1. Department of Immunology
IMMUNOHEMATOLOGY By
E. Salehi Ph.D.
Assistant prof.
Department of Immunology
History
ABO System Phenotype
ABO System Genotype
Rh system
Other Blood Groups
Blood Group detection and incompatibility
Hereditary Newborn Disease HDN
Blood Transfusion

2. Karl Landsteiner (1868-1943) Discovered ABO blood groups, 1900
Nobel Prize, 1930

4. Red Blood Cell Membrane Components

5. Biological Functions of Blood group Systems
Functional Diversity
Transporters/Channels
Transporting Water-Soluble molecules/compounds
Rh, Colton, Diago, Kx, Kidd
Receptors
Biological
Duffy, Knops, Indian
Microbial
MNS, P, Lewis, Duffy, Cromer
Adhesion Molecules
Leuthran, Xg, L-W, Indian
Role in Complement Pathway
Chido/Rodgers, Cromer, Knops
Enzymes
ABO, P, Lewis, H
Structural Proteins
Maintain Shape
MNS, Diago, Gerbich

6. Type .2
Type .1 3
*PS = oligosaccharide chain attached to either glycosphingolipid (RBC) or glycoprotein (secretions).

7. Type 2 Precursor Chain

8. Formation of H Antigen

10. ABO Antigen Genetics LOCATION
The presence or absence of the ABH antigens on the red blood cell membrane is controlled by the H gene
The presence or absence of the ABH antigens in secretions is indirectly controlled by the Se genes.

11. H Antigen
The H gene codes for an enzyme that adds a sugar (Fucose) to the terminal sugar of a Precursor Substance (PS*). The biochemical structure below constitutes the H Antigen. (h gene is an amorph.)
H gene acts on a Precursor substance(PS)* by adding Fucose
*PS = oligosaccharide chain attached to either glycosphingolipid (RBC) or glycoprotein (secretions).

12. The H antigen is found on the rbc when you have the Hh or HH genotypes but NOT from the hh genotype.
The A antigen is found on the rbc when you have the Hh, HH, and A/A, A/O or A/B genotypes.
The B antigen is found on the rbc when you have the Hh, HH, and B/B, B/O or A/B genotypes.

13. Possible Blood group Genotypes
Parent
Allele A B O AA AB AO BB BO OO

14. ABO Subgroups
ABO subgroups differ in the amount of antigen present on the red blood cell membrane, specifically, they have less - it is quantitative.
Subgroups are the results of less effective enzymes! Not as efficient at converting H antigens to A or B antigens so fewer are present on the rbc.
Subgroups of A are more common than Subgroups of B.

15. Subgroups of A The two principle subgroups of A are: A1 and A2
Both react strongly with reagent anti-A.
To distinguish A1 from A2 red blood cells test with plant lectin: Dolichos biflorus
Approximately 80% of Group A and Group AB persons red cells are agglutinated by Dolichos biflorus and can be designated A1 and A1B.
The remaining 20% are A2 and A2B.

16. ABO Subgroups A2 Phenotype
A2 persons produce anti-A1 allo-antibodies (%1-8)
A2B persons produce anit-A1 allo antibodies (%22-35)
Allo-Anti-A1 can cause ABO Discrepancies (How?) and incompatibility in crossmatching. It is not considered clinically significant if it does not react at 37oC.

17. Number of A antigen A1=800000 A2=250000 A3=35000 Ax=4800 Aend=3500
Am=700

18. فعالیت کم و ایده آل درPH=7A2 A1
خصوصیات
+++
++++
واکنش با آنتیA
200000
800000
تعداد -
واکنش بالکتین رقیق شده
TYPE2
TYPE1,2
ساختمان آنتی ژن
فعالیت کم و ایده آل درPH=7
فعال تر وایده آل درPH=6
فعالیت
ترانسفرازی
تنهاMg
Mn,Mg
فلز موردنیاز
6-7
9-10
نقطه فوکوسینگ ایزوالکتریکی

19. Amount of H Antigen according to ABO Blood Group
Blood Group O people have red blood cells rich in H antigen. Why?
Neither the A or B genes have converted the H antigens to A or B antigens - just a whole bunch of H!
Least Amount of H
Greatest Amount of H
O > A2 > B > A2B > A1 > A1B
Lectin
O cells
A2 cells
A2B cells
B cells
A1 cells
A1B cells
Bombay cells
lectin-H 4+ 3+
2-3+ 2+
weak to negative
negative
Lectin-A1
positive

20. Formation Of ABO Antigens In SecretionsHh
ABO
PS2
ABO on Cells
H Antigen
ABO
PS1
ABO in secretions
H Antigen
Se se

21. Bombay (Oh) Phenotype Results from the inheritance of hh genotype
Red blood cells lack H, A and B antigens
First discovered in Bombay, India
Red cells are NOT agglutinated with anti-A, Anti-B or Anti-H (Ulex europaeus - lectin)
Serum has strong anti-A, Anti-B and anti-H so they agglutinate ALL ABO blood groups
ParaBombay (Ah) Phenotype

