This document summarizes an experiment conducted in the Henderson Lab at Boston University to determine the effect of delayed fixation on kidney histopathology. The experiment tested different fixation delay times at both room temperature and 4°C and analyzed changes to glomeruli and tubules under light microscopy. Results showed glomeruli were more resistant to damage than tubules. At 4°C, changes were seen after 4 hours of delay, while at room temperature, changes occurred within 30 minutes. Immunohistochemistry analysis found antigen degradation occurred at similar delay times.

1. The Effect of Delayed
Fixation on Kidney
Histopathology
Catherine Wallace de Melendez
Boston University
Henderson Lab
BLCS Fall 2015

2. Contents
• Henderson Lab Overview
• Kidney Overview
• Externship Goals
• Tissue Processing for Paraffin Embedded Slides
• Processing Errors and Complications
• Experiment Objectives
• Experiment Design and Execution
• Results and Conclusions
• Where Do We Go From Here?
• References and Acknowledgements

3. Henderson Lab
• Overseen by Joel Henderson, MD, PhD
– Renal Pathologist for BMC and Assistant Professor
at BUSM
• Studies the role of mechanical stresses in
Glomerular injury
– Main focus on the Podocyte
• Additional focus on developing new ways to
observe and measure physical changes at the
micro- and nano- level

4. The Kidney:
Anatomy
• Retroperitoneal Organs
• Around 12 cm long x 6 cm wide x 3 cm deep
• Main functions: remove waste, maintain fluid
homeostasis and electrolyte balance

5. The Kidney:
Functional Unit
• Nephron is the Functional Unit
of the Kidney
• Around 1 million nephrons per
kidney in humans
• Receive ¼ of Cardiac Output:
about 1 L per minute
• Waste, ions, and fluid removed
from blood in Glomerulus
– Water and ions returned in
tubules
Image modified from "2611 Blood Flow in the Nephron" by OpenStax College
http://cnx.org/content/col11496/1.6/, Jun 19, 2013

6. Externship Goals
1) Develop Experience and Proficiency in a
variety of tissue processing and analysis
techniques
– Tissue Processing, Standard and Special Staining,
Immunohistochemistry Staining, Electron
Microscopy Techniques
2) Learn how the experimental method is put
into practice in a working lab
– Observe, question, and learn from lab mentors
– Develop and execute an original experiment

7. Acquisition, Grossing,
& Fixation of Tissue
• Tissue from various
sources:
– Autopsy/ Necropsy
– Biopsy
– Surgical Specimens
• Most common fixative
for paraffin embedding
is 10% Neutrally
Buffered Formalin
Image from http://www.newcomersupply.com/products/formalin-grossing-spill-pads

8. Processing, Embedding,
& Sectioning of Tissue
Processing
• Dehydration
• Clearing
• Infiltration
Embedding & Sectioning
• Tissue is surrounded by paraffin
• Tissue is exposed and then 3-5μm
slices are cut and put on slides
• Tissue is ready to bake and stain

9. Processing Errors &
Complications
Pre-Analytic Errors can
cause major and minor
issues in the ability to
analyze the tissue
• Fixative to tissue ratio
• Wrong orientation during
embedding
• Thickness of sections
• Folds in Sections
• Length of time between
removal of tissue from
body and fixation (delayed
fixation)
Delayed Fixation
• Tissue does not always go directly
from the patient or research
animal directly to fixative
• This post mortem damage can
make it more difficult to determine
the state of tissue at the time it
was removed

10. Autolysis
au·tol·y·sis (aw-tol'i-sis),
1. Enzymatic digestion of cells
(especially dead or degenerate)
by enzymes present within
them.
autolysis. (n.d.) Farlex Partner Medical Dictionary. (2012).
Retrieved November 29 2015 from http://medical-
dictionary.thefreedictionary.com/autolysis
Places in the kidney we
expect to see autolytic
damage
• Glomeruli
• Proximal Tubules
– Brush Border loss
– Nuclear changes
• All Tubules
– Flattened/Sloughed Cells
– Necrotic Cell Debris
– Lumen Distension

11. Experiment
Objectives
• Determine the point at which a delay in
fixation effects the quality of renal tissue
specimens.
• Determine the early signs of tissue damage
and necrosis.
• Look at the difference the storage
temperature make in the rate of tissue
damage.

12. Experiment
Overview
*Measured in hours post euthanasia
Fixation
Time*
0.5 1 2 4 24
RT (21C)
4C
Specimen Acquisition
Processing/
Sectioning/Staining
Analysis
0

13. Results:
Low Mag 20x
30 min 1 hr 2 hr 4 hr 24 hr
4C
RT
Baseline
*All Slides Stained with Periodic Acid-Schiff
Provides an overall look at how the architecture of the
tissue changed over time before focusing in on individual
structures.

14. Results:
Glomeruli at 60x
30 min 1 hr 2 hr 4 hr 24 hr
4C
RT
Baseline
*All Slides Stained with Periodic Acid-Schiff
C
B
Looking at structure of Capillary loops in the Glomerulus
(C) and the morphology of the Basement Membrane(B).

