Table of Contents. Preface... xv. Editorial Comments... xvii. About the Authors... xix

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1 Preface xv Editorial Comments xvii About the Authors xix Section I. Routine Tube Methods I-A. Typing for ABO and Rh I-B. Testing for Unexpected Antibodies: LISS Additive Procedure I-C. Crossmatching by LISS Antiglobulin I-D. Weak D Testing Procedure Table I-D-1. Interpretation Recommendations for Direct and Indirect Tests with Anti-D I-E. Crossmatching by Immediate-Spin I-F. Grading and Scoring Serologic Reactions Table I-F-1. Grading Serologic Reactions I-G. Intralaboratory Competency Testing: IAT Reading and Grading Procedure Section II. Antibody Detection II-A. Testing by Albumin Addition/Layering II-B. Testing by Albumin Antiglobulin II-C. Testing by Enzyme Antiglobulin II-D. Testing by LIP Antiglobulin II-E. Testing by LISS Wash Antiglobulin II-F. Testing by LISS-Ficin Antiglobulin II-G. Testing by Polyethylene Glycol Antiglobulin II-H. Testing for Cold-Reactive Antibodies by Direct Agglutination II-I. Testing by Saline Indirect Antiglobulin Section III. Enzyme Techniques III-A. Detecting Antibodies with the Bromelain One-Stage Method III-B. Treating RBCs with -Chymotrypsin vii-

2 Judd s Methods in Immunohematology III-C. Detecting Antibodies with the Ficin One-Stage Method III-D. Treating RBCs with Ficin III-E. Treating RBCs with Neuraminidase (T-Activation) III-F. Detecting Antibodies with the Papain One-Stage Method III-G. Treating RBCs with Papain III-H. Treating RBCs with Pronase III-I. Treating RBCs with Trypsin (Crude Enzyme) III-J. Treating RBCs with Trypsin (Purified Enzyme) Section IV. Elution Techniques IV-A. Washing Before Eluting IV-B. Eluting Antibodies with Chloroform IV-C. Eluting Antibodies with Chloroform/Trichloroethylene IV-D. Eluting Antibodies with Citric Acid IV-E. Eluting Antibodies with Cold Acid IV-F. Eluting Antibodies from Stroma IV-G. Eluting Antibodies with Ether IV-H. Eluting Antibodies by Freezing-Thawing (Lui Method) IV-I. Eluting Antibodies by Freezing-Thawing (Wiener Method) IV-J. Eluting Antibodies by Heat IV-K. Eluting Antibodies with Methylene Chloride IV-L. Eluting Antibodies with Microwaves IV-M. Eluting Antibodies from Placental Tissue IV-N. Eluting Antibodies Using Ultrasound IV-O. Eluting Antibodies with Xylene/D-Limonene IV-P. Dissociating of IgG with EDTA-Glycine-HCl IV-Q. Dissociating of IgG with Chloroquine Diphosphate Section V. Cell Separation Methods V-A. Harvesting Autologous RBCs by Direct Centrifugation V-B. Harvesting Autologous RBCs Using Phthalate Esters V-C. Cell Separation Using Percoll-Renografin Procedure V-D. Harvesting Autologous RBCs in Hemoglobin S or Sickle Cell Disease viii-

3 V-E. Separating Mixed-Cell Populations with Antibodies V-F. Mononuclear Cell Separation Procedure Section VI. Alternative Serologic Methods for Antibody Detection VI-A. Capillary: Saline Agglutination Procedure VI-B. Capillary: Albumin One-Layer Procedure VI-C. Capillary: Albumin Two-Layer Procedure VI-D. Capillary: Papain One-Stage Procedure VI-E. Capillary: Ficin One-Stage Procedure VI-F. Capillary: IAT on Precoated RBCs Procedure VI-G. Capillary: Precoating for IAT Procedure VI-H. Microplate: ABO and Rh Typing Procedure VI-I. Microplate: Antibody Detection Using LISS Procedure VI-J. Microplate: LIP Technique Procedure VI-K. Gel: Preparing Buffered Gel Columns VI-L. Gel: Preparing Anti-IgG Columns VI-M. Gel: Detecting Antibodies Using Buffered Gel Columns VI-N. Gel: Detecting Antibodies Using Anti-IgG Columns Appendix VI-1. Capillary Methods: General Considerations Appendix VI-2. Microplate Tests: General Considerations Appendix VI-3. Sephadex Gel Beads: Background Information Appendix VI-4. Agglutination Reactions in Gel Columns Section VII. Reagent RBCs (Non-Enzyme-Treated) VII-A. Preparing C3b/C4b-Coated RBCs VII-B. Preparing IgM/C3b-Coated RBCs VII-C. Preparing C3b-Coated RBCs: Fruitstone Method VII-D. Preparing C4b-Coated RBCs VII-E. Preparing Immunoglobulin-Coated RBCs VII-F. Preparing IgG-Coated RBCs VII-G. Treating RBCs: AET Procedure VII-H. Treating RBCs: DTT Procedure VII-I. Inactivating S Antigen: Sodium Hypochlorite Treatment ix-

