Immunohematology / ISBB / IH Lesson 3: HH & ABO Blood Group System PDF

IMMUNOHEMATOLOGY / ISBB / IH LESSON#3: HH & ABO BLOOD GROUP SYSTEM PRELIMS | A.Y. 2023 - 2024 | SIR JASON “AB” CHUA Hh AND BLOOD GROUP SYSTEM Cartwright (Yt)...

IMMUNOHEMATOLOGY / ISBB / IH LESSON#3: HH & ABO BLOOD GROUP SYSTEM PRELIMS | A.Y. 2023 - 2024 | SIR JASON “AB” CHUA Hh AND BLOOD GROUP SYSTEM Cartwright (Yt) YT 011 Blood group antigens are part of the red cell membrane Xg XG 012 ○ Expression depends on the inheritance of specific blood Scianna SC 013 Dombrock DO 014 group gene Colton CO 015 ○ Classified to be as carbohydrates linked to either lipids Landsteiner-Wiener LW 016 (termed as glycolipids) or proteins (termed as Chido/Rodgers CH/RG 017 glycoproteins) Hh H 018 Exposure to such antigens through blood transfusion / pregnancy Kx XK 019 can lead to the production of antibodies towards the antigen that Gerbich GE 020 the recipient is lacking Cromer CROM 021 In anatomy, red cells are carrying specific sets of antigens that is Knops KN 022 unique and different from another individual Indian IN 023 In immunology, a specific antigen could lead to the formation of a Ok OK 024 specific antibody → leads to the destruction of the cells that Raph RAPH 025 contain such antigen JMH JMH 026 I IGNT 027 Globoside GLOB 028 Gil GIL 029 Rh-associated RHAG 030 glycoprotein ISBT, International Society of Blood Transfusion. 36 registered blood group systems up to date ○ ISBT organized the gene name and number mainly because the terminology for red blood cell antigens were inconsistent before and there were actually some confusions with regards to the antigens of a particular blood group ○ In order to standardize and organize, they created a working committee on the terminology for red cell antigens in the year 1980. Red cells are mainly composed of proteins (both in external and ○ The goal is not to replace terminology, but rather, to internal surface) provide additional terminology suitable for computer Protein Blood Group Antigen software use BAND 3 Diego Hh blood group system is included with ABO blood group system GLYCOPHORIN A & B because the two have a special relationship MNSS (tallest rbc membrane protein) ○ ABO blood group antigen would not exist without H ABO GLYCOLIPIDS gene LEWIS POLYPEPTIDES Rh glycoprotein Remember: KARL LANSDSTEINER Blood group antigens are residing on a specific red blood cell membrane proteins; The presence of blood group antigens are mainly DEPENDENT on the inherited gene → leads to variations in red blood cell membrane protein in terms of forms, sizes, as well as length Blood group antigens are expressed differently on the red cell membrane depending on which red blood cell protein they are going to included ISBT BLOOD GROUP SYSTEM ASSIGNMENTS BLOOD SYSTEM NAME ISBT GENE NAME ISBT NUMBER ABO ABO 001 Discovered the ABO blood groups in 1901 MINS MNS 002 ○ Paved the way for the study of immunohematology and PIPK P1 003 blood transfusion medicine Rh RH 004 First person who performed ABO forward and reverse blood Lutheran LU 005 grouping Kell KEL 006 ○ Drew blood from himself and his five associates, he Lewis LE 007 separated the cells and the serum, and mixed each cell Duffy FU 008 sample with each serum Kidd JK 009 ○ Recognized different patterns of agglutination when Diego DI 010 human blood samples were mixed in random pairings ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 1  ○ Assigned blood groups as A, B, and O Make use of KNOWN RED CELLS Karl Landsteiner Law - there is presence of agglutination in the ○ are identified to be red cells that contain A antigen and serum of individuals who lack the corresponding ABO antigen. red cells that contain B antigen. example: LANDSTEINER’S LAW the antibody of the patient will react with the A antigen, therefore it Normal, healthy individuals possess ABO antibodies to the ABO has Anti-A = Blood type b antigen absent from their red cells ○ If you are going to have a specific ABO antigen, then you FREQUENCY DISTRIBUTIONS OF ABO PHENOTYPES (U.S. POPULATION) are going to have an antibody against an antigen that is ABO PHENOTYPE WHITE (%) BLACK (%) ASIAN (%) not present on your red cell A (2) 40 (2) 27 (2) 28 ○ A person cannot produce an antibody against his own B (3) 11 (3) 20 (3) 27 antigen. AB (4) 4 (4) 4 (4) 5 example: (least frequent) A type A individual lacks the B antigen, then most likely, the O antibody present in his plasma is against the B antigen (termed as (1) 45 (1) 49 (1) 40 (majority) anti-B antibody) A type B individual lacks the A antigen, thus, the antibody present A B AB O in his plasma is against the A antigen (anti-A antibody) White (40) Asians (27) Asians (5) Black (49) Asians (28) Black (20) White (4) White (45) Black (27) White (11) Black (4) Asians (40) For blood