Pigments and accumulations. Necrosis v. Apoptosis Decreased ATP production Increased mitochondrial permeability Leakage of Ca++ Ca-activated hydrolases.

Pigments and accumulations

Necrosis v. Apoptosis• Decreased ATP production• Increased mitochondrial

permeability• Leakage of Ca++ • Ca-activated hydrolases

degrade lipid, protein, RNA, DNA

• Denatured nucleic acid and protein may be large or small

• After initial “blebbing” of ER or plasma membranes, membranes burst

• ATP depletion initiates necrosis

• Inflammation elicited

• Bax/Bak channels open• Increased mitochondrial

permeability • Leakage of CytC• CytC/Apaf-1-activated

caspases cascade to activate DNase

• Condensed nucleosomal DNA is cut into 200 bp fragments

• After initial “blebbing,” condensed apoptotic bodies form

• ATP required for apoptotic enzyme activity

• No inflammation

Hemoglobin

Oxyhemoglobin

• Oxygen-bound hemoglobin, Hb or HbO2, is red• Mucous membranes and nail beds look pink• Lack of Hb is called anemia

Too few erythrocytes• Hemmorhage or i.v.cell-free fluids (Ringer’s)• Quantified as hematocrit (packed cell volume)

Erythrocytes are hypochromic• Dietary iron deficiency

Anemic tissues are pale; less red, more colorless

Hypochromic anemia

Deoxyhemoglobin• Reduced hemoglobin, HHb is dark purple-red• Mucous membranes and nail beds look blue (cyan)• Presence of HHb is called cyanosis

Poor systemic circulation due to shock, heart disease Poor oxygenation of Hb due to low oxygen tension,

pneumonia, COPD

• Acrocyanosis is limited to distal extremities (acro = extreme) Cold-induced vasular constriction Reynaud’s syndrome

• Primary may be instigated by extreme temperature or emotional stress

• Secondary to other disorders, CREST syndrome– calcinosis, Raynaud's phenomenon, esophageal dysmotility,

sclerodactyly, and telangiectasia

Pallor and cyanosis

Methemoglobin• Ferrous (oxidized) hemoglobin, Hb(III), is brown• Mucous membranes and nail beds look brown or darkly

cyanotic with chocolate-colored arterial blood• Cyanosis absent cardiopulmonary symptoms caused by

elevated circulating Hb(III) is called methemoglobinemia Hb(III) is normally <2% total; normally produced Hb(III) reduced to HHb with oxidation of NADH by one of three

isotypes of cytochrome b5 reductase (cytb5r, EC 1.6.2.2) Hb(III) has altered conformation and does not bind O2 Symptoms appear at >30% total hemoglobin Death occurs at around 70%

• Hines bodies within erythrocyes consist of precipitated Hb(III)

• Scabs (and old meat) look brown due to oxidized Hb(III)

Methemoglobinemia• Certain drugs and nitrites oxidize HHb to Hb(III)

antimalarials (chloroquine, primaquine), topical anesthetics (benzocaine, lidocaine, prilocaine), inhaled nitric oxide, sulfonamides, etc.

• Hereditary Hb mutations that stabilize iron in ferric state• Hereditary cytochrome b5 reductase deficiency

Type-I deficiency is limited to red cell isoform Type-II affects all isoforms in all tissues and presents as severe

encephalopathy, microcephaly, generalized dystonia, movement disorders and mild cyanosis

• Toxic methemoglobinemia is treated with low dose of methylene blue NADPH-dependent cyb5r (slow) is activated by methylene blue

• Blue dye reduces NADP+ to NADPH• Enzyme reduces Hb(III) to HHb with oxidation of NADPH to

