Immunohematology Lab - 03 Methods for Rh and MN Blood System

IMMUNOHEMATOLOGY LABORATORY • LABORATORY METHODS FOR RH BLOOD GROUP & MN TYPING OUTLINE • The Rh Blood Group System o Historical Perspective o Rh Blood Group System o Rh Nomenclature o Rh Typing o Rh Typing Procedures • Weak D Testing o Lab Discussion o Weak D Variations o Testing of Weak D • M-N Typing o Clinical Significance o Lab Discussion o M-N Typing Procedures • THE RH BLOOD GROUP SYSTEM HISTORICAL PERSPECTIVE • • • • Before 1939 - ABO Blood Group System is the only clinically significant blood group system o 1901 - ABO blood group system was discovered As 1930s ended - 2 important discoveries made in the field of Immunohematology 1939 o Levine and Stetson described a hemolytic transfusion reaction involving an obstetrical patient. o After delivering to a stillborn child, the mother needed blood transfusion, so the husband of the patient was chosen to give blood. The ABO blood type of the husband was compatible to the wife. After transfusion of blood, the wife developed transfusion reaction. In this case, they found out that the husband possess antigen that was not found on the RBC of the wife. o In the same year, they discovered the “D” antigen 1940 o Landsteiner and Wiener reported on the antibody produced by rabbit and guinea pigs when Rhesus Monkey’s (Rh) RBCs were transfused to them o Monkey red cells are foreign to guinea pigs and rabbits so they will produce antibodies against the monkey red cells. The abs produced by these animals was able to react to 85% of human’s population. The antibody produced is known as “Anti-Rh” o The same ab produce by guinea pigs and rabbits that were able to react with animal cells was later on named “Anti-LW” RH BLOOD GROUP SYSTEM • • • • • • Is a clinical blood group system just like ABO In routine blood typing RH specifically, D antigen is routinely tested (identifying presence of D ag) Considered as most complex and most extensive because this blood group system is comprised of some 50 different antigens unlike ABO system that only have 3 ags (A, B, H). Out of 50 different antigens, there are 5 principal antigens of clinical significance such as D, C, E, c, e. The inheritance of these antigens are similar to ABO which is through codominance For the 5 principal antigens, there are 3 genes involve for the regulation and expression of these antigen o RHD o RHCE o RHAG • RHD o Responsible for the presence or absence of D ag o If D ag is present, the person is Rh (+) (85% of people) o If D ag is absent, the person is Rh (-) (15% of people) ▪ Rh (-) is common among Caucasians o Rh negative phenotype is clinically significant among female patient on their child-bearing years because of risk for hemolytic disease of fetus and newborn (HDFN) which may cause severe anemia, jaundice o Found in chromosome 1 which is same locus for the hematologic condition known as hereditary elliptocytosis RHCE o Responsible for expression & regulation of C, E, c, e o Found in chromosome 1 o Considered as alleles w/ RHD as they have same locus RHAG o AKA co-expressor gene because it is important for the expression of the principal Rh antigens. o Allows full expression of the 5 principal antigens o Responsible for the formation of Rh-antigen like polypeptides on RBCs o These polypeptides are critical for the expression of principal antigens because without it, they principal antigens are not expressed. o They are present in Chromosome 6 RH NOMENCLATURE • 2 Classification: o Genetically-associated ▪ It has genetic basis ▪ Based on the 5 principal antigens ▪ Example: Fisher-Race (DCE), Weiner (Rh-Hr) o Non-genetically associated ▪ It has no genetic basis ▪ Example: Rosenfield, ISBT FISHER-RACE/ DCE TERMINOLOGY • • • • • The 5 principal antigens are produces by 3 closely link sets of alleles (D,C,E). The five major antigens defined are D, C, E, c, e Out of the 5 antigens only D antigen lacks the antithetical partner. “d” antigen in fisher-race terminology is not considered a true antigen. o “d” - represent Rh negative It is used to denote absence of D antigen. Therefore, d antigen can be used to represent Rh negative. Why DCE? Because the C is present between D & E allele. o Based on order of discovery o D - more clinically significant among the 5 WIENER/ Rh-Hr Terminology • • • • • Rh antigen has series of blood factors, in