group A individuals, their red cell contains the A antigen ○ Cannot produce an antibody against self-antigen Based on the data gathered by the National Voluntary Blood Services Program (NVBSP) ○ Can produce an antibody against antigen absent in one’s ○ the most common blood type is BLOOD GROUP O self (B antigen) → contains anti-B antibody ○ O > A > B > AB For blood group B individuals, their red cell contains the B antigen Ranking is the same as the frequency in the ○ Cannot produce an antibody against self-antigen US population. ○ Can produce an antibody against antigen absent in one’s ○ Majority of the Filipinos are Rh positive while only 1% self (A antigen) → contains anti-A antibody has Rh negative blood. For blood group AB individuals, their red cell contains both A and ○ Blood Group O = Universal Donors So if there is a lack of supply of A and B B antigen packed red cells, the Group O packed red cells ○ Cannot produce an antibody against self-antigen = DOES can be transfused to A and AB individuals. NOT CONTAIN ANY ANTIBODY For blood group O individuals, their red cell DOES NOT CONTAIN INHERITANCE OF A, B, AND O ANTIGENS both A and B antigen Bernstein, 1924 ○ Contains BOTH ANTI-A AND ANTI-B ANTIBODY ○ Multiple allele theory the inheritance of A, B, and O Antigens so BLOOD TYPING that individual is going to inherit one (1) ABO gene from each parent Determines which blood group an individual belongs These two genes will determine which ABO FORWARD TYPING antigens are present on the red cell Front type / shift to the right typing / Direct cell typing membrane. ○ Determines the antigens present on the red cell surface ○ Remember: the expression of the A,B, and O antigens Make use of ANTISERA as reagent will depend if you have inherited(from your parents. ○ Mainly contains antibodies specific for the antigens that could be present on the red cell One locus in chromosome 9 Sample used is red blood cells A,B, O genes Interaction of (3) genes at 3 separate loci (Basis for expression or REVERSE TYPING formation of ABO antigens.) Back type / shift to the left typing / Indirect cell typing ○ ABO ○ To determine what are the antibodies that could be ○ Hh present on the patient’s plasma. Sample = Plasma or serum ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 2  ABO cannot be expressed in the red cell if there is no Hh antigen/gene. ○ Se ABO could not be expressed in the secretion if there is a lack of secretor (Se) gene. TYPE 1 PRECURSOR SUBSTANCE seen on secretions (plasma, saliva) & body fluids Simple mendelian genetics If one parent gave you the A gene and one parent contains the O gene, you are going to have the phenotype A. ○ Remember: A is dominant over O because O is the recessive gene. Last three molecules of paragloboside: ○ The phenotype of an individual depends on the genotype of the parent. ○ Galactose (2) If homozygous With regards to the terminal sugar of your the double dose of the gene precursor substance which is galactose, it has If heterozygous the same beta-1, 3 linkage. two different genes The 1st carbon of the galactose is ○ Could either be that both genes are dominant or one is attached to the 3rd carbon of dominant and the other one is recessive. Genotype: The gene that was taken from the parent N-acetylglucosamine. ○ Genotype = AO ○ N-acetylglucosamine (GlcNAc) if you have A and O = Heterozygous is attached to the previous sugar molecule Phenotype: The antigen that was presented in the red cell. (Galactose) via Beta-1,3 linkage ○ expressed in your red cells These are found in the body fluids and secretions. ○ The dominant gene can be your basis for your ○ mainly because the terminal galactose is attached to the phenotype. preceding sugar which is the N-acetylglucosamine via the Beta 1-3 linkage. THE PRECURSOR SUBSTANCE Remember: where is beta found? The inheritance of ABO gene is not actually directly going to Sugar molecules have 2 forms, it could be an alpha or beta produce an antigen but rather it is mainly going to produce a depending on the position of the OH on the first carbon or specific enzyme in which they are termed as GLYCOSYL anomeric carbon. TRANSFERASES. ○ Beta = OH is above the ring while the H is below the ring PARAGLOBOSIDE ○ Alpha = OH is below the ring while the H is above the an oligosaccharide chain attached to a protein or lipid molecule ring basic precursor substance of the A, B and H antigens. they are mainly composed of oligosaccharide chains TYPE 2 PRECURSOR SUBSTANCE The precursor substance that is expressed on the red cell surface OLIGOSACCHARIDE CHAINS is type 2 precursor substance. The oligosaccharide chain which is the paragloboside will be seen on red cell membrane converted into ABO antigen by the addition of a specific sugar made possible by the enzyme (glycosyltransferases) that is produced by the gene. GLUCOSYLTRANSFERASES [Glycosyl =sugar | Transferases = transfer] enzyme produced by the gene transfer sugar molecules on a precursor substance in order to make it a specific ABO antigen and that precursor substance is Type 2 is the same with type 1 which is the Beta 1, 3 but known as PARAGLOBOSIDE. differ on the attachment of the terminal sugar galactose to the N-acetylglucosamine sugar via BETA 1,4 LINKAGE. ○ the 1st carbon of the terminal galactose is attached to the 4th carbon of the N-acetylglucosamine. Hence, it is called Beta-1,4 linkage. FORMATION OF H ANTIGEN ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 3  The only antigen found in the H blood group system. ○ Alpha-2-L-fucosyltransferase will transfer the fucose to The formation of H antigen is mainly through the inheritance of the 2nd carbon of the terminal galactose. the H gene ○ So, the 1st carbon of the fucose is attached to the 2nd ○ H gene is closely linked to Se gene, in which they are in carbon of the terminal galactose. This then forms the H the same loci found in Chromosome 19 antigen, and with the H antigen, the fucose is now the The alleles of H gene is H and h immunodominant sugar. “h” - the recessive allele ○ But because the A gene is also inherited, then “H” - dominant allele Alpha-3-N-acetylgalactosaminyltransferase is coded, in double dose of the h allele (hh) = no which it will transfer the N-acetyl-d-galactosamine in the expression of the H antigen on the red cell. 3rd carbon of the terminal galactose, which now makes The inheritance of the H gene will not directly form the H antigen, it the immunodominant sugar of A gene. rather it will code for a specific enzyme known as Immunodominant sugar Alpha-2-L-fucosyltransferase. ○ N-acetyl-D-galactosamine Gene Product: Alpha-2-L-fucosyltransferase FORMATION OF B ANTIGEN ○ transfer a fucose sugar to the 2nd carbon of the terminal sugar, galactose. Expression of B antigen is dependent on the inheritance of ABO ○ So, the 1st carbon of the fucose is attached to the 2nd gene, particularly B allele. carbon of the terminal galactose. BB (homozygous) or BO (heterozygous) Fucose will now be identified as the ○ In order to express the B antigen, the individual must Immunodominant sugar. inherit the B allele. So it could occur with the double ○ Note: for paragloboside, the immunodominant sugar is dose of B (BB) or the combination of an amorph the terminal galactose. allele(BO) But paragloboside is converted to H antigen, Found in Chromosome 9 another sugar is added (fucose) becomes the Alleles: immunodominant sugar. ○ A, B and O Immunodominant sugar (terminal sugar) The inheritance of the B gene will not directly from the B antigen. ○ L-fucose Rather, it will produce a specific enzyme in the form of Amorph Alpha-3-D-galactosyltransferase. ○ If you have inherited a double dose of the amorph allele Gene Product: Alpha-3-D-galactosyltransferase of h gene(hh)= no secretion of the ○ transfer the galactose sugar to the H antigen. alpha-2-L-fucosyltransferase. The paragloboside should first be converted to no addition of fucose to the terminal the H antigen. galactose of the paragloboside. Alpha-2-L-fucosyltransferase will transfer the All paraglobosides will remain as is and fucose to the 2nd carbon of the terminal conversion to H antigen will not occur. galactose. ○ Bombay phenotype (hh) So, the 1st carbon of the fucose is attached to silent h allele the 2nd carbon of the terminal galactose. The first individual found to have such But because the B gene is also inherited, then phenotype was found in Bombay, India. Alpha-3-D-galactosyltransferase is coded, in which it will transfer the D-galactose via 1,3 attachment to the terminal galactose of the H FORMATION OF A ANTIGEN antigen. Forming now the B antigen, in which The A antigen expression is dependent on the inheritance of the the immunodominant sugar is the D-galactose ABO gene. Immunodominant sugar AA (homozygous) or AO (heterozygous) ○ D-galactose (Gal) ○ able to express A antigens on their red cell. ABO gene BIOCHEMICAL STRUCTURES ○ Found on Chromosome 9 Alleles: A, B, and O ○ In order to express the A antigen, the individual must have the A gene. But it could also occur with the combination of A and the amorph ABO gene(O). The inheritance of the A gene will not directly from the A antigen. Rather, it will produce a specific enzyme in the form of Alpha-3-N-acetylgalactosaminyltransferase. Gene Product: Alpha-3-N-acetylgalactosaminyltransferase ○ Alpha-3-N-acetylgalactosaminyltransferase will transfer the sugar, N-acetyl-D-galactosamine. ○ The paragloboside must be converted first to H antigen before it is converted to A antigen. That’s why the formation of the ABO antigens are dependent on the presence of the H gene BIOCHEMICAL STRUCTURES Inheritance of A or B antigen while lacking the IMMUNODOMINANT GENE PRODUCT H gene will prevent the paraglobosides from SUGAR being converted to H antigen. Therefore also preventing the formation of A and B antigen. ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 4  L-fucosyltransferase or SECRETOR GENES H alpha-2-L-fucosyltransferase SECRETOR NON-SECRETOR GENE(rbc) (SeSe or Sese) (sese) & (responsible for the transfer of the L-fucose Se GENE fucose sugar via 1-2 linkage to the ✔ alpha-2-L-fucosyltransferase ✖ alpha-2-L-fucosyltransferase (secretion terminal galactose of your s) paragloboside or your precursor ANTIGEN LOCATION substance) N-acetylgalactosaminyltransferase or INTEGRAL PARTS alpha 3 N-acetyl-D-galactosamine The ABO antigens are not exclusively found on the RBC transferase membranes alone mainly because they also contain the A GENE N-Acetylgalactosamine paragloboside or the precursor substance of your ABO antigens. (transfer the N- acetyl galactosamine It could also be found in other parts of the body such as: sugar to the terminal galactose via ○ RBCs 1-3 linkage) ○ Endothelial cells D-galactosyltransferase ○ Platelets B GENE (transfer D-galactose sugar via 1-3 D-galactose ○ Lymphocytes linkage to the terminal galactose of h ○ Epithelial cells antigen) The number of antigens is going to vary, in which the antigens found in the other parts are not as abundant compared to the Remember: number of the antigens that are present on the RBC membrane. a and b antigen formation will be dependent on the presence of h antigen, APPLICATION ○ h antigen is lacking -> all paraglobosides will remain as ANTIGEN EXPRESSION is = no formation of a and b antigen GENE INHERITED RBC SALIVA AB HH Sese A, B, H A, B, H MOLECULAR BASIS OF ABO BLOOD TYPES ✔ AB gene A and B Transferase differ from the substitutions of the amino ✔ H gene Alpha-2-L-fucosyltransferase acids on particular amino acid sequence on A and B transferases converting all paragloboside to A, B, and H is A, B, and H is Amino Acid Substitutions in A and B Transferases: become H-antigen. expressed expressed AMINO ACID NUMBER H-antigen converted to become PHENOTYPE either A or B antigen 176 235 266 268 ✔ Se gene (Secretor) A Arg Gly Leu Gly AB Hh sese A, B H NONE B Gly Ser Met Ala ✔ AB gene They have the same amino acid sequence except for the particular ✔ H gene amino acids in this position 176th, 235th, 266th, and 268th, so Alpha-2-L-fucosyltransferase because of these differences iba iba ang sugar na tinatransfer nila. converts the type 2 paragloboside Absence of H that is only found in the RBC A, B, and H is antigen in the saliva ○ For A → N-acetylgalactosamine which will convert to the A and B expressed since it is a ○ For B → D-galactose non-secretor antigen. ✖ Se gene (Non secretor) FORMATION OF ABH SOLUBLE ANTIGENS AND SECRETOR STATUS Only RBC is able to express the A For ABO/ABH antigens to be expressed in secretion you need to and B antigens OO HH Sese H H inherit the secretor genes ✔ O gene Does not code for any enzyme. SECRETOR GENES Therefore, there will not be any A H is only expressed Alleles or B antigens converted. in RBC H antigens is ✔ H gene expressed because ○ Se H antigen will remain No AB antigen b/c secretor ○ Se (silent gene) ✔ Se gene (Secretor) only O is inherited Gene product: Alpha-2-L-fucosyltransferase Type 1 precursor substances converted to H antigens. ○ Secretor gene does not directly produce the ABH OO HH sese H NONE antigens in the secretions but rather it is going to form ✔ O gene an enzyme, alpha-2-L-fucosyltransferase ✔ H gene H is only expressed ○ Alpha-2-L-fucosyltransferase will transfer L-fucose in the Alpha-2-L-fucosyltransferase in RBC converts type 2 precursor H is absent in saliva 2nd carbon of the terminal galactose → H gene b/c non secretor substances found in RBC to No AB antigen b/c ○ Only transfers L-fucose on Type 1 paragloboside only O is inherited become H antigen. In which it has the beta 1,3 linkage of the ✖ Se gene (Non secretor) terminal galactose to the preceding sugar (NAG). NOTE: Not all H antigens will be converted to A or B. Some are Unconverted Immunodominant sugar: L-fucose particular paragloboside: type I ABO ANTIBODIES Se enzyme cannot convert the paragloboside on the red cell Naturally occuring membrane, only on the secretions ○ Acc to Karl Landsteiner’s law - an individual is not going ○ At the same time, the paragloboside produced by H to produce an antibody against an ABO antigen that is antigen could not convert the paragloboside found on present on their red cells but ABO antibodies are said to the secretions but only on the red cell membrane. be naturally occurring. ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 5  ○ No prior exposure to the antigen is needed to produce ➔ Perform cross matching first to identify ABO blood type of ABO ab in the serum/plasma. recipient and donor to ensure safety of transfusion. Recall: ab are produced mainly to the exposure of an antigen(Ie. blood transfusion, pregnancy) If you are Type A -> anti-B is already present in plasma even w/out exposure via transfusion or pregnancy. ○ The reason why you have your naturally occurring antibodies is mainly b/c the bacteria in nature tend to have A/B -like antigens and b/c we are exposed to them so that’s why it lead to the production of anti-a and anti-b present in the blood of the px. ○ Production is not mainly due to exposure to a particular antigen but they are just there. ○ Only ABO can be performed in reverse typing. ABO immunoglobulins (IgG & IgM) ○ Both react preferentially at room temperature(20-24oC or below) Parents Offspring ○ Both efficiently activate complement at 37oC (AA x AA) IgM homozygous (BB x BB) Predominant antibody type in ABO Homozygous (OO) Reacts at room temperature No A or B antigens present on rbc IgG Both homozygous (OO x OO) Group O individuals: only H ag are IgG forms of Anti-A and Anti-B that present on the rbc could be present in the plasma of A & B are both dominant. Both are (AA x BB) an individual. expressed Reacts at body temperature. Homozygous and (AA x AO) ○ In Type O individuals -> Anti-AB (IgG form) Homozygous or heterozygous heterozygous (BB x BO) Anti-A and Anti-B are present b/c they have One of the offsprings could be: no A & B antigens BUT, they have the anti-AB Homozygous (AA or BB) Reacts on both A & B antigens but Both heterozygous (AO x AO) Heterozygous (AO or BO) (BO x BO) on different epitopes from where double dose of amorph Anti-A and Anti-B reacts. gene (OO) produces strong agglutination RECALL: Initiated at birth Forward typing ○ Forward typing only on newborns ○ Identify antigens present on RBC ○ Titers of ABO ab at birth are very low until the baby ○ Reagent: Antibodies reaches 3-6 months of age Reverse Typing ○ Identify antibodies in plasma Since there is already the presence of antigen ○ Reagent: RBC w/ either A or B antigens upon birth, we can identify their ABO blood type. FORWARD TYPING HOWEVER, it is not wise to perform reverse typing on newborns bc the titer is very low - SLIDE METHOD undetectable Results obtained from reverse typing are actually ab coming from the mother or maternal in origin which cannot support your forward typing ○ Although there is a presence of ABO ag on the rbc upon birth, the number of ag is only 25-50% compared to those of the adult. Prepare the slide ○ 3 - 6 months after birth, there is enough ab to be ○ Label it with name , age , sex detected ○ Label Anti A and Anti - B Ab declines later in life - elderly people usually (1) Place antiserum have very low levels of anti-A and anti-B which ○ The purpose of placing the antiserum first, is for you to gives problems in reverse typing. identify that you have already added the serum, ○ Ab production peaks at 5 - 10 years until adulthood otherwise if you place blood first , it is not easy check Cause intravascular hemolysis /recall if you already have put the antiserum ○ Through complement activations via blood transfusions ○ The other reason, antiserum act as cushion to the blood of the wrong ABO blood type to a particular px. b/c upon dropping there is an impact that could cause NOTE: transfusing the wrong blood type to a particular px will activate a hemolysis. complement from the naturally occurring ab regardless if it’s IgM or IgM ○ Anti-serum color indicators: which could lead to immediate lysis of transfused red cells which is fatal to Anti A - Bromthymol blue [blue Angel] the px. Anti B - Acriflavine yellow [yellow Bird] ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 6  (2) Add one drop of patient’s red cell in each antiserum ○ Reverse typing : the patient has antibody A ○ Use different applicator stick to mix each of the According to the law of Karl Landsteiner - you antiserum with blood to avoid cross reaction cannot produce an antibody against your own ○ Rotate the slide manually for a few turns , no need to use antigen but can produce an antibody against a rotator. an antigen that is lacking on the red cell Read the results Since the patient has antigen B , normally it is ○ Observe for the agglutination going to produce antibody A indicate the presence of antigen on the RBC. Reverse typing checks or confirms the forward typing Based on the picture above, there is a homogeneous mixture of the red cell and the antisera. ○ Anti -A=No agglutination , this indicates that there is no A antigen in the red cell of the patient because the anti- a did not react ○ Anti B = there is agglutination, which means there is antigen B present on the red cell of the patient since Anti- B