Immunohematology - 04 Rh Blood Group System

• IMMUNOHEMATOLOGY RH BLOOD GROUP SYSTEM OUTLINE • Rh Blood Group System o Historical Perspective o Rh Blood Group System o Nomenclature o Rh Inheritance o Eh Antigens o Ah Antibodies o Rh Null Syndrome o Hemolytic Disease of the Fetus and Newborn • • 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 by Landsteiner 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 codominant manner • • 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 the D antigens o If D antigen is present on RBC, the person is typed as Rh positive (D+). 85% of the population possess this antigen. o If D antigen is absent, the person is phenotyped as Rh negative (D -). About 15% of the population lacks D antigen which is commonly found among Caucasians. o Rh negative phenotype is clinically significant among female patient on their child-bearing years because of the risk for HDFN (Hemolytic Disease of the Fetus or Newborn) on their subsequent offspring. o Found in Chromosome 1 which is the same locus for the hematologic condition known as Hereditary Elliptocytosis RHCE o Responsible for the presence or absence of the C, E, c, e antigens o Found in chromosome 1 o If RHCE and RHD genes are both present in the same locus they are considered as allele because they occupy the same position RHAG o A.K.A. co-expressor gene because it is important for the principle Rh antigens o Responsible for the formation of Rh antigen-like polypeptides on RBC o This polypeptides are critical for the expression of principal antigens because without it, they principal ags are not expressed. o They are present in Chromosome 6 NOMENCLATURE • 2 Classification: o Genetically-associated ▪ It has genetic basis ▪ Example: Fisher-Race (DCE) and Weiner (Rh-Hr) o Non-genetically associated ▪ It has no genetic basis, standardized ▪ Example: Rosenfield (alpha-numeric) and 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 o WEINER/ Rh-Hr TERMINOLOGY • • • • • • Weiner vs Weiner Shorthand o Weiner: DCe = Rh1 o Weiner Shorthand: DCe = R1 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 o Blood factor: Rho, hr’, hr” • ROSENFIELD / ALPHA NUMERIC TERMINOLOGY • • • • It has no genetic basis and is based according to the D antigen is 1st discovered among other ags (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: Application using antisera: Anti D = + Anti C = Anti E = + Anti c = + Anti e = + Rosenfield manner of reporting: (spaced) Rh 1 -2 3 4 5 Sample: Application using antisera: Anti D = + Anti C = Anti E = N/A Anti c = + Anti e = + Rosenfield manner of reporting: Rh 1 -2 4 5 *If antiserum is not available in the laboratory, omit the number because it is not tested. 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 o 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 = Ro Ex. 2: DCE = Rz Ex. 3: dCe = r’ Ex. 4: dce = r Ex. 5: DCe/dcE = R1r’’ Ex. 6: DCE/dCE = Rzry Ex. 7: R2r’ = DcE/dCe Fisher-race D (Rh +) Absence of D (Rh -) C E CE c e ce (ISBT) INTERNATIONAL SOCIETY OF BLOOD TRANSFUSION • • • • • • • • CONVERSION: FISHER-RACE TO BLOOD FACTOR • • • • 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 (AngMNSRhea) • ABO = 001 • MNS = 002 • Rh = 004 ▪ Last 3 digits = Specific antigens • D = 001 • C = 002 • E = 003 • c = 004 • e = 005 • • • • • • • Fisher-Race D (Rh +) C E c e C c E e D = Rho C = rh’ E = rh” c = hr’ e = hr” Ex. 1: DCE = Rhorh’rh’’ Ex. 2: dce = hr’hr’’ Blood factors Rho rh hr ‘ ‘’ RH BLOOD GROUP NOMENCLATURE CONVERSION • Difference between Weiner and Shorthand: Shorthand doesn’t have “h” Weiner Shorthand Fisher-Race Rho Ro Dce Rh1 R1 DCe Rh2 R2 DcE Rhz Rz DCE rh r dce rh’ r’ dCe rh” r” dcE rhy ry dCE Ror Dce/dce R1R2 DCe/DcE ryr” dCE/ dcE • • • Blood Factors Rhohr’hr’’ Rhorh’hr’’ Rhohr’rh’’ Rhorh’rh’’ hr’hr’’ rh’hr’’ hr’rh’’ rh’rh’’ Rhohr’hr’’/hr’hr’’ Rhorh’hr’’/Rho rh’rh”/ hr’rh” Rosenfield VARIATIONS OF THE Rho (D) ANTIGENS Rh: 1-2-345 Rh: 1 2 3 4 5 Rh: -1 2 3 4 -5 • R1 or DCe/dce - most common among Caucasian (white) approximately 35% Ror or DCE/dce - most common in Africans (black) approximately 42% RH INHERITANCE • • Rh locus - located on chromosome 1 (along with the genes for elliptocytosis) This chromosome position is also the same position for metabolic protein enzyme such as: o 6-PGD (6-Phosphogluconate dehydrogenase) o PGM (Phosphoglucomutase) o PPH (Phosphopyruvate Hydratase) o They are also synthesized in the same position in Chromosome 1 • RH ANTIGENS • • Characterized as non-glycosylated proteins on red cell membrane meaning, carbohydrate structure is not present in the molecule/antigen. (Cannot be found on secretions) It is well-developed at birth. It is already expressed during fetal development. Because of this, it is associated with HDFN (Hemolytic Disease of the Fetus and the Newborn) D ANTIGEN • • • • • • Determination of the Possible Offspring Phenotype RZ r r’ Rzr’ rr’ r’’ Rzr’’ rr’’ o 50% - Rh+, heterozygous (due to R and r) o 50% - Rh-; heterozygous o RzR1 - Rh +; homozygous o Ror - Rh+; heterozygous o rr - Rh-; homozygous Considered as most clinically significant of all non-ABO ags It is so highly immunogenic. Small incompatibility can elicit rapid immune response and destruction of RBCs The only Rh antigen that undergoes routine testing, except in the case of investigation of unexpected antibodies If it is present in RBC, this group of individual is known as “Rh positive.” About 85% of population possess D antigen For “Rh negative” without D antigen. About 15% of the population doesn’t possess D antigen Rh Phenotype and Distribution in the Population Fisher-Race Weiner Gene Frequency (Asians) Rh positive DCe R1 40% DcE R2 16% Dce Ro 2% DCE Rz 0.08% Rh negative dce r 38% dCe r' 1% dcE r" 1% dCE ry o R1 haplotype is commonly encountered about 40% among Rh positive. • Du phenotype o Weakened expression of the D antigen o Rarely found among Caucasian but common among Africans o Gives weak or negative reaction with anti-D. ▪ Weak reaction with anti-D is classified as High Grade type (2+, 1+, mf) ▪ Negative reaction with anti-D is classified as Low Grade type (0) o Detected by performing IAT (Indirect Antihumanglobulin Test) ▪ Anti-D is negative do not report it as Rh negative instead, perform IAT. If IAT (Coomb’s test) is positive, it is considered to be Rh positive because IAT is used to confirm the presence of low grade Du antigen Grades of Du o High Grade Du ▪ Not passed on to future generations ▪ It is not inherited because it is the product of the development on rare r’ or ry chromosomal arrangement. ▪ When ry and r’ are combined with Rh positive haplotype (R1r’/R1r’), the chromosomal arrangement of the genes given these haplotypes can cause the formation of High Grade Type. ▪ It is the chromosomal arrangement and not the actual gene causing the weakened D expression ▪ High Grade Du reacts with the anti-D reagent (wk+). Therefore, IAT is rarely require. o Low Grade Du ▪ Direct product of inherited gene. Since, it is because of the gene causing the weakened D expression, it is transferred or passed on to future generation ▪ Common among Africans ▪ It is not detected by anti-D. it is only detected by the IAT or the indirect AHG test Mechanism for Du Phenotype Occurrence o Genetic Weak D/ Genetic Du/ Low Grade Du ▪ An inherited weakerned expression of D antigen ▪ In this mechanism, antigens are complete but few in number ▪ This is the mechanism of Low grade type ▪ In the molecular level, the D antigen molecule undergo mutation o C trans/ Position effect/ Gene Interaction Effect ▪ This the mechanism for High grade type ▪ The Gene interaction effect because of the chromosomal arrangement of the genes ▪ The allele carrying D is trans to an allele carrying C (positional effect) o o o o o D mosaic antigen ▪ According to Weiner, the Rh antigens particularly the D antigen is a mosaic type. The D antigen has the ff subunits: • A subunit • B subunit • C subunit • D subunit o D=DA DB DC DD ▪ These 4 subunits must be present for the D antigen to be strongly expressed. There 4 possible combination of the subunits that could cause the formation of the weak D antigen type: • aBCD - DA is missing • AbCD - DB is missing • ABcD - DC is missing • ABCd - DD is missing In these 4 combinations, the most common product is Weak D antigen Mosaic antigen is an antigen that is composed of different subunits. Meaning for the antigens to be strongly expressed, the subunits must be complete. Otherwise, the expression of antigen is weak. There is missing components of D antigen subunits The anti-D formed by D mosaic individual can cause HDN and HTR RH ANTIBODIES • Rh ANTIBODIES • DETERMINATION OF D STATUS • • • ANTI-D INDIRECT AHG INTERPRETATION + No need to perform Rh (+) + Rh (+) RH (-) Anti-D o The screening presumptive test for the D status o Anti-D or Anti-Rho is the commonly used typing reagent for Rh typing IAT o Used for confirmatory testing for weak D specifically the low grade type Immunogenicity of Common Rh antigens D>c>E>C>e o This determines the ability to cause disease • • • • • G ANTIGENS • • • Are produced by the ssame Rh gene complex that produces C and D antigens Most C-positive and D-positive RBCs are also G-positive. Because they share the same complex, they have similar serologic reaction with typing serum when tested in the laboratory. In the laboratory, if the result of the sample is positive with C and D, then it is positive also for G antigen UNUSUAL/ RARE Rh PHENOTYPE • • • • Composed of compound antigen This compound antigens although composed of 2 Rh specificity, these antigens are considered to be f(ce) - a compound antigen of c and e rhi(Ce) - a compound antigen of C and e 3 Varieties of Rh Antibody: o First-Order Rh Antibody ▪ AKA Complete Divalent/Bivalent Saline Reacting Rh Antibody ▪ It is complete because it is a pentamer ▪ This is IgM which is an unusual type of Rh ab ▪ This antibody reacts optimally at low temperature in saline medium with Rh antigens ▪ These are rarely observed o Second-Order Rh Antibody ▪ AKA Incomplete Monovalent High protein/Albumin Reacting Antibodies ▪ IgG type ▪ Reacts optimally at warm temperature (37C) in high protein media with Rh antigens ▪ Clinically significant o Third-Order Rh Antibody ▪ AKA Typical AHG-Reacting Antibody ▪ IgG type ▪ Reacts optimally at 37C in AHG medium ▪ One of the most commonly observed • Are IgG or IgG3 RBC-stimulated either during transfusion or during pregnancy o IgG - can cross placenta Usually do not agglutinate in saline, if it does it is the unusual type of Rh antibody which is the Reacts best at 37C and can be demonstrated by testing in high-protein media or by the indirect antiglobulin test Reaction is enhanced by the use of enzyme-treated RBCs o Enzyme reagents are able to enhance/improve the reaction o Enzyme reagents used in the laboratory: ▪ Ficin - derived from Fig tree ▪ Bromelain - derived from pineapple extract ▪ Papain - derived from Papaya extract ▪ Trypsin - derived from human instestinal tract Do not usually bind complement because Anti-Rh antibodies are distant from one another on RBC These antibodies can only activate complement extravascularly that is why Rh antibodies are associated with Delayed-Type Hemolytic Transfusion reaction because the site of lysis is extravascular. They cross the placenta and can cause HDFN Rh Null SYNDROME • • The individual characteristically