Interactive Lecture

1. Prof Mulazim Hussain Bukhari
MBBS,DCP,CHPE,Mphil,FCPS,PhD
HOD Pathology, UCMD, University of Lahore
Intracellular Accumulations

2. Intracellular Accumulations
•One of the manifestations of metabolic derangements in
cells is the intracellular accumulation of abnormal amounts
of various substances

3. 3 categories
Normal cellular component accumulated in excess:
Water, lipids, glycogen, carbohydrates
Abnormal substance:
Exogenous (mineral, infectious agents) or
endogenous (abnormal synthesis during metabolism)
Pigments:
Tattooing
INTRACELLULAR ACCUMULATIONS

4. EXCESS of NORMAL CELLULAR
COMPONENTS
 Fatty Change
 Cholesterol Accumulation
 Protein Accumulation
 Glycogen Accumulation

5. FATTY CHANGE
Fatty change is the
accumulation of
triglycerides within the
cytoplasm.

7. Atherosclerosis, Oil red - O

8. Liver and heart are the commonest organs for Fatty Change
FATTY CHANGE

9. CAUSES OF FATTY CHANGE

10. MORPHOLOGY
Intracellular accumulations of water or polysaccharides
(e.g., glycogen) may produce clear vacuoles, and it becomes
necessary to resort to special techniques to distinguish three
types of clear vacuoles.
 The identification of lipids requires the avoidance of fat
solvents commonly used in paraffin embedding for routine
hematoxylin and eosin stains.

11.  To identify the fat, it is necessary to prepare frozen tissue
sections of either fresh or aqueous formalin-fixed tissues.
 The sections may then be stained with Sudan IV or Oil Red-O,
 both of which impart an orange-red color to the contained lipids.
 The periodic acid-Schiff (PAS) reaction is commonly
employed to identify glycogen, although it is by no means
specific.
 When neither fat nor polysaccharide can be
demonstrated within a clear vacuole, it is presumed to
contain water or fluid with a low protein content.

12. CHOLESTROL ACCUMULATION

13. ATHEROSCLEROSIS
In atherosclerotic plaques, smooth muscle cells and
macrophages within the intimal layer of the aorta and
large arteries are filled with lipid vacuoles, made up of
cholesterol and cholesterol esters.
Such cells have a foamy appearance (FOAM CELLS),
and aggregates of them in the intima produce the yellow
cholesterol-laden atheromas characteristic of this
serious disorder.

14. Some of these fat-laden cells rupture, releasing lipids
into the extracellular space.
The extracellular cholesterol esters may crystallize in
the shape of long needles, producing quite distinctive
clefts in tissue sections.

18. Erruptive xanthomas

19. Xanthelasma

21. Niemann-Pick Disease

22. Niemann-Pick Disease

24. PROTEIN ACCUMULATION
Intracellular accumulations of proteins usually appear as
rounded, eosinophilic droplets, vacuoles, or aggregates in the
cytoplasm.
By electron microscopy, they can be amorphous, fibrillar, or
crystalline in appearance.
In some disorders, such as certain forms of amyloidosis,
abnormal proteins deposit primarily in the extracellular
space

30. Morphology
• Alzheimer's disease is characterised by loss of neurons and
synapses in the cerebral cortex and certain subcortical regions.
• This loss results in gross atrophy of the affected regions,
including degeneration in the temporal lobe, parietal
lobe,parts of the frontal cortex, cingulate gyrus and brainstem
nuclei like the locus coeruleus

32. GLYCOGEN ACCUMULTION
Glycogen masses appear as clear vacuoles within the
cytoplasm.
Best preserved in nonaqueous fixatives; for its localization,
tissues are best fixed in absolute alcohol.
Staining with Best’s carmine or the periodic acid schiff (PAS)
reaction imparts a rose-to-violet color to the glycogen,
diastase digestion of a parallel section before staining serves
as a further control by hydrolyzing the glycogen.
Example…..Diabetes Mellitis

35. LIPOFUSCIN
Insoluble pigment,
also known as
lipochrome and wear-
and-tear or aging
pigment.
Polymers of lipids and
phospholipids
complexes with
protein

36. Derived through lipid peroxidation of polyunsaturated
lipids of subcellular membranes.
Telltale sign of free radical injury and lipid peroxidation.
Appears as a yellow-brown, finely granular
intracytoplasmic, often perinuclear pigment
Prominent in the liver and heart of aging patients or
patients with severe malnutrition and cancer cachexia.
On electron microscopy, the granules are highly
electron dense, often have membranous structures in
their midst, and are usually in a perinuclear location.

