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Continuous-specimen-flow, high-throughput, 1-hour tissue processing: a system for rapid diagnostic tissue preparation. (New Technology in Laboratory Medicine).



A waiting period of 1 day or longer for a pathologic diagnosis following any surgical procedure is customary. (1,2) Patient anxiety, the delay to plan or institute treatment, and other adverse aspects related to the delay between surgery and diagnosis are consequences of constraints 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 processing method (CPM) is the toxicity of reagents used, especially formaldehyde and xylene xylene (zī`lēn) or dimethylbenzene (dī'mĕthəlbĕn`zēn), C6H4(CH3)2 . Formaldehyde, for instance, 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 oropharynx /oro·phar·ynx/ (-far´inks) the part of the pharynx between the soft palate and the upper edge of the epiglottis.

o·ro·phar·ynx
n.
 and the respiratory tract respiratory tract
n.
The air passages from the nose to the pulmonary alveoli, including the pharynx, larynx, trachea, and bronchi.


Respiratory tract 
. (3,4) Furthermore, because the study of diseases at the molecular level in archival tissue is being greatly expanded, severe degradation of RNA RNA: see nucleic acid.
RNA
 in full ribonucleic acid

One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic
 by the conventional method constitutes a major disadvantage for practitioners. (5)

During the last 2 decades, as a consequence of the introduction of microwave energy into histology laboratories, progress has been made in the development of rapid processing methods and the elimination of toxic reagents. (6-10) 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 method 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 annually 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 Jackson Memorial Hospital (also known as "Jackson" or abbreviated "JMH") is a non-profit, tertiary care teaching hospital and the major teaching hospital of the University of Miami Leonard M. Miller School of Medicine in Miami, Florida. , and the remaining are from the University of Miami This article is about the university in Coral Gables, Florida. For the university in Oxford, Ohio, see Miami University.

The University of Miami (also known as Miami of Florida,[2] UM,[3] or just The U
 Hospital and Clinics, including the Sylvester Comprehensive Cancer Center. About 110 000 paraffin blocks are prepared every year from these tissues. Small biopsy specimens are received in the laboratory in containers partially filled with 10% sodium phosphate-buffered formalin formalin /for·ma·lin/ (for´mah-lin) formaldehyde solution.

for·ma·lin
n.
An aqueous solution of formaldehyde that is 37 percent by weight.
 (4% formaldehyde). Large specimens, such as uteri, breasts, intestines, and placentas, are usually received fresh or in various-sized containers with formalin. Until September 1997, the tissues were processed overnight 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, Torrance, Calif).

During the developmental stages of our continuous-throughput processing method (CTPM CTPM Content Traffic Presell Monetize
CTPM CTP Manager (GCCS)
CTPM Common Tactical Picture Manager
CTPM Critical Technical Parameter Matrix
CTPM Continuous Time Packet Model
), we used samples from residual large surgical specimens for evaluation. As soon as the efficacy of the new methodology was established, we began routine processing 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 require 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 x 5.0-cm metallic plate on a 45.0 x 30.0 x 2.5-cm flat plastic slab. The metallic plate has two 1.5-mm-deep cutouts, measuring 3.0 x 2.5 cm and 2.5 x 1.8 cm. Tissues are placed over either of the cutouts and slid horizontally along the slicing plate. During 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 processing, an additional 3.0-mm-deep slot was cut in the metallic plate, which can procure thicker sections if needed.

[FIGURE 1 OMITTED]

Processing

The CTPM as presently practiced in the Department of Pathology 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 acetone (ăs`ĭtōn), dimethyl ketone (dīmĕth`əl kē`tōn), or 2-propanone (prō`pənōn), CH3COCH3 , polyethylene glycol, mineral oil, paraffin, and minute amounts of glacial acetic acid glacial acetic acid
n.
Acetic acid that is at least 99.8 percent pure.
 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:

[FIGURE 2 OMITTED]

Step 1.--The samples are microwaved at 62 [degrees] C for 15 minutes in 1000 mL of a solution of acetone, isopropyl alcohol, polyethylene glycol, glacial acetic acid, and dimethyl sulfoxide. Air bubbling produces agitation.