have the Rh null phenotype (---/---) or absence of the principal antigens (D, C, E, c, e) 2 Mechanisms in the development of Rhnull phenotype: o Regulator type mechanism ▪ The issue is with the co-expressor ▪ RHD and RHCE are expressed (normal) ▪ The RHAG is either mutated/deleted/absent o Amorphic type mechanism ▪ RHAG is expressed ▪ RHD & RHCE are either Mutated/Deleted/Absent • • It expresses no Rh antigen on red cell and the phenotype is expressed as ---/---. The ff are the symptoms: o Stomatocytosis o Reticulocytosis o Compensated hemolytic anemia o Increased HbF, Bilirubin specifically B1 (unconjugated bilirubin) o Decreased Haptoglobin, Osmotic Fragility tets o Slight decreases of Hb and Hct NOTE: In the serum of patient without the principal antigens, they possess the anti Rh:29 o It is the broad specific antibody. It is described as Anti-total Rh (it can react with many Rh antigens that could cause immune response). • • FALSE REACTION WITH Rh TYPING • • False positive reaction o Decrease in specificity o May be caused by: ▪ Positive DAT - most common cause of Rh-typing (Du typing) discrepancies ▪ Rouleaux ▪ Cold agglutinins False negative reaction o Decrease sensitivity o May cause by: ▪ Incorrect cell suspension (too heavy or too light zonal phenomenon) • Postzone - excess antigen o Solve by cell washing • Prozone - excess antibodies o Solve by serial dilution ▪ Improper procedure PATHOGENESIS OF HDFN • • • HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN (HDFN) • Definitions: o Also known as erythroblastosis fetalis o Abnormal condition characterized by early destruction of fetal/neonatal RBC due to maternal incompatible abs o Mother is Rh (-), baby is Rh+ (due to father’s) • • CLINICAL FEATURES OF HDFN • • • Anemia o Most immediate severe complication among fetus o If moderate to severe: Jaundice occurs within 24 hrs o If mild anemia: Jaundice occurs late after birth Hyperbilirubinemia (Jaundice) o Increased unconjugated bilirubin/ pre-hepatic/B1 o Most immediate severe complication among newborns o Among neonates only Kernicterus o Severe complication of hyperbilirubinemia o Characterized by the increased accumulation of B1 in the brain tissues and brain nuclei ▪ Increased B1 - toxic for the brain o Most severe form of hyperbilirubinemia o Present only in babies because blood brain barrier is still permeable o Predominant WBC in CSF of newborns: Monocytes o Predominant WBC in CSF of adults: Lymphocytes o Can cause seizures, brain damage, deafness, death Hydrops Fetalis (Fetal Edema) o Seen at 18-20 weeks of gestation o Demonstration using ultrasound o Conditions promoting Fetal Edema: ▪ Fetal hypoalbuminemia ▪ Severe anemia ▪ Congestive heart failure ▪ Severe liver diseases/ hepatic failure Hepatosplenomegaly o Enlargement of the liver and the spleen due to increased infiltration of abnormal fetal/neonatal red cell in the reticuloendothelial system o Abnormal Cells - cell with positive sensitization and maternal antibodies o In HDFN, erythroblasts are seen in peripheral blood of stressed erythropoiesis causing hemolysis due to abnormal cells • It all starts when there is blood incompatibility between the mother and the baby o The mother lacks the antigen that the baby possess (D/Rh antigen) o The red cell antigen of the aby due to the father must be well-developed at birth. Therefore, causing HDFN Antigens causing HDFN: o ABO o Rh o Kell o Duffy o Kidd o i o Ss Transplacental hemorrhage (TPH) occurs o Also known as fetomaternal bleeding/hemorrhage o Blood of the fetus combines with the blood of mother o Occur at 28 weeks of gestation or during delivery Antibody production o Production of maternal antibodies directed against antigen (+) fetal cells causing sensitization leading to hemolysis or immune response IgG