23. A Mystery….Why “preformed” ?
3-6 mo
5-10 yr
Ab titer

24. ABO Blood Grouping Reagents
Forward Grouping
Reagent Anti-A and Anti-B
IgM class Monoclonal antibody reagent
Reverse Grouping
Reagent A1 and B cells (3-5% suspension)
Routine tests on donors and patients must include both the forward and reverse grouping

26. Frequency of ABO Blood Groups
Group O 47%
Group A 42%
Group B 8%
Group AB 3% 5

27. The Rh Blood Group System
Described by Landsteiner in 1940
Antibodies produced as a result of pregnancy or transfusion
Immune antibodies - IgG
Can cause haemolytic disease of the newborn and transfusion reactions 6

29. Inheritance of Rh genes
Fisher-Race theory of inheritance
Rh antigens produced by three closely linked alleles C or c, D or d, E or e. (these alleles are located in 2 locus RHD & RHCE
We inherit these genes in groups of three from each parent
A common combination is CDe/cde
Other individuals have combinations of cDE, cde, Cde, cdE 8

31. Rh System D Positive are either D/D or D/d D Negative are d/d
85% of the population are D Positive
15% of the population are D Negative
Other Rh antigens discovered and named C,c,E and e
Weak D phenotype
Rhnull 7

32. Weak D Phenotype (Du)
The weak D phenotype is thought to occur by one of three mechanisms:
(a) inheritance of an RHD gene encoding for a weakened expression of D (DCe or DcE)
(b) interaction of the D gene with other genes (Dce/Ce)
(c) inheritance of an RHD gene missing some epitopes. (lack of part of D)

34. Hemolytic Disease of the Newborn (HDN)
(Erythroblastosis fetalis)

35. Background
A French midwife was the first to report hemolytic disease of the newborn (HDN) in 1609.
In 1932, Diamond and colleagues described the relationship of fetal hydrops, jaundice, anemia, and erythroblasts in the circulation, a condition later called erythroblastosis fetalis.
Levine later determined the cause after Landsteiner and Weiner discovered the Rh blood group system in 1940.
In 1953, Chown subsequently confirmed the pathogenesis of Rh alloimmunization to be the result of passage of Rh-positive fetal red blood cells after transplacental hemorrhage into maternal circulation that lacked this antigen.

37. Rh Incompatibility Expression is limited to RBCs
Rh positive: 45% are homozygous and 55% are heterozygous
Rh incompatibility is a condition which develops when there is a difference in Rh blood type between that of the pregnant mother (Rh negative) and that of the fetus (Rh positive).
After the initial exposure to a foreign antigen, the maternal immune system produces antibodies of the immunoglobulin M (IgM) isotype that do not cross the placenta, and later it produces antibodies of the IgG isotype that traverse the placental barrier.

38. in type O mothers, the antibodies are predominantly IgM in nature
ABO incompatibility
ABO incompatibility is limited to type O mothers with fetuses who have type A or B blood
in type O mothers, the antibodies are predominantly IgM in nature
Because A and B antigens are widely expressed in a variety of tissues besides RBCs, only small portion of antibodies crossing the placenta is available to bind to fetal RBCs. In addition, fetal RBCs appear to have less surface expression of A or B antigen, resulting in few reactive sites—hence the low incidence of significant hemolysis in affected neonates.

39. Causes Rh system antibodies ABO system antibodies Uncommon causes
Common causes for HDN
Rh system antibodies
ABO system antibodies
Uncommon causes
Kell system antibodies
Rare causes
Duffy system antibodies
MNS and s system antibodies
No occurrence in HDN
Lewis system antibodies
P system antibodies

40. BEFORE BIRTH
Antibodies cause destruction of the red cells
Anemia
heart failure
fetal death

42. AFTER BIRTH
Antibodies cause destruction of the red cells
Anemia
Heart failure
Erythroblastosis
General edema Called hydrops fetalis and erythroblastosis fetalis
Build up of billirubin
Kernicterus
Severe retardation

44. Kernicterus due to hyperbilirubinemia due to erythroblastosis fetalis due to Rh incompatibility

45. بیلی روبین:
(1) interruption of normal neurotransmission (inhibits phosphorylation of enzymes critical in release of neurotransmitters)
(2) mitochondrial dysfunction
(3) cellular and intracellular membrane impairment (billirubin acid affects membrane ion channels and precipitates on phospholipid membranes of mitochondria
(4) interference with enzyme activity (binds to specific billirubin receptor sites on enzymes).