Step 2.--The samples are heated for 15 minutes at 62 [degrees] C 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 ag·i·tate  
v. ag·i·tat·ed, ag·i·tat·ing, ag·i·tates

v.tr.
1. To cause to move with violence or sudden force.

2.
 by bubbling.

Step 3.--The samples are heated for 5 minutes at 62 [degrees] C 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 solution is agitated by bubbling.

Steps 4 and 5.--The samples are incubated at 65 [degrees] C in 2 consecutive 1000-mL solutions of a mixture of low-viscosity mineral oil and paraffin. A vacuum of 640 mm Hg is applied for 5 minutes to each bath.

Steps 6 through 9.--Impregnation is accomplished by incubation at 65 [degrees] C 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 impregnation /im·preg·na·tion/ (im?preg-na´shun)
1. fertilization.

2. saturation (1).


impregnation

1. the act of fertilizing or rendering pregnant.

2. saturation.
 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 fairly constant since the processing system was adapted in 1997, the manner in which these agents are applied has been modified. 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 creation of a specifically designed microwave applicator ap·pli·ca·tor
n.
An instrument for applying something, such as a medication.


applicator,
n a device for applying medication; usually a slender rod of glass or wood, used with a pledget of cotton on the end.
 (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. Treatment 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 des·ic·cate  
v. des·ic·cat·ed, des·ic·cat·ing, des·ic·cates

v.tr.
1. To dry out thoroughly.

2. To preserve (foods) by removing the moisture. See Synonyms at dry.

3.
 resting in a glycerin glycerin /glyc·er·in/ (-in) a clear, colorless, syrupy liquid used as a laxative, an osmotic diuretic to reduce intraocular pressure, a demulcent in cough preparations, and a humectant and solvent for drugs. Cf. glycerol.  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-impregnation 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 [micro]m and stained with hematoxylin-eosin. When indicated, histochemical and immunohistochemical stains are performed following standard techniques.

For RNA extraction, 20 ribbons (20 [micro]m thick) of paraffin-embedded tissue were deparaffinized with xylene twice for 15 minutes 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 GIBCO Grand Island Biological Company (tissue culture media enterprise)  BRL BRL

In currencies, this is the abbreviation for the Brazilian Real.

Notes:
The currency market, also known as the Foreign Exchange market, is the largest financial market in the world, with a daily average volume of over US $1 trillion.
, Grand Island, NY) using power homogenizer. Two microliters of Pellet Paint (Novagen, Madison, Wis) fluorescent dye-labeled coprecipitant was added to each sample. The RNA was isolated following the Trizol protocol (GIBCO), isopropanol isopropanol, isopropyl alcohol, or 2-propanol (ī'səprō`pənōl, ī'səprō`pĭl), (CH3)2CHOH, a colorless liquid that is miscible with water.  precipitation, air drying, and dissolution in 40 mL ribonuclease-free sterile water. Concentration was estimated by spectrophotometry spectrophotometry

Branch of spectroscopy dealing with measurement of radiant energy transmitted or reflected by a body as a function of wavelength. The measurement is usually compared to that transmitted or reflected by a system that serves as a standard.
, and the integrity of total RNA was tested by assessing the resolution of 28S and 18S ribosomal RNA in 0.8% agarose agarose

more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments.
 gel stained with ethidium bromide. The complementary DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 was generated from 100 ng of total RNA with random hexamer primers using a Gene-Amp RNA polymerase chain reaction (PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
) kit (Perkin Elmer, Foster City, Calif) and was amplified by reverse transcriptase (RT)-PCR using glyceraldehyde glyceraldehyde /glyc·er·al·de·hyde/ (glis?er-al´de-hid) an aldose, the aldehyde form of the three-carbon sugar derived from the oxidation of glycerol; isomeric with dihydroxyacetone.  phosphate dehydrogenase dehydrogenase /de·hy·dro·gen·ase/ (de-hi´dro-jen-as?) an enzyme that catalyzes the transfer of hydrogen or electrons from a donor, oxidizing it, to an acceptor, reducing it.

de·hy·dro·gen·ase
n.
 (GAPDH GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase (also seen as G3PDH) ) primers with AmpliTag DNA polymerase (Perkin Elmer). The cycle parameters for the PCR were 30 cycles at 94 [degrees] C for 1 minute, 55 [degrees] C for 1 minute, and 72 [degrees] C for 1 minute with a 1-time final extension at 72 [degrees] C 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 sections 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 hematoxylin-eosin. Histochemical and immunohistochemical assays were similarly performed using these double-mounted slides.

