MLS Review - Blood Banking - Test for RBCs

MEDICAL LABORATORY SCIENCE REVIEW 3. Incubation ▪ May depend on the temp. require ▪ Sensitization • 37C (for IgG) • Room temp (for IgM) (eg. ABO typing sera) 4. Centrifugation (~5 mins) ▪ A special type of centrifuge is used for microtube ▪ Agglutinates will be trapped ▪ When there is no clumping, the RBCs will settle at the bottom due to centrifugal force & gravity 5. Results (>10 mins) ▪ Results are checked ▪ Results are stable up to 3 days • Can be used to check unusual results with other consultants/specialists to confirm and interpret the results BLOOD BANKING TECHNIQUES: TESTS FOR RBCS OUTLINE • • Gel Test Antihuman Globulin Test (Coomb’s Test) BLOOD BANKING TECHNIQUES GEL TEST • • • • • Was developed by French doctor, Yves Lapierre, in 1985 o Wanted to find a way to trap RBC agglutinate/clump during a standardized sedimentation-centrifugationagglutination procedure ▪ Eventually developed using gel to do so Uses microtubes in hard plastic cards Microtube o Gel is already integrated in the microtube o Dextran polyacrylamide gel - gel used for microtube o Has 2 parts ▪ Reaction chamber - Upper part/chamber • Wider than the tube itself ▪ Column - Lower part/chamber • Long narrow portion • Contains the gel Principle of the Test: based on controlled centrifugation of RBCS through a dextran acrylamide gel medium containing a predispensed reagent o The gel acts as a sieve to capture or trap RBC agglutinates/clumps after an ag-ab reactions o The sieve action of the gel uses size exclusion chromatography ▪ The molecules of the gel are slightly apart from each other due to its semisolid nature ▪ The space between molecules allows RBCs (unagglutinated) to pass through ▪ When RBCs agglutinate/clump, its size increases ▪ Increase in size make RBCs trapped by the gel • Bigger clump = higher position of RBC clumps on the gel matrix ▪ When RBCs are unagglutinated and small, they pass through the gel and they would settle at the bottom of the microtube Procedure (Low-ionic Antiglobulin): 1. Addition of cells (source of ag) 2. Addition of plasma/serum (source of ab) • Types Of Gel Test: o Specific - the reagent is incorporated/integrated in the gel matrix; the reagent is already predispensed ▪ Only requires to add the sample and follow other procedures (need for incubation, additional testing, etc.), before proceeding to centrifugation ▪ Specific for a particular target antibody/antigen o Neutral/Plain - gel is not yet predispensed w/ reagents ▪ Requires addition of reagent along with the sample in the microtube ▪ There is no reagent in the gel matrix 1 o • • Low Ionic Antiglobulin - specific for sensitization procedure (AHG) ▪ AHG reagent is added into the gel matrix o To Differentiate specific & neutral ▪ Specific - direct/forward/cell typing • The antisera are already predispensed in the gel matrix; just add RCS/sample • For detection of antigen ▪ Neutral - reverse/backward/indirect serum typing • Known RCS (reagent) & serum (sample) are added • For detection of antibody Advantages: o Standardization - major advantages of gel test tech ▪ In performing conventional methods in lab, medical technologists vary in performing techniques • One common varying technique is tube shaking to confirm presence of agglutination • Varying tube shaking can cause varying reading, grading, & interpretation ▪ Gel test technology no longer require tube shaking to confirm the presence of clump/sediment • The gel will capture/trap agglutinates ▪ Standardization increases accuracy of results o Provides a stable well-defined endpoints of agglutination reaction ▪ The gel matrix allows trapping of RBC clumps that is stabilized by the gel matrix ▪ The agglutination reactions in the gel medium can be reviewed/observed up to 3 days ▪ Slide method only allow reading within a few minutes due to rapid drying up ▪ Tube method only allow reading