Technical Faults in Histopathology Lab

Fixation Tissue carryover

1. The major problems related to fixation are delayed or  Small pieces or fragments of tissue may be carried from
incomplete fixation 1 tissue to the next at the embedding table, resulting in
2. Autolysis is caused by delayed fixation cross-contamination
Prevention:
In H& E section, the tissue may show: 1. Carefully clean forceps used at the embedding table between
specimens
 Loss or total disappearance of nuclear chromatin.
2. Open only the cassette with the tissue to be embedded
 Some cells may disappear (intestinal mucosa), shrink and leave
artifactual space around.
Tissue not embedded at the same level
Prevention:
If the tissue is not properly flattened by pressing it down
- Add fixative to the specimen as soon as possible
uniformly when it is placed in the embedding mold, or multiple
tissues to be placed in the same mold are embedded at different
Incomplete fixation
levels
• The cells characterized by smudge nuclei (indistinct nuclear
Prevention
pattern), nuclear bubbling
1. Press the tissue uniformly
Prevention:
2. Keep the paraffin molten enough to get all pieces embedded at
1. Prolong fixation time
the same level.
2. Thin gross section
3. Work very fast when embedding multiple pieces
3. Fresh formalin solution
4. Cassette should not be tightly backed
5. Agitation of cassettes in the fixative
Troubleshooting in microtomy
Processing
Crooked ribbons
 Most problem encountered in processing is related to either
 Result when the horizontal edges (top and bottom) of the
over processing or under processing
block are not parallel.
Over dehydration  If the lower block edge is not parallel to the knife edge.
Prevention:
 Due to excessive dehydration, which results in micro-chatter
1. The upper and lower edge should be parallel
around the edges of the tissue on H & E Stain.
2. The lower block edge is parallel to the knife edge
Prevention:
3. No problem in the blade edge
- Small biopsies processed separately
- Shorten the dehydration time
Block face unevenly sectioned
Poor processing:  Occur when the block holder is not parallel to the blade.
• Due to improper dehydration (water in tissue), impaired  One side of the block is exhausted while attempting to get a
clearing, clearing agent in paraffin, too much heat during complete section of the block face
processing Prevention:
Prevention: 1. Ensure at the beginning of the sectioning that the block holder
- No cassettes condensation is adjusted so that the block face and the blade are perfectly
- Absolute alcohol is fresh parallel.
- Free of Water Fluid are changed according to the schedule
Heat is used only for paraffin & E Stain Thick section
Prevention:
1. Adjust the thickness

Embedding and specimen orientation Vertical scratches

 Refers to casting or blocking (paraffin blocking)  Caused by defect in the blade edge, calcium, bone or hard
Hints: material in the specimen
 Specimen orientation Prevention:
 Proper pressure force applied 1. Ensure at the beginning of the sectioning that the block holder
 To the entire specimen during orientation and initial is adjusted so that the block face and the blade are perfectly
solidification to obtain flat tissue parallel.
 Technical Faults in Histopathology Lab

Holes in the section Pale nuclear staining

 Occur when block is faced too aggressively.  Slide not exposed to the hematoxylin long enough
 The specimen is either excessively dehydrated or  Exhausted (over oxidized or depleted) hematoxylin
improperly processed.  Over differentiation
Prevention: Prevention:
1. Ensure to chill the block with ice before cutting and discard 1. Leave the slide longer
ribbons until the hole disappear 2. Use fresh hematoxylin • Time the differentiation • •
2. Facing the block less aggressively with smaller micrometer
advances of the block for each section removed Dark nuclear staining
 Slide exposed to the hematoxylin too long
Failure of ribbon to form  Section is too thick
 Commonly caused by dull blade.  Differentiation step is too short
 Result from too hard paraffin or too much blade tilt Prevention:
Prevention: 1. Thin sections
1. Paraffin with lower melting point 2. Decrease hematoxylin exposure
2. Decrease blade tilt 3. Increase time for differentiation
3. Change room temperature •
Red or red-brown Nuclei
Wash boarding or Undulation in the section  If the nuclei are stained red or reddish brown instead of blue,
 Commonly occurs in very hard tissue such as uterus or in either the hematoxylin is breaking down or the bluing step
over fixed tissue. was not properly done
 It is the macroscopic type of chatter commonly caused by Prevention:
loose clamping of blade or block. 1. New hematoxylin
Prevention: 2. Proper bluing
1. Proper clamping of blade and block
2. Ensure the block holder shaft is not over extended Blue black precipitate
3. Ensure the microtome is in good working order  Hematoxylin precipitate
4. Decrease the blade tilt Prevention:
1. Filter the hematoxylin
Chatter or microscopic Vibration
 Commonly caused by over dehydration or lack of Pale Cytoplasmic staining
moisture in the tissue.  Eosin pH is over 5
 It could also result from dull blade or too much blade tilt  High pH may result from carryover of the bluing agent.
which cause the section to be scrabed rather than cut.  The section may be too thin
 Cutting too rapidly.  Left long in the dehydration
Prevention: Prevention:
1. Proper processing 1. Check Eosin pH
2. Restore moisture by facing the block down on an ice tray 3. 2. Completely remove bluing agent before transferring the slides to
Decrease the blade tilt eosin to allow stained slides to stand in the lower concentration of
4. Decrease cutting speed: one wheel per second is considered alcohols after the eosin.
reasonable speed. 3. The more water in the alcohol, the more eosin that will be
removed