reacted ○ Therefore , this patient will going to have B blood type BLUE - ANGEL (reaction with blue reagent = blood type A) If there is reaction in anti A ,you have presence of A antigens on YELLOW- BIRD (reaction with yellow reagent = blood type B) the RBC, but no reaction with anti-B. Therefore the patient is TYPE A REVERSE TYPING If there is no reaction in anti -A, but there is agglutination in Anti B ,therefore the RBC has B antigen; the patient is TYPE B SLIDE METHOD If there is both agglutination in anti A and anti- B, then there is A antigen and B antigen; the patient is TYPE AB Prepare the slide : If there is no reaction or agglutination in both anti-A and anti-B, ○ name of the patient , age, sex therefore the patient lacks antigen A and antigen B; classified as ○ Label with KA ( known A cells ) KB ( known B ) TYPE O Reagents for reverse typing Note: THERE ARE NO COLOR INDICATORS IN REVERSE TYPING Prepared in red cell suspension ( 1) Place the patient’s serum first ○ This is to ensure that you have added the serum , otherwise you may forget if you added the patient’s serum once you place the red cell first. ○ Acts as cushion (2) Place the known cells ○ Mix with applicator stick ○ Rotate for few times (3)Read the results ○ Observe agglutination reactions In reverse typing if there is reaction in known A cells and Known B cells, therefore it indicates that the patient has antibodies against A antigen and B antigen; this is for patients with TYPE O since they lack both antigen , therefore they can produce both antibody Based on the example above, against the lacking antigen. : there is Anti- A and Anti B in RBC ○ there is agglutination in known A cells, therefore the If there is reaction in known A cells and no reaction in known B patient has antibody A. cells, therefore the patient lack antibody B but has antibody ○ Known A = There is agglutination, px has Antibody A (anti-A); classified as TYPE B ○ known B = there is no reaction, this indicates absence of If the reaction is only in known B cells , therefore the patient has anti B antibody of the patient. antibody B ( anti-B) ; classified as TYPE A If there are no reaction in both known A and B cells ,therefore the it indicates absences of antibody A and antibody B in the serum /plasma of the patient towards towards both antigens; classified as TYPE AB; since they have both A and B antigens therefore it lacks A and B antibody thus no reaction. Base the results of the example: FORWARD TYPING ○ Forward typing result : The patient is blood group B ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 7  TUBE METHOD 1st row set of tubes ○ Forward typing: There is a homogenous mixture of serum and red cells. It also indicates the absence of antigens on red cells mainly because anti-A Ab and anti-B Ab did not cause any agglutination reactions. Therefore, absence of A and B antigen = Blood group O Prepare 2 tubes ○ Reverse typing: If agglutination reaction is present on ○ (1) label with anti- A and (1) label with anti-B known A and known B = px serum has antibody on both ○ Indicate the name of the patient , age and sex A antigen and B antigen – typical for individuals with O ○ Ideal labeling is in vertical manner blood group Add reagent first prior to red cell 2nd row set of tubes ○ Place 1 drop of anti-A and anti-B respectively ○ Forward typing: There is an agglutination reaction ○ Purpose of which are for easy determination or which indicates that the px has antigen on red cells checking and to provide cushion to the RBC to avoid specifically A antigens therefore a blood group A. hemolysis upon impact ○ Reverse typing: Px serum has reacted with the known B Add 1 drop of the patients RBC that is prepared on a suspension cells indicating that it produced an antibody against an Shake it gently for a few times antigen that is absent on its red cells – typical for Centrifuge the tube for 10 seconds only individuals with A blood group Observe for agglutination 3rd row set of tubes ○ Forward typing: There is an agglutination reaction with antibody B, therefore the px’s red cells contain B REVERSE TYPING antigens TUBE METHOD ○ Reverse typing: Its plasma contains anti-A since it reacted on the known A cells – typical for individuals with B blood group 4th/last row set of tubes ○ Forward typing: There is an agglutination reaction on both Anti-A and Anti-B reagents indicating the presence of both A and B antigens on the red cell of px ○ Reverse typing: No agglutination reaction is observed on both known A and known B since AB individuals lack antibodies against their own antigens – typical for individuals with AB blood group 1. Prepare (2) tubes a. label it with KA for the known A cells and KB for the REMEMBER: Reverse typing is the confirmation of forward typing. known B cells ○ If