Molecular techniques

Different molecular techniques such as fluorescent in situ hybridization, Southern blot, etc... can be used to detect genetic diseases.

AMYLOIDOSIS

Definition. Extra cellular  deposition of an eosinophilic hyaline homogenous material in Various organs, occurring in a variety of clinical  states.

Staining reactions

Iodine :- Brown, turning blue on addition of H2SO4 (gross and microscopic Stain).
P.A.S. – Positive  (Magenta pink).
Congo Red -Orange red which on polarisation gives green birefringence.
Von Geison's –Khaki colour.
Thioflavin T -Yellow fluorescence.

Amyloid is called typical if it given the above staining  reactions Other wise it is termed atypical or para-amyloid.

Classification 

1.    Systemic  amyloidosis associated with underlying disease (secondary),

(A) Chronic infections like 

- Tuberculosis.
- Bronchiectasis.
- Lung abscess.
- Osteomyelitis.
- Syphilis.

(B) Chronic inflammations of varied etiology:

- Rheumatoid arthritis.
- Ulcerative colitis.
- Regional enteritis.
- Lupus erythematosus.

(C) Neoplastic proliferations:

- Of immune system – Multiple myeloma, Hodgkin’s disease.
- Cancers like Renal cell carcinoma etc.

II Systemic primary amyloidosis  with no underlying cause.

III Heredofamilial types.

- Amyloidosis with mediterranean fever.
- Amyloid polyneuropathy.
- Amyloid nephrophathy
- Familial cardiac amyloidosis
- Familial cutaneous amyloid.
- Lattice corneal dystrophy

IV. Localised amyloidosis:

- Senile - in heart, brain, seminal vesicles.
- Amyloidoma of tongue, bronchial tree, skin.
- In islets of Langerhans in Diabetes mellitus.
- In medullary thyroid carcinoma.

Deposition sites
In relation to reticulin  and collagen fibres and to basement, membranes especially
subendothelial. 

Organs involved commonly are : 

Secondary amyloidosis

- Liver.
- Spleen.
- Kidney
- Lymph nodes.
- Adrenals.

Primary amyloidosis

- Heart
- Tongue and gingiva.
- Gastro intestinal tract.
- Lung.
- Wall of small vessels.

Nature and pathogenesis of amyloid
It is primarily made up of protein arranged in two patterns

- There are filaments twisted together to from the fibrils. These chemically resemble light chains of immunoglobulins
- Rods composed of stacked rings. These are made up of alpha globulin components of plasma proteins (P-components)

- In addition to these, extracts of crude amyloid contain  mucopolysacharides complement and gamma globulins.

- Origin of amyloid :- current concept is that it is a direct product of cells of the immune sustem with some abnormality in their immune response

The abnormality may be due to :
- A genetic enzyme defect.
- Prolonged antigenic challenge.
- Neoplastic transformation
- Supression of normal. Response as in induced tolerance.

CARCINOMA IN SITU

Epithelial malignancy which has not yet invaded even -the local confines viz basement membrane is termed as carcinoma in situ (intra epithelial neoplasia, pre-invasive cancer)

This lesion merges morphologically with severe dysplasia

Common sites for carcinoma-in-situ :

• Cervical squamous epithelium
• Oropharynx
• Bronchial epithelium.
• Breast ducts and lobules.
• Skin, in the form of Bowen's disease.
• Glans penis and vulva in the form of Erythroplasia of Queyrat

Immunoglobulins. (Ig)

 These are made up of polypeptide chains. Each molecule is constituted by two heavy and two light chains, linked by disulfide (S-S) bonds. The h~ chains are of 5 types, with corresponding, types or  immunoglobulin. IgG (gamma), IgM (mu µ ), IgA(alpha α), IgD(delta ), IgE(epsilon)

Each of these can have light chains of either kappa (k) or lambda type.Each chain has a constant portion (constant for the subtype) land a variable portion (antigen specific).

Enzyme digestion can split the Ig molecule into.2 Fab (antibody binding) fragments and one Fc (crystallisable, complement binding ) fragment.

 IMMUNO PATHOLOGY

Abnormalities of immune reactions are of 3 main groups

• Hypersensitivity,
• Immuno deficiency,
• Auto immunity.

Hypersensitivity (ALLERGY)

This is an exaggerated or altered immune response resulting in adverse effects

They are classified into 4 main types.

