2. AP Biology 1 The Basics Of Blood  Controlled by genes at unknown No. of chromosomal loci.  Appearance by 40 days of I.U. Life- unchanged till death.  Also present in tissues & tissue fluids.  Blood group system: A group of antigens controlled by a locus having a variable no of allele genes. Antigens: -

3. AP Biology 2 The Basics Of Blood  > 15 blood group systems are recognised :  ABO, Rh, Kell, Duffy, MN, P, Lewis, Lutheran, Xg, Li, Yt, Dombrock, Colton, Public antigens & Private antigens.  Blood type- means individual antigen phenotype which is the serological expression of the inherited genes  Most of these blood group antigens have been found to be associated with hemolytic disease.  However– ABO & Rh account for 98% Antigens: -

4. AP Biology 3 The Basics Of Blood Alloantibodies / Agglutinins Natural IgM Iso / immune antobodies IgG Formed in response to foreign R.B.C. or soluble blood group substance. Antibodies: -

5. AP Biology 4 The Basics Of Blood  Antibodies are formed against most of the major group antigens & present in almost all individuals when the antigen is absent.  In most other minor systems, natural antibodies to the antigens are found occassionally but as their anitgenicity is low, the immune antibodies are also rare ( except –Kell & Duffy)  Mostly of them are IgM type.  React poorly at body temp. ( except anti-A & anti- B), but agglutinate R.B.C.s at 5-20°C  Usually do not cross placenta. Natural Antibodies: -

6. AP Biology 5 The Basics Of Blood  In contrast the immune or isoantibodies are IgG.  Best react at body temp. & readily cross placenta.  Most antibodies are complement binding notable exceptions being Rh & MN. Immune Antibodies: -

7. AP Biology 6 Antibodies Can Be Detected by: - a. Saline agglutination test (SAT). b. Tests using cells suspended in colloid media. c. Tests using enzyme-treated cells- Rh & occasional antobodies. d. Indirect antiglobulin ( Coomb’s test) - wide spectrum.  Antibodies may be Complete / Incomplete  IgMIgG Detected by  SATb, c, d

8. AP Biology ABO Blood Grouping

9. AP Biology ABO Basics  Blood group antigens are actually sugars attached to the red blood cell.  Antigens are “built” onto the red cell.  Individuals inherit a gene which codes for specific sugar(s) to be added to the red cell.  The type of sugar added determines the blood group.

10. AP Biology Introduction to Blood ABO blood groups found on outside of cell

11. AP Biology Introduction to Blood ABO blood groups found on outside of cell

12. AP Biology Introduction to Blood  RBC = no DNA; WBC = yes DNA All blood cells have blood groups on outside of cell. Red Blood Cells contain the protein hemoglobin, which carries oxygen

13. AP Biology Introduction to Blood Hemoglobin picks up and drops off oxygen

14. AP Biology This diagram illustrates the terminal sugar for each blood group.

15. AP Biology ABO Type Frequencies In U.S. ABO TypePer Cent O45% A40% B11% AB4%

16. AP Biology Landsteiner’s Rule  Individual’s will form immune antibodies to ABO blood group antigens they do not possess.  Substances are present in nature which are so similar to blood group antigens which result in the constant production of antibodies to blood group antigens they do not possess.  Critical for understanding compatibility between ABO blood groups.

17. AP Biology Antibody clinical significance  Immunizations are frequently done to protect us from disease.  Receive Hepatitis B immunization.  Actual bits of hepatitis virus injected.  Body recognizes as foreign and produces an immune antibody.  Subsequent exposure to real Hepatitis B virus will result in destruction of the virus by immune antibodies.  ABO antibodies are immune and will result in destroying incompatible cells which may result in the death of the recipient.

18. AP Biology Inheritance  Blood group antigens are “codominant”, if the gene is inherited, it will be expressed.  Some aberrant genotypes do occur but due to the rarity will not be discussed.  Understanding of basic inheritance important.

19. AP Biology Genetics  Two genes inherited, one from each parent.  Individual who is A or B may be homozygous or heterozygous for the antigen.  Heterozygous: AO or BO  Homozygous: AA or BB  Phenotype is the actual expression of the genotype, ie, group A  Genotype are the actual inherited genes which can only be determined by family studies, ie, AO.

20. AP Biology Example of Determining Genotype  Mom’s phenotype is group A, genotype AO  Dad’s phenotype is group B, genotype BO BO AAB 25%AO 25% (Group A) OBO 25% (Group B)OO 25% (Group O)

21. AP Biology Other Examples MomDadOffspring Blood Group AABB100% AB BOOO50% each of B or O OO 100% O OOAO50% each of A or O

22. AP Biology Group O  Approximately 45% of the population is group O.  No A or B antigens present, think of as “0” antigens present.  These individuals form potent anti-A and anti-B antibodies which circulate in the blood plasma at all times.

