microwave in 1000 mL of a solution similar to that used in
step 2, but with a higher concentration of mineral oil. The solu-
tion is agitated by bubbling.
Steps 4 and 5. The samples are incubated at 658C in 2 con-
secutive 1000-mL solutions of a mixture of low-viscosity mineral
oil and paraffin. A vacuum of 640 mm Hg is applied for 5 min-
utes to each bath.
Steps 6 through 9. Impregnation is accomplished by incu-
bation at 658C in 4 baths of molten paraffin applying a vacuum
of 640 mm Hg. Tissue sections are transferred from 1 paraffin
bath to the next at 5-minute intervals, for a total impregnation
time of 20 minutes.
Because step 1 lasts 15 minutes, the system can be accessed
with additional samples every 15 minutes, as more specimens
become available.
Although the physical agents and reagents have remained fair-
ly constant since the processing system was adapted in 1997, the
manner in which these agents are applied has been modified.
Arch Pathol Lab MedVol 126, May 2002
Microwave Vacuum Agitation Volume, L
62 ... Bubbling 1
62 ... Bubbling 1
62 ... Bubbling 1
... Yes ... 1
... Yes ... 1
... Yes ... 1
... Yes ... 1
... Yes ... 1
... Yes ... 1
Initially, commercial microwave tissue processors (H2500 or
H2800, Energy Beam Sciences, Inc, Agawan, Mass) were used for
the first 3 steps of the procedure. Toward the development of a
fully automated system, a transitional phase resulted in the cre-
ation of a specifically designed microwave applicator (Microwave
Materials Technologies, Inc, Knoxville, Tenn), which was used
from late 1999 to early 2000. With either system, the samples
were manually transferred from one microwave to another. Treat-
ment of tissue samples with the mineral oil/paraffin mixture
(steps 5 and 6) and the 4 final impregnation steps were carried
out in a large desiccator resting in a glycerin bath. In January
2000, we began using a robotic instrument that fully automated
the processing system. Main features of this instrument are (1) a
robotic arm that transfers the samples from one chamber to the
next; (2) 3 microwave units, the chambers of which serve as the
container for the 1000-mL solutions; and (3) paraffin-impregna-
tion stations providing heat and vacuum. The microwave units
allow for uniform distribution of the microwave energy, thus
avoiding damage to the processed tissue.
Following paraffin impregnation and embedding, the sections
are cut at 3 to 5 mm and stained with hematoxylin-eosin. When
indicated, histochemical and immunohistochemical stains are
performed following standard techniques.
For RNA extraction, 20 ribbons (20 mm thick) of paraffin-em-
bedded tissue were deparaffinized with xylene twice for 15 min-
utes and washed in 100% ethanol 2 times for 10 minutes. After
brief air drying, the tissue sections were homogenized with 1 mL
of Trizol reagent (GIBCO BRL, Grand Island, NY) using power
homogenizer. Two microliters of Pellet Paint (Novagen, Madison,
Wis) fluorescent dyelabeled coprecipitant was added to each
sample. The RNA was isolated following the Trizol protocol
(GIBCO), isopropanol precipitation, air drying, and dissolution
in 40 mL ribonuclease-free sterile water. Concentration was esti-
mated by spectrophotometry, and the integrity of total RNA was
tested by assessing the resolution of 28S and 18S ribosomal RNA
in 0.8% agarose gel stained with ethidium bromide. The comple-
mentary DNA was generated from 100 ng of total RNA with
random hexamer primers using a Gene-Amp RNA polymerase
chain reaction (PCR) kit (Perkin Elmer, Foster City, Calif) and was
amplified by reverse transcriptase (RT)-PCR using glyceralde-
hyde phosphate dehydrogenase (GAPDH) primers with
AmpliTag DNA polymerase (Perkin Elmer). The cycle parameters
for the PCR were 30 cycles at 948C for 1 minute, 558C for 1 min-
ute, and 728C for 1 minute with a 1-time final extension at 728C
for 7 minutes.
Comparison With CPM
To compare CTPM and CPM, 44 duplicate sets of tissue slices
were taken from 38 randomly selected surgical specimens. One
set was processed by CPM and the other by CTPM. Histologic
sections were double-mounted on the same slide; the CTPM sec-
Rapid Diagnostic Tissue PreparationMorales et al 585
Figure 2. Schematic representation of specimen flow and total time required to prepare hematoxylin-eosinstained slides from fresh tissue,
comparing 5 currently available processing methods: 1, continuous-throughput processing method; 2, short version of the conventional processing
method (CPM); 3 and 4, microwave methods described by Visinoni et al10 and Kok et al,8 respectively; and 5, CPM. The color-coded steps are as
follows: green, tissue dissection; pink, fixation; orange, processing; and blue, a combination of embedding, microtomy, and routine staining. The
red arrow depicts the waiting period before a new batch of specimens can be reloaded into the system.