I. Type I-(reaginic, anaphylactic). This is mediated by cytophylic Ig E antibodies, which get bound to mast cells. On re-exposure, the Ag-Ab reaction occurs on the mast cell surface releasing histamine.

Clinical  situations

I. Systemic anaphylaxis, presenting with bronchospasm oedema hypertension, and even death.

2. Local (atopic) allergy.

• Allergic rhinitis (hay fever)
• Asthma
• Urticaria.
• Food allergies.

2. Type II. (cytotoxic). Antibody combines with antigen present on-cell surface. The antigen may be naturally present on the surface or an extrinsic substance (e.g.drug) attached to cell surface.

The cell is then destroyed by complement mediated lysis (C89) or phagocytosis of the antibody coated cell.

Clinical situations

• Haemolytic anemia.
• Transfusion reaction
• Auto immune haemolytic anemia.
• Haemolysis due to some drugs like Alpha methyl dopa

Drug induced thrombocytopenia (especially sedormid).

Agranulocytosis due to sensitivity to some drugs.

Goodpasture’s syndrome-glomermerulonephritis due to anti basement membrane antibodies.

3. Type III. (Immune complex disease). Circulating immune complexes especially

small soluble complexes tend to deposit in tissues especially kidney, joints, heart and

arteries.

These then cause clumping of platelets with subsequent release of histamine. and

serotonin resulting in increased permeability. Also, complement activation occurs which

being chemotactic results in aggregation of polymorphs and necrotising vasculitis due to

release of lysosmal enzymes

Clinical situations

• Serum sickness.
• Immune complex glomerulonephritis.
• Systemic lupus erythematosus.
• Allergic alveolitis.
• Immune based vasculitis like
  • Drug induced vasculitis.
  • Henoch – Schonlein purpura

4. Type IV. (Cell mediated). The sensitized lymphocytes may cause damage by

cytotoxicity or by lymphokines and secondarily involving macrophages in the reaction.

Clinical situations

I. Caseation necrosis in tuberculosis.

2. Contact dermatitis to

• Metals.
• Rubber.
• Drugs (topical).
• Dinitrochlorbenzene (DNCB).

5. Type V. (stimulatory) This is classed by some workers separately and by other with

cytotoxic type (Type II) with a stimulatory instead of toxic effect

Clinical Situations :

LATS (long acting thyroid stimulator) results in thyrotoxicosis (Grave’s disease)

Jaundice, or icterus

a. Characterized by yellowness of tissues, including skin, eyes, and mucous membranes. 
b. Caused by excess conjugated and/or unconjugated serum bilirubin. (increased levels of bilirubin in the blood)
lcterus is visible when the serum bilirubin exceeds 2 mg/dl. In unconjugated hyperbilirubinemia, bilirubin is not excreted into the urine because of tight protein binding in serum. In conjugated hyperbilirubinemia, small amounts of bilirubin are excreted in the urine because
it is less tightly protein bound. 

 NOTE: Concentration of bilirubin in blood plasma does not normally exceed 1 mg/dL (>17µmol/L). A concentration higher than 1.8 mg/dL (>30µmol/L) leads to jaundice.
 
 The conjunctiva of the eye are one of the first tissues to change color as bilirubin levels rise in jaundice. This is sometimes referred to as scleral icterus.

c. Types and causes include:
(1) Hepatocellular jaundice—caused by liver diseases such as cirrhosis and hepatitis.
(2) Hemolytic jaundice—caused by hemolytic anemias.
(3) Obstructive jaundice—caused by blockage of the common bile duct either by gallstones (cholelithiasis) or carcinomas involving the head of
the pancreas. 

Differential diagnosis 

Jaundice is classified into three categories, depending on which part of the physiological mechanism the pathology affects. The three categories are:

Pre-hepatic → The pathology is occurring prior to the liver.
Hepatic → The pathology is located within the liver.
Post-Hepatic → The pathology is located after the conjugation of bilirubin in the liver. 

