GEL TECHNOLOGY By Dr Neelam Shah • Different methods for Antibody detection. • #Tube tests • #Column agglutination technology . includes • -Gel technology • - In another column agglutination technology, a column of glass microbeads in a diluent is used instead of gel. As with the gel test, the beads may either entrap agglutinated cells. • Immunofluorescence • Solid-Phase Red Cell Adherence Tests • Enzyme-Linked Immunosorbent Assay Agglutination Reactions Stage 1 Sensitization: attachment of Antibody to Antigen on the RBC membrane. Stage 2 • Lattice formation: formation of bridges between the sensitized red cells to form the lattice that constitutes agglutination. Ab molecule cross link RBCs forming lattice INTRODUCTION • Gel technology system developed by Dr.Yves Lapierre of France,gives more reproducible and standardied test results. • This technology utilizes the differential migration of RBC agglutinates through a small microtube containing Dextran Acrylamide gel,size exclusion gel column. Fully automated system USES • For any immunohematolgical tests that has hemagglutination as its endpoint. -ABO and Rh typing -Typing for other blood group system-Antibody screening and identification -Compitability testing including cross matching ADVANTAGES OF GEL TECHNLOGY • • • • • • • Simple ,reliable ,rapid , very sensitive. No need to multiple washing of red cell before adding into Antihuman globulin (AGH) serum. and no need to add sensitized controlled cell to all negative AGH tests. Less specimen volume needed. Greater uniformity between repeated tests. No variations among technologist in reading and grading agglutination. Provision of centrifuge calibarted to optimal speed for fix and corrected length of time , reduces potential error during this phase. The cards can be digitally photographed , downloaded as permanent laboratory data. DISADVANTAGES • Special centrfuge to accommodate the microtube card. • Special incubators to incubate microtube cards. • Pipettes to dispose serum and red cell suspention. • Expesive. Centrifugation of microtubes ABOUT GEL CARD • Gel held in a microtubes contain in a plastic card. • Each microtubes contain cephadex(dextran acrylamide) gel prepared in a buffer solution such as LISS or Saline. • Preservatives –Sodium azide • Specific reagents –Agh or RBC specific antisera is added. • Sedimenting agent is bovine serum albumin. • Reagent is uniformly dispersed throughout gel column. ABO/Rh(D) Group Card • Human red blood cell antigen can be divided into four groups A, B, AB and O depending on the presence or absence of corresponding antigens on the red blood cells. • The Anti-A, Ant-B reagent are used to detect the presence or absence of corresponding antigens on red blood cells. - - - - Gel-card of Rh system • In Newborn's the antigens are not completely developed, thus weaker reaction may be shown when compared to adults , when tested. • In adults the antigens and respective antibodies are present, but in newborns the antibodies appear after 4 to 6 months of birth. • Also human red blood cells are classified as Rh (D) positive or Rh (D) negative depending upon the presence or absence or of Rh (D) antigen. • The D antigen and weak D (Du) are fully developed at birth. If the mother is Rh (D) negative, it is very much important to determine the D antigen and weak D of the newborn. PRINCIPLE • Serum and cell reaction takes place in a microtube consisting of a reaction chamber that narrower to become column. • The card containing red blood cells is centrifuged under specific conditions, the red blood cells possessing corresponding antigen will agglutinate in the presence of the antibody directed towards the antigen in the gel matrix and will be trapped in the gel column. • The red cells, which do not react, are not trapped in the gel column and get settled at the bottom of the column. The reactions are visually read and graded according to their reactivity pattern. • REAGENTS • The Matrix ABO/RhD Group Card contains six microtubes , pre-filled with gel in a suitable buffer containing specific Monoclonal Anti-A, Anti-B and Anti-D antibodies. • SAMPLE COLLECTION • No special preparation of the patient is required prior to sample collection. • Samples should preferably be tested as soon as possible. If delay in testing occurs sample should be stored at 2-8°C or may be used within 7 days of collection. Preferably, blood sample should be drawn into citrate, EDTA or CPD-A anticoagulant. Do not used hemolysed or contaminated samples or those contaning clots. SAMPLE PREPARATION • • • • • • • • • FOR FORWARD GROUPING Prepare a 5% suspension in LISS 1.bring Liss at room temp before testing. 