4 Judd s Methods in Immunohematology VII-J. Freezing RBCs: Glycerol Preservation and Recovery VII-K. Freezing RBCs: Liquid Nitrogen Preservation and Recovery Section VIII. Antibody Identification VIII-A. Identification of Single or Multiple Antibodies (Process) Figure VIII-A-1. Considerations in investigating unexpected antibodies Figure VIII-A-2. Considerations in investigating blood samples with a positive autocontrol Table VIII-A-1. Serologic Characteristics of Some Alloantibodies to RBC Antigens VIII-B. Management of Samples and Patients with Cold Agglutinins (Process) VIII-C. Evaluation of Panel Results: Crossing Out (Process) Figure VIII-C-1. Antigens crossed out on the top of the antigen profile Figure VIII-C-2. Antigens crossed out on each individual panel cell VIII-D. Selected Cells Process VIII-E. Selection of Antigen-Negative Blood for Transfusion (Process) Table VIII-E-1. Selection of Antigen-Negative Blood for Transfusion VIII-F. Determination of Probability Levels (Process) Table VIII-F-1. Probability Values VIII-G. Acidifying Sera VIII-H. Using a Combined Adsorption/Elution Procedure VIII-I. Detecting Trypsin-Resistant NVG Receptors VIII-J. Using Soluble Blood Group Substances in an Inhibition Procedure Table VIII-J-1. Antibody Inhibition by Blood Group Substances VIII-K. Inhibiting Rouleaux with DIDS VIII-L. Identifying Anti-N form VIII-M. Detecting Complement-Fixing Antibodies by a Two-Stage EDTA-IAT Appendix VIII-1. Evaluation of Initial Antibody Identification Panel Appendix VIII-2. Approaches to Identify/Confirm Antibody Specificity Section IX. Investigating Antibodies to High-Prevalence Antigens Table IX-1. Effect of Papain and Thiol Reagents on Reactions of Antibodies to High-Prevalence RBC Antigens Table IX-2. Effects of Enzymes and Chemicals on Reactions of Antibodies to High-Prevalence RBC Antigens x-

5 Figure IX-1. Investigation of antibodies to high-prevalence antigens IX-A. Inhibiting Antibodies with Pooled Plasma or Serum IX-B. Identifying Anti-Ch/Rg: Rapid Procedure IX-C. Recognizing Antibodies To HLA Determinants IX-D. Using Allogeneic C4 for Adsorption of Anti-Ch/Rg IX-E. Using 2 M Urea for the Screening/Confirmation of Jk(a-b-) RBCs Section X. Prenatal and Perinatal Testing X-A. Tests During Pregnancy (Process) X-B. Rh Immune Globulin Administration (Process) X-C. Evaluation of HDFN at Delivery (Process) Figure X-B-1. Flow diagram for Rh Immune Globulin administration X-D. Prenatal Antibody Titration Procedure X-E. Screening for Fetal Rh-Positive RBCs in Maternal Rh-Negative Samples X-F. Quantifying Anti-D with the Enzyme-Linked Antiglobulin Test X-G. Screening for Large Fetomaternal Hemorrhage (Modified Kleihauer-Betke Acid-Elution Test) X-H. Quantifying Fetomaternal Hemorrhage with the Enzyme-Linked Antiglobulin Test X-I. Analyzing Amniotic Fluid by Spectrophotometry X-J. Treating Cord Blood Contaminated with Wharton's Jelly Section XI. Investigating Samples Containing Autoantibodies Table XI-1. Specificity of Cold-Reactive Autoantibodies: Comparison of Reactions with Untreated O, I+ RBCs XI-A. Investigation of Cases of Immune Hemolysis and/or a Positive DAT (Process) XI-B. Antiglobulin Testing: Direct Tests with Anti-IgG and Anti-C XI-C. Antiglobulin Testing: Direct Tests with Anti-IgM and Anti-IgA XI-D. Antiglobulin Testing: Direct Tests with Polybrene XI-E. Antiglobulin Testing: Enzyme-Linked DAT XI-F. Dispersing Autoagglutination XI-G. Testing the Thermal Amplitude of a Cold-Reactive Autoantibody XI-H. Diagnostic Testing of a Cold-Reactive Autoantibody by Titration XI-I. Determining Specificity of a Cold-Reactive Autoantibody by Titration xi-