23. AP Biology Group A  Approximately 40% of the population is group A.  No B antigens present.  These individuals form potent anti-B antibodies which circulate in the blood plasma at all times.

24. AP Biology Group B  Approximately 11% of the population is group B.  No A antigens present.  These individuals form potent anti-A antibodies which circulate in the blood plasma at all times.

25. AP Biology Group AB  Approximately 4% of the population is group AB.  Both A and B antigens present.  These individuals possess no ABO antibodies.  NOTE: This slide is in error as it only illustrates presence of one antigen not 2.

26. AP Biology Hemolysis  If an individual is transfused with an incompatible blood group destruction of the red blood cells will occur.  This may result in the death of the recipient.

27. AP Biology Summary Blood GroupAntigens on cellAntibodies in plasma Transfuse with group AAAnti-BA or O BBAnti-AB or O ABA and BnoneAB, A, B or O ONoneAnti-A & BO

28. AP Biology ABO & Rh(D) 27 The Rh(D) Antigen  RH is the most complex system, with over 45 antigens  Discovered in 1940 after work on Rhesus monkeys  Subsequently discovered to be unrelated to monkeys  RH gene located on short arm of chromosome 1

29. AP Biology 28 Rhesus Blood Group System  First demonstrated by testing human blood with rabit anti sera against red cells of Rhesus monkey & classifying Rh negative & Rh positive.  However the underlying biochemical genetics is not well understood and the genotyping & phenotyping remains little confused  The genotype is determined by the inheritance of 3 pairs of closely linked allelic genes situated in tanderm on chromosome 9 & named as D/d, C/c, E/e (Fisher- Race theory)

30. AP Biology 29 Rhesus Blood Group System  The gene ‘d’ is an amorph & has no antigenic expression. So there are only five effective antigens.  But Weiner postulates a series of allelic genes at a single locus Rho (D), rh (C),rh (E), hr © & hr (e)  The updated system of Rosenfield refers these antigens as – Rh1, Rh2, Rh3, Rh4, Rh5  Subsequently less common antigens Cw, Du, Es have been found  The foetus inherits one gene from each group as a haplotype such as sets of Cde, cde etc from each parent

31. AP Biology 30 Rhesus Blood Group System  12 sets of combinations & 78 genotypes are possible. Most frequent genotypes are –  Cde/cde(33%), Cde/cDe(18%), Cde/cDE(12%) cDE/cde(11%), cde/cde(15%), cdE/cde(1%), Cde/cde(1%)  Though several Rh genotypes and phenotypes have been described, for clinical & all practical purposes it is enough to know whether one is Rh POSITIVE or NEGATIVE against anti D sera.

32. AP Biology 31 Rhesus Blood Group System  Incidence of Rh negative varies in different races:  Mongoloids- nil, Chinese & Japanese- 1-2%, Indians-5%, Africans-5-8%, Causcasians-15- 17% & Basques-30-35%.  The antigenic expressions of these genes are dependent on an interaction between R.B.C. membrane protein & phospholipid molecules resulting in a set of antithelical epitopes, the coresponding antigens, consisting of C/c, D/d, E/e.  The antigenic determinants form an intrinsic part of the red cell membrane protein structure.

33. AP Biology 32 Rhesus Blood Group System  C/c & E/e are weak antigens and impractical to match.  ‘D’ is by far the most immunogenic in the Rh system excepting those that have the natural antibodies.  There is a rare type of Rh negative called Rh null who lack all known Rh antigens.  ‘D’ antigen has no natural antibody while C & E have the coresponding natural antibodies, though weak & found infrequently.

34. AP Biology 33 Rhesus Blood Group System  A single transfusion of + ve blood to a – ve person has a 50% chance of forming anti Rh D antibodies (IgG)  Anti Rh antibodies are of three categories-  1st order – saline / bivalent / complete antibodies  2nd order - albumin active / univalent / incomplete antibodies  3rd order – atypical / antiglobulin active / incomplete antibodies

35. AP Biology ABO & Rh(D) 34 Simple Genetics of Rh(D)  86% of caucasians are Rh(D) pos  The antithetical antigen d has not been found  The d gene is recessive:  Dd, dD, DD, persons are Rh(D) pos  Only dd persons are Rh(D) neg

36. AP Biology ABO & Rh(D) 35 Distribution of Rh(D) Types PopulationRh(D) posRh(D) neg Caucasian86%14% African- American 95%5% Oriental>99%<1%