forward and reverse typing do not coincide by using ○ Ideally, labeling of px’s; name, age and sex is written in the sample of the px, most likely there is a vertical position discrepancies or problems such as; 2. Place first serum on tube and add (1) drop of known A cells on KA Underlying conditions tube and (1) drop of known B cells on KB tube Technical problems 3. Shake it gently and place it on centrifuge and spin it for 10 Problem in the reagent seconds Problem in the px’s sample 4. Take tubes out of centrifuge and read for the results by trying to ○ Resolve the discrepancy first before releasing it as a observe any agglutination present result AGGLUTINATION IN BLOOD TYPING METHOD FORWARD & REVERSE TYPING If an antigen is present in a red cell, the specific antibody against the antigen is going to react on it forming the lattice formation. ○ hemagglutination reaction for blood typing. TUBE METHOD RESULT Explanation: The possible reactions on forward and reverse typing of ABO determination using tube method are as follows; ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 8  RH antibodies ○ Last 3 tubes - Control, A1 and B have no reagent, only gel. FORWARD TYPING: ○ A, B, Rh REVERSE TYPING: ○ A1 and B PRINCIPLE If there is any agglutination reaction, the cell with the antibody that has reacted to it is going to be trapped along the gel and could not go at the bottom of the gel if there is any agglutination It is necessary to grade reaction either 4+, 3+, 2+, 1+ or 0 reaction. Hemolysis is also determined during testing ○ In this case, hemolysis could not always be a positive PROCEDURE reaction mainly because it could also be indicating that 1. A, B Rh: Add a drop of the red cell suspension of the patient the problem is actually on the sample of the patient. sample. It could be that the sample is hemolyzed prior 2. Control/auto control: mixture of patient’s to red cell suspension or during the red cell serum or plasma with red cells. suspension ○ Done to determine if the patient ○ Hemolyzed sample = Repeat testing has antibodies against the Often times, the strongest reaction is 4+ and 3+; the weakest is antigen present on the red cells 2+ and 1+ ○ Commonly encountered with: If there is no agglutination reaction, 0 will be written on the result Individuals who have sheet multiple transfusions With autoimmune DIFFERENT AGGLUTINATION REACTION IN THE TUBE METHOD disorders 3. A1 and B: ○ Place 1 drop of the patient’s serum ○ Add known A cells in A1 and known B cells in B tube. 4. Incubate (if necessary) 4+ - Red cell button is a solid agglutinate 5. Centrifuge - Clear background ○ Specialized centrifuge to accommodate the gel card. To confirm if true agglutination: shake tube slightly and see if it remains agglutinated ○ Different from the usual centrifuge we commonly use. 6. Observe red cell button formation at the bottom of the gel tube. 3+ - Several large agglutinate Specialized centrifuge to accommodate the gel card. - Clear background 2+ - Many medium-sized agglutinate - Clear background 1+ - Medium and small-sized agglutinate - Background is turbid with many free red cells 0 - No agglutinated red cells are visible - Red cells are observed flowing off the red cell button during the process of grading FORWARD TYPING: FORWARD & REVERSE TYPING: GEL METHOD Tube 1: Presence of red cell at the bottom of the gel tube. ○ No antigen and antibody complexes that were formed since the red cells were able to diffuse through the agarose gel. ○ DOES NOT CONTAIN THE A ANTIGEN Tube 2: Red cell has formed a layer at the top portion (trapped) ○ There is an antigen and antibody reaction, since they’re trapped at the top portion of the gel. ○ POSITIVE FOR B ANTIGEN (Blood type B) HAVE REAGENTS | NO REAGENTS Tube 3: to determine Rh positive ○ It contains Anti-Rh antibodies. Widely used ○ A layer of red cells forming indicates antibody Utilizes a gel card which contains 6 tubes complexes. ○ gels are also incorporated with the reagents ○ RH POSITIVE ○ First 3 tubes - contains reagents that has; CONTROL TUBE: Anti A Anti B ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 9  ○ Red cell has diffuse through the agarose gel, hence no antibody against its own red cells. ○ NO AUTOIMMUNE DISORDER. REVERSE TYPING: A1 Tube: Red cell trapped at the top ○ Has Anti-A against. ○ Confirming its blood type B. B Tube: Red cell at the bottom ○ The 1st carbon of the terminal Galactose on the ○ No reaction, since it diffuses at the bottom. precursor substance is attached to the 4th carbon of ○ HAS NO ANTI B N-acetylglucosamine in a beta 1 → 4 linkage. This is now a paragloboside/precursor RESULT: Patient has Blood type B, Rh positive. substance IN REPORTING, SPELL OUT Rh POSITIVE/NEGATIVE ○ Immunodominant sugar: Galactose Same method is also applied in cross