which each factor is an antigen recognized by an antibody The blood factor is considered as antigen that is recognized by a specific antibody. The gene in Weiner is a mosaic comprising of number of blood factors. Blood factors comprise the Rh antigen. Example: Antigen - Rho Blood factor: Rho, hr’, hr” o ROSENFIELD / ALPHA NUMERIC TERMINOLOGY • • • • • • It has no genetic basis and is based according to the order of discovery. D antigen is 1st discovered among other antigen (1939) thus, o D = Rh 1 (space between “Rh” and “1”) o C = Rh 2 o E = Rh 3 o c = Rh 4 o e = Rh 5 Sample 1: o (+) (+) (-) (-) (-) o Rh 1 2 -3 -4 -5 Sample 2: o (+) (-) (+) (+) (-) o Rh 1 -2 3 4 -5 Sample 3: o Application using antisera: Anti D = + Anti C = Anti E = + Anti c = + Anti e = + o Rosenfield manner of reporting: (spaced) ▪ Rh: 1 -2 3 4 5 o *If antiserum is not available in the laboratory, omit the number because it is not tested. Sample 4: o (+) (+) (N/A) (-) (+) o Rh 1 2 -4 5 (ISBT) INTERNATIONAL SOCIETY OF BLOOD TRANSFUSION • • • • • It aims to provide machine and eye readable system of reporting or expressing red blood cell antigen It aims to standardize and unify the manner of reporting of the red blood cell antigen. It has also no genetic basis. Old ISBT o Has 6 digits o Numerical code was used for reporting ▪ First 3 digits = Blood group system • ABO= 001 • Rh= 004 ▪ Last 3 digits= Specific antigens • D = 001 • C = 002 • E = 003 • c = 004 • e = 005 o Thus, for Rh system: ▪ D = 004001 ▪ C = 004002 ▪ E = 004003 ▪ c = 004004 ▪ e = 004005 New ISBT o Alpha numeric code is used same with Rosenfield o The alphabetic name of the blood group system is used in upper case. Ex. Rh= RH o Antigens are represented by number ▪ D=1 ▪ C =2 ▪ E=3 ▪ c=4 ▪ e =5 Thus, for Rh system: ▪ D = RH1 (no space between “RH” and “1”) ▪ C = RH2 ▪ E = RH3 ▪ c = RH4 ▪ e = RH5 CONVERSION: WEINER TO FISHER-RACE • • • • • • • • • • • • • • • • • • • Weiner R r w/ 1 or ‘ w/ 2 or “ w/ z or y w/o 1 or ‘ w/o 2 or “ w/ o Remember: o R = subscripts (1,2,z,o) o r = superscripts (‘,”,y) Ex. 1: DCe = R1 Ex. 2: DcE = R2 Ex. 3: DCE = Rz Ex. 4: Dce = Ro Ex. 5: DcE/Dce = R2Ro Ex. 6: DCE/DCe = RzR1 Ex. 7: dCe = r’ Ex. 8: dcE = r’’ Ex. 9: dCE = ry Ex. 10: dce = r Ex. 11: dcE/dCE = r’’ry Ex. 12: dCe/dce = r’r Ex. 13: DcE/dcE =R2r’’ Ex. 14: DCE/dce = Rzr Ex. 15: R1r’ = DCe/dCe Ex. 16: Ror = Dce/dce Ex. 17: Rzry = DCE/dCE Ex. 18: r’’r = dcE/dce Fisher-race D (Rh +) Absence of D (Rh -) C E CE c e ce CONVERSION: FISHER-RACE TO BLOOD FACTOR • • • • • Fisher-Race D (Rh +) C E c e C c E e D = Rho C = rh’ E = rh” c = hr’ e = hr” Blood factors Rho rh hr ‘ ‘’ • • • • • • • • Ex. 1: DcE = Rhohr’rh’’ Ex. 2: DCE = Rhorh’rh’’ Ex. 3: Dce = Rhohr’hr’’ Ex. 4: dcE = hr’rh’’ Ex. 5: R2R1 = DcE/DCe = Rhohr’rh’’/Rhorh’hr’’ Ex. 6: Rzr = DCE/dce = Rhorh’rh’’/hr’hr’’ Ex. 7: DcE/Dce = Rh 1 2 3 4 5 Ex. 8: DCE/dCe = Rh 1 2 3 -4 5 PROCEDURE • RH TYPING • • • • • This term usually refers to the determination of the presence or absence of the D antigen on the red cells under test. Because of its clinical importance, the term “Rh positive” and “Rh negative” are used depending on the presence or absence of agglutination with the ant-D serum. In general, tests for other antigens in the Rh system are performed in the same manner, by the saline tube test, the slide test, or the modified tube test. The Rh slide test should be done with a high concentration of both protein and red cells. Therefore, the typing serum must have a protein concentration of 6 percent or more, preferably 25 to 30 percent and the cell suspension should have 40-50 percent concentration in plasma or serum. False positive test sometimes occurs due to formation of small fibrin clots in serum or plasma. If blood is drawn without anticoagulant, sufficient time must be allowed for adequate clotting to occur. If anticoagulant samples are tested, the blood should be promptly and thoroughly mixed with anticoagulant. o Rh types are proteins o Small fibrin clots indicate agglutination reaction The viewing box should have a light source that will bring the temperature on the plate to 45-50°C, so that the test surface approximates 37°C. The light should be left on so that the temperature is constant. Tests should be performed on glass slide, not directly on the viewbox plate. o Rh have IgG antibodies (optimum temp at 37C) Rh (D) typing is based on the