NADP+

Signs of methemoglobinemia

Carboxyhemoglobin

• Carbon monoxide-bound hemoglobin, COHb, is bright red

• Mucous membranes and nail beds look cherry red, along with veinous blood

• COHb does not transport oxygen CO is essentially permanently bound to the heme

• 200-fold greater binding affinity than oxygen

Chronic exposure leades to hypoxia Acute exposure may be deadly

• Symptoms start at 20% saturation• Death may occur at 70% saturation

• Hemoglobinemia RBCs break apart releasing Hb into plasma Plasma looks pink

• Hematuria Whole RBCs in urine

• Hemoglobinurea Lysed RBCs and Hb in urine Hb is nephrotoxic Affected tubules show reddish-orange crystals

of Hb upon autopsy

• Hb imbibition (post-mortem only) Heart and vessels appear pale pink due to

post-mortem RBC rupture

Hemolysis and Hemoglobinuria

Hemoglobin recycling• Ruptured RBCs in tissues are phagocytosed by

macrophages spleen, liver• Old RBCs accumulate in spleen and liver to be

processed by macrophages and Kuppfer cells• Iron is removed to ferritin which may oxidize to

hemosiderin• Iron-free heme is converted to biliverdin (green)• Biliverdin converted to bilirubin (yellow)• Bilirubin is insoluble, transported to liver through

plasma bound to albumin• Hepatocytes conjugate bilirubin with glucuronic

acid making it water soluble• Conjugated bilirubin is excreted in bile

Hepatic bile ducts

Colors of bruising• Initial hemorrhage of RBCs into tissue is cleared by

macrophages, which process Hb Oxyhemoglobin and Deoxyhemoglobin Deoxyhemoglobin and Biliverdin Biliverdin and Bilirubin Bilirubin and Hemosiderin Hemosiderin

• When iron is completely cleared, tissue resumes normal color

• Accumulation of hemosiderin is hemosiderosis• Hemochromatosis is severe, chronic accumulation

Colors of bruising

Excessive accumulation of iron• Accumulation of hemosiderin is hemosiderosis• Hemosiderin is normally found in marrow, spleen,

liver• Hemosiderosis in tissues is secondary to:

iron intake oveload (enteral or parenteral), long-term hemodialysis or transfusions, blood disorders

• Hemochromatosis is severe, chronic accumulation in liver, pancreas, myocardium Primary, genetic hemochromatosis most frequent in men

of northern European descent

• Hemochromatosis results in oxidative damage and inflammation

Hemosiderosis, hemochromatosis

Jaundice, icterus

• Bilirubin build-up in tissues Prehepatic or hemolytic: due to excessive

hemolysis• build-up of unconjugated bilirubin

Hepatic or hepatocellular: due to failure of at least 80% of liver function

• both conjugated and unconjugated bilirubin accumulates

Posthepatic or obstructive: due to failure of bile to drain into GI

• Conjugated bilirubin accumulates

Porphyrins• Porphyra = purple

• Porphyrins, aka tetrapyrroles, are intermediates of heme synthesis

• Conjugated bonds in ring structures are photoactive, fluorescent

• Porphyrias result from inborn errors of heme metabolism

• Seven separate types of porphyria

• Most common disorder, acute intermittent porphyria, is an autosomal dominant deficiency of the third enzyme in the heme synthesis pathway (porphobilinogen deaminase)

• Several intermediates in the pathway, including the first, aminolevunlinic acid, are neurotoxic

• Symptoms of accumulation include UV sensitivity and neurological dysfunction

Variegate porphyria• Autosomal dominant acute hepatic porphyria• Protoporphyrinogen oxidase gene on bands 1q22-23• Termed “South African” as it occurs in South Africans of

Boer descent with an incidence of 1 case per 330• All are descendants of Gerrit Jansz and Ariaantje Jacobs

who married at the Cape of Good Hope in 1688• Similar founder effects explain the high prevalence in the

houses of Stuart, Hanover, and Prussia (British royal family)• Symptoms begin in the second or third decade of life• Cutaneous manifestations are present in 80% of patients• Acute attacks are seen in approximately 50% of patients

causing neuropsychiatric, gastrointestinal, and cardiovascular manifestations as a result of the increase in porphobilinogen (PBG) and aminolevulinic acid (ALA)

