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(For kidney and other see Services Index)
A. FOR COMBINED HRLM-TEM STUDIES WE NEED ONE SPECIMEN ONLY.
1. Needle biopsies and small
specimens (Fig # 1A): put the intact specimen (s)
immediately in HRLM-TEM fixative:
Paraformaldehyde-Glutaraldehyde (PARAFOR-G), provided by us
upon request. DO NOT MINCE THE SPECIMEN. One of the
dimensions of the sample must be 2 mm or less. LABEL THE
CONTAINER WITH THE NAME OF THE PATIENT.
2. FROM LARGER SPECIMENS (Fig #1 B): Cut the specimen in 7
mm fragments. One of the dimensions of the specimen MUST BE
2 mm OR LESS. The other dimension may be as large as 7 mm.
Put the samples IMMEDIATELY IN PARAFOR-G provided by us upon
request. DO NOT MINCE THE SPECIMEN. LABEL THE CONTAINER WITH
THE NAME OF THE PATIENT.
3. From full organs or large
surgical specimens (tumors) (Fig #1C): Take wedge samples
from three different representative areas and proceed as
with the larger biopsies (Fig 1B).
4. If all of the specimen is already in formalin fixative
and you need HRLM-TEM studies, take three 7 mm samples from
the periphery (best fixed area). One of the dimensions of
the sample must be 2mm or less.
5. If all of the specimen has been embedded in a paraffin
block and you need HRLM-TEM studies, submit the paraffin
block and a representative H&E slide marking the areas
from which you want HRLM-TEM studies done. The paraffin
block will not be returned.
6. HRLM-TEM studies from a selected area of an H&E
section are also possible. Submit the slide marked with the
area you want HRLM-TEM studies on. The H&E slide will
not be returned.
7. LABEL THE CONTAINER WITH THE NAME OF THE PATIENT. SEE
ALSO VII BELOW.
B. SAMPLING FOR IMMUNOHISTOLOGY (IH):
IN OUR LABORATORIES WE PERFORM
FLUORESCENT MICROSCOPY (FM):
1. FOR NEEDLE BIOPSIES OR SMALL SPECIMENS OTHER THAN KIDNEY
(2 mm or less in one dimension) FM ONLY (Fig # 2A): Place
the sample IMMEDIATELY in FM transport media, Zeus or
Michelle's, (provided by our LAB upon request). LABEL THE
VIAL WITH THE NAME OF THE PATIENTOR PATHOLOGY NUMBER.
2. NEEDLE BIOPSIES OR SMALL SPECIMENS FOR COMBINED FM and
HRLM-TEM (Fig # 2B) : Take two samples from both ends of the
needle biopsy for FM and sent the central part for HRLM-TEM.
LABEL THE CONTAINER WITH THE NAME OF THE PATIENTOR PATHOLOGY
NUMBER.
3. LARGER SPECIMENS FOR COMBINED
FM-HRLM-TEM (Wedge biopsies) (Fig # 2C): Dissect the
specimen in to 2 mm slices, each one containing
representative areas of the lesion. Put one slice
IMMEDIATELY in FM transport media. Put the other slices in
Parafor-G for HRLM-TEM studies. From full organs or larger
surgical specimens take three wedge biopsies and proceed as
in 3 above. LABEL THE VIAL WITH THE NAME OF THE PATIENT.
4. NOTE: This transport media will preserve the specimen for
FM for 5 days at room temperature. THE TRANSPORT MEDIA IS
NOT A FIXATIVE!! DO NOT USE THIS TRANSPORT MEDIA FOR
HRLM-TEM SPECIMENS!!!
5. INFORMATION THAT MUST BE SUBMITTED WITH THE SPECIMENS :
See VII below.
II. PREPARATION OF
WHITE BLOOD CELLS FOR HRLM-TEM
STUDIES.
1. Withdraw about nine
(9) ml of blood in a tube with heparine (1%) or EDTA
(5%).
2. Immediately add one (1) ml of our HRLM-TEM fixative
(Parafor-G)(Submitted by us on request) and mix.
3. Centrifuge for 20 minutes at 2,500 rpm.
4. Gently remove the plasma standing on the top of the buffy
coat. Do not disturb the buffy coat!!!
5. Very gently add (without disturbing the buffy coat) more
Parafor-G fixative.
6. Fix for at least three hours (may stay for 24 hours).
7. Send the buffy coat (now solidified) in Parafor-G
fixative to the lab.
8. Fill out the requisition/information form with the
clinical information and differential diagnosis and submit
it with the specimen to our laboratory (IMPORTANT: See VII
below).
9. LABEL THE VIAL WITH THE NAME OF THE
PATIENT.
III. PREPARATION OF
CELL SUSPENSIONS (Peritoneal fluid, pleural fluid, gastric
fluid, etc.) FOR HRLM-TEM.
1. Use fresh specimens
(immediately after drawing).
2. Add one (1) part of Parafor-G fixative to nine (9) parts
of the fluid.
3. Centrifuge at 5,000 - 10,000 rpm for 20 minutes.
4. Discard the supernate very gently without disturbing the
sediment.
5. Add Parafor-G fixative very gently without disturbing the
sediment. Fix overnight.
6. Send the pellet (sediment) in Parafor-G fixative to the
lab.