tions were placed toward the frosted end of the slide and the
CPM sections on the opposite end. Thus, tissue sections were
subjected to identical conditions during staining with hematox-
ylin-eosin. Histochemical and immunohistochemical assays were
similarly performed using these double-mounted slides.
Hematoxylin-eosinstained slides were evaluated for integrity
of tissue architecture, as well as for details of nuclei and nucleoli,
preservation of cytoplasmic structure, cellular secretions, stroma/
epithelium interface, and possible presence of crenation of red
blood cells and hemolysis.
We also evaluated the impact of the new method on our sur-
gical pathology turnaround times by comparing the release of
final reports during a period of 6 months (January 1 to June 30,
1997) that preceded implementation of the new procedure with
that of a comparable recent period (January 1 to June 30, 2000).
To compare the preservation of RNA using CTPM and CPM,
6 specimens were procured fresh, immediately upon surgical re-
moval from patients. These specimens consisted of normal testis,
normal ovary, leiomyoma of the uterus, hyperplastic prostate
gland, and 2 breast excisions, one with carcinoma and the other

Figure 3. Normal uterus. Low-power view demonstrating widely patent lymphatics in tissue processed using the continuous-throughput processing
method (a) compared to the same specimen processed using the conventional processing method (b) (hematoxylin-eosin, original magnification 325).
Figure 4. In situ lobular neoplasia of breast. The morphology of epithelial and stromal elements is practically identical between matched spec-
imens prepared using the continuous-throughput processing method (a) and the conventional processing method (b) (hematoxylin-eosin, original
magnifications 3100).
Figure 5. Normal small intestine. The pattern and intensity of mucicarmine stain is identical in matched specimens prepared using the continuous-
throughput processing method (a) and the conventional processing method (b) (mucicarmine, original magnification 3100).
Figure 6. Infiltrating ductal carcinoma. Uniform strong immunohistochemical reaction for estrogen receptor in the nuclei of tumor cells. The
reaction is the same in the specimens prepared using the continuous-throughput processing method (a) and the conventional processing method
(b) (immunoperoxidase for estrogen receptor, diaminobenzidine plus cupric sulphate color intensification and fast green counterstain, original
magnification 3100).
586 Arch Pathol Lab MedVol 126, May 2002 Rapid Diagnostic Tissue PreparationMorales et al
Arch Pathol Lab MedVol 126, May 2002 Rapid Diagnostic Tissue PreparationMorales et al 587
 Table 3. Comparison of Diagnostic Turnaround Times
of Surgical Specimens*
JanuaryJune 1997 JanuaryJune 2000
CPM, No. (%) CTPM, No. (%)
Same day 56 (0) 4919 (36)
1 day 5139 (44) 4198 (31)
2 days 3029 (26) 2027 (15)
3 days 1887 (16) 1006 (7)
.3 days 1534 (13) 1440 (11)
Total 11 645 13 590
* CPM indicates conventional processing method; CTPM, continu-
ous-throughput processing method.
with fat necrosis. One sample from each of these surgical speci-
mens was processed by CTPM and CPM. An additional sample
from each specimen was also processed by CTPM, but with
0.05% diethyl pyrocarbonate (DEPC) added to the solutions in
steps 1, 2, and 3. The total RNA was isolated and amplified by
RT-PCR using primer for the housekeeping gene GAPDH (see
Processing) and run on the agarose gel.
RESULTS
The CTPM method reduced the processing of both fresh
and fixed tissue samples to approximately 60 minutes.
Compared to the conventional method, blocks of tissue
prepared by the CTPM are relatively softer, hence easier
to cut and make ribbons. Evaluation of the quality of his-
tology sections and staining properties of the double-
mounted slides revealed comparable results with CTPM
and CPM (Figures 3 through 6). In general, however, sec-
tions prepared by CTPM exhibited brighter staining with
eosin and stronger reaction with hematoxylin. We have
since reduced the exposure times for hematoxylin-eosin
to one half of what was required with CPM. Other differ-
ences observed included widely patent uterine lymphatics
in tissue processed using CTPM (Figure 3). The overall
tissue architecture, stroma, secretory products, cell mor-
phology, and nuclear morphology, however, appeared
practically the same. Histochemical stains were also com-
parable in the double-mounted slides of tissues processed
by CPM and CTPM (Figure 5). The intensity of immuno-
histochemical reactions and pattern of distribution of the
antigens were for the most part identical in slides pro-
cessed by CPM and CTPM (Figure 6).