Symptoms of porphyria

Lipofuscin

• Fuscus = brown• Inclusions of lipid peroxides, phosphates,

proteins • Not harmful• Colors ear wax• Accumulates in liver, heart from normal “wear

and tear”• Pigment accumulates near nucleus • Indicative of age, oxidative damage• Does not stain blue with Prussian blue or Perl’s

iron

Lipofuscin

Melanin

• Melas = black• Synthesized from tyrosine by tyrosinase• Reaction confined to melanosome compartment

of melanocytes in dermis• Whole melanosome is transferred to

keratinocytes in epidermis• Blocks UV radiation• Benign accumulations (freckles, moles) are

called nevi (pl., singular: nevus or naevus = from birth; birthmark)

• Alternative name, lentigo, like a lentil or pea

Histological accumulations

• Endogenous pigments Hemoglobin, bilirubin, biliverdin, porphyrin,

iron, hemosiderin, melanin, lipofuscin

• Compounds that absorb histological stain Lipid and cholesterol Hyaline Amyloid Proteins Calcium complexes (soaps, CaPO4)

• Exogenous pigments

Hyaline

• Histologic appearance of pale, glassy,diffuse pink in H&E staining

• Eosine binds free amino groups N-ends, Lys, Arg May indicate protein breakdown or influx of

plasma proteins

• Accumulation of staining, not itself a pigment

Amyloid

• Amyl = starch• Misfolded proteins in B-pleated sheet

formation that resist digestion and accumulate as inclusions

• Amyloid accumulations in glomeruli stain brown with iodine, similar to the iodine reaction with glycogen

• Amyloid and glycogen stain pink under white light with Congo red, but polarized light bounces off amyloid and looks green

Protein accumulations

• Pink in H&E staining

• Proteins may accumulate as aggregates in vacuoles or in extracellular spaces

• Protein droplets may stain brightly in proximal tubules

• Secretory granules budded from ER may stain brightly as Russell bodies

• Misfolded proteins aggregate, such as 1-antitrypsin in hepatocytes

Dystrophic calcification

• Associated with necrosis, aging or damaged heart valves

• Precipitated calcium salts look white

• Basophilic when stained with H&E

• Blue granular crystals

Calcified bicuspid

Metastatic calcification

• Associated with imbalances in phosphorous equilibrium or hypercalcemia Hyperparathyroidism stimulates resorption of Ca from

bone Accelerated bone turnover due to immobility,

metastatic cancer, leukemia, Paget disease Vitamin-D intoxication, sarcoidosis Renal failure

• Phosphate retention• Hyperparathyroidism

• Principally affects acid secreting cellls Gastric mucosa, kidneys, lungs, systemic arteries,

pulmonary veins

Sarcoidosis

• Exogenous pigment accumulations Foreign matter, colored or refractive in

microscope slides• Anthracosis – coal miner’s black lung (anthrako =

charcoal, carbon)• Silicosis – another occupational hazzard

• Accumulations of matter that stains Proteins

• Extracellular matrix and cytoskeletal fibers

Glycogen Fat, lipid, cholesterol

• Actually a void in H&E due to solvent

• Exogenous pigment accumulations Foreign matter, colored or refractive in

microscope slides• Anthracosis – coal miner’s black lung (anthrako =

charcoal, carbon)• Silicosis – another occupational hazzard

• Accumulations of matter that stains Proteins

• Extracellular matrix and cytoskeletal fibers

Glycogen Fat, lipid, cholesterol

• Actually a void in H&E due to solvent

Reversible damage – fatty change

Intracellular accumulations of a variety of materials can occur in response to cellular injury. Here is fatty metamorphosis (fatty change) of the liver in which deranged lipoprotein transport from injury (most often alcoholism) leads to accumulation of lipid in the cytoplasm of hepatocytes.