Lecture 2 - Concepts in Mol Bio - Ati Globulin Test PDF

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LECTURE 2 – Concepts in MolecularBiology; Antiglobulin TestCONCEPTS IN MOLECUL AR BIOLOGYMOLECULAR GENETICS Deoxyribonucleic Acid (DNA) ○ Backbone of heredity ○ Made up of phosphates and sugars and contains: Cytosine, Guanine, Adenine, Thymine (difference with RNA: RNA has additional Uracil) ○ H-bo...

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in Molecular st CONCEPTS IN MOLECULAR BIOLOGY ○ MOLECULAR GENETICS  Deoxyribonucleic Acid (DNA) ○ Backbone of heredity ○ Made up of phosphates and sugars and contains: Cytosine, Guanine, Adenine, Thymine (difference with RNA: RNA has additional Uracil) ○ H-bond, Nitrogenous Bases (2 A-T / 3 CG) –responsible to held together and form double helix and have its parallel strands ○ DNA and gene are NOT the same. (Gene is just part of the DNA and responsible for protein build up ○ REPLICATION: semi-conservative (new strand is made up of old and the new ones) Process: 1. During each replication it consists of 2 set of adenine and thymine and will act upon the enzyme helicase (for unzipping/uncoiling the DNA strands) 2. Enzyme topoisomerase (prevents further coiling due to presence of tension) 3. after the process it will have now copy of leading strand and then another enzyme responsible for copying the lagging strand is RNA polymerase. 4. Then, there will be another copy from replication thus creating DNA Polymerase 1 and 2 to proofread the copied DNA. 5. After that, all of those enzymes and RNA products and substances are joined together by DNA thus creating replication. ○ REPAIR:  Photoreactivation (where thymine separates bec of exposure to UV rays),  Excision (There series of codes are added wrongly then it will be cut and removed and replaced by correct ones),  Mismatch (The purine is being paired by another purine),  Recombinational (there is removal of new strand to maintain the original template),  SOS Repair (The damage is not contained on the nucleus and extends all

throughout the cytoplasm and throughout the cell) MUTATION: Any change in the structure of the cell or sequence of DNA it could be caused by physical factor or biochemical factor Types of mutation:  Transition – Pyrimidine is exchange with another pyrimidine. There is exchange of purine to purine  Transversion – purine is replaced with pyrimidine then vice versa  Deletion and Insertion – both can cause frameshift meaning the message is not sent properly thus resulting to deletion and insertion

 Ribonucleic Acid (RNA) ○ Single stranded ○ Uracil is attached to Adenine ○ Contains Ribose sugar ○ Function: Transmit genetic information Will undergo the ff. processes: ○

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TRANSCRIPTION – The DNA is copied to the RNA and can produce mRNA (messenger RNA which transports and is a disposable form of DNA) PROCESSING – maintains the integrity of the mRNA TRANSLATION – RNA strands are transformed to peptides and proteins

ANT IGL OBULIN TEST  Aka Coomb’s Test named after Dr. Coomb (in this testing we are obtaining presence of immunoglobulins from immunized human species)  Source of rgnt: Animals (e.g. rabbits)  Antibodies directed against the human globulin  Detects IgG and complement-sensitized RBCs  Connects the two IgG RBCs to visualize agglutination. MODE OF ACTION  AHG (antihuman globulin) + RBC sensitized w/ IgG Ab = Hemagglutination of sensitized cells

in Molecular st AHG REAGENT  Reagent: “green” in color; Janus green  Polyspecific ○ CONTENT: Antibody to human IgG and C3d complement (responsible for activating the complement system) ○ ACTIVITY: against IgA and IgM heavy chains  Monospecific ○ CONTENT: Either anti-IgG or antibody to complement PREPARATION OF AHG  Process ○ Human globulin injected to rabbit = Triggered Immune Response  production of antibodies  Mice injected with inducer [monoclonal ] or rabbit, goat, or sheep [polyclonal]  Monoclonal – one animal used  Polyclonal – two or more animal used  Monospecific vs Polyspecific ○ Uses HYBRIDOMA TECHNOLOGY – can be used to produce monoclonal anti-globulin and is derived from one clone of plasma cells and recognized a single epitope (injected with either complement or immunoglobulin [MONOSPECIFIC] injected with both complement and immunoglobulin [POLYSPECIFIC]  Kohler and Milstein – developer of Hybridoma Technology PRINCIPLE OF ANTIGLOBULIN TEST 1. Antibody molecules and complement components are globulins. 2. Injecting an animal with human globulin stimulates the animal to produce antibody to the foreign protein. 3. AHG reacts with human globulin molecules. 4. Washed RBCs coated with human globulin are agglutinated by AHG.