2.dispense 0.5 ml of Liss into a clean glass test tube. 3.Add 50 µl of whole blood or 25 µl of pack cell and mix gently. FOR REVERSE GROUPING Prepare a 0.8% suspension in Liss 1.Collect known A1 and B cells. 2.Wash the cells twice with 0.9% normal saline and discard the supernent. • 3.dispense 0.5 ml of Liss into a clean glass test tube. • 4.Add 5 µl of pack cell and mix gently. SAMPLE PREPARATION • PLASMA OR SEUM FOR REVERSE GROUPING • If serum is used instead of plasma , the serum must be cleared by centrifuging at 1500g for 10 min , so as to avoid presence of fibrin residues , which might interfere with test results. • If delay in testing occurs sample should be stored at 2-8 degree c. after separation and may be used up to 48 hrs or stored frozen a -20 to -80 degree c. TEST PROCEDURE • 1. Allow samples and reagent to reach room temperature. • 2. Label the appropriate microtubes of the Gel card with patient name / identification number. • 3. Pipette 50 μl of 0.8% A1 cell suspension to the microtube 5. • 4. Pipette 50 μl of 0.8% B cell suspension to the microtube 6. • 5. Pipette 50 μl of patient’s plasma or serum to the microtube 5 and 6. • 6. Take care to ensure that the micropipette tip does not touch the reagent in the microtube. • 7. allow the card to incubate 10 min at room temp. • 8. Pipette 10 to 15 μl of 5% patient’s cell suspension to the microtube 1 to 4. • 9. Centrifuge the cards for 10 minutes in the card centrifuge. • 10. Read and record the results. INTERPRETATION OF RESULTS • Positive Reaction : Agglutinated red blood cells form a clear line on the surface of the gel or get dispersed in the gel. • Negative Reaction: Unagglutinated red blood cells settle at the bottom of the microtube. • Reading and interpretation of results must be done after centrifugation process only. • The reaction strength may be recorded as follows: Strength of reaction • 4+ Agglutinated red blood cells form a solid band at the top of the gel column. • 3+ Most agglutinated red blood cells remain in the upper half of the gel column. • 2+ Agglutinated red blood cells are observed throughout the length of the column. A small button of red blood cells may also be visible at the bottom of the gel column. • 1+ Most agglutinated red blood cells remain in the lower half of the column. A button of cells may also be visible at the bottom of the gel column. Strength of reaction • ± Most agglutinated red blood cells are in the lower third part of the column. • Negative (0) All the red blood cells pass through and form a compact button at the bottom of the gel column. • Mixed field agglutination Agglutinated red blood cells form a band at the top of the gel and nonagglutinated red blood cells form a compact button at the bottom of the gel column. • H Hemolysis Strength of reaction Readin & grading of hemagglutination in gel system EXPECTED REACTIVITY PATTERN FOR ABO GROUPING Anti-A Anti-B Blood Group 1+to 4+ Negative A Negative 1+to 4+ B 1+to 4+ 1+to 4+ Ab Negative Negative O EXPECTED REACTIVITY PATTERN FOR Rh(D) TYPING Anti-D Rh(D) type 1+ to 4+ Rh positive Negative Rh negative REACTION FOR REVERSE GROUPING A1 B Blood group 1+ to 4+ Negative B Negative 1+ to 4+ A 1+ to 4+ 1+ to 4+ O Negative Negative AB • Human red blood cells that shows weak reaction with anti-A and anti-B probably indicates subgroups of A and B and further testing is recommended. • Very weak D/partial D type human red cells may give negative reaction . such cells should be retested with coombs card. • The microtube control must show a negative reacion . if control is positive , then the ABO determination is not valid . repeat the procedure. REVERSE GROUPING CARD WITH AUTOCONTROL • Card contain neutral gel in all microtubes. • 0.1% sod azide as preservative. • All procedure is same as mention early. AHG (Coombs) Test Card AHG (Coombs) Test Card • Generally antibodies involved in transfusion reactions are of two types, namely the complete and the incomplete. • The complete antibodies agglutinate in vitro human red blood cells in saline medium. • The incomplete type of antibodies sensitize red blood cells without agglutination. • Usually IgM