within 1 hour • After 1 hour, the specimen dries out o Decreased Sample volume needed for testing ▪ Microsampling is utilized ▪ Microsample - volume of the sample is <1 mL o Enhanced sensitivity and specificity of the result ▪ Cell washing is no longer performed • Cell washing - used to remove unbound abs & proteins which can interfere with detection ▪ Gel matrix will prevent excess proteins/abs to interfere with the reaction of antigens with specific antibody ▪ Removal of cell washing saves time and improves TAT Grading of Reactions o Only agglutinates are detected in gel test o Hemolytic reactions are not detected in gel test ▪ When RBCs are lysed and cellular content are released, Hgb is not trapped by the gel matrix which will settle at the bottom of the tube o Tube Method Grade % Ag Score Description of Appearance 4+ 100% 12 One solid clump, no free cells, clear supernatant 3+ 75% 10 Several large clumps, clear supernatant 2+ 50% 8 Several/Many medium-sized clumps, cloudy red supernatant 1+ 25% 5 Numerous small-sized clumps, cloudy red supernatant +/<25% 3 Numerous very small clumps easily (weak) dispersed, cloudy red supernatant Grade % Ag Score Description of Appearance 4+ 100% 12 One solid clump, no free cells, clear supernatant 3+ 75% 10 Several large clumps, clear supernatant 2+ 50% 8 Several/Many medium-sized clumps, cloudy red supernatant + <25% 1 Appears negative macroscopically, but agglutination visible microscopically 0 0 0 Negative, no agglutination reaction seen macroscopically H 0 Complete hemolysis, no intact RBCs remaining PH 0 Partial hemolysis, some RBCs still intact but hemolysis visible in supernatant mf 0 Mixed field-mixtures of agglutinated and agglutinated cells o o • ▪ % Ag - % of antigen reacted w/ specific antibody Gel Test ▪ 4+ - presence of RBCs in top most portion of the gel matrix • Indicates strong reaction, large clump/agglutinate formed in the reaction chamber ▪ 3+ - presence of RBCs on upper part of gel matrix ▪ 2+ - RBCs are dispersed/distributed on different parts of the matrix with most cells at the center ▪ 1+ - RBCs are found on lower part of gel matrix ▪ Weak (+/-) - RBCS are found near the bottom of the microtube ▪ 0 (Negative) - RBCs form cell button at the bottom of the tube; RBCs settled at the bottom Example ▪ Only the patient red cells were added Applications o Used in Blood banking o Used in serology in Immunology ▪ If the reagent is known ab, antigen can be detected o As long as the result is RBC agglutination, gel test can be used in any test 2 ANTIHUMAN GLOBULIN TEST (COOMB’S TEST) • Principle: Technique for detecting cell-bound immunoglobulin. It is used to detect incomplete antibodies (IgG). o Incomplete IgG antibodies are only capable of sensitizing RBC antigen ▪ Sensitizing - binding or coating red cell antigens o They cannot form lattice sequence o In the presence of AHG, cell-bound incomplete IgG are linked together to form lattice sequence o Lattice Formation - occur when antibodies cross-link with one another adjacent to their antigen ▪ Responsible for agglutination reaction (end product of lattice) ▪ Only a few antibodies can form lattice (IgM) • Its large size allows efficient lattice formation ▪ IgG - short antibody, cannot cross-link with another IgG • AHG will link IgG on cell surface to form lattice and produce agglutination • AHG makes it possible for sensitized IgG to form lattice and product agglutinates • IgM vs IgG o IgM ▪ Naturally occurring • Antigenic stimulation not required for production; no prior immunogenic stimulation required ▪ Complete (pentamer) • Biggest antibodies ▪ Agglutinating • Best agglutinating antibodies ▪ Cold-reactive (1-6C) ▪ Saline-reactive ▪ Ex. ABO ab ▪ Complement binding (more potent) • Eg. classical complement pathway o IgG ▪ Immune antibody • Requires immune stimulation for IgG synthesis ▪ Incomplete (Monomer) • Smallest antibody ▪ Coating/sensitizing • Due to its small size, it cannot form lattice on its own ▪ Warm-reacting (body temp;37C) • Clinically significant ▪ Albumin/protein & AHG-reactive ▪ Ex. Rh antibody ▪ Complement binding ▪ Subclasses in order of most to least potent • IgG 3 → IgG 1 → IgG 2 ▪ Focus in AHG testing AHG REAGENTS (COMMERCIALLY PREPARED) • Polyspecific AHG Reagents – consists of a pool of rabbit anti-human IgG & mouse monoclonal anti-C3b & anti-C3d. o Also referred to as broad spectrum Coombs reagent o Method of Preparation: Conventional/classic method ▪ Aka Hyperimmunization of rabbit (conventional) ▪ Preparation: • Rabbit is the test animal • Purified human sample is prepared and injected to the rabbit o Purified human sample contains human antibodies (IgG) and complement o Human proteins are immunogenic to the rabbit causing reaction • Rabbits’ immune system will produce AHG due to immunologic reactions o Polyspecific - the rabbit ab can react with human complement • The rabbit serum is collected and used as source of Polyspecific AHG reagent • Green dye is added to be distinguished with other types of reagent • Monospecific AHG Reagents – contains only one antibody specificity. o Either: ▪ Anti-IgG ▪ Anti-C3b or C3d o Method Of Preparation: Hybridoma Technique ▪ Aka Kohler and Milstein Technique ▪ Mouse plasma cell and myeloma cells are combined to fuse to produce hybridoma cell ▪ Hybridoma cell - immortalized ab-forming cell • Responsible for producing monospecific antibody (AHG) • Immortality is due to characteristic of cancer cell/malignant myeloma cell • Ab-forming capacity - due to mouse plasma cell STAGES OF ANTIGEN-ANTIBODY INTERACTION • Sensitization o The first stage o Occurs when antibodies react with antigens on the cells and coat the cells. o Fab portion of antibody interacts with epitope (ab attachment site) of the antigen ▪ Fab - antigen binding fragment 3 • Agglutination o The second stage of the reaction o Occurs when antibodies on coated cells form crosslinkages between cells resulting in visible clumping. o Crosslinking between antibodies (lattice formation) o Lattice formation can only occur if the abs are able to link with another ab adjacent to the ag o IgM - second stage is easy because it is long and thick o IgG - second stage is impossible ▪ For the second stage to occur, AHG will facilitate the crosslinking of IgG for lattice formtion ▪ o TYPES OF AHG PROCEDURES • Direct AHG Test (DAT) o Detects in vivo sensitization of red cells with IgG and/or complement. ▪ In vivo sensitization - RBCs of the px is sensitized with abs in the circulation o Useful in the ff. situations: ▪ Investigation of transfusion reactions (e.g.HTR) • Immediate TR - During or after transfusion of blood, within 24 hours, signs and symptoms manifest • Delayed TR - manifestation of signs and symptoms after 24 hours • Transfusion reactions during transfusion can be severe and fatal o Due to ABO incompatibility • Cause of TRs are investigated o Blood is collected from px and donor o Post-transfusion px blood is tested for DAT ▪ Confirms that the red cells are not entirely of px that there may be a mixture of px and donor’s blood o (+) DAT - px abs are sensitized with recently transfused donor cells ▪ Diagnosis of HDFN • Tests for red cells of the fetus collected through cord blood • HDFN - fetal RBCs are prematurely lysed/ destroyed by incompatible maternal antibodies • (+) DAT - confirmatory for HDFN; single most important procedure for HDFN diagnosis o Denotes presence of maternal antibodies (IgG) in fetal blood o Fetus and newborns have underdeveloped immune system; don’t have the capacity to produce own abs o If abs are detected to be attached on fetal RBCs, these are regarded as maternal antibodies (IgG) o Maternal IgG can actively cross placenta and actively sensitize the fetal cells o Maternal antibodies can provide protection to the baby if the abs are present in