TYPES OF ANTIGLOBULI N TEST: DIRECT ANT IGLOBULIN T EST  Detect in-vivo (inside the body) sensitization of RBC  100-500 IgG/RBC; 400-1100 C3d/RBC  Specimen: Washed Red Cells  Reagent: AHG  Disease Associated: AIHA (Autoimmune Hemolytic Anemia), HDFN (Hemolytic Disease of Fetus and Newborn), HTR (Hemolytic Transfusion Reaction)

 PROCEDURE 1. 1 drop of 3-5% patient’s RCS (Red cell suspension) 2. Add 2 drops of Polyspecific / AHG Reagent 3. Mix well and centrifuge for 1 min. @ 1500 rpm 4. Resuspend the cells by gentle agitation Note: if results showed positive, evaluate first the px with the ff:  EVALUATION: Knowledge of Patient’s Diagnosis, Drug Therapy, Recent Transfusion History.  Control: Monospecific, Polyspecific or Saline (to detect any spontaneous agglutination of cells or any form of reaction that may occur even without addition of AHG reagent)

TYPES OF ANIGLOBULIN TEST: INDIRECT ANT IGLOBULIN T EST  Detects in-vitro (outside the body) sensitization of RBC  100-200 IgG or C3d / RBC  Specimen: Serum  Reagent: AHG and Red Cell  Application: Ab detection (xmatch, Ab screen, Ab panel), Ab Titration  PROCEDURE 1. Incubate RBCs with antisera 2. Perform washing 3x 3. Add 2 drops of AHG reagent 4. Centrifuge 5. Examine for agglutination and grade Note: If results are neg (-), need to perform confirmatory test by the ff:  EVALUATION ○ Add antibody coated RBCs to NEGATIVE reaction (confirmatory) – dapat nay reaction; ○ If no hemagglutination after confirmatory, test is INVALID FACTORS AFFECTING ANTIGLOBULIN TEST  Ratio of Serum to Cells ○ 40:1 (2 drops serum:1 drop 5% RCS) – in order to have equilibrium. ○ If Increase ratio of serum, there is increase sensitivity to the test; might result to false neg/false pos  Reaction Medium ○ Albumin – come closer contact w/ cells for aggregation ○ LISS (low ionic strength saline) – it can enhance antibody uptake therefore allowing

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decrease incubation time (so from 30- 120 min ma 15-30 min nalngg) ○ POLYETHYLENE GLYCOL – increase antibody uptake; there is removal of water to cells thereby concentrating the antibody Temperature ○ Incubation temp. @ 37 degree Celsius Incubation Time ○ 30-120 mins. (bec majority of clinically significant antibodies, 30 minutes pa mag react) ○ If remaining time is extended it can cause to elution of RBC (the mode of action of RBC is decrease; less sensitivity) Washing of RBCs ○ Wash 3x (to free unbound serum immunoglobulins) ○ Inadequate = FLASE (-) (due to neutralization of AHG reagent by unbound serum immunoglobulins) Saline for Washing ○ pH 7.2 – 7.4 ○ Fresh preparation (if NSS stored in plastic container for long periods the pH would decrease and decrease the sensitivity of antibodies; 1 a week of preparation) Addition of AHG ○ Added to cells immediately after washing (to minimize the chance of antibody elution onto the cell; mag detach and antibody sa cell) Centrifuge Reading ○ 1000 RCFs for 20 secs (the higher RCF, the higher the sensitivity) ○ Inadequate = FALSE (+); Vigorous = NEGATIVE

ANTIGLOBULIN TEST SOURCES OF ERROR

FALSE POSITIVE  Improper specimen collection or storage may activate complement in-vitro. (e.g. sample collected is clotted; wrong temp req)  Autoagglutinable Cells  Bacterial Contamination  Cells positive with DAT used for IAT  Dirty glassware  Over centrifuge and reading  Potentiators

FALSE NEGATIVE       

Inadequate or improper washing of cells. Deteriorated or neutralized AHG rgt (non-reactivation) AHG rgt not added Prozone or Postzone phenomenon Inadequate incubation Poor reading technique Low ph of saline...