Hematoxylin-eosin-stained slides were evaluated for integrity of tissue architecture, as well as for details of nuclei and nucleoli nucleoli

plural form of nucleolus.
, preservation of cytoplasmic cytoplasmic

pertaining to or included in cytoplasm.


cytoplasmic inclusions
include secretory inclusions (enzymes, acids, proteins, mucosubstances), nutritive inclusions (glycogen, lipids), pigment granules (melanin, lipofuscin,
 structure, cellular secretions, stroma/ epithelium interface, and possible presence of crenation of red blood cells and hemolysis hemolysis (hĭmŏl`ĭsĭs), destruction of red blood cells in the bloodstream. Although new red blood cells, or erythrocytes, are continuously created and old ones destroyed, an excessive rate of destruction sometimes occurs. .

We also evaluated the impact of the new method on our surgical 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 removal from patients. These specimens consisted of normal testis testis (tĕs`tĭs) or testicle (tĕs`tĭkəl), one of a pair of glands that produce the male reproductive cells, or sperm. , normal ovary ovary, ductless gland of the female in which the ova (female reproductive cells) are produced. In vertebrate animals the ovary also secretes the sex hormones estrogen and progesterone, which control the development of the sexual organs and the secondary sexual , leiomyoma of the uterus, hyperplastic prostate gland, and 2 breast excisions, one with carcinoma and the other with fat necrosis. One sample from each of these surgical specimens was processed by CTPM and CPM. An additional sample from each specimen was also processed by CTPM, but with 0.05% diethyl pyrocarbonate (DEPC DEPC Diethyl Pyrocarbonate
DEPC Down East Partnership for Children
DEPC Data Evaluation and Publication Committee
) 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 histology sections and staining properties of the double-mounted slides revealed comparable results with CTPM and CPM (Figures 3 through 6). In general, however, sections prepared by CTPM exhibited brighter staining with eosin eosin /eo·sin/ (e´o-sin) any of a class of rose-colored stains or dyes, all being bromine derivatives of fluorescein; eosin Y, the sodium salt of tetrabromofluorescein, is much used in histologic and laboratory procedures.  and stronger reaction with hematoxylin hematoxylin /he·ma·tox·y·lin/ (he?mah-tok´si-lin) an acid coloring matter from the heartwood of Haematoxylon campechianum; used as a histologic stain and also as an indicator. . We have since reduced the exposure times for hematoxylin-eosin to one half of what was required with CPM. Other differences observed included widely patent uterine lymphatics in tissue processed using CTPM (Figure 3). The overall tissue architecture, stroma stroma /stro·ma/ (stro´mah) pl. stro´mata   [Gr.] the matrix or supporting tissue of an organ.stro´malstromat´ic

stro·ma
n. pl. stro·ma·ta
1.
, secretory secretory /se·cre·to·ry/ (se-kre´tah-re) (se´kre-tor?e) pertaining to secretion or affecting the secretions.

se·cre·to·ry
adj.
Relating to or performing secretion.
 products, cell morphology, and nuclear morphology, however, appeared practically the same. Histochemical stains were also comparable in the double-mounted slides of tissues processed by CPM and CTPM (Figure 5). The intensity of immunohistochemical reactions and pattern of distribution of the antigens were for the most part identical in slides processed by CPM and CTPM (Figure 6).

[FIGURES 3-6 OMITTED]

Table 3 summarizes the impact of CTPM on turnaround times for our surgical pathology reports. The reported turnaround times apply to all cases, including those requiring deeper sections, histochemical or immunohistochemical stains, and intradepartmental consultations.

The integrity of extracted RNA following CTPM, particularly 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 significantly weaker than amplification product obtained in CTPM-processed tissue (Figure 7).