the neonatal circulation o Maternal ab sensitization of fetal cell lead to hemolysis ▪ Due to incompatible abs o Maternal antibodies should not sensitize fetal cells o o o o Diagnosis of autoimmune and drug-induced hemolytic anemias • Autoantibodies formed against self-antigen can sensitize px red cells in vivo • (+) DAT - presence autoimmune Cells used for DAT should be collected into either EDTA or citrate containing anticoagulant to minimize the possibility of in vitro attachment of complement components. ▪ EDTA & citrate have anti-complementary activity • For calcium ion chelation o C1 molecular complex is deactivated ▪ C1 - largest complement protein • Recognition unit • Inactivity will not allow in vitro complement attachment with RBC o Prevent positive reactions • Compose of C1q C1r C1s subunits • Held together by calcium ions • When calcium is absent, the C1qrs subunits are dissociated making the C1 molecule to be inactive The direct antihuman globulin test (DAT) is needed to demonstrate antibodies in the event of in vivo erythrocyte sensitization. Thus, antibodies or complement components already fixed to the patient's erythrocytes are detected. Following a triple washing process with the sensitised cells, the AHG serum is added. Conventional Method ▪ o Patient blood is collected in EDTA (lavender)/Citrate (l. blue) tube ▪ The blood is washed 3 times • Washing - to eliminate unbound/unsensitized antibodies (which can neutralize reagent AHG) ▪ Add reagent immediately ▪ Centrifugation ▪ Check for red cell agglutination Importance of Washing in DAT ▪ Presence of other unbound abs (non-blue) • Target of washing ▪ Washing phase • 3-5x - venous blood • 6-8x - cord blood (contains Wharton’s Jelly) o Wharton’s Jelly - a mucopolysaccharide which can promote rouleaux formation 4 ▪ Addition of AHG reagent • Immediate addition after washing • If there is a delay in the addition of reagent, antibodies may undergo spontaneous elution process (detachment of antibodies from ag) o Detachment will lead to false negative o ▪ IgG Bridge Formation • AHG reagent will bridge human IgG together to produce lattice formation • AHG - serves antibody o Aka anti-antibody o Antibody that would react to another ab • Human IgG - serves as antigen o Antibody in nature o In presence of AHG, it functions as ab • Multiple crosslinking of AHG will cause bridge formation or lattice formation to produce Agglutination o o o ▪ Agglutination Procedure ▪ Immediate spin ▪ Incubation/sensitization • Px serum and donor red cell are tested (major x-match) • Determines if the px has ab that will sensitize the donor red cells ▪ Addition of AHG reagent • Centrifuge → check for agglutination • (+) agglutination = (incompatible) • (-) = compatible 2 different samples from two individuals Weak D antigen testing ▪ ▪ IAT confirms Low-grade weak-D If patient is IAT confirmed with low-grade weak-D, the patient is reported as Rh (+) • Classified as Rh (+), belongs to 85% w/ D ag FACTORS AFFECTING THE AHG TEST • • Indirect AHG Test (DAT) o A two-step procedure (sensitization and agglutination that determines in vitro sensitization of red cells ▪ In vitro sensitization - outside the body in test tubes ▪ Sensitization - incubation stage/thermophase ▪ Agglutination - AHG phase o Useful in the ff. situations: ▪ Detection of incomplete antibodies in compatibility testing or to screening cells in antibody screen ▪ Identification of antigen specificity, using a panel of red cells ▪ Determination of red cell phenotype using known antisera (e.g. Du testing) ▪ Titration of incomplete antibodies (IgG) • • Ratio of serum to cells o Minimum ratio 40:1 = 2 drops serum and 1 drop of 5%v/v cell suspension o Prozone - excess antibody; false negative o Postzone - excess antigen; false negative o Unproportional ratio can lead to false negative o Always maintain proper proportion Temperature - Optimal: 37ºC (IgG) o