Dark Cytoplasmic staining

H & E staining
Incomplete Deparaffinization
 White spots may be seen in tissue sections after the
deparaffinization step.
 Usually caused by water left in the tissue
 Incomplete drying
Prevention:
1. Dry section properly before beginning deparaffinization
2. Change fluids according to the schedule
3. If the slides have been stained, decolorize and restain
 If the cytoplasm is overstained and the differentiation is poor,
the contrast between the nucleus and the cytoplasm is lost
Prevention:
1. Avoid overconcentration of eosin
2. Dilute eosin solution
3. Do not leave sections in eosin for long
4. Allow enough time in dehydration solution, specially 70% alcohol,
to allow good eosin differentiation
5. Check section for proper thickness
Dark basophilic staining of nuclei and cytoplasm, especially
around tissue edges
 Laser and electrocautery techniques denature
macromolecules and produce heat artifact, generally marked
by dark basophilic staining in nuclei and cytoplasm
Prevention:
1. Nothing to be done
Technical Faults in Histopathology Lab
Oogenesis: refers to the process of oocyte
development and maturation from primary growth
oocyte to hydrated oocyte.
Primary growth oocyte stage includes the Chromatin
nucleolar and the Perinucleolar stages.
Chromatin nucleolar oocytes:
have a big nucleus that may contain several small
nucleoli, although often there is an outstanding one.
The scant cytoplasm is basophilic and homogeneous
Perinucleolar oocytes:
Nucleus contains several peripheral nucleoli, located
around the nuclear membrane. The cytoplasm
gradually
loses its basophilia
Secondary growth oocyte stages start with the
Cortical alveoli stage and continues with the
Vitellogenic stages.
Before the formation of the Cortical alveoli in some
species small lipid droplets can already be detected on
the perinuclear ooplasm.
Cortical alveoli stage
Oocytes show granular and more acidophilic
cytoplasm. The highly acidophilic zona radiata
become apparent.
1st Vitellogenic stage
Appearance of small yolk granules on the periphery of
the cytoplasm.
The cortical alveoli have a greater distribution than in
the previous stage and the lipid droplets, when
present, increase greatly in size.
2nd Vitellogenic stage
Increase in number, size, and distribution of the yolk
granules, which occupy virtually all the cytoplasm.
3rd Vitellogenic stage
Show much thicker yolk granules and lipid
droplets. The oocyte maturation process is included
in the oocyte
secondary growth phase although some authors
Gametogenesis: hich diploid germinal
cells undergo meitoic division and differentiation to
form mature haploid gametes.
Maturation: ends with the migration of the
nucleus towards the animal pole
its breakdown and a further rapid size increase of the
oocyte caused by the uptake of fluid
Ovulation: occurs when the follicle breaks after hydration.
The remaining follicles are then called postovulatory
follicles (POFs).
The postovulatory follicles appear as structures folded
over onto themselves and degenerate through several
stages
This mechanism is considered to differ from the atretic
process
Spermatogenesis
Germinal epithelium: normally composed of
spermatocytes
that are formed when a single clone of primary
spermatogonia is enclosed by Sertoli cells.
The germ cells develop synchronously inside these cysts.
At the end of the process, the cysts open and the
spermatozoa are released into the lobular lumen.
Spermatocysts can open and release developing germ
cells into the lobular lumen before they become
spermatozoa.
Primary spermatogonia: appear individually or
in small groups, having a major round
nucleus which can contain one or few nucleoli.
Secondary spermatogonia: greater in number and
always enclosed into a cyst surrounded by Sertoli cells.
The nucleus may still contain nucleolus, but it is smaller
than in the previous stage.
Primary spermatocytes: nucleus varies as the cell
proceeds through the prophase of the meiosis, being the
most easily identifiable phase the pachytene stage,
marked by the synaptonemal complexes
Secondary spermatocytes: smaller nuclear size, are very
difficult to observe because the time between the first
and second division is short.
Spermatids: condensation of the chromatin, which
implies a smaller size of the nucleus. The cytoplasm also
reduces considerably.
The flagellum starts to growth
Spermatozoa: have a more or less rounded and small head
and the flagellum
Wallace & Selman (1981) and West (1990)
 Technical Faults in Histopathology Lab
GSI
 (SFSM) individuals with gonads close to spawning
condition occurred in the same months that GSI was
highest
Sex Ratio
 Fish sex ratio was tested monthly and annually for statistical
differences (p < 0.05) using chi-square (Χ2)
 213 were females (39.4%) and 327 (60.6%) were males with
these latter. Males were more abundant in the total sample
(Χ2 = 24.0666, p < 0.05, df = 11), which produced a male
biased sex ratio (1.6 males: 1.0 females). Males were more
abundant in February (Χ2 = 5.4615, p < 0.05, df = 1) and
August (Χ2 = 6.4606, p < 0.05, df = 1)
 Sex ratios above 1:1 for majority of months along year make
us to opinion in a paired spawning behavior, also due the
spawning aggregation in off shore reefs where traps are
setting.
 Proportion of males to females remained generally constant
throughout the sampling period, apart from minor
fluctuations at times when sampling was particularly poor.
For the total of all fish examined during this study, the ratio
was very close to 1:1 (0.49 females: 0.51 males).
 Sex ratio for S. lemuru in WA coastal waters was close to
unity.
 Besides, maturation, and subsequently growth, usually
differs among males and females due to the different
physiological implications that this process has in both
sexes,
 Stock assessment generally assume a sex ratio of 1:1. Often
skewed within and between cohorts and spawning
aggregations
 Causes for skewed sex ratios have been suggested to stem
from differences in size and age-linked natural mortality
between the sexes
 Changes in sex ratios can affect stock dynamics and
reproductive success in many ways. Fertilization success of