Table 3 summarizes the impact of CTPM on turnaround
times for our surgical pathology reports. The reported
Figure 7. Reverse transcriptasepolymerase chain reaction (RT-PCR) using glyceraldehyde phosphate dehydrogenase primers. The total RNA was
prepared from specimens of normal testis (lanes A), normal ovary (lanes B), leiomyoma of the uterus (lanes C), prostate (lanes D), and 2 breast
excisions (lanes E and F). Tissue was processed as follows: lane 1, continuous-throughput processing method (CTPM); lane 2, CTPM processing
with 0.05% diethyl pyrocarbonate; and lane 3, conventional processing method. The dash indicates negative RT-PCR control and the M indicates
the 1-kb BRL-GIBCO DNA marker. The position of the 500-bp amplified product is indicated on the right side.
588 Arch Pathol Lab MedVol 126, May 2002
turnaround times apply to all cases, including those re-
quiring deeper sections, histochemical or immunohisto-
chemical stains, and intradepartmental consultations.
The integrity of extracted RNA following CTPM, partic-
ularly with addition of DEPC, was significantly improved,
although 28S and 18S ribosomal bands were degraded in
most samples. Nevertheless, when 100 ng of the same RNA
was used for RT-PCR amplification (using GAPDH primers)
the expected 500-base pair (bp) band was obtained in all
RNA specimens extracted following CTPM, but in only 3
of the 6 specimens extracted after CPM. When amplification
was obtained from CPM-processed tissue, the intensity of
amplified product on ethidium bromide gel was signifi-
cantly weaker than amplification product obtained in
CTPM-processed tissue (Figure 7).
COMMENT
Because of the 8 hours or longer presently required to
prepare tissues for histology, diagnosis of biopsy speci-
mens on the day after receipt of the specimen is custom-
ary. The diagnosis of complex specimens may take even
longer. Figure 2 schematizes the various steps, relevant
methods, and their approximate time requirements from
receipt of tissues in the laboratory to availability of micro-
scopic sections for diagnosis. The CPM is particularly ac-
countable for delaying the evaluation of surgically re-
moved tissue, as this is the lengthiest of the stages of di-
agnostic tissue preparation. The various steps required by
CPM are responsible for this long process, that is, incu-
bation in separate solutions of formalin for fixation, a se-
ries of increasing concentrations of alcohol for dehydra-
tion, and xylene for clearing tissue of alcohol before im-
pregnation. This 8-hour or longer process is usually per-
formed overnight in automated instruments. The
pathologist, therefore, is unable to render a diagnosis
based on microscopic examination of tissue sections until
the next day at the earliest, almost 24 hours after the spec-
imen arrives in the laboratory.
Attempts are being made to reduce the time for tissue
processing, either by shortening the cycle of automated
CPM processors or by using microwave-assisted methods.
A shortened method of the CPM is presently practiced. To
that effect, tissues are usually fixed for a minimum of 30
minutes in formalin before placing them in automated
processors, which are programmed for a cycle that lasts
from about 70 to 150 minutes (Figure 2). However, only
Rapid Diagnostic Tissue PreparationMorales et al
 Table 4. Comparison of Continuous-Throughput Processing Method (CTPM) With Other Currently
Used Processing Methods*
CTPM
Prefixation required No
Specimens added every 15 min Yes
Fresh tissue to paraffin in 1 h Yes
Formalin used in processing No
Xylene used in processing No
High-quality HC and IHC Yes
Relative RNA integrity Yes
Complete automation Yes
HC indicates histochemistry; IHC, immunohistochemistry; and CPM, conventional processing
Please see references 610.
small specimens are amenable to this procedure. More-
over, the processor cannot be accessed with additional
samples until completion of the entire cycle. Because of
these constraints, the shortened method is used almost
exclusively for the handling of emergency biopsies.
