ENZYME HISTOCHEMISTRY jps

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ENZYME HISTOCHEMISTRY :

ENZYME HISTOCHEMISTRY Dr. Jinesh P. S. 04/07/2012

HISTOCHEMISTRY :

HISTOCHEMISTRY Histochemistry : Based on chemical reactions between cell components and stains. The end products of reaction are permanent , colored precipitates that can be viewed under the microscope. There are stains specific to each component of the cell, based on the basic or acidic nature of the dye.

BASIC PRINCIPLES OF HISTOCHEMISTRY :

BASIC PRINCIPLES OF HISTOCHEMISTRY Histochemistry combines the methods of histology with those of chemistry or biochemistry, to reveal the biochemical composition of tissues and cells beyond the acid-base distribution shown by standard staining methods ( Hx & E ), without disrupting the normal distribution of the chemicals.

ENZYME HISTOCHEMISTRY - HISTORY :

ENZYME HISTOCHEMISTRY - HISTORY In 1786 Plenk emphasized importance of vital reactions for differentiating PM and AM wounds Earlier – signs of wound healing & histological examinations Raekallio in 1960 reported enzyme activity changes in the edges of vital wounds inflicted at least 1 h before death Fazekas and Viragos- Kis described increases of histamine and serotonine levels in vital skin wounds Immuno histochemistry – in 1996

ENZYME HISTOCHEMISTRY:

ENZYME HISTOCHEMISTRY Enzymes – proteins catalyzing biological reactions. EHC – Visualizing Chemicals and Enzymes in Tissue. Enzyme histochemistry – a link between biochemistry and morphology. It mirrors early metabolic imbalance of a pathological tissue lesion.

PROCEDURE:

PROCEDURE The wounds or its parts are excised with their surroundings about half an inch in each direction. One half is fixed overnight in neutral 10% formalin at 4 0 C for the demonstration of esterase and phosphatase activity. Other half is fresh frozen with isopentane chilled with dry ice for detecting adinosine triphosphatases & aminopeptidases. The sections are incubated in solutions containing substrates for the enzymes of interest and reagents that yield insoluble colored or electron-dense precipitates at the sites of enzyme activity.

E H C of INJURIES:

E H C of INJURIES Two zone – around ante mortem injury Central zone – 200 to 500 μ deep, shows decreasing enzyme activity - considered as early sign of imminent necrosis – negative vital reactions Peripheral zone – 100 to 300 μ deep – increase in enzyme activity – considered as adaptive defense mechanism of local connective tissue cells as enzymatic response to injury – positive vital zone Post mortem wounds – no vital reaction around

OCCURRENCE OF ENZYMES :

OCCURRENCE OF ENZYMES Vital wound age ATPase Esterase Amino peptidase Acid phosphatase Alkaline phosphatase Poly morpho nuclear cells Mono nuclear cells > 16 hours + + + + + + + > 8 hours + + + + + + > 4 hours + + + + > 2 hours + + + > 1 hour + +

ATPase :

ATPase Catalyzes hydrolysis of ATP to ADP, releasing energy Functions : co-transporters, pumps & Transmembrane ATPases – cell metabolism and export of toxins, wastes, solutes In muscle biopsy – used at different pH to distinguish bet. different types of fibers Diagnostically important – muscle diseases have characteristics patterns of loss of specific fiber types or sub-types Elevated within 1 hour of Injury

ESTERASES :

ESTERASES A hydrolase – that splits esters into an acid and an alcohol Elevated within 1 hour after injury Some non-specific esterases can be used for identifying macrophages in necrotic fibers and abnormal lysosomal activity in muscle fibers Cholinesterase can be used to demonstrate intramuscular nerve twigs

AMINO PEPTIDASES :

AMINO PEPTIDASES Aminopeptidases cleave amino acids from the N-terminus of proteins and peptides They function in protein maturation and degradation and in many biological processes such as antigen processing and presentation Elevated within 2 hours after injury

ACID PHOSPHATASE :

ACID PHOSPHATASE Is a hydrolase enzyme used to free attached phosphate groups from other molecules during digestion Optimum action in acid medium Stored in lysosomes & functions when fuse with endosomes Elevated within 4 hours after injury Lysosomes can distinguish from other cytoplasmic organelles

ACID PHOSPHATASE – clinical use other than E H C:

ACID PHOSPHATASE – clinical use other than E H C Human acid phosphatase Clinical importance Lysosomal (LAP) LAP deficiency: an inherited autosomal recessive disorder affecting metabolism Prostatic (PAP) Serum marker for cancer of the prostate Semen marker used in cases of sexual abuse Erythrocytic (EAP) Increased susceptibility to developmental disturbances and haemolytic favism with certain alleles Paternity testing Macrophage (MAP) Gaucher's disease: an inborn error of cerebroside metabolism Osteoclastic ( OcAP ) Metastasis to the bone Bone resorption conditions

ALKALINE PHOSPHATASE :

ALKALINE PHOSPHATASE is a hydrolase enzyme responsible for removing phosphate groups from nucleotides, proteins, and alkaloids. Optimum action in alkaline medium Elevated within 8 hours after injury Iso enzymes CLINICAL CONDITIONS LIVER ALP 1 Cholestasis , cholecystitis , cholangitis , cirrhosis, Primary Biliary Cirrhosis, hepatitis, fatty liver, sarcoidosis , liver tumor, liver metastases, drug intoxication BONE ALP 2 Paget disease, osteosarcoma , bone metastasis, renal osteo dystrophy, multipla myeloma & osteomalecia , rickets, vitamin D deficiency

ADVANTAGES :

ADVANTAGES Enzyme histochemistry gives a biologic timetable useful in timing of ante mortem wounds Vital reactions are recognizable several days after death Diagnostic of some diseases

LIMITATIONS :

LIMITATIONS Cannot be used for real time in vivo analysis of any tissue Uses in humans limited to biopsied & P M tissues For looking at changes in tissue over time, each point in time requires a new tissue sample Tissue preparation and enzyme histo -chemical analysis may alter specimen morphology or chemistry depending on the methods and materials used Far advanced senility, cachexia, and very severe & multiple injuries may delay the local reactivity of the skin (in PM sample)

THANK YOU :

THANK YOU

REFERENCES :

REFERENCES Reinhard B. Dettmeyer , Forensic Histopathology: Fundamentals and Perspectives, Ed 1, 2011 C. G. Tedeschi , E. G. William, L. G. Tedeschi , Forensic medicine – A study in trauma and environmental hazards, Vol 1, 1977 C. Hernhndez-Cueto, D.N. Vieirab, E. Girela, E. Marquesb, E. Villanueva, F. Oliveira Sib, Diagnostic ability of D- dimer in the establishmentof the vitality of wounds; Forensic Science International 76 (1995) 141-149 H Bull, P G Murray, D Thomas, A M Fraser, P N Nelson; Acid phosphatases , Mol Pathol . 2002 April; 55(2): 65–72

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