matching, some ○ HARMENING: Sugars occupying the terminal positions laboratories would have a slide and tube method. of this precursor chain and conferring blood group ○ Prone to drying, you cannot retrieve it for future specificity are called the immunodominant sugars references. DISADVANTAGE: ○ Somewhat expensive ADVANTAGE: ○ If there is a need to review the results, you may do so. ○ You can utilize it for a month RESULTS Reactions in the Gel method are GRADED ○ 0: negative H GENE ○ 1+: small amount of complexes formed INHERITANCE OF THE H GENE: ○ 2+: presence of aggregates at the top and small ○ able to code an α-2-L- fucosyltransferase (enzyme). amount at the bottom that transfers the sugar L-fucose in the ○ 3+: few red cell antigens were able to reached the terminal galactose of type 2 chains. bottom You will have a 1-2 Linkage of fucose to the ○ 4+: No red cell were able to diffuse/strong agglutination terminal galactose. ○ Hemolysis: based on color, reddish tint. This is now the H antigen. Because of complement activation, ○ the immunodominant sugar L-fucose. hemolysis would indicate a positive reaction HARMENING: L-fucose is the sugar or due to hemolyzed red cells in the red cell responsible for H specificity. suspension. To ensure hemolysis reaction, repeat the procedure. REMEMBER: ALL paragloboside must be converted to become H RECALL OF THE FORMATION OF A, B, AND H ANTIGENS antigen before it is converted to A or B antigen or both, depending 2 types of precursor substances: type 1 & type 2 on the gene you inherited. TYPE 1 A GENE Found in the secretions A gene is inherited. HARMENING: beta 1 → 3 linkage between galactose and ○ Able to code for production of N-acetylglucosamine. α-3-N-acetylgalactosaminyltransferase transfers an N-acetyl-galactosamine sugar via 1-3 linkage via 1-3 linkage on the D-galactose (Gal) sugar after H antigen. TYPE 2 Found in the red cell membrane Classified because of the linkage of terminal galactose with the preceding sugar and that is the N-acetylglucosamine. ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 10  B GENE Inherit a B gene ○ that codes for the production of CELLS(forward) SERUM(reverse) ABO GROUP α-3-D-galactosyltransferase (enzyme). ANTI-A ANTI-B A CELLS B CELLS transfer the galactose (Gal) sugar molecule 4+ 0 0 4+ A via 1-3 linkage on the 3rd carbon of the 0 4+ 4+ 0 B terminal galactose. 4+ 4+ 0 0 AB 0 0 4+ 4+ O If there is a reaction with ANTI-A but no reaction with ANTI-B results, the antigen present on the red cell of the patient is A antigen. ○ It can be corrected or confirmed with the reverse typing, the reaction is on B cells. Therefore, the patient serum has an antibody against the B antigen. ○ Which indicates that it has antigen A on its red cell and AB INDIVIDUALS ANTI-B on their plasma or serum. Hence, A Blood type. Both A and B genes are inherited. If the forward reaction is on ANTI-B, it indicates B antigen. ○ They are Codominant genes Confirm it with the reverse typing, 4+ in A cells therefore the ○ Both will be expressed on the red cell surface patient has A cell. Hence, B Blood type. membrane. If the forward reaction shows both ANTI-A and ANTI-B, indicating the presence of both A and B antigens on the red cell. No O GENE agglutination reactions against known A cells and B cells since If you have inherited the h gene then you are able to convert your they cannot produce antibodies since its present on their red cell. paragloboside into H antigen. But, if you have inherited the O gene ○ AB Blood type of the ABO blood group, you are not able to produce any enzymes If the forward reaction shows no reaction on both ANTI-A and that are going to convert h antigens to become A or B antigen. ANTI-B, indicates both absence of antigens on the red cell of the ○ H antigens remains untouched patient. This enables them to make antibodies against them. ○ Blood type O has no A or B antigens, only H antigens Hence the reaction in the reverse typing. present on the red blood cell. ○ O Blood Group The O blood group has the highest concentration of H antigen [END] compared to A and B. Se GENE The expression of A, B, and H antigens are not only limited on the red cell membrane but can also be found in the secretions. inheritance of Se gene codes for the production of the transferase α-2-L-fucosyltransferase (enzyme). se gene if there is no coding of such enzyme. REMEMBER: TYPE ONE ○ Found in the secretions ○ HARMENING: beta 1 → 3 linkage between galactose and N-acetylglucosamine. The α-2-L-fucosyltransferase (enzyme). ○ Transfer the fucose molecule via 1-2 linkage on the terminal galactose of the precursor substance of type 1 precursor substance or paragloboside. ○ To form the H antigen that is found on the secretions. ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 11