6 Judd s Methods in Immunohematology XI-J. Adsorbing Cold-Reactive Autoantibodies with Autologous RBCs XI-K. Adsorbing Cold Autoantibodies with Heterologous (Rabbit) RBCs XI-L. Testing for PCH Using the Donath-Landsteiner Test XI-M. Determining the Specificity of Warm-Reactive Autoantibodies by Titration XI-N. Adsorbing Warm-Reactive Autoantibodies with Enzyme-Treated Autologous RBCs XI-O. Adsorbing Autoantibodies with ZZAP-Treated Autologous RBCs XI-P. Adsorbing Autoantibodies with ZZAP-Treated Autologous RBCs (Micromethod) XI-Q. Adsorbing Warm-Reactive Autoantibodies with Allogeneic RBCs Section XII. Investigating Drug-Induced Hemolysis Table XII-1. Drugs Reported to Cause Immune Hemolysis and a Positive DAT XII-A. Dissolving Drugs in Solution XII-B. Preparing Urine for Detecting Metabolite-Dependent Antibodies XII-C. Detecting Drug-Dependent Antibodies Reacting with Drug-Treated RBCs XII-D. Detecting Antibodies to Semisynthetic Penicillins XII-E. Detecting Cephalothin-Dependent Antibodies XII-F. Detecting Drug-Dependent Antibodies Reacting in the Presence of Drug XII-G. Detecting Metabolite-Dependent Antibodies Reacting in the Presence of Drug Section XIII. Investigating ABO Typing Problems Table XIII-1. Causes of ABO Typing Discrepancies Table XIII-2. Some Anomalous or Uncommon ABO Typing Results Figure XIII-1. Flow chart for the investigation of ABO discrepancies XIII-A. Investigation of Mixed-Field Reactions (Process) XIII-B. Investigation of Nonconcordant ABO Typing Reactions (Process) XIII-C. Evaluating an ABO Discrepancy XIII-D. Using Protease-Treated RBCs for ABO Typing Resolution XIII-E. Confirming Weak A Or B Antigen Expression by Adsorption and Elution XIII-F. Detecting Minor RBC Populations and ABO Mixtures XIII-G. Determining ABH Secretor Status XIII-H. Determining H Antigen Expression xii-

7 XIII-I. Separating A:O Mixtures XIII-J. Detecting A and B Transferases by Conversion of Group O RBCs XIII-K. Selecting Plasma Containing High-Titer Anti-A and/or Anti-B Section XIV. Investigating RBC Polyagglutination Table XIV-1. Reactions of Polyagglutinable RBCs with Lectins XIV-A. Detecting Polyagglutination with AB and Cord Blood Serum XIV-B. Preparing Crude Extracts of Lectins XIV-C. Preparing Solutions of Purified Lectins XIV-D. Aggregating RBCs with Polybrene XIV-E. Acetylating RBCs XIV-F. Inhibiting Acquired-B Reactivity XIV-G. Testing RBCs with Acidified Anti-B Section XV. Miscellaneous Methods XV-A. Testing for PNH: Acid Hemolysis (Ham's) Test Table XV-A-1. Acid Hemolysis Test Procedure XV-B. Testing for HEMPAS: Modified Acid Hemolysis (Ham's) Test XV-C. Converting IgG Antibodies to Direct Agglutinins XV-D. Assessing RBC Survival Using 51 Cr-Labeled RBCs XV-E. Differentiating IgM from IgG Antibodies Using DTT Table XV-E-1. Interpretation of 2-ME/DTT Studies to Determine Immunoglobulin Class of Antibody XV-F. Differentiating IgM from IgG Antibodies Using 2-ME XV-G. Performing Dosage Studies Using the Enzyme-Linked Antiglobulin Test XV-H. Predicting Clinical Significance Using the Monocyte Monolayer Assay XV-I. Screening for PNH: Sucrose Hemolysis Test XV-J. Isolating Genomic DNA from Blood XV-K. Converting Plasma to Serum Appendices Index xiii-

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