37. AP Biology ABO & Rh(D) 36 Significance of Rh(D)  80% of Rh(D) neg persons exposed to Rh(D) pos blood will develop anti-D  Anti-D can also be stimulated by pregnancy with an Rh(D) positive baby  Sensitisation can be prevented by the use of anti-D immunoglobulin, antenatally and post natally  Rh(D) neg females of childbearing potential should never be given Rh(D) positive blood products

38. AP Biology ABO & Rh(D) 37 Inheritance  ABO & RH genes are not linked  ABO & Rh(D) type are inherited independently For example: An A Rh(D) pos mother and a B Rh(D) pos father could have an O Rh(D) neg child

39. AP Biology ABO & Rh(D) 38 Inheritance of ABO and Rh(D) Mother Group A AO Rh(D) pos Dd Father Group B BO Rh(D) pos Dd Group A AO Rh(D) pos Dd Group B BO Rh(D) pos Dd Group O OO Rh(D) neg dd

40. AP Biology Hemolytic Disease of the Neborn – How it Occurs  A child is Rh pos  B during pregnancy fetal Rh pos rbc’s escape into maternal circulation  C Mother produces antibodies to Rh (D) antigen  D Second pregnancy with Rh (D) pos child results in destruction of fetal D pos rbcs

41. AP Biology 40 Pathogenesis Of Rh Iso-immunisation  Rh Negative Women Man Rh positive (Homo/Hetero)    Fetus   Rh Neg Fetus No problem Rh positive Fetus   Rh+ve R.B.C.s enter Maternal circulation  Mother previously sensitized Secondary immune response   ? Iso-antibody (IgG)  Non sensitized Mother Primary immune response  Fetus  unaffected, 1 st Baby usually escapes. Mother gets sensitised?   Fetus Haemolysis   ?

42. AP Biology 41 Pathogenesis Of Rh Iso-immunisation  Chances of T.P.H/F.M.H. are only 5% in 1st trimester but 47% in 3rd trimester, many conditions can increase the risk.  Chances of primary sensitization during 1st pregnancy is only 1-2%, but 10 to 15% of patients may become sensitized after delivery.  ABO incompatibility and Rh non-responder status may protect.  Amount of antibodies that enter the fetal circulation will determine the degree of haemolysis

43. AP Biology 42 Pathology Of Iso-immunisation HAEMOLYSIS  IN UTEROAFTER BIRTH  BILLIRUBIN  ANAEMIA   MAT. LIV NO EFFECT   HEPATIC ERYTHROPOESIS & DYSFUNCTION  PORTAL & UMBILICAL VEIN HYPERTNSION, HEART FAILURE       BIRTH OF AN AFFECTED INFANT - Wide spectrum of presentations. Rapid deterioration of the infant after birth.May contiune for few days to few months. Chance of delayed anaemia at 6-8 weeks probably due to persistance of anti Rh antibodies.  Jaundice Kernicterus Hepatic Failure  DEATH ERYTHROBLASTOSIS FETALIS    IUD     

44. AP Biology 43 Prevention of Rh Incompatibility  Premarital counseling? Ambitious?  Proper matching of blood particularly in women before childbearing.  Blood grouping must for every woman, before 1st pregnancy.  Rh+ve Blood transfusion- 300mcg Immunoglobulin (minimum).  Proper management of unsensitised Rh negative pregnancies.

45. AP Biology 44 Management of Unsensitised Pregnancy  Blood typing at 1st visit, If negative husband’s typing. If husband is also negative then no treatment  If husband is positive, if possible, Homo/Hetero?  Do Indirect Coomb’s test of mother –  Negative-good.  Repeat ICT at 28 weeks – Negative- ICT at 35 weeks - Negative- Observe  Positive  Sensitised - 300mcg Rh immunoglobulin

46. AP Biology 45 Management of Unsensitised Pregnancy  In Abortion, Ectopic, CVS-  Pregnancy < 12 weeks- 50mcg Anti D  Pregnancy >12 weeks- 300mcg Anti D  APH, IUD, Amniocentesis, Abdominal trauma, Foetal-maternal hemorrhage - 300mcg Anti D  At birth- cord blood for ABO & Rh typing  Baby Rh negative – Be happy

47. AP Biology 46 Management of Unsensitised Pregnancy  If Rh positive- Test mother’s blood for ICT & Infant’s for DCT  Negative or weakly reactive- 300mcg immunoglobulin  Positive – Sensitised–Hb & Bilirubin Estimation of the infant -Treat the infant  ?Prophylactic Anti D administration during antenatal period to all negative mothers at 28weeks and again at 34 / 36 weeks.