principle of agglutination. Normal human red blood cells, possessing antigens, will clump in the presence of antibody directed toward the antigens. Agglutination of patient or control red blood cells with antiD serum and no agglutination with the control reagent is a positive test result, which indicates the presence of the D antigen on the red blood cells. Absence of agglutination is a negative test result, which indicates the D antigen is not demonstrable. • • INTERPRETATION OF RESULT • • RH TYPING PROCEDURES MATERIALS • • • • • • • • • Test tubes 10 or 12 x 75 mm Slides Applicator sticks Viewing box Expired blood Serofuge Incubator Saline solution Anti-Rh (anti-D) typing sera (detects D antigens) Slide Test 1. Prepare a 40-50% suspension of the cells to be tested in their own or group compatible serum or whole blood may be used. 2. Add 2 drops of the cells suspension to a slide prewarmed to 40-50°C on a slide viewing box. 3. Add 1 drop of Anti-Rh slide test serum. 4. Mix well and tilt the view box back and forth. 5. Examine agglutination. Interpret as negative those cells which are not agglutinated within 2 minutes. Do not observe longer than two minutes. o RCS prep → 2RCS → Anti-D → Observe Saline Tube test 1. Prepare a 2% suspension in saline of washed cells to be tested. 2. Add 1 drop of anti-Rh saline test serum to a small test tube (10 x 75 mm) 3. Add 2 drops of the 2% cell suspension. 4. Incubate at 37°C for 60 minutes. 5. Examine sedimented cells for agglutination. 6. If there is no agglutination, shake the tube and centrifuge at 1000-2000 r.p.m. for 1 minute. 7. Resuspend the cells and examine for agglutination. If desired, readings may be confirmed with the aid of small hand lens or magnifying mirror. Modified Tube Test 1. Prepare a 2% suspension of cells in their own group compatible serum. 2. Add 1 drop of anti-Rh slide test serum to a small test tube (10 x 75 mm) 3. Add 2 drops of the 2% cell suspension. 4. Mix and centrifuge at 1000-2000 r.p.m. for 1 minute. 5. Examine for agglutination. If desired, readings may be confirmed with the aid of small hands lens or a magnifying mirror. • • • Sample Patient Result: A+ 2+ 0 3+ According to the image: There is agglutination in Anti-A typing serum and Anti-D antiserum, No agglutination in Anti- B antiserum. Therefore, the blood type of the patient is "A" Rh+. Rh typing is usually performed alongside with ABO Forward and Reverse typing. (+) Agglutination: Rh + (-) Agglutination: Do not report it yet as Rh negative. Presumptively, it is Rh negative but a confirmatory test (Coomb’s Test) must be performed before you report it as Rh (-). WEAK D TESTING INTERPRETATION OF RESULTS LAB DISCUSSION • • The presence of variant Rh (Du) can best be detected by incubating the red cell suspension with anti-Rh₀ (D) followed by antiglobulin testing (indirect Coomb’s test) Although the Du factor is modified D antigen, frequently non-reactive on the slide or in the tube, it is antigenic when injected into the D negative recipient. A screening test must be routinely performed on all blood samples tested as D negative in the usual slide or tube test. This test is based on the antigen’s ability to absorb antibody. o Rh has high antigenicity o Incompatibility may cause transfusion reactions • WEAK D VARIATIONS • • • • • Weak expression due to alteration of RhD antigen o Weakened reaction or negative C trans - Steric arrangement of C affects the expression of D antigen (Dce/dCe) Weak D - Mutations in the RHD genes Partial D - Epitopes of D antigen is missing or altered Del - Extremely low (el) number of Rh antigens in the RBC surface (can only be detected by elution) • • TESTING OF WEAK D MATERIALS • • • • • • • 10 or 12 x 75 mm test tube Serofuge Saline solution Expired blood Antiglobulin (Coomb’s/AHG reagent) Serum incubator (for IgG antibodies - 37C) Anti-Rh (D) serum • • PROCEDURE • 1. 2. 3. 4. 5. 6. 7. 