Immunohematology Lab - 01 Intro to Blood Banking

IMMUNOHEMATOLOGY LABORATORY INTRODUCTION TO BLOOD BANKING; BLOOD BANKING APPARATUS, EQUIPMENT, AND REAGENTS; CELL WASHING; & PREPARATION OF RED CELL SUSPENSION OUTLINE • • • • Introduction • o Laboratory Safety o Grading Agglutination Reactions Blood Banking Apparatus, Equipment, And Reagents o Commonly used Blood Banking Equipment and Apparatus o Reagents Commonly used in Blood Bank Laboratory Introduction to Cell Washing o Why is Cell Washing Performed? o Cell Washing Techniques in Preparing Red Cell Suspension o Discussion o Preparation Cell Suspension GRADING AGGLUTINATION REACTIONS • • • • • • INTRODUCTION LABORATORY SAFETY STANDARD PRECAUTIONS AND LABORATORY TEST • • Universal precautions are infection control guidelines designed to protect workers from exposure to diseases spread by blood and certain body fluids. Universal precautions stress that all patients should be assumed to be infectious for blood-borne diseases such as AIDS and Hepatitis B, since all patients infected with blood-borne pathogens cannot easily be readily identified. The clinical laboratory contains a variety of safety hazards, many of which are capable of producing serious injury or life-threatening disease. To work safely in this environment, laboratory personnel must learn what hazards exist; the basic safety precautions associated with them, and must apply the basic rules of common sense required for everyday safety. Some hazards are unique to the health-care environment and others are encountered routinely throughout life. SAFETY PRECAUTIONARY MEASURES IN THE LAB THAT YOU HAVE TO KEEP IN MIND (SUMMARY) 1. 2. 3. 4. 5. 6. 7. 8. Consider ALL patients’ blood and body fluids to be biohazardous regardless of diagnosis. Always wash hands before and after contact with patients. Wear gloves and waterproof aprons, mask and goggles if splashing can occur during wound sterilization, endoscopy, dialysis or postmortem procedures. Wear gloves when handling blood, body fluids, tissues or contaminated surfaces. Dispose of all needles in puncture-proof boxes, which must be accessible in all rooms. Minimize mouth-to-mouth transmission by keeping mouthpieces readily available on crash carts and in all areas where this need is possible. Clean blood and body fluid spills with a solution bleach (10%) and water or with hospital disinfectant. Immediately report all needle sticks, accidental splashes and contamination of wounds by body fluids • It is essential to learn how to properly grade agglutination reactions since most of the tests performed in Blood Bank laboratories involve agglutination reaction as an end product of the procedure. Antigen = Agglutinogen Antibody = Agglutinin Principle o The purpose of grading reactions is to allow comparison of reaction strengths. This purpose is beneficial in detecting multiple antibody specificities or antibodies exhibiting dosage. Materials o Centrifuged serologic tests for agglutination. o Agglutination viewer Procedure 1. Gently shake or tilt the tube to resuspend the red cell button in the tube. The tilt technique uses the solution meniscus to gently dislodge the red cell button from the wall of the tube. 2. Observe the way that cells are dispersed from the red cell button. 3. Record reactivity by comparing the agglutinates to the descriptions in Table 1-8-1.The reactivity should be assessed when the red cells have been completely resuspended from the button. Interpretation of Agglutination Reactions Macroscopically Observed Findings Designation Score One solid agglutinate 4+ 12 Several large agglutinates 3+ 10 Medium-size agglutinates, clear 2+ 8 background Small agglutinates, turbid background 1+ 5 Very small agglutinates, turbid background 1+w 4 Barely visible agglutination, turbid w+ or +/2 background No agglutination 0 0 Mixtures of agglutinated and mf unagglutinated red cells (mixed field) Complete hemolysis H Partial hemolysis, some red cells remain PH BLOOD BANKING APPARARATUS, EQUIPMENT, AND REAGENTS • Laboratory equipment and