Delayed test reaction due to improper temp o High temp - cause protein denaturation ▪ Can lead to destruction of abs = false neg o Low temp - IgG is inactive, there will be no sensitization; false neg Incubation Time – In saline suspension: 30-120 minutes o Low Ionic Saline Solution (LISS) suspension: 10-15 mins ▪ LISS - a potentiator which reduce zeta-potential ▪ Potentiators - shortens incubation period by increasing sensitivity by reducing zeta potential • Enhance/increase interaction/reaction of antigens and antibodies 5 ▪ • • • • • Zeta potential - force of electric repulsion between cells; delays reactions • ↑ ZP = RBCs repel each other o Normally, there is high ZP • ↓ ZP = RBCs react rapidly o ↓ ZP = = ↓ incubation time = ↑ sensitivity o Absence of potentiator do not affect the reaction but the incubation time is lengthened Reaction medium o 60-minute saline test = 30-minute albumin technique o 22% Albumin – 2 drops 22% albumin + 2 drops serum + 1 drop 3-5% cell suspension ▪ is said to reduce the zeta potential between o RBCs thus increasing the rate of antibody uptake on the cell o LISS – 2 drops 3% RBC suspension in LISS + 2 drops serum ▪ also increases sensitivity and shortens incubation times Washing of cells – minimum of three times o 3-5x - venous blood o 6-8x - cord blood o For conventional method o Not used for gel test o Improper/Incomplete washing - false negative due to neutralization of unbound abs to the reagent AHG Saline for washing – should be fresh and buffered to a pH of 7.2-7.4 o Extreme pH can cause protein denaturation ▪ Antibodies may be inactivated (false neg) Addition of AHG reagents should be added to washed cells immediately after washing. o Delay in addition can cause spontaneous elution that can lead to false negative Centrifugation - 1000 rcf for 15-20 seconds, o High speed and long time - false positive o Slow speed and short time - false negative • AUTOMATED AHG TECHNOQUE: • • SOURCES OF ERROR IN THE AHG TECHNIQUE • • False-Positive Results (decreased specificity) o Autoagglutinable cells o Bacterial contamination or other contamination in cells or saline o Cells with a positive DAT used for IAT o Overcentrifugation and overreading o Polyagglutinable cells o Dirty glass wares o Saline contaminated with silica or heavy metals False Negative Results (decreased sensitivity) o Inadequate or improper washing of cells (most common cause) ▪ AHG neutralized by unbound or unsensitized abs o AHG reagent nonreactive owing to deterioration or neutralization o AHG reagent not added o Serum not added in the indirect test o Serum nonreactive owing to deterioration of complement o Inadequate incubation conditions o Postzone and Prozone (cell suspension either too weak or too heavy) o Under centrifugation o Poor reading technique Importance of Washing • Low Ionic Polybrene Technique (LIP): o Polybrene - rouleaux promoting agent ▪ Enhance/promote interaction o Low ionic environment allow sensitized RBCs to interact with one another o Interaction of sensitized RBCs are enhanced Enzyme-Linked Antiglobulin Test (ELAT): o RBCs with IgG are used as primary antibody o Anti-Human IgG contains labeled enzyme o AHG + sensitized RBC = activation of labeled enzyme + substrate = change in color ▪ Substrate will change in color when there is AHG sensitized red cell binding ▪ Intensity of substrate color change is directly proportional on the quantity of the sensitized abs present in the sample o Also used to quantitate abs bound to red cells o Uses colorimeter or spectrophotometer to measure absorbance and quantitate antibodies sensitizing or bound to RBCs ▪ Color reaction determine the quantity/ concentration of antibodies bound to RBCs Solid Phase Method (Direct And Indirect): o Occurs in microwells predispensed with antigens o Antigens are bound to the bottom of the well o Px plasma/serum is incubated into the well ▪ Source of antibody 6