How to submit biopsy specimens for histopathology

1. Dr. MD. SAIDUZZAMAN SAYID
MBBS, BCS (Health)
Lecturer
Department of Pathology
Dinajpur Medical College, Bangladesh

2. Overview
 Introduction
 Submission Form
 Tissue Fixation
 Packaging
 Submitting multiple
sites
 Endoscopic biopsies
 Denoting margins
 Things to avoid
 Other things to know
 Contacting us

3. Introduction
Cell Tissue Organ
Structure Biochemical Function
Anatomy Biochemistry Physiology
Any change from normal Pathology
Disease

4. Introduction
Pathology is the study of the structural,
biochemical and functional changes in
cells, tissues and organs that underlie
disease.

5. Introduction
 It serves as the bridge between the basic sciences and
clinical medicine, and is the scientific foundation for
all medicine.

6. Introduction
 Clinical correlation is important for optimal
diagnosis.

7. Introduction
Histopathological examination is
used to provide diagnostic
information that is important for
timely diagnosis of disease to
determine treatment plan.
 Fresh tissue is extremely fragile &
subject to autolysis.

8. Introduction
Loss of specimen is a tragic result
both for patient & pathologist
Handle all specimens with care &
respect.
Handle quickly & correctly.

9. Tissue for study obtained
from
Biopsies
Autopsies

10. Requirement to send specimen
Requisition form/submission form
Container
Fixative

11. Submission
form

12. Submission form
One slip for one patient
Fill properly including clinical history, pre-operative,
operative, post-operative diagnosis, organ or tissue.
More than one specimen for same patient.
Label specimen name with letter A,B,C,D etc.
Label specimen container similarly.
e.g. A- tissue from right cheek
B- tissue from left cheek.

13. Submission Form
Help Us Help You
** Please provide anatomical site, lesion description,
and pertinent clinical information on the submission
form**
 Anatomical location, as well as critical clinical
information may allow your pathologist to provide you
with the best possible diagnosis and/or differentials

14. Submission Form
 If you have a list of differentials you’d like
to rule out, please mention such.
Again, please make every effort to provide necessary
information in the designated areas on biopsy
submission form. It will help us help you help your
patients.

15. Submission Form
 First of all & most importantly surgeon should take
adequate care to avoid contamination of tissue with
tissue from other patient.
 This may happen in operation room, clinic, or in
pathology lab.
Things to be taken care for during & after
biopsy surgery

16. Specimen container
 Plastic or glass jar
 Label matching requisition slip
 Reg no.
 Full name
 Age, Sex
 Word no, Bed no
 Site & side
 More specimen mark as A, B, C, D etc.
 Signature of doctor with date

17. Tissue Fixation
1. Should prevent autolysis & putrefaction of the cell
2. Should penetrate evenly and rapidly
3. Should harden the tissues
4. Increase the optical density
5. Should not cause shrinkage or swelling of the cells
6. Must not react with the receptor sites & thus must
not interfere with the staining procedure
7. Must be cheap and easily available
Aims

18. Fixation
Small intestine well preserved Autolyzed Small intestine
Good fixative is most important in the production of
satisfactory results in histopathology

19. Simple Fixatives
 Formalin

20. ADVANTAGES & DISADVANTAGES
ADVANTAGES
1. Rapid penetration
2. Easy availability & cheap
3. Does not over harden the tissue
4. Fixes lipids for frozen sections
5. Ideal for mailing

21. ADVANTAGES & DISADVANTAGES
DISADVANTAGES
1. Irritant to the nose, eyes and mucous
membranes
2. Formation of precipitate of paraformaldehyde
which can be prevented by adding 11- 16 %
methanol
3. Formation of black formalin pigment, Acid
formaldehyde hematin

22. Formulae
10% Formalin (Unbuffered)
 40% Formaldehyde 100 ml
 Tap Water 900 ml

23. Formulae
10% Neutral Buffered Formalin (NBF)
 40% Formaldehyde 10 ml
 Tap Water 90 ml
 Sodium Hydrogen phosphate 0.4gm
 Disodium Hydrogen phosphate 0.65gm
 pH 7.2-7.4
Buffered formalin prevents formation of pigment acid
formaldehyde hematin formed from hemoglobin at
acidic pH.

24. Tissue Fixation
Specimen submit in 10% Formalin
Formalin tissue ratio 10:1
No other fixative should be used
Specimen should be in a container that
can be sealed & will not leak

25. Tissue Fixation
 Frozen section
 Cultures
 Renal & skin tissues for immunoflurescence
 Flow cytometry
 Chromosome studies
 Electron microscopy
Submit fresh tissue
Not in Formalin

26. Tissue Fixation
This is an example of
an 20 cm diameter
mass lesion which
was fixed at the clinic
and subsequently sent to
the lab in a plastic,
labeled, zip lock bag
devoid of any formalin.