Several investigators, notably Boon and colleagues,79
Leong,6 and Visinoni et al,10 have pioneered the use of
microwaves to expedite fixation and/or processing of tis-
sue for microscopy. Because dielectric solutions and tissue
sections absorb microwave energy, it is translated into
heat, both in the solutions and within the tissues im-
mersed in those solutions. The resulting effect is acceler-
ated diffusibility of the solutions and denaturation of pro-
teins; both phenomena lead to shortened processing time.
Boon and colleagues79 and Visinoni and associates10 have
developed procedures that reduce the number of steps
and the time customarily required to process fixed biop-
sies and larger samples. This reduction was accomplished
primarily by combining vacuum and microwave energy
and using mixtures of reagents different from those used
in conventional methods. In these procedures, the pro-
cessing time for fixed tissue varies from 30 minutes to 2
hours, depending on the size or thickness of the samples.
These methods use a specially designed microwave oven
into which the processing solutions are manually brought
in and out.
Although the aforementioned microwave-assisted and
conventional shortened methods reduce processing time
significantly, they are attendant to a number of technical
difficulties that would seem to preclude their widespread
acceptance. The requirement to fix specimens for a num-
ber of hours prior to entering the processing stage in mi-
crowave methods would make them impractical for same-
day diagnostic tissue preparation. Furthermore, none al-
low for adding specimens until the entire cycle is com-
plete; specimen batching is therefore required. It is also
cumbersome to adjust the timing of each step according
to the size of the specimens, as required by some of the
protocols mentioned.9,10 Moreover, microwave methods
heretofore reported are not automated, another significant
impediment for the daily practice of histology.
The method we describe provides for continuous flow
of specimens from dissection to wax impregnation. This
system can accept new specimens every 15 minutes, lead-
ing to availability of microscopic slides for diagnosis
throughout the day and, most importantly, on the same
day that those specimens are received. Since the imple-
mentation of CTPM, more than one third of our surgical
pathology specimens are diagnosed and reported on the
Arch Pathol Lab MedVol 126, May 2002
Other
Regular Short Microwave-
CPM CPM Based Methods
Yes Yes Yes
No No No
No No No
Yes Yes No
Yes Yes No
Yes Yes Yes
No No Not reported
Yes Yes No
method.
same day that they are received. This rate compares very
favorably with our previous practice and with that of oth-
ers as reported in surveys of departments of pathology.1,2
This important improvement in our practice has caused
no compromise in the quality of the diagnostic material,
because routine histochemical and immunohistochemical
stains obtained by CTPM are comparable to CPM.
Another advantage of CTPM is that it requires only
about 10% of the total volume of reagents, which also are
considerably less toxic than those used in CPM. Further-
more, lack of requirement for prefixation and complete
elimination of formalin and xylene from the system are
important for the safety of personnel; the method also
minimizes the cost of recycling or disposal of these chem-
icals. Moreover, there is no need to monitor the instrument
overnight, as is the case with CPM because of the possi-
bility of instrument failure. Because the method is com-
posed of well-defined steps with standard volumes, times,
pressure, and temperatures, it has led to the development
of a robotic processing instrument, thus converting a pre-
viously manual procedure to a fully automated one. A
comparison of CPTM with other currently used process-
ing methods is summarized in Table 4 and Figure 2.
Notwithstanding the aforementioned benefits provided
by CTPM, we are far from realizing the full potential of
this method. One of its most important attributes may re-
side in providing high-quality slides for histopathologic
diagnosis together with superior RNA preservation. Al-
though the integrity of RNA in our preliminary work was
partially compromised, it was of sufficient quality that
segments up to 500 bases could be consistently amplified,
provided that the processed tissue was submitted fresh,
immediately after surgical removal. As most PCR-ampli-
fied fragments in molecular pathology are smaller than
500 bases, it suggests that CTPM-RNA can be used for
most PCR amplifications. A prerequisite for RNA preser-
vation in CTPM-processed tissue is to either obtain freshly
removed surgical specimens or use a special preservative
that maintains morphologic integrity and RNA quality.
Such a solution is currently being evaluated for transpor-
tation and storage of surgical specimens.
In summary, we have developed and are now using a
method for preparing tissues for diagnosis that offers
many advantages over the long-established tissue-pro-
cessing procedures. Because this method facilitates contin-
uous flow of tissue samples, it may eventually lead to the
automation of all steps of diagnostic tissue preparation,
akin to the handling of liquid tissue samples in the clinical
laboratory.
Rapid Diagnostic Tissue PreparationMorales et al 589
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Rapid Diagnostic Tissue PreparationMorales et al