37. ANTHRACOSIS
• Most common exogenous pigment is carbon or coal dust.
• When inhaled, it is picked up by macrophages within the
alveoli, transported through lymphatic channels to the regional
lymph nodes in the tracheobronchial region.
• Accumulations of this pigment blacken the tissues of the lungs
(anthracosis) and the involved lymph nodes.
• In coal miners, the aggregates of carbon dust may induce a
fibroblastic reaction or even emphysema and thus cause a
serious lung disease known as coal worker's pneumoconiosis.

39. MELANIN
Melanin, derived from the Greek (melas = black), is an
endogenous, non-hemoglobin-derived, brown-black
pigment formed when the enzyme tyrosinase catalyzes the
oxidation of tyrosine to dihydroxyphenylalanine in
melanocytes.

42. Clinical scenario
• A 20-year-old student, was brought to the emergency room l by friends,
with a abdominal pain. Questioned, he admitted to being a "fairly
heavy" drinker, and his friends confirmed this.
• At that time, he was noted to have a mild increase in abdominal girth
and hepatomegaly (enlarged liver); also mild elevation in his blood
levels of serum aspartate-aminotransferase (AST) and gamma-glutamyl-
transferase (GGT). Both of these enzymes are transaminases found in
liver cells that “leak” into the blood when liver cells are injured and
hence are indicators of liver cell injury.
• He was diagnosed with alcoholic steatosis.
• A year after this incident, he was killed in an automobile accident while
under the influence, and his body was autopsied, as required by
law. His case is reviewed in the following paragraphs.

43. WHAT LIPID TYPES FOUND IN CELLS CAN FORM
DEPOSITS?
• Lipids in abnormal deposits belong to the types of lipid normally found in the body:
• Lipids normally found in the body are:
• a) Triglycerides (TGs) are the main lipids of fat cells.
• • TGs accumulation in liver cells is very frequent and is called steatosis.
• • In liver cells TGs form droplets that fuse into one, single large droplet, not
bound by a membrane.
• b) Free fatty acids (FFA) form small, uniform droplets that do not fuse.
• c) Cholesterol and its esters are components of cell membranes.
• • Accumulate most frequently in atherosclerotic arteries.
• • Three types of cells: macrophages, endothelium, smooth muscle cells.
• d) Phospholipids are components of cell membranes.
• • Form myelin figures which are abnormal membranous structures within cells
or in the tissue spaces.

44. HOW ARE LIPIDS VISUALIZED IN THE MICROSCOPE?

45. HOW ARE LIPIDS VISUALIZED IN THE MICROSCOPE?
• Lipids are microscopically elusive.
• They are lost forever during paraffin embedding, which requires
soaking the tissues in fat solvents (ethanol, xylene).
• Therefore to search for fats, unprocessed tissue must be cut to
frozen state using a special type of microtome and then treated with
a fat stain ( oil red O, Sudan III, Sudan IV, Sudan Black).
• All these are dyes are specific for fats but not for the different types
of fats.
• They can rule out proteins and water-soluble materials, but we do
not know which type of fat is present: triglyceride or cholesterol
ester.
• Definitive identification must rest on chemical analysis.

47. Steatosis is the accumulation of Triglycerides (TGs).
• TG storage requires a supply of free fatty acids (FFA) from other
tissues. FFAs from adipose tissue or from ingested fat are normally
transported into hepatocytes (liver cells).
• In the liver FFAs are processed into three ways:
– FFAs can be burned as fuel to produce ATP (by mitochondria).
• (Liver, myocardium, skeletal muscle and kidney derive their energy from the
oxidation of FFA.)
– FFAs can combine with glycerol to give rise to Triglycerides.
– FFAs can combine with glycerol + choline + phosphate giving rise to
phospholipids.