8. Beforehand, perform screening test using Anti-D TS Place a drop of Anti-Rh (D) serum into a labeled test tube. Add 2 drops of a 2-5% suspension of RBCs to be tested. Mix well and incubate at 37°C for 15-30 minutes. Add another drop of the cell suspension to a tube labeled “control”. This will be direct antiglobulin test. No incubation needed. Wash the cell suspension of both tubes for the antiglobulin procedure. After the final washing, remove the saline and add 1 drop of antiglobulin serum to each tube. Shake and mix. Centrifuge at 1000-2000 r.p.m. for 1 minute Gently dislodge cell sediment and observe for agglutination. If the test is positive and the control (a direct antiglobulin test) is negative, the individual variant Rh activity and his phenotype is Rh (Du). If the test is negative and the control is negative, the blood is truly negative for Rh₀ (D) and can be labeled Rh-negative. If the control is positive, the result of the anti-globulin test for Rh₀ (D) cannot be interpreted since the cells are already coated with globulin. Blood with a positive direct antiglobulin test should not be used for transfusion. In blood donors, who should be a symptomatic by history and hemoglobin estimation, a positive antiglobulin test sometimes signals an onset of hematologic disease, but some asymptomatic individuals have chronically coated red cells for no discernible reason. A common cause of positive antiglobulin test is medication with the antihypertensive drug alpha-methyldopa. If the Rh₀ (Du) testing is done on potential recipients and the control is positive, the patient should be given Rhnegative blood, and the physician should be notified that the patient has a positive antiglobulin test. Interpretation Anti-D TS (screening) AHG (IAT) Interpretation (+) No need Rh (+) (-) (+) Rh (+) Du (-) (-) Rh (-) M-N TYPING • • • • Significance of the Test: Medico-legal work (paternity testing or paternity disputes) Reagent: Anti-M and Anti-N antisera (from rabbits) o Lectins can also be used Two procedures: o Slide test o Tube test NOTE: Anti-M and Anti-N in the blood are only clinically significant if they react at 37 degrees Celsius o Anti-M & Anti-N - react at room temp (not CS) o Anti-M & Anti-N - no agglutination at room temp is clinically significant (37) CLINICAL SIGNIFICANCE • • Anti-M o Hemolytic transfusion reaction o Decreased RBC survival (hemolytic anemia) o HDFN (erythroblastosis fetalis) Anti-N o Dialysis associated anti-N (px with renal failure) → Anti-Nf (formaldehyde anti-N) development LAB DISCUSSION • • • • The use of M-N typing is usually restricted to medico legal work, such as in paternity testing or paternity disputes. It can be used also in other investigative work and to select donors for the blood transfusion recipient who lacks on or the other M-N antigens and has produced antibodies. Blood typing for M and N is usually performed with a reagent prepared in rabbits. The tests may be carried out on the slide or in the test. Lectins can be used. Careful attention must be given to time and temperature of incubation, as well as the amount of cell suspension and reagent used. o Prevent prozone & post-zone phenomenon MN blood group - generally IgM non clinically significant antibodies that generally react at room temp (37C) and optimally at 4C o Part of antibody identification panel M-N TYPING PROCEDURES PROCEDURES • • • Based on direct agglutination test Tube Test 1. Prepare a 2 % suspension in saline of washed, fresh cells to be tested. 2. Add 1 drop of anti-M to a properly identified test tube, and 1 drop of anti-N to a second tube 3. Add 2 drops of the cells suspension to each tube. 4. Mix well and allow to stand 30 minutes at room temperature. No incubation required. 5. Examine for agglutination. Slide test 1. Prepare a 10% suspension in saline of washed, fresh cells to be tested. 2. Add 1 drop of anti-M to the left half of the slide and 1 drop of anti-N to the right half of the slide. 3. Add 2 drops of cells suspension to each half of slide. 4. Mix well and tilt the slide back and forth. 5. Mix for agglutination. Do not observe longer than 1 min. THINGS TO REMEMBER IN M-N TYPING: • • • • • RCS/Saline suspensions must be prepared from fresh blood. o All specimens & reagents in BB should be freshly prepared to ensure proper and optimum results Fresh cell suspension must be used. Centrifugation in the tube test will cause false agglutination causing false positive Delay in reading will cause non-specific results. Adhere to prescribed time limits. Control cells of types M, N and MN should be used. INTERPRETATION OF RESULTS • • • Positive Result Upon Addition of the Reagent = (+) Agglutination, grade according to agglutination Negative Result Upon Addition of the Reagent = (-) Agglutination Reaction with: Anti-M Anti-N Phenotype + M / MM + + MN + N / NN •