7. If unable to form a pellet, add a dilute solution of
gelatin. For more information, call the laboratory.
8. Fill out the requisition/information form with the
clinical information and differential diagnosis and submit
it with the specimen to our laboratory
9. LABEL THE VIAL WITH THE NAME OF THE PATIENT.
(IMPORTANT: See VII below).
IV. PREPARATION OF
BONE MARROW FOR HRLM-TEM.
1. Segregate soft (no
bone) marrow particles from the aspirated material and place
them in Parafor-G fixative.
2. Fill out the requisition form and submit it to our
laboratory.
3. LABEL THE VIAL WITH THE NAME OF THE
PATIENT.
V. PREPARATION OF FINE
NEEDLE ASPIRATES FOR HRLM-TEM:
1. Segregate the largest
particles from the aspirated material and place them in
Parafor-G fixative.
2. Fill out the requisition form and submit to to our
laboratory.
3. If there are no large particles, process as described
above for cell suspensions (III).
4. LABEL THE VIAL WITH THE NAME OF THE PATIENT. (IMPORTANT:
See VII below).
VI. PREPARATION OF
URINARY SEDIMENT FOR HRLM-TEM.
1. Use a well mixed
fresh urine sample. PROCESS IMMEDIATELY!!!
2. Place nine (9) parts of urine in a clean conical tube and
add one (1) part Parafor-G fixative.
3. Centrifuge at 5,000 to 10,000 rpm for 20 minutes.
4. Discard the supernate very gently without disturbing the
sediment.
5. Add Parafor-G fixative very gently without disturbing the
sediment.
6. Submit the conical tube or tubes with the sediment in
fixative to the lab.
7. Fill out the requisition/information form with the
clinical information and differential diagnosis and submit
it with the specimen to our laboratory
8. LABEL THE VIAL WITH THE NAME OF THE PATIENT.
(IMPORTANT: See VII below).
VII. INFORMATION
NEEDED IN OUR LABORATORY: SUBMIT THE FOLLOWING INFORMATION
WITH THE SPECIMEN:
1. Fill our requisition
form with the clinical information. Please fill both sides
of our requisition form. SEND YOUR SURGICAL PATHOLOGY
REPORT, A DIFFERENTIAL DIAGNOSIS, OR THE REASON WHY YOU WANT
FM, AND/OR HRLM-TEM STUDIES.
2. Send an H & E slide or results from other pathology
studies if available.
3. INFECTIOUS MATERIAL MUST BE CLEARLY LABELED AS SUCH.
4. AUTHORIZATION FROM YOUR DEPARTMENT OF PATHOLOGY. Most
hospitals and clinics require that all specimens being send
out for consultation or diagnosis, are authorized by their
Department of Pathology.
5. BILLING INFORMATION (SEE ATTACHED FEE SCHEDULE PG
R1).
VIII. REASON FOR NOT ACCEPTING SPECIMENS IN OUR LABORATORIES:
Our laboratory is CAP certified
and CLIA registered. To keep the high performance level of
our services we must follow established regulations. We can
not accept specimens if:
1. The vial with the sample is not labeled with the
patient's name.
2. There is no requisition form with the sample.
3. The requisition form has not been completed. Please fill
both
4. The container with the sample has been broken or
leaking.
5. The specimen is not in an adequate fixative or transport
media.
6. No billing information.
IX. PROCESSING TIME
AND REPORTS.
1. HRLM STATS are
reported the same day. Cut off time is 11 am. See Fee
Schedule for additional fee for Stats (request).
2. IH and HRLM studies alone are usually reported within 24
hours.
3. Combined studies and TEM reports are sent within 5
working days after biopsy is received.
4. Any additional delays will be reported by telephone.
5. The original report with color photographs is submitted
to the direction filled first in our requisition form.
6. A black and white Xerox copy of the report and
photographs will be submitted to the direction filled second
in our requisition form.
7. Color copies of the original report are available for an
additional fee (see fee schedule Pg R1).
8. Black and white copies (Xerox copies) will be send upon
request at no additional fee.
REFERENCES / REFERENCIAS
Bancroft, J.D. and Stevens, A.: Theory and Practice of Histological Techniques, Churchill Livingstone. third Ed., 1990.
Flores TR, Hoffmann EO and Coover J: Improved High Resolution Light Microscopy and Transmission Electron Microscopy Techniques in Diagnostic Pathology. J Histotec (#1):45-52, 1997. (Click here for complete article).
Flores TR, Hoffmann EO and Velasquez M: Stat high resolution light microscopy (HRLM) - Transmission Electron Microscopy (TEM) Techniques for transplant renal biopsies. J. Histortec 21 (#3): 213-217, 1998. (Click here for complete article).
Glauert, A.M.: Fixation, Dehydration and Embedding of Biological Specimens. North Holland Publishing Co., New York, 1975.
Hoffmann E.O., Coover J, Flores TR, et al: Improvements for routine renal pathology. Lab Med 16: 237, 1985.
Holliday MA, Barratt TM, Avner ED (1994): Pediatric Nephrology, 3rd edn. Williams and Wilkins, Baltimore.
McDowell, E.M., and Trump, B.F.: Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Path Lab Med 100:405, 1976.