48. Lipid
• To be exported out of the liver cell TGs and phospholipids
need to be assembled in a particle called lipoprotein.
• Lipoprotein is a globule filled with triglycerides, free
cholesterol and cholesteryl esters, wrapped in a membrane of
phospholipids built from apoproteins.
• The amount of FFS and TGs in the liver depends on the balance
between the processes of delivery and removal, and without
the apoprotein, the lipoprotein cannot be built and its lipid
components cannot be exported from the cell.

49. Steatosis due to toxic effects of exogenous agents.
• Toxic effects on protein synthesis also result in steatosis.
• Various toxic agents can induce steatosis by depressing protein
synthesis.
• As in malnutrition, the principal effects of blocking protein
manufacture are decreased export of lipoproteins from the
liver and consequent liver steatosis.

50. Toxic agents such as
– ethionine,
– puromycin,
– tetracycline,
– toxin of the highly piosonous mushroom Amanita phalloides,
amanitin.)
– Intoxication with carbon tetrachloride (CCl4), a common industrial
and household solvent, blocks protein synthesis by causing free
radical damage to the endoplasmic reticulum membranes.

51. Consequences of steotosis

52. Type of Hepatic injury
• 1. Simple liver steatosis is a mild, asymptomatic disease and is reversable in
about 10 days or 1 to 6 wks.
– Clinical tests of liver function show little change.
• 2. Alcholoic Hepatitis results from long-term excessive use of
alcohol, which eventually overtaxes the liver's ability to detoxify alcohol, and cell
death will occur. When liver cells die, the liver displays an inflammatory reaction
• 3. Cirrhosis: Prolonged inflammation eventually causes scarring by
proliferation of connective tissue,or cirrhosis. Loss of functional parenchymal
hepatocytes increases the stress on surviving cells, which in turn leads to more
cell death, more scarring,
– Daily intake of more than 60g/d alcohol for 10-20 years leads to liver cirrhosis. Only 10-
15% of alcoholics, however, develop liver cirrhosis. Cirrhosis is an irreversible process, it
progresses slowly toward death.

53. Case 2
• A 32-year-old woman, Three months ago her brother was found to
be carrying the genes for hereditary hemochromatosis in a
national health screen.
• His ferritin level was 2499 µg/L (10X the normal range: 40-200).
• When she heard this news she was concerned about the possibility
that she too might have this condition, she requested testing.
• Her doctor agreed and ordered the iron tests and gene tests, and
found that she too is homozygous for the C282Y mutation; but her
ferritin level was 24 µg/L.

54. WHAT IS HEREDITARY HEMOCHROMATOSIS?
• hereditary hemochromatosis is a disorder due to
deposition of hemosiderin in the parenchymal cells,
principally in liver, heart, pituitary gland, and
pancreas).
• Deposition of hemosiderin causes tissue damage and
dysfunction.
• Hemochromatosis is a condition of "iron overload

55. PICTURE OF A LIVER OF A PATIENT WITH
HEREDITARY HEMOCHROMATOSIS: GROSS VIEW

56. Herediatry Spherocytosis

57. WHAT IS THE PRINCIPAL IRON-CONTAINING
PIGMENT INCLUSION?
• The two principal forms of iron-containing body pigments are
ferritin and hemosiderin.
• The rusty color of these two closely related intracellular pigments
reflects their vital function: iron storage.
• The total body iron content is about 6 gram in men and 4 gram in
women with about 80% of the total body iron is in functional state,
found in hemoglobin, myoglobin, and iron-containing enzymes such
as catalase and the cytochromes.
• The storage pool (20% of the total body iron) is represented by
hemosiderin and ferritin in organs such as liver, spleen and bone
marrow.

58. Iron in the body
• Free iron is extremely toxic because it induces free radical
formation and the pool of storage is tightly bound to either
ferritin or hemosiderin which are the normal iron storage
products.
• There is no regulated pathway of iron excretion which is limited
to 1-2 mg per day lost by shedding of mucosal and skin epithelial
cells. Iron balance therefore is maintained largely by regulating
the absorbtion of dietary iron.