crosses placenta (maternal) o Antibodies transported across the placenta: IgG1 and IgG3 - 2nd trimester Hemolysis of cells o Maternal antibodies causes fetal red cell destruction by the reticuloendothelial system in the spleen or liver MAJOR TYPES OF HDFN • ABO HDFN o More common type of HDFN but are less severe compared to Rh o Occurs when ▪ Mother is type O and baby is type A ▪ Mother already has anti-A ab that will react with baby’s A antigen resulting to immediate agglutination reaction ▪ There is no need for previous exposure o Natural phenomenon ▪ Sensitization is already occurring even at 1st pregnancy o Can cause extravascular hemolysis o • Mild anemia is manifested ▪ Mother’s antibodies may react with baby’s antigens or other tissues ▪ Has few attachment to RBCs ▪ Reaction with RBCs are weak o Characterized by weakly positive or negative DAT o Used for in vivo sensitization o Anemia is absent and very mild but spherocytosis and reticulocytosis are increased o Jaundice / Hyperbilirubinemia, if present, appears 2448 hours after delivery or 1-2 days ▪ Resolved by phototherapy o ABO HDFN is seen most often in Group A or Group B infants who have Group O mother (with anti-A/anti-B) o Mild form of HDFN o 2 Mechanism that Promote Mild HDFN ▪ Presence of A and B soluble substances that can actively neutralize maternal antibody ▪ ABO antigen (specifically A antigen) is weakly expressed in fetal/neonatal RBC Rh HDFN o Most severe HDFN particularly of the D antigen but less common o Characterized by (+) DAT o Anemia is present and severe, and reticulocytosis increases o Jaundice/ Hyperbilirubinemia appears within the 1st 24 hours o Moderate to severe manifestation o Can be prevented by administration of Rhogam/ RhIg (neutralizes the antibody to inhibit reaction between maternal antibody and fetal antigen to prevent hemolysis) o Usually the Rh positive 1st born infant of Rh negative mother is not affected because mother has not yet been immunized o 1st born is responsible for immunizing the mother, 1st born may have Rh+ which may be foreign o In the 2nd baby, do not usually live o IgG (IgG1/IgG3) crosses the placenta in the 2nd trimester o Anti – D produced by the mother against the D antigen of the baby promoted HDFN o 1st Pregnancy ▪ Mother is Rh- and Father is Rh+ ▪ There is a chance of the baby being Rh+ ▪ The 1st pregnancy is not affected ▪ After delivery, there is an occurrence of transplacental hemorrhage where some of fetal RBC ag will move into mother’s Rh- blood ▪ Rh+ blood from baby will enter the mother’s Rhcirculation which causes exposure of mother to Rh+ causing sensitization and development of Rh antibodies ▪ In rare cases, hemolysis occurs when there is an accident requiring transfusion that will allow prior exposure to Rh+ even before pregnancy o • Subsequent Pregnancies ▪ Mother is Rh-, Father is Rh+, baby may be Rh+ ▪ After 1st pregnancy, there have been sensitization that produced antibodies against Rh+ ▪ Transplacental migration occurs for passive immunity for the baby ▪ Mother’s Rh abs will migrate to the circulation and react with the Rh ag of fetal RBCs causing agglutination which will eventually cause the destruction of fetal RBCs ▪ With RBC destruction, the baby becomes anemic causing low oxygen carrying capacity of blood leading to compensation of the heart to protect the baby ▪ The heart compensation can lead to fetal cardiac failure ▪ B1 is released during hemolysis, which can be toxic to the brain leading to brain damage causing kernicterus ▪ RBC production in liver & spleen in compensation to destruction of RBCs cause hepatosplenomegaly characterized by decrease or failure in liver function causing low albumin levels (edematous) ▪ the baby is jaundiced (increased b1) and edematous (hypoalbuminemia) and