cod was shown to depend on male abundance where
proportion of fertilized eggs declined with reduction in
number of spawning males per female
 Population growth declines when population size falls below
a threshold value
 Differences in size or behavior, where one sex is more active
than the other, may also lead to skewed estimates of sex
ratios,
 Growth, maturation and mortality are known to be sexually
dimorphic in many species and this can severely impact the
perceptions of the stock status.
 Depende rin sya sa location “males, generally are more
abundant in reef areas around islands while females are
more abundant in coastal areas due to their spawning
behavior”
Size at first Maturity
L50
 Adhering to minimum fishing size rules for species
with different body forms
 Minimum landing sizes considered which sex had the
greater length, because of the need to allow
reproductive activity.
 When L50 was estimated by using relative frequency
of reproductive animals identified through testes or
ovarian morphology,
 Estimates of the size at first maturity (L50) were
made by visual identification of gonadal development
and through the gonadosomatic index (GSI). L50 was
estimated as 12.5 cm for males and 13.0 cm for females
when applied visual identification of gonads
development. L50 estimates increased to 13.05 than
females
 L50 is the average size of an age-group when it starts
to reproduce for the first time
 Estimated for L50, the shift in the growth rate may
represent a process of adjustment of physiological
parameters for the incoming sexual maturation.
 Sardinella lemuru reaches 50% sexual maturity at
between 140- and 150-mm FL.
 Maturity ogive is used to determine the proportion of
individuals that are mature or immature at certain age or
size.
 Size at maturity are key parameters in fisheries stock
assessment and can vary not only between cohorts but
also between sexes.
 Proportion of fish that is mature at a certain length
 Represents the length at which 50% of the fish are
mature
 Collected on a length-stratified basis can be used
directly without causing bias because within each length
class the individuals were randomly sampled.
 The estimation of maturity ogives for each sex
separately shows, in most of the species, that males
generally mature at younger ages than females
 Validating macroscopic data used for maturity ogive
estimation is always recommended.
 Mistake obviously overrates the proportion of
mature individuals compared with the true
population, and consequently leads to an
overestimation of the spawning stock biomass.
 Technical Faults in Histopathology Lab
Additional Info
1. SFSM
 The maturity of the males could also not be determined
because of insufficient sample size and range of
sampling length. (pdf1126)
 A critical period of fish life is encountered when sexual
maturation (L50) is attained and when fish must
allocate resources for survival, growth, and
reproduction
 The length–frequency sample and the status of the
population.
 Two major problems were involved: (1) how big the GSI
should be to consider the ovary as engaged in
reproductive activities
 This recognition is usually visual, and subjective
descriptions of macroscopic aspects of ovaries and
testes at different maturation stages
 Size at first sexual maturity (L50) was estimated from a
logistic curve based on the relative frequency of adults
in each length class (SL), according to the formula Mf =
exp [a+(b)*SL)/(1+exp (a+ (b)*SL)], where Mf is the
fraction of adult specimens. All individuals in
vitellogenesis were considered as adults.
 size at first sexual maturity for snappers corresponds
approximately to 50% of the maximum size of the
species.
Histology
 Simultaneous presence of post ovulatory follicles, yellow
bodies and hydrated oocytes in ripe spawning females
 Microscopic maturity staging is a useful tool to validate
the macroscopic maturity staging and to describe gonad
developmental stages
 Give a more reliable estimate of the maturity ogive as it
avoids misinterpretation, microscopic maturity staging is
considered as ground truth for the determination of
staging errors.
\
Morphology
 Visual assessment of the
developmental stage was made based on the
macroscopic appearance of the gonads.
 Gonad morphology is the most common and direct
index to determine gonad developmental stage
 Provide a more accurate analysis of the reproductive
characteristics and the annual reproductive cycle of the
species.
 Macroscopic inspection is based on alterations in ovary
size and appearance, whereas histological methods
evaluate changes in oocyte stages at cellular level.
 Macroscopical judgement does not always mirror the
actual gonadal status as some features may not be
discernable with the naked eye and consequently
misinterpreted.
 Validate a macroscopic observation
 Needs to be validated by the means of a more accurate
method.
Significance
 Significant to study the reproductive biology of fish
useful in the prediction of spawning grounds, spawning
seasons and for exploitation of fishable stocks.
 Knowledge about spawning habits and maturation
cycle is necessary to know the periodic replenishment
of fishable stock.
 Spawning season gives an idea about peak of maturity
in fishes.
 Fecundity is regulated in response to environmental
conditions, food availability nutrition, predation, and
energetic resources to reproductive success
 Propose management and conservation strategies for
this endemic species.
 Overfishing activities of large-scale fishing industries
pose a big threat to the breeding season of the small
pelagic fishes and its body shape is a great factor for
their survival and ability to migrate.
 Successful tool in relating the shape of
S. lemuru to the overfishing activities.
 Overfishing is most prevalent in small
pelagic fishes such as S. lemuru)
because they are concentrated into the
shallow areas of the sea where they
can be easily captured
 Small pelagic fishes lack the necessary
body-shape for them
to be able to migrate.
 Incapable of migrating into the deeper
portion of the sea.
 Technical Faults in Histopathology Lab