Pre-hepatic
Pre-hepatic jaundice is caused by anything which causes an increased rate of hemolysis (breakdown of red blood cells).
Certain genetic diseases, such as sickle cell anemia, spherocytosis, thalassemia and glucose 6-phosphate dehydrogenase deficiency can lead to increased red cell lysis and therefore hemolytic jaundice. 
 Commonly, diseases of the kidney, such as hemolytic uremic syndrome, can also lead to coloration. Defects in bilirubin metabolism also
present as jaundice, as in Gilbert's syndrome (a genetic disorder of bilirubin metabolism which can result in mild jaundice, which is found in about 5% of the population) and Crigler-Najjar syndrome.
In jaundice secondary to hemolysis, the increased production of bilirubin, leads to the increased production of urine-urobilinogen. Bilirubin is not usually found in the urine because unconjugated bilirubin is not water-soluble, so, the combination of increased urine-urobilinogen with no bilirubin (since, unconjugated) in urine is suggestive of hemolytic jaundice. 

Laboratory findings include:
• Urine: no bilirubin present, urobilinogen > 2 units (i.e., hemolytic anemia causes increased heme metabolism; exception: infants where gut flora has not developed).
• Serum: increased unconjugated bilirubin.
• Kernicterus is associated with increased unconjugated bilirubin. 

Hepatocellular 
Hepatocellular (hepatic) jaundice can be caused by acute or chronic hepatitis, hepatotoxicity, cirrhosis, drug induced hepatitis and alcoholic liver disease. Cell necrosis reduces the liver's ability to metabolize and excrete bilirubin leading to a buildup of unconjugated bilirubin in the blood.

Laboratory findings depend on the cause of jaundice.
• Urine: Conjugated bilirubin present, urobilirubin > 2 units but variable (except in children). Kernicterus is a condition not associated with increased conjugated bilirubin.
• Plasma protein show characteristic changes.
• Plasma albumin level is low but plasma globulins are raised due to an increased formation of antibodies. 

Bilirubin transport across the hepatocyte may be impaired at any point between the uptake of unconjugated bilirubin into the cell and transport of conjugated bilirubin into biliary canaliculi.

Post-hepatic  

Post-hepatic jaundice, also called obstructive jaundice, is caused by an interruption to the drainage of bile in the biliary system. The most common causes are gallstones in the common bile duct, and pancreatic cancer in the head of the pancreas. Also, a group of parasites known as "liver flukes" can live in the common bile duct, causing obstructive jaundice. Other causes include strictures of the common bile duct, biliary atresia, cholangiocarcinoma, pancreatitis and pancreatic pseudocysts. A rare cause of obstructive jaundice is Mirizzi's syndrome. 

Pathophysiology 

When RBCs are damaged, their membranes become fragile and prone to rupture. As each RBC traverses through the reticuloendothelial system, its cell membrane ruptures when its membrane is fragile enough to allow this. 

Hemoglobin, are released into the blood. The hemoglobin is phagocytosed by macrophages, and split into its heme and globin portions. The globin portion, a protein, is degraded into amino acids and plays no role in jaundice. 

Two reactions then take place with the heme molecule. 
The first oxidation reaction is catalyzed by the microsomal enzyme heme oxygenase and results in biliverdin (green color pigment), iron
and carbon monoxide. 
The next step is the reduction of biliverdin to a yellow color tetrapyrol pigment called bilirubin by cytosolic enzyme biliverdin reductase. 

This bilirubin is "unconjugated," "free" or "indirect" bilirubin. Approximately 4 mg of bilirubin per kg of blood is produced each day.[11] The majority of this bilirubin comes from the breakdown of heme from expired red blood cells in the process just described.

However approximately 20 percent comes from other heme sources, including ineffective erythropoiesis, and the breakdown of other heme-containing proteins, such as muscle myoglobin and cytochromes.

Hepatic events

The unconjugated bilirubin then travels to the liver through the bloodstream. Because bilirubin is not soluble, however, it is transported through the blood bound to serum albumin. 
In Liver, it is conjugated with glucuronic acid (to form bilirubin diglucuronide, or just "conjugated bilirubin") to become more water soluble.
The reaction is catalyzed by the enzyme UDP-glucuronyl transferase.

This conjugated bilirubin is excreted from the liver into the biliary and cystic ducts as part of bile. Intestinal bacteria convert the bilirubin into urobilinogen. 

Urobilinogen can take two pathways. It can either be further converted into stercobilinogen, which is then oxidized to stercobilin and passed out in the feces, or it can be reabsorbed by the intestinal cells, transported in the blood to the kidneys, and passed out in the urine as the oxidised product urobilin. 

Stercobilin and urobilin are the products responsible for the coloration of feces and urine, respectively.