class of antibodies and IgG1 and IgG3 type of IgG antibodies fix complement. • Cell lysis , in vivo, is mediated through the complement system and the complement C3b is metabolized to C3d. • In the direct antiglobulin test, Anti Human Globulin reagent is used to detect antibodies adsorbed to the red blood cells in vivo. • In the indirect antiglobulin test, Anti Human Globulin reagent is used to detect antibodies adsorbed to the red blood cells in vitro. • Anti Human Globulin reagent is useful for compatibility testing, antibody detection, antibody identification, umbilical cord red blood testing and the detection of weak D/partial D. • REAGENTS • AHG (Coombs) Test cards contain six microtubes prefilled with gel in a suitable buffer containing anti-human IgG and monoclonal antiC3d. • PRINCIPLE • As the gel card containing red blood cells is centrifuged under specific conditions, the red blood cells sensitized with antibody will agglutinate in the presence of the Anti Human Globulin reagent in the gel card and will be trapped in the gel column. • The red cells, which do not react are not trapped by the gel matrix and are pelletted to the bottom of the microtubes. The reactions are visually read and graded according to their reactivity pattern. • SAMPLE COLLECTION • (1) For DAT blood dawn in EDTA is preferred but citrated whole blood may be used. • (2) For IAT serum no more than 48 hours should be used . donor units may be tested up to the end of their dating. • (3) To avoid presence of fibrin residues, serum or plasma may be cleared by centrifuging at 1500 g for 10 minutes • (4) Do not use hemolysed or contaminated blood samples, or those containing clots. • SAMPLE PREPARATION • Preparation of 0.8 % red blood cell suspension is as follows: • 1. Bring the Liss to room temperature before testing. • 2. Add 0.5ml of Liss into a clean glass test tube. • 3. Add 5 μl of packed cells to the Liss and mix gently. • 4. The red blood cell suspension so obtained should be used for testing. • 5. Set up as many gel cards as may be required A. DIRECT ANTIGLOBULIN TEST (DAT) 1. Label the appropriate microtubes with patient name or identification number. Remove the aluminium foil carefully. 2. Pipette 50 μl of the 0.8% patient’s red cell suspension. 3. Centrifuge immediately for 10 min; read and record the results. A. DIRECT ANTIGLOBULIN TEST (DAT) • INTERRETATION • Negative reaction indicates absence of detectable IgG or C3d component on the red cells. • Positive reaction indicates that red blood cells are sensitised with IgG or complement componant C3d. B. ANTIBODY SCREENING IAT • 1. Label the appropriate microtubes with patient name or identification number. Remove the aluminium foil carefully. • 2. Pipette 50 μl of the 0.8% red cell suspension. • 3. Immediately add 25 μl of patient/donor serum or plasma. The interval between cell and serum/plasma transfer should not exceed 10 minutes. • 4. Incubate 15 minutes at 37 C in an incubator. • 5. Centrifuge for 10 min; read and record the results. B. ANTIBODY SCREENING IAT • INTERRETATION • Negative reaction indicates Absence of detectable iregular antibodies in the patient’s or donor’s serum/plasma. • Positive reaction indicates Presence of irregular antibodies. • Further testing is recommended to identifybthe antibody specificity. FOR COMPATIBILITY TEST • 1.Pipette 50 μl of the 0.8% of donor red cell suspension. • 2.Pipette 50 μl of the 0.8% of patiet’s red cell suspension added to other microtube,this serves as an autocontrol. • 3.add 25 μl of patient serum or plasma. The interval between cell and serum/plasma transfer should not exceed 10 minutes. • 4. Incubate 15 minutes at 37 C in an incubator. • 5. Centrifuge for 10 min; read and record the results. FOR COMPATIBILITY TEST • INTERPRETAION • The auto control should negative to validate results. • Negative reaction indicates Compatibility of donor blood with the recipient. • Positive reaction indicates incompatibility of donor blood with the recipient. • After incubation hemolysis observe in upper portion of column , it should be interpreted as positive reaction. • PERFORMANCE • Evaluation of AHG (Coombs) Test cards for Antibody Screening / Antibody Identification and Autocontrol was performed by an external Immunohaematology Laboratory in comparison to a Reference method. • 1.Total 551 samples were tested for Antibody Detection.. Test card Reference method Sensitivity: 