[FIGURE 7 OMITTED]

COMMENT

Because of the 8 hours or longer presently required to prepare tissues for histology, diagnosis of biopsy specimens on the day after receipt of the specimen is customary. 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 microscopic sections for diagnosis. The CPM is particularly accountable for delaying the evaluation of surgically removed tissue, as this is the lengthiest of the stages of diagnostic tissue preparation. The various steps required by CPM are responsible for this long process, that is, incubation in separate solutions of formalin for fixation, a series of increasing concentrations of alcohol for dehydration, and xylene for clearing tissue of alcohol before impregnation. This 8-hour or longer process is usually performed 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 specimen 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 small specimens are amenable to this procedure. Moreover, 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, (7-9) Leong, (6) and Visinoni et al, (10) have pioneered the use of microwaves to expedite fixation and/or processing of tissue for microscopy. Because dielectric solutions and tissue sections absorb microwave energy, it is translated into heat, both in the solutions and within the tissues immersed in those solutions. The resulting effect is accelerated diffusibility of the solutions and denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures.  of proteins; both phenomena lead to shortened processing time. Boon and colleagues (7-9) and Visinoni and associates (10) have developed procedures that reduce the number of steps and the time customarily required to process fixed biopsies 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 processing 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 number of hours prior to entering the processing stage in microwave methods would make them impractical for same-day diagnostic tissue preparation. Furthermore, none allow for adding specimens until the entire cycle is complete; 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, leading to availability of microscopic slides for diagnosis throughout the day and, most importantly, on the same day that those specimens are received. Since the implementation of CTPM, more than one third of our surgical pathology specimens are diagnosed and reported on the same day that they are received. This rate compares very favorably with our previous practice and with that of others 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. Furthermore, 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 chemicals. Moreover, there is no need to monitor the instrument overnight, as is the case with CPM because of the possibility of instrument failure. Because the method is composed 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 previously manual procedure to a fully automated one. A comparison of CPTM CPTM Corporación Parque Tecnológico de Mérida (Merida's Technology Park Corporation)
CPTM Commonwealth Partnership for Technology Management (UK)
CPTM Capacity Planning Tools & Metrics
 with other currently used processing 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 reside in providing high-quality slides for histopathologic diagnosis together with superior RNA preservation. Although 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-amplified 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 preservation 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 transportation 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-processing procedures. Because this method facilitates continuous 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.
Table 1. Conventional Processing Method

                                         Heat,
                  Concentra-   Time,   [degrees] C
Step   Solution    tion, %      min    (Convective)

  1    Formalin       10        120         40
  2    Formalin       10        120         40
  3    Alcohol        80         30         40
  4    Alcohol        95         30         40
  5    Alcohol        95         45         40
  6    Alcohol       100         45         40
  7    Alcohol       100         45         40
  8    Alcohol       100         45         40
  9    Xylene        100         45         40
 10    Xylene        100         45         40
 11    Paraffin      ...         30         60
 12    Paraffin      ...         30         60
 13    Paraffin      ...         30         60
 14    Paraffin      ...         30         60

       Pressure/   Agita-   Volume,
Step    Vacuum      tion       L

  1       Yes       Yes        4
  2       Yes       Yes        4
  3       Yes       Yes        4
  4       Yes       Yes        4
  5       Yes       Yes        4
  6       Yes       Yes        4
  7       Yes       Yes        4
  8       Yes       Yes        4
  9       Yes       Yes        4
 10       Yes       Yes        4
 11       Yes       Yes        4
 12       Yes       Yes        4
 13       Yes       Yes        4
 14       Yes       Yes        4
Table 2. Continuous-Throughput Processing Method

                              Heat, [degrees]
                                     C

                      Time,   Convec-   Micro-
Step     Liquids       min     tive      wave

 1     Solution I *    15       ...       62
 2     Solution II     15       ...       62
         ([dagger])
 3     Solution II      5       ...       62
         ([dagger])
 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       ...

                            Volume,
Step   Vacuum   Agitation      L

 1      ...     Bubbling       1
 2      ...     Bubbling       1
 3      ...     Bubbling       1
 4      Yes        ...         1
 5      Yes        ...         1
 6      Yes        ...         1
 7      Yes        ...         1
 8      Yes        ...         1
 9      Yes        ...         1

* Solution of isopropyl alcohol, acetone, polyethylene
glycol, glacial acetic acid, and dimethyl sulfoxide.