15. Results:
Glomeruli at 4C Over
First 2 hours
Baseline 30 min 1 hr 2 hrs
*All Slides Stained with Periodic Acid-Schiff
• No obvious changes in either the Capillary loops (C) or
Basement membrane(B) of tissue stored at 4C for up to
2 hours before fixation.
C
B

16. Results:
Glomeruli at 4C From 4
to 24 hours
*All Slides Stained with Periodic Acid-Schiff
• Changes in Basement membrane(B) visible in tissue
stored at 4C for 4 hours before fixation
• Obvious changes in Golmeruli at 24 hours of delay
Baseline 4 hrs 24 hrs
C
B

17. Results:
Glomeruli at Room Temp
Over First Hour
*All Slides Stained with Periodic Acid-Schiff
• Changes in Basement membrane (B) visible in tissue
stored for as little as 30 minutes at Room Temperature
Baseline 30 min 1 hr
C
B

18. Results:
Glomeruli at Room Temp
From 2 to 24 hours
*All Slides Stained with Periodic Acid-Schiff
• Obvious changes in Golmeruli of tissue stored at Room
Temperature for as little as 2 hours before fixation
Baseline 2 hrs 4 hrs 24 hrs
C
B

19. Results:
Tubules at 60x
30 min 1 hr 2 hr 4 hr 24 hr
4C
RT
Baseline
*All Slides Stained with Periodic Acid-Schiff
Looking at Brush Borders (B) and Nuclei (N) of Proximal
Tubules as well as looking for Necrotic cell debris in
Lumens (L) of both Proximal and Distal Tubules.
L
L
N
B

20. Results:
Tubules at 4C Over First
Hour
*All Slides Stained with Periodic Acid-Schiff
• No obvious changes in Tubules of Tissue stored at 4C
for up to 1 hour before fixation.
Baseline 30 min 1 hr
L
L
N
B

21. Results:
Tubules at 4C From 2 to
24 hours
*All Slides Stained with Periodic Acid-Schiff
• Changes in Brush border (B) visible in tissue stored at
4C for as little as 2 hours before fixation
• Loss of cytoplasmic volume (V) and difficulty resolving
cell walls at 4 hour delay in fixation
• Necrotic cell debris in lumens by 24 hours (D)
Baseline 2 hrs 4 hrs 24 hrs
B
DV

22. Results:
Tubules at Room Temp
Over First Hour
*All Slides Stained with Periodic Acid-Schiff
• Changes in Brush border (B) visible in tissue stored for
as little as 30 minutes at Room Temperature
• Loss of cytoplasmic volume (V) and difficulty resolving
cell walls at 1 hour delay in fixation
Baseline 30 min 1 hr
B
V

23. Results:
Tubules at Room Temp
From 2 to 24 hours
*All Slides Stained with Periodic Acid-Schiff
• Necrotic cell debris (D) in lumens of room temperature
stored tissue with as little as 2 hours delay in fixation
• Nuclear changes (N) visible in proximal tubules by 24
hours delay
Baseline 2 hrs 4 hrs 24 hrs
ND

24. Conclusions from
PAS Stained Slides
Based on these results I concluded the following:
• Glomeruli are much more resistant to post mortem
tissue necrosis than tubules
– Earliest sign of Glomerular damage is changes in the
Basement membrane.
– Capillary network loses its structure much later
• Earliest sign of Tubular damage is loss of cytoplasm
volume, followed by damage to brush border.
– Late sign of Tubular damage is change in nuclear
appearance of proximal tubule cells
• Tissue stored for up to two hours before fixation at
4C maintains structure at light microscopy levels

25. Answers… and
More Questions
• This determination of the time limit for storage
of renal tissue before fixation could guide
protocols for labs on how they handle their
specimens, both clinical and research
• It also raised another question: Tissue often also
requires Immunohistochemical analysis in
addition to light microscopy observations.
– Do these time cutoffs also apply to tissue used for IH
analysis? Will the antigen binding sites on the renal
proteins degrade faster than the overall morphology?

26. IH Procedure
Deparaffinize and
Rehydrate
Antigen Retrieval
Blocking
Primary Antibody
(Synaptopodin)
Secondary Antibody
(Texas Red)

27. IH Results: 4C
• At 4 hours of storage in 4C before fixation,
focal loss of fluorescence is visible
Baseline 30 hr 1 hr 2 hr 4 hr

28. IH Results: Room
Temp
• Focal loss of antigen binding for synaptopodin
visible in as little as 30 minutes delay at room
temperature before fixation
• Widespread loss of binding sites at a 4 hour
delay in fixation
Baseline 30 hr 1 hr 2 hr 4 hr

29. IH Conclusions
• For tissue stored at 4C, 4 hours is the first time
point I noted focal loss of fluorescence. It is also
when I started noticing changes to the basement
membrane with PAS stained slides.
• For Room Temp stored tissue, I first noted a focal
loss in fluorescence at 30 minutes, which was the
same point I started to see changes in the
basement membrane with the PAS stained slides.
• In order to state that IH analysis is reliable up to
the point that the tissue shows light microscopy
changes I would need to look at more antibodies.

30. References
Cleveland Clinic. (2015, September 23). Chronic Kidney Disease. Retrieved November 6,
2015, from Diseases and Conditions: https://my.clevelandclinic.org/health/
diseases_conditions/hic_chronic_kidney_disease
Kumar, V., Abbas, A. K., & Aster, J. C. (2013). Robbins Basic Pathology (9th ed.).
Philadelphia, PA: Elsevier Saunders.
National Diagnostics. (2011). Factors Affecting Fixation. Retrieved August 29, 2015, from
Histology: https://www.nationaldiagnostics.com/histology/article/factors-affecting-
fixation
Rolls, G. (2011, May 26). An Introduction to Specimen Processing. Retrieved September
28, 2015, from Leica Biosystems:
http://www.leicabiosystems.com/pathologyleaders/an-introduction-to-specimen-
processing/
The Jackson Laboratory. (2015). C57BL/6J. Retrieved November 6, 2015, from Mouse
Strain Datasheet: https://www.jax.org/strain/000664

31. Acknowledgement
s
• Dr. Chris Andry
• The Henderson Lab
– Dr. Joel Henderson
– Dr. Mostafa Belghasem
– James Stevenson
• Boston Medical Center Department of
Pathology
• Boston University Metropolitan College and
School of Graduate Medical Sciences