anemic which can lead to problems in the hollow organs ▪ in severe cases, these can lead to uterine death Comparison of ABO HDFN and Rh HDFN ABO HDFN Rh HDFN Type of Naturally occurring Immune IgG Antibody IgG or Immune IgG Ex. Anti-D Ex. Anti-A/B/AB in group O serum Obstetric 1st born may be 1st is unaffected, History affected disease is more severe in subsequent offspring, still birth is common Anemia Absent or Mild Moderate to Severe Bilirubinemia Peak at 1-3 days Rapid rise (rises slowly) DAT Weakly positive or Positive negative Blood Microspherocyte No microspherocyte Picture Prevention None (Phototherapy Rh Ig (Rhogam) for jaundice) ASSESSMENT OF FETOMATERNAL BLEEDING • Rosetting Test o A qualitative test that distinguishes Rh positive fetal cells from Rh negative maternal cell o Used to determine the severity of HDFN o Specimen: 1hr post-delivery maternal whole blood o Target cell: Rh D (+) o Fetal cells are sensitized by Anti-D reagent o Anti- D coats D indicator cells o Incubate Maternal cell suspension with Anti- D reagent o If the rosette screen is positive, a quantitative procedure must be performed to determine the number of vials of RhIg that should be given o ▪ D indicator cell ▪ Composed of blood type “O” R2R2 (DcE/DcE) ▪ Preferred because it has the most antigenic site on red cells Solution 1: FMH = # fetal cell x MBV # maternal cell (2000-30) FMH = 30 x 5000 mL 1970 FMH = 76.14 mL Dose RhIg = FMH / 30 Dose RhIg = 76.14 / 30 Dose RhIg = 2.5 ≈ 3 + 1 = 4 vials ▪ • Kleihauer – Betke Stain (Acid Elution Technique) o A quantitative test that distinguishes hemoglobin F containing fetal RBCs from those adult cells that contain Hgb A o Antibody Titer – the more severe is HDFN ▪ Principle: Hgb F resist acid elution ▪ Therefore, Hb-F containing cells take up the stain and the Hb A containing cells appear as ghost cells o Acid reagents: ▪ Phosphoric acid or ▪ Citric acid buffered at pH 3.3/3.2 o Solution 2: % fetal cells = (# FC counted / 2000) x 100 % fetal cells = (30 / 2000) x 100 % fetal cells = 1.5 x 50 FMH = 75 Dose RhIg = 75/30 Dose RhIg - 2.5 ≈ 3 + 1 = 4 vials Rules: ▪ For decimals less than 5, round down and add one dose. • *Ex. 2.4 round down to 2 + 1 dose = 3 vials ▪ For decimals more than 5, round up and add one dose • *Ex. 2.7 round up to 3 + 1 dose = 4 vials ▪ Why +1 dose? 1 dose is always added to ensure complete neutralization of Fetal cells in the maternal circulation. o DIAGNOSIS AND TREATMENT/ MANAGEMENT OF HDFN o o o Vol. of FMH (mL) = # of fetal cells x Maternal blood vol. # of maternal cells ▪ # of maternal cells = 2000 - # fetal cells ▪ MBV - maternal blood volume ▪ FMH - Fetal Maternal Hemorrhage ▪ 2000 cells are to be counted ▪ Count the cell on the feather edge where the cells are not overlapping ▪ If maternal blood volume is not mentioned, use 5000 mL (An average volume of a woman in child bearing age is 5L or 5000 mL) % of the Fetal cells x 50 = FMH ▪ % Fetal cells = (# fetal cells counted/2000) x 100 ▪ To determine the # of vials, you divide FMH by volume by 30 ▪ Note: 1 vial (std dose) of Rhogam neutralizes 30 mL of FMH Sample Problem: An acid elution stain was made using a 1 hour-post-deliver maternal blood sample. 2000 cells were counted and 30 of these cell appeared to contain HbF. Calculate the # number of vials of Rhogam that would be indicated under this circumstance: ▪ FMH = ? ▪ Dose RhIg = ? • Amniocentesis o Amniotic fluid is collected o Tested for bilirubin o Change in Optical Density at 450 nm ▪ *450-700nm – visible light o Result is plotted vs. gestational age of the fetus starting at 27 weeks of gestation o Using Lileys’s graph o 3 Zones: ▪ Zone 1 – lower part of the graph; mild HDFN/absent/no medical intervention required ▪ Zone 2 – middle part; moderate HDFN/ medical intervention required ▪ Zone 3 – upper part/danger zone/ sever HDFN/ immediate medical intervention is required • Corocentesis/Precutaneous Umbilical Blood Sampling (PUBS) o Cord Blood ▪ Cord Blood Hemoglobin ▪ <10g /dl = fetal anemia ▪ < 8g /dl = severe fetal anemia o • • • • Direct Anti-Human Globulin Test(DAT) ▪ Single most significant serologic test in diagnosing of HDFN ▪ Detect in vivo sensitization of maternal antibodies on fetal RBCs ▪ (+) antibody of the mother that crossed the placenta o Antibody Screening/ Identification ▪ Identification of HDFN other than ABO and Rh o Reticulocyte Count ▪ Determine the presence of Hemolytic Anemia Intrauterine Transfusion o Delicate procedure which is carried out if the following are present: ▪ Hydrops fetalis ▪ Amniotic fluid bilirubin is at high/upper zone 2 and zone 3 of Lileys’s graph ▪ Cord blood hemoglobin is <10 g/dL Phototherapy o Prevent Kernicterus o Used to correct hyperbilirubinemia/jaundice of newborn o Two Actions ▪ Promotes photoconversion/ oxidation of its less toxic form of biliverdin ▪ Promotes destruction of excess B1 in circulation Early Delivery o To stop further transplacental crossing of maternal antibody to the fetal circulation and prevent further sensitization that could cause further damage preventing uterine death Exchange Transfusion o The simultaneous withdrawal of blood and infusion of compatible donor blood to infants with HDFN o To decrease bilirubin levels (B1 = toxic) o To correct anemia o To remove the infant’s sensitized RBCs to prevent further hemolysis o To decrease concentration of incompatible antibodies that could lead to further sensitization & hemolysis o Donor Blood Characteristics ▪ Must be group specific or must be negative for the antigen against which the mother’s antibodies are directed ▪ Should be group O of the same ABO group as the mother and infant, if both are the same ▪ Must not have any unexpected antibodies • Detected by antibody screening ▪ Must be less than 7 days old • To maximize 2,3-DGP level of donor unit and prevent hyperkalemia (hyperpotassemia) ▪ Should be negative for cytomegalovirus (CMV) • Exclusively resides in WBCs ▪ Should be negative for Hbs • Prevented sickling of RBCs ▪ Recommended to irradicated to prevent Graft versus Host Disease (GVHD) • T cells are the most immunocompetent WBCs in terms of immune response, thus must be inactivated to prevent GVHD PREFERENCE OF SPECIMEN FOR COMPATIBILITY TESTING • • • Mother’s Serum – preferred for testing; has the highest maternal Ab titer An eluate prepared from the infant’s red cell Infant’s serum – low titer ab PREVENTION OF HDFN • • • Rh Immune Globulin (Rhogam) o Concentration and purified anti-D gamma globulin o Acts as immunosuppressant and is given to nonimmunized Rh- mother who have given birth to Rh+ infants to suppress the development of anti-D o Standard Criteria for the Administration of RhIg ▪ The mother must be Rh negative ▪ The infants must be Rh positive ▪ The mother must not be immunized to the D antigen (the mother doesn’t have anti-D yet) Indications: o Post – Partum ▪ Rh negative mother should receive Rh Ig soon after delivery of an Rh-positive infant ▪ The recommended time interval is within 72 hours after delivery o Antenatal RhIg ▪ Should be given month in the 3rd trimester or about 28 weeks/7 months of gestation ▪ NOTE: All unsensitized Rh (-) women who have spontaneous abortion of miscarriage at up to 12 weeks gestation should receive RhIg (50 ug/microdose/millidose) Dose and Administration of RhIg o 300 ug Dose Rh Ig (Conventional) ▪ Can neutralize the effects of up to 15 mL of Rh positive packed RBCs or 30 mL of whole blood (>12 weeks of gestation) o 50 ug Dose Rh Ig (Microdose) ▪ Is sufficient for abortion, amniocentesis and ectopic rupture at up to 12 weeks gestation