27. Tissue Fixation
Incomplete parallel cuts
minimum of 2 cm apart
(bread loafing) can be
utilized to assist with
appropriate tissue fixation
for solid organ.
Be sure to avoid complete transection or too many cuts which
can both result in loss of tissue orientation!
Large solid specimens

28. Tissue Fixation
 Hollow specimen like cystic cavities:
Hollow specimen cavity either opened or filled
with formalin by syringe or catheter or packed
with gauge or cotton soaked in formalin.
Cystic lesions are injected with formalin after
removal of original fluid.

29. Tissue Fixation
When tissue float
Large specimen that
floats on fixative
should be covered by a
thick layer of gauze.

30. Tissue Fixation
Large flat tissue
Large, flat, heavy specimen
that rest on bottom of the
containers, the gauze should
be placed between the
container bottom and
specimen.

31. Tissue Fixation
Large samples can be held to
fix (at least 24 hrs) at your
clinic prior to submitting to
the lab to help avoid
shipping large volumes of
formalin which may be
costly and hazardous

32. Submitting specimens
 The container should be large enough to
accommodate the specimen and filled with
enough formalin to completely cover & surround
the specimen.
 The specimen should be float freely in the
container for adequate fixation.

33. Submitting specimens
 Submit whole specimen in a single laboratory
 Don’t divide specimen to submit in different laboratories
Material on which diagnosis
is made (slides, blocks) can
be stored for long time &
can be evaluated by
different observers or by the
same observer at different
time. Lobular carcinoma in fibroadenoma

34. Submitting specimens
Don’t discard any tissue removed from the body
Submit for histopathology
Apparently innocent looking tissue
may contain ugly behavior
(malignancy)

35. Packaging
• Container should have large enough
opening
• Fresh tissue is malleable and can
manipulate to fit into container
• Upon fixation tissue becomes rigid
and can not remove easily without
cutting or breaking the container.
No

36. Packaging
Formalin filled jars containing specimens should be placed
in a plastic bag, box, or other container with absorbent
material to absorb any leakage
YES
The container should be
couriered or brought to the
laboratory in a biohazard bag
with a completed requisition

37. Packaging
 Paperwork should be placed in a separate plastic bag to
avoid contact with formalin if leaking does occur. Such
contact can result in altered and illegible paperwork.
NO

38. Submitting Multiple Sites
 Submit multiple specimen of same patient in
multiple separate appropriately labeled jar.
YES

39. Submitting Multiple Sites
 If multiple specimens are submitted in a single
container (which is less ideal) there needs to be some
method of tissue identification (i.e. suture) to denote
respective anatomical sites.
YES

40. Endoscopic Biopsies
All fragments submit in a
same container
YES

41. Endoscopic Biopsies
screen cassette
 The optimal method to submit endoscopic biopsy is
to place it in a screen cassette after which the cassette
should be placed in an appropriately labeled formalin
filled jar. If individual cassettes are labeled properly
(sharpie or no. 2 pencil), multiple cassettes can be place
in one jar.
YES

42. Endoscopic Biopsies
 Do not submit endoscopic biopsies wrapped in
gauze. Specimens may become lost or may be
crushed during the attempted retrieval process.
NO

43. Denoting Margins
- Ink the area of interest
- Ink prior to bread loafing (if needed)
- Allow ink to begin drying before placing
the specimen in formalin
 Surgical Ink

44. Denoting Margins
- Used to indicate margins or for orientation
- Use variable numbers and/or colors of suture
- Provide a clear description on the submission
form denoting what the sutures indicate (i.e. one
suture = cranial margin)
 Tagging

45. Denoting Margins
* Submission of samples from the post-surgical bed
* Any tumor / neoplastic cells in these specimens is
evidence of remaining microscopic disease
* Similar to “submitting multiple sites” clearly label
and submit each region individually
 Tumor Bed Samples

46. Mapping on a larger scale
Cystoprostatectomy Prostate Posterior
bladder
Anterior
bladder
Ureter
Red = Right
Green = Left

47. Things to Avoid
 Please help keep our technician’s fingers
safe and DO NOT submit specimens with
needles for any reason!
NO

48. Things to Avoid
 Please do no staple or suture tissue to
cardboard. It can damage tissue and prevent
appropriate margin assessment
NO

49. Other Things to Know
It is important for you to realize that after all is
said and done the pathologist typically evaluates 1
to 4, 5µm thick sections from the entire specimen
which is submitted.
Images depicting a mass from
which a section is taken,
embedded in paraffin, and
subsequently sectioned to a
thickness of 5µm for
microscopic evaluation.

50. Specimen identification and labeling
Tissue specimen received in the laboratory have a request form that
lists the patient information, history & description of the site of origin.
 The specimen are labeled by giving
ID number in the laboratory.

51. Specimen identification and labeling
 Any discrepancies of specimen identification
noted by pathology assistant should contact
with pathologists and/or clinician if there are
any questions.

52. Causes of rejection of specimen
 Specimen not in formalin
 Unlabeled or improperly labeled
container
 Without requisition slip or incomplete
requisition slip.

53. Other Things to Know
Our Staffs is here
working hard for
you!
 Our staffs and pathologists are here to assist you

54. Contacting Us
If you have any questions about how to best
submit your sample or have questions
regarding any other issues, please contact
the laboratory
Mobile No - 0171-3337793
0171-1361198