59. Iron forms
• Ferritin is essentially a protein, apoferritin, complexed with iron. Ferritin is
always found within cells (as opposed to circulating in blood) and it is located on
cytosol.
• Ferritin is found in all tissues, but particularly in liver, spleen and bone marrow.
• Another molecule, transferrin, is a serum glucoprotein that serves as a carrier
for iron.
– Transferrin acts as a shuttle between the sources of iron and cells that need it. Iron
absorbed from the gut passes into the blood where it is taken up by transferrin and is
carried to the storage organs such as liver, spleen and hematopoietic bone marrow.
• Hemosiderin is a complex of degraded protein shells of ferritin, denaturated
ferritin, and other material within lysosomes. It's common to find hemosiderin in
macrophages that are cleaning up a localized hemorrhage, for example.

60. Difference between two form
• In fact the distinction between ferritin and hemosiderin is
academic, because both have the same significance: stored
iron.
• Under normal conditions, small amounts of hemosiderin are
always visible in macrophages of the bone marrow, spleen and
liver, all of which are actively engaged in red cell breakdown.
Hemosiderin can be visualized in tissues by the Perl’s staining.
•

62. Case III
• A retired Army officer, age 87, lives in an apartment in an assisted-
living facility. At the age of 12, in 1929, he had rheumatic fever.
• He has good medical care by his physician for a variety of
conditions.
• On his last physical exam the doctor noted that he has a heart
murmur, and referred Harry to a cardiologist.
• Harry's left ventricle is somewhat enlarged, he has a crescendo-
decrescendo systolic murmur of aortic stenosis, which is heard at
the second intercostal space in the right upper sternal border;
ultrasound imaging shows calcification of the aortic valve.

63. Diagnosis: Senile Degenerative Calcific Aortic
Stenosis.

64. PATHOLOGIC CALCIFICATION
• Abnormal tissue deposition of calcium salts, together with
smaller amounts of iron, magnesium, and other mineral salts
• There are two forms of pathologic calcification.
I. When the deposition occurs locally in dying tissues, it is
known as dystrophic calcification; it occurs despite
normal serum levels of calcium and in the absence of
derangements in calcium metabolism.
II.The deposition of calcium salts in otherwise normal tissues
is known as metastatic calcification, and it almost always
results from hypercalcemia secondary to some disturbance
in calcium metabolism.

65. DYSTROPHIC CALCIFICATION
• Macroscopic deposition of Ca salts in dead or degenerated
tissue(injured tissues).
• It represents extracellular deposition of calcium from circulation or
interstitial fluid.
• Its often visible to naked eye and ranges from gritty sand like grains
to firm rock hard material
• It requires persistence of necrotic tissue.

66. Dystrophic calcification
• Caseous necrosis in tuberculosis is the most commen site and
has no functional consequences.
• Dystrophic calcification may also occur in crucial locations such
as in mitral or aortic valves after rheumatic fever.
• In such instances calcification leads to impeded blood flow
because it produces inflexible valve leaflets and narrowed
valve orifices(mitral and aortic stenosis).

70. PSAMMOMA BODIES
Single necrotic cells may constitute seed crystals that
become encrusted by the mineral deposits.
The progressive acquisition of outer layers may create
lamellated configurations, called psammoma bodies.

72. Dystrophic calcification
• in artherosclerosis leads to narrowing of vessels. It also plays a
role in diagnostic radiography.
• Mammography is based on detection of calcification in breast
cancer.
• Diagnosis of congenital toxoplasmosis, an infection involving
the CNS is suggested by visualization of calcification in infant
brain.

73. WHAT IS CALCIFICATION?
• Calcification is the process of deposition of calcium salts,
especially in the form of hydroxyapatite [Ca10 (PO4)6 • (OH)2],
or for convenience, just "apatite."
• Calcium is ubiquitous in tissue, a vital component for many
functions, not just structural support.
• As with other nutrients, it's carried in the blood, and the blood
calcium level is a fair indicator of its availability.

74. CAN CALCIFICATION BE A NORMAL PROCESS?
• Yes. The deposition of apatite is a normal process in the
formation and maintenance of bone. Another example is the
accumulation of small calcifications in the pineal body of the
brain. These have no known function, nor clinical importance,
but they're always present.
• The main structural difference between bone and pathologic
calcifications is the nature of the calcified substrate:
– in bone, where calcification is essential to function, the substrate is
osteoid, a specific organic matrix characteristic of that tissue.