reagents play a key role in experiments and for you to understand the concepts of Blood Banking and Transfusion Medicine. COMMONLY USED BLOOD BANKING EQUIPMENTS AND APPARATUS • • Incubator/Water bath o used for setting the reaction temperature to 37 degrees Celsius (for IgG) o also used to inactivate complement proteins at 56 degrees Celsius o temperature must be maintained to ensure agglutination reactions Viewing box o used in Rh typing to visualize antigen-antibody reactions o has a surface temperature of 37 degrees Celsius • Microscope - demonstrate microscopic reactions such as small agglutinates Test tubes - are used for macroscopic antigen-antibody reactions; for storage/container Glass slides - for macro and microscopic reactions; to contain sample for short time Magnifying glass - used to demonstrate minute reaction in absence of microscope Graduated cylinder - used to measure large volume of fluid Pipet/Droppers - used to transfer small volume of liquids/ fluids Calibrated centrifuge tube o type of tube with calibration marks used to measure the portions of blood after centrifugation Applicator stick - used the mix the sample and reagents • • • • • • REAGENTS COMMONLY USED IN BLOOD BANK LABORATORY • Typing Sera o Used to detect unknown antigens on patient red cell o Has known antibodies o It is used in forward or direct blood typing o Note: Typing sera contains antibodies (Known) therefore it detects antigen (unknown) on patient red cell o Specimen of Choice: Patient red cell suspension o Examples: ▪ Anti-A typing serum - detects A antigen (azul) ▪ Anti-B typing serum - detects B antigen (banana) ▪ Anti-AB typing serum - detects AB antigen, also useful for ABO subgroupings • Known Red Cell Suspension o Used to detect unknown antibodies in the patient’s serum; antigen is known o Specimen: serum sample o Used in indirect/Reverse blood typing o Known RCS is only prepared daily because it is only valid for 24 hours o Known A RBCs - used to detect Anti-A antibodies o Known B RBCs - used to detect Anti-B antibodies o Known O RBCs - used to detect Anti-A, B, and AntiAB antibodies • AHG Reagent (Antihuman globulin reagent) o Aka Coomb’s reagent o It is used to detect cell bound incomplete IgG antibody sensitization o Green in color Serofuge o type of centrifuge used to separate liquids from solid components from the blood • • o • • Lectins o these are antibody-like agents derived from plant extracts that reacts with a particular antigens o Anti-H Lectin derived from Ulex europeus o Anti-A Lectin derived from Dolichos biflorus o Anti-M Lectin derived from Iberis amara o Anti-N Lectin derived from Vicea graminea 22% Bovine albumin/Low Ionic Strength Solution (LISS) o also known as potentiators o Potentiators promotes decrease in zeta potential (lower agglutination rate) and incubation time which promote test sensitivity ▪ ↑ ZP = ↑ incubation = ↓ sensitivity ▪ ↓ ZP = ↓ incubation = ↑ sensitivity o Potentiators increase test sensitivity TECHNIQUES IN PREPARING RED CELL SUSPENSION • DISCUSSION • • INTRODUCTION TO CELL WASHING • In Blood Banking, it is essential to perform cell washing prior to performing series of procedures. In this module, you will be able to know about the concepts of cell washing and why is it a vital procedure in blood bank laboratories. WHY IS CELL WASHING PERFORMED? • • • To remove excess antibodies present in the sample To remove excess proteins present in the cell surface. Tests that require cell washing o ABO Typing o Rh typing o Antibody Screening o Crossmatching/Compatibility Testing o MN Typing o AHG/Coomb’s Test CELL WASHING • • Cell washing is a common procedure performed in the laboratory. This is a series of careful steps taken to wash red blood cells normally three times intermittently with centrifugation and decanting. o NSS → Centrifuge → Decant Overwashing may remove antigens MATERIALS • • • • 10 or 12 x 75 mm test tubes Serofuge Saline (NSS) Fresh blood (expired blood for experiment) PROCEDURE 1. 2. 3. 4. 5. 6. 