92.9% 97.6% Specificity: 97.2% 98.8% • • 2. Total 544 samples were tested for Autocontrols Test card Reference method Sensitivity: 94.5% 97.6% Specificity: 94.5% 96.2% • 3. Total 43 samples were tested for Antibody Identification Sensitivity: Test card Reference method 96% 94% REMARKS • (1)Freezing or Evaporation of the gel card due to exposure to heat may impede the passage of unagglutinated red blood cells through the gel card. • (2) Gel cards showing damaged aluminum foil should be discarded. • (3) Bubbles in the gel card may interfere the passage of unagglutinated red blood cells. gel cards with bubbles entrapped in the gel may be centrifuged before testing. gel cards with resisting bubbles should be discarded. • (4) Gel cards showing decreased volume or cracked gel should be discarded. • (5) Usage of red blood cell concentrations other than those described may interfere the test results. • (6) Usage of hemolysed samples may interfere the final interpretation of results. • (7) Bacterial or other contamination of reagents during use may cause false positive or false negative results. • (8) Fibrin residue in the serum or red cell aggregation in the red cell suspension can trap non agglutinated cells presenting a pink line on top of the gel when most cells pellet to the bottom after centrifugation. • (9) Do not use lipemic, icteric and hyperproteic samples. • (10) Gel card that exhibit drying if used ,can lead to erroneous result. - LISS SOLUTON • The antigen-antibody interaction in blood group serology is dependant on antigen density, concentration of antibody, pH, ionic concentration of reaction medium and temperature. • Reducing the ionic concentration of the reaction medium especially enhances the uptake of weak antibodies by the red blood cell antigens. • Also, usage of LISS (Low Ionic Strength Solution) is helpful in detection of weak antibodies during cross match techniques, antibody screening and antibody identification. Liss • REAGENT • LISS is a buffered low ionic strength solution of appropriate sodium chloride molarity useful in serological applications. • STORAGE AND STABILITY • Store the reagent at 2-8°C. Do not freeze. • The shelf life of the reagent is according to the expiry date indicated on the label. Do not use beyond expiry date LISS(LOW IONIC STRENGTH SALT SOLUTION) • Contain 0.2% Sodium chloride. • Consist of 0.17M saline180 ml • 0.15M phosphate buffer-20 ml • 0.3M sodium glycinate,pH 67-800ml • All anibodies are not equally sensitive to liss.anti A and antiB remains unaffected. • To prevent lysis of red cells at such a low ionic strength, a nonionic substance such as glycine is incorporated in the LISS • Most laboratories use a LISS additive reagent, rather than LISS itself. These commercially available LISS additives may contain albumin in addition to ionic salts and buffers. liss • PRINCIPLE • In blood group serology, the ionic concentration of reaction medium is largely dependant on the concentration of sodium and chloride ions contributed by isotonic saline. • When optimum concentration of antibody is present, antigen-antibody interaction occurs even though the sodium and chloride ions are present in sufficient quantity. • But when weak antibodies are present, sodium and chloride ions may interfere with binding of antibody to the antigens present on the red blood cell membrane. • By lowering the ionic concentration of salt, the ionic strength is reduced which increases the rate of antibody uptake by red blood cells. liss • PERFORMANCE • The performance of LISS was evaluated on over 100 samples (from donors, patients and neonates) drawn on recommended anticoagulants. • The evaluation demonstrated 100% specificity and sensitivity of the reagent versus the expected results with common known ABO , Rhesus phenotypes, Cross match, DAT and Autocontrol. liss • REMARKS • 1. Aged or stored red blood cells may show weaker reactivity than freshly collected cells. • 2. Usage of red blood cell concentrations other than those described may interfere the test results. • 3. Usage of hemolysed samples may interfere the final interpretation of results. • 4. Bacterial or other contamination may cause false positive or false negative results. • 5. Red cell aggregation in the red cell suspension may interfere the passage • • • • • References WHO manual AABB manual Henry’s clinical diagnosis and management www.DiaMed.com • THANK YOU……….