75. WHEN IS CALCIFICATION PATHOLOGICAL?
• Broadly speaking, pathological calcification means calcification
in the wrong place.
• Dead tissues are favorite targets for calcification but even live
cells and the extracellular matrix can calcify.
• The chemical form of pathologic calcification and bone is the
same, hydroxyapatite (or apatite), i.e. basic calcium phosphate
Ca10(PO4)6 · OH2

76. Two main types of calcium deposits
• 1. When the calcium phosphate deposits occur locally in
dying tissues, this circumstance is known as dystrophic
calcification.
• 2. When the calcium phosphate deposits occur body-wide in
otherwise normal tissues, it is known as metastatic
calcification

77. Examples of dystrophic calcification:
• (a) Calcification of necrotic tissue.
• (b) Calcification of tuberculous granuloma.
• (c) Calcification of the aortic valve.
• (d) Calcification of atherosclerotic arteries
• (e) Extreme Dystrophic Calcification: Lithopedion
• (f) Psammoma Bodies

78. The “Law of Askanazy" and metastatic
calcification:
• The preferred location of calcium metastases are gastric mucosa,
kidneys and lungs, cornea, systemic arteries and pulmonary veins
• The systemic arteries and pulmonary veins calcify because they
carry arterial blood in which carbonate is at its lowest; systemic
veins and pulmonary arteries are relatively spared because their
blood is more acid, thanks to dissolved carbon dioxide.
• As is obvious, in all these situations, the tendency is to drive the
system to an alkaline condition, favoring precipitation of calcium
salts.

79. Hypercalcemia has many causes:
• In hospitals it is seen most often in patients whose skeleton is
rapidly destroyed by metastases of malignant tumor.
• Another classic but less common cause is hyperparathyroidism
which is due to a functional tumor of the parathyroid glands.
• A third cause is excess intake of vitamin D.
• High plasma phosphate concentrations also lead to metastatic
calcification, because phosphate rises as a result of chronic
renal failure.

80. Findings
• Autopsy findings in cases of severe hypercalcemia are striking:
• Calcified tissues appear chalky.
• The gastric mucosa can appear strangely white, and feels like a
rasp, grating under the knife.
• The lungs become similar to a fine bathing sponge, because
alveolar walls become very stiff with calcium deposits
• Microscopically, mitochondria of normal cells are the first
target of hypercalcemia.

82. Self learning

83. 1. Which of the following is the correct technique for
identifying intracellular accumulation of triglyceride in
tissue sections?
• Sudan IV staining of paraffin sections
• H&E
• PAS
• Oil red O staining of frozen sections
• Prussian blue

84. 2. A patient, whose liver biopsy shows
alcoholic hepatitis, and who continues to
abuse alcohol, may develop liver cirrhosis.
• a. True
• b. False

85. 3. Donna, a 65-year-old woman presents with enlarged liver and chronic
elevations in serum aspartate aminotransferase (AST), and alanine
aminotransferase (ALT) to 3-4 fold the upper limits of normal. She has a
history of alcohol abuse but does not take any medications. Her complete
blood count, viral serologies, autoimmune markers, ferritin and the
remainder of her liver function tests are all negative or normal. Liver biopsy
is performed and reveals an intracellular accumulation of:
• Cholesterol
• Water
• Glycogen
• Hemosiderin
• Triglycerides

86. 4. Which of the following best explains the pathogenesis
of Donna’s liver disease?
• Increased transport of lipoproteins from the liver
• Decreased fatty acid supply from adipose tissue
• Increased β-oxidation of fatty acids in mitochondria
• Fatty acid biosynthesis stimulated by the excess NADH over
NAD+
• Increased protein intake
•

87. 5. All of the following statements are correct concerning iron metabolism and iron
overload except:
• Ferritin is identifiable by electron microscopy
• “Black eye” is an example of local iron overload.
• Primary hemochromatosis is a condition of generalized iron
overload secondary to massive blood transfusions.
• Hemosiderin can be visualized in tissues by the Perl’s staining.
• Body iron is stored in the form of ferritin or hemosiderin

88. 6. Which of the following is an example of
metastatic calcification?
• Calcific aortic stenosis
• Psammoma bodies
• Calcification of necrotic cells of a myocardial infarction
• Nephrocalcinosis
• Lithopedion

89. Mucopolysaccharidoses - 1

90. Waterhouse-Friderichsen Syndrome

91. Wegener’s granulomatosis

92. E N D