48. AP Biology 47 Management of Sensitized Pregnancy  Causes of sensitization-  Misinterpretation of maternal Rh type  Rh +ve blood transfusion  Unprotected preg. & labour  Inadequate dose / improper use of IgG on previous occasions  Immunization to cross-reacting antigen

49. AP Biology 48 Management of Sensitized Pregnancy  Careful planning during antepartum, intrapartum & neonatal period  Father’s blood type & Rh antigen status  Knowledge of maternal antibody titer to the specific antigen  Intrauterine foetal monitoring with repeated ultrasound examination, cordocetesis / amniocentesis

50. AP Biology 49 Management of Sensitized Pregnancy  Fetus Rh Negative: - Observation  Fetus Rh Positive: -  Intrauterine transfusion of ‘Rh Neg’ blood as indicated  Timely delivery any time after 32 weeks  Management of the infant up to 8 weeks  In cases of severely sensitized women, consider medical termination of pregnancy and sterilization.

51. AP Biology Blood Typing  There are 2 components to blood typing:  Test unknown cells with known antibodies  Test unknown serum/plasma with known rbc’s  The patterns are compared and the blood group is determined.

52. AP Biology Slide Blood Typing  Very rudimentary method for determining blood groups.  CANNOT be used for transfusion purposes as false positives and negatives do occur.  A “false positive” is when agglutination occurs not because the antigen is present, but cells may already be clumpled.  A “false negative” is one in which the cells are not clumped because there are too many cells or not enough reagent.

53. AP Biology Slide Blood Typing - continued  The slide is divided into halves.  On one side a drop of anti-A is added, this will attach to and cause clumping of rbcs possessing the A antigen.  On the other side a drop of anti-B is added which will cause clumping of rbcs with the B antigen.  A drop of rbcs is added to each side and mixed well with the reagent.  The slide is tilted back and forth for one minute and observed for agglutination (clumping) of the rbcs

54. AP Biology Interpretation of Slide Typing Testing with Anti-A Anti-Serum  If an rbc contains the A antigen the red blood cells will be agglutinated by anti- A, a positive reaction.  If an rbc does not have the A antigen there will be no clumping, a negative reaction.

55. AP Biology Interpretation of Slide Typing Testing with Anti-B Anti-Serum  If an rbc contains the B antigen the red blood cells will be agglutinated by anti-B, a positive reaction.  If an rbc does not have the B antigen there will be no clumping by anti-B, a negative reaction.

56. AP Biology Slide Blood Typing Group A  An unknown rbc suspension is added to known anti-sera.  The left hand of the slide contains anti-A which reacts with the unknown cell.  The right hand side contains anti-B which does not react with the cell.

57. AP Biology Slide Blood Typing Group B  An unknown rbc suspension is added to known anti-sera.  The left hand of the slide contains anti-A does not react with the unknown cell.  The right hand side contains anti-B which reacts with the cell.

58. AP Biology Slide Blood Typing Group O  The left hand of the slide contains anti-A does not react with the unknown cell.  The right hand side contains anti-B does not react with the unknown cell.

59. AP Biology Slide Blood Typing Group AB  The left hand of the slide contains anti-A which reacts with the unknown cell.  The right hand side contains anti-B which reacts with the unknown cell.

60. AP Biology Summary of Slide Typing Anti-AAnti-BBlood Group NEG O POSNEGA POSB AB

61. AP Biology ABO & Rh(D) 60 The ABO Antigens  Added to Proteins or Lipids in Red Cells  Substrate Molecule is H (fucose)  A antigen is N-acetyl-galactosamine (GalNAc)  B antigen is Galactose (Gal)  A and B genes code for transferase enzymes

62. AP Biology ABO & Rh(D) 61 ABO Antibodies  A and B substances very common  Antibodies produced to “non-self”  Produced after first few months of life  A & B people have mainly IgM  O people have IgG  May fade in old age

63. AP Biology ABO & Rh(D) 62 Distribution of ABO Groups Population OABAB Aborigines613900 Basques514441 Blackfoot (N. Am. Indian)178201 Bororo100000 Chinese-Canton4623256 Chinese-Peking29273213 English474283 Hawaiians376121 Irish5235103 Mayas98111 Navajo (N. Am. Indian)732700 Peru (Indians)100000 United Kingdom (GB)474283 USA (blacks)4927204 USA (whites)4540114

64. AP Biology ABO & Rh(D) 63 Distribution of the A allele

65. AP Biology ABO & Rh(D) 64 Distribution of the B Allele

66. AP Biology ABO & Rh(D) 65 Distribution of the O Allele