 Tend to be concentrated to the areas where they can be

easily caught.
 Fishing too much leads to the degradation of the fish
population.
 In the Philippines, during the months of November until
March is the spawning period for the S. lemuru.
 Body shape of the pelagic fishes is a great factor for their
survival and ability to migrate.
 This study aims to relate the body shape of S. lemuru to its
survival and the threats of overfishing activities
 Despite the abundance of aquatic resources, the over
fishing activities have always been a threat and factor of
decreasing the populations of pelagic fishes, including S.
lemuru.
 The duration of peak spawning was relatively short and
variable between years.
 Monitoring of catches should continue so that a longer
time series of biological information can be gathered.
 Spawning season may be relatively short, being restricted
to the January-March period.
 Relative fecundity (the number of eggs per gram of
ovary-free body weight) was also calculated
 Assuming that increases in water temperature provide the
stimulus to these internal physiological changes in gonad
activity, the apparent shift in period of peak spawning
between years suggests interannual variability in the
pattern of increases in water temperature during summer.
 If a rise in water temperature occurs at the beginning of
the spawning season, an early stimulus for gonad
development could lead to an ‘early’ spawning.
 Although the duration of the reproductive period was two
to three months, the peak period was quite short.
 Reproductive ecology of fish populations is essential for an
effective stock assessment and fisheries management