7. Place 3 cc of blood into a small test tube (10 x 75 mm). Fill the test tube with saline solution. o Use fresh NSS to prevent hemolysis Centrifuge for 1 minute at 1000-2000 r.p.m. Decant the supernatant saline solution in a quick manner to avoid disturbing the packed RBC. Fill again the test tube with saline solution. Repeat procedures 3 and 4. Do this 2-3 times. The remaining packed RBC in test tube is washed RBC. The reactivity of blood group antigens may be weakened or lost after prolonged storage. The length of time test red cells may be stored is determined by the type of anticoagulant used and the storage conditions. Anticoagulants in use include oxalate, EDTA, ACD, CPD, Alserver’s, etc. Storage stability is enhanced by glucoses. Red cells should be stored 4°C and bacterial contamination and hemolysis should be avoided. Red cells may be made up in different suspension mediums; i.e., saline, albumin. Group AB serum. etc. Dilute suspensions should be freshly prepared each day and stored at 4°C often contain preservatives (antibacterials) which permit longer storage (consult manufacturer’s directions). Suspension should be washed free of any hemoglobin. If hemolysis is excessive, red cell stroma which would compete for antibody. The concentration of the red cell suspension needed for blood group will vary depending on the technique being used. For routine procedures, it is not necessary to prepare accurately measured suspensions. Periodically, an accurate suspension of known strength should be prepared in the size of test tube usually used. This may then be compared with the estimated suspensions as customarily utilized. PREPARATION OF RED CELL SUSPENSION PRINCIPLE • PRINCIPLE OF THE TEST • Before we proceed to certain blood bank procedures, it is important that we must know how to prepare the red cell suspension correctly if we want to observe accurate results. A red cell suspension is a common reagent used for many serologic procedures. Red cell suspensions provide the appropriate serum to cell ratio to allow for grading and interpretation of tests results. MATERIALS • • • • • • • • • • Big test tubes centrifuge 10 or 12 x 75 mm test tubes serofuge Applicator sticks 22% bovine albumin Droppers AB serum Cylinder expired blood PROCEDURES • RESULTS OF THE EXPERIMENT • Ideally, a prepared RCS must have a "tomato red" color • • • • In practice, 2-5% RCS volume per volume concentration is being prepared. Diluent used for RCS preparation o NSS (Isotonic)- 0.85 to 0.9% salt solution o In absence of NSS, you will use: ▪ Blood type AB serum ▪ Bovine albumin In preparing RCS, you will need: o Washed Packed RBC o Diluent (NSS) In calculating the concentration desired in preparing red cell suspension: o To calculate the Total Volume of RCS TV RCS = PCV x 100 % RCS Concentration ▪ NOTE: • TV RCS = Total Volume of RCS (mL) • PCV = Packed Cell Volume (mL) • % RCS= Desired RCS concentration o To calculate the volume of the diluent TV of diluent = TV RCS - PCV ▪ NOTE: • TV RCS = Total Volume of RCS • PCV = Packed Cell Volume Sample Problem: A medical technologist wants to prepare a 2% blood type A RCS using 0.1 ml of PRBC o Given % RCS conc. = 2% PCV = 0.1 mL o TV RCS TV RCS = PCV x 100 % RCS Concentration TV RCS = 0.1 mL x 100 2% TV RCS = 5 mL o TV diluent TV diluent = TV RCS - PCV TV diluent = 5 mL - 0.1 mL TV diluent = 4.9 mL Let us derive the formula: o After computing the tv of rcs, let us now compute for the tv of the diluent: o RCS PREPARATION • o • • Conclusion: To prepare a 5mL of 2% RCS, you need to add 4.9 mL of NSS to 0.1 mL of RBC Ideal outcome of your prepared red cell suspension o The suspension must be “TOMATO RED” in color o Too light suspension= excess NSS o Too dark suspension= excess packed RBCs Practice: A medical technologist was assigned in the blood bank to prepare a 5% blood type O RCS using 0.5 ml of PRBC. Show your solution and complete conclusion. o TV RCS = (0.5 mL / 5%) x 100 = 10 mL o TV diluent = 10 mL - 0.5 mL = 9.5 mL o Conclusion: To prepare a 10 mL of 5% RCS, you need to add 9.5 mL of NSS to 0.5 mL of RBC.