([dagger]) Solution containing same reagents as step 1,
with the addition of low-viscosity mineral oil.
Table 3. Comparison of Diagnostic Turnaround
Times of Surgical Specimens *

           January-June 1997   January-June 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,
continuous-throughput processing method.
Table 4. Comparison of Continuous-Throughput Processing Method
(CTPM) With Other Currently Used Processing Methods *

                                                             Other
                                                           Microwave-
                                                             Based
                                         Regular   Short     Methods
                                  CTPM     CPM      CPM    ([dagger])

Prefixation required              No       Yes      Yes    Yes
Specimens added every 15 min      Yes      No       No     No
Fresh tissue to paraffin in 1 h   Yes      No       No     No
Formalin used in processing       No       Yes      Yes    No
Xylene used in processing         No       Yes      Yes    No
High-quality HC and IHC           Yes      Yes      Yes    Yes
Relative RNA integrity            Yes      No       No     Not reported
Complete automation               Yes      Yes      Yes    No

* HC indicates histochemistry; IHC, immunohistochemistry;
and CPM, conventional processing method.

([dagger]) Please see references 6-10.


References

(1.) Zarbo RJ, Gephardt GN, Howanitz PJ. Intralaboratory timeliness of surgical pathology reports. Arch Pathol Lab Med. 1996;120:234-244.

(2.) Novis DA, Zarbo RJ, Saladino AJ. Interinstitutional comparison of surgical biopsy diagnosis turnaround time. Arch Pathol Lab Med. 1998;122:951-956.

(3.) OSHA Regulations (Standards--29 CFR CFR

See: Cost and Freight
). Medical surveillance--formaldehyde. Standard Number 1910.1048AppC.

(4.) Warmington AR, Wilkinson JM, Riley CB. Evaluation of ethanol-based fixatives as a substitute for formalin in diagnostic clinical laboratories. J Histotechnol. 2000;23:299-308.

(5.) Liotta L, Petricoin E. Molecular profiling of human cancer. Nature Rev. 2000;1:48-55.

(6.) Leong AS-Y. Microwave irradiation in histopathology his·to·pa·thol·o·gy
n.
The science concerned with the cytologic and histologic structure of abnormal or diseased tissue.


Histopathology
The study of diseased tissues at a minute (microscopic) level.
. Pathol Annu. 1998; 23(pt 2):213-233.

(7.) Boon ME, Kok LP, Ouwerkerk-Noordam E. Microwave-stimulated diffusion for fast processing of tissue: reduced dehydrating, clearing and impregnation times. Histopathology. 1986;10:303-309.

(8.) Kok LP, Boon ME. Ultrarapid vacuum-microwave histoprocessing. Histochem J. 1995;27:412-419.

(9.) Boon ME, Wals-Paap C, Visinoni FA, et al. The two-step vacuum-microwave method of histoprocessing. Eur J Morphol. 1995;33:349-358.

(10.) Visinoni F, Milios J, Leong AS-Y, et al. Ultra-rapid microwave/variable pressure induced histoprocessing: description of a new tissue processor. J Histotechnol. 1998;21:219-224.

(11.) Essenfeld E, Essenfeld H, Morales AR, inventors; University of Miami, Miami, Fla, assignee. High quality, continuous throughput, tissue fixation--dehydration--fat removal--impregnation method. US patent 6 207 408, B1. March 2001.

Accepted for publication January 2, 2002.

From the Department of Pathology, University of Miami/Jackson Memorial Medical Center, Miami, Fla (Drs Morales, Vincek, and Nadji and Ms Duboue); and the Essenfeld Pathology Laboratory, Policlinica Metropolitana, Caracas, Venezuela (Drs H. Essenfeld and E. Essenfeld).

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).
COPYRIGHT 2002 College of American Pathologists
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2002 Gale, Cengage Learning. All rights reserved.

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Author:Morales, Azorides R.; Essenfeld, Harold; Essenfeld, Ervin; Duboue, Maria Carmen; Vincek, Vladimir; N
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:May 1, 2002
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Use of FTA Gene Guard filter paper for the storage and transportation of tumor cells for molecular testing.
Slide preparation aids accuracy.
Product information.
Product information.
Sputum, the final frontier: PCR from the research bench to the MRSA diagnostic trench.

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