46. PREVENTION Before birth After birth
Work up mother for risk and evaluation of complications
After birth
Rh immune globulin - IgG anti-D given to prevent primary immunization

47. Before birth workup Identify women at risk
ABO - Rh -(Du) - Antibody screen
based on results continue testing (Handout)
IgM antibodies are insignificant
IgG antibodies - titer - freeze and store - retiter with a second sample - looking for a 1:32 rise or change in titer

48. Before birth workup titer identifies mothers who need amniocentesis
titer every 4 week until 24th week - then every 2 weeks
amniocentesis is performed after 21st week on high titer - high mortality

49. Amniocentesis Analyze pigment that indicates increased hemolysis
Measure OD from and plot as a function of wavelength
Draw straight line and obtain difference in OD at 450

50. Amniocentesis

51. Intrauterine transfusions Bilirubin
Hb is below 11 g/dL
Usually O and compatible with mother’s antibody
CMV, Hb S, and leukocyte negative
immediate correction of anemia and resolution of fetal hydrops, reduced rate of hemolysis and subsequent hyperinsulinemia, and acceleration of fetal growth for nonhydropic fetuses who often are growth retarded

52. After birth Rh Immune Globulin Give antenatal 28 -32 weeks
also after amniocentesis - IUT - abortion - ectopic pregnancy - miscarriage
Each vial contains 300 ugm and will prevent sensitization by 15 ml RBC or 30 ml whole blood

53. Post Natal Laboratory Studies Mother
ABO - Rh - Du (micro) - Antibody screen - Antibody identification if necessary
Baby
ABO - Rh - Du - DAT for IgG antibodies - elute DAT positive and identify antibody
CBC
Imaging studies

54. TREATMENT
Exchange transfusion
Phototherapy

55. Phototherapy

56. The following are requirements for exchange transfusion :
Severe anemia (Hb <10 g/dL)
Rate of bilirubin rises more than 0.5 mg/dL/hr despite optimal phototherapy
Hyperbilirubinemia
DAT

57. Exchange Transfusions Objectives
Decrease serum billirubin and prevent kernicterus
Provide compatible red cells to provide oxygen carrying capacity
Decrease amount of incompatible antibody
Remove fetal antibody coated red cells

58. Potential complications of exchange transfusion include the following:
Cardiac - Arrhythmia, volume overload, congestive failure, and arrest
Hematologic - Overheparinization, neutropenia, thrombocytopenia, and graft versus host disease
Infectious - Bacterial, viral (CMV, HIV, hepatitis), and malarial
Metabolic - Acidosis, hypocalcemia, hypoglycemia, hyperkalemia, and hypernatremia
Vascular - Embolization, thrombosis, necrotizing enterocolitis, and perforation of umbilical vessel
Systemic - Hypothermia

59. Blood banking & transfusion

60. Blood in History China, 1000 BC The soul was contained in the blood.
Egyptians bathed in blood for their health.
Pliny and Celsus describe Romans drinking the blood of
fallen gladiators to gain strength and vitality and to cure epilepsy.
Taurobolium, the practice of bathing in blood as it cascaded
from a sacrificial bull, was practiced by the Romans.

61. Pope Innocent VIII “…a Jewish daring innovator,
whose name has not come
down to us in memory of
his deed, proposed to find
the pontiff a fountain of
jouvenance in the blood of
three youths who died as
martyrs to their own
devotion and the
practitioners zeal.”
Drinkard, 1870

62. HISTORY James Blundell, Obstetrician
Harvey Discovered Circulation of Blood
1628
1665-’66
Wilkins & Lower Transfusions from dog to dog
1667
Jean-Baptiste Denis Performed first recorded blood transfusions from animals to humans
James Blundell, Obstetrician
1818
First transfusion of human to human

63. James Blundell

64. Early lamb blood transfusion
Animal to Human Transfusion
Early lamb blood transfusion

65. The Kimpton-Brown
transfusion apparatus was
commonly used before
citration. It consisted of a
paraffin-coated gradient glass
cylinder with a horizontal
side tube for suction. It was
in use until approximately 1918.

66. Lewisohn’s Method of Transfusion
Blood is collected in a citrated flask….…...and immediately transfused.

67. Early transfusion: Paris, France

68. Donors must be: 17 years of age in good health weigh at least 40 kg
weigh at least 40 kg
pass a physical and health history examination prior to donation

69. Who should not donate blood?
Anyone who has ever used illegal intravenous (IV) drugs 
Hemophiliacs 
Anyone with a positive test for HIV 
Anyone who has had hepatitis since his or her eleventh birthday 

71. Transfusion
Autologus transfusion : it refers to those transfusions in which the blood donor & transfusion recipient are the same.
Allogeneic transfusion: It refers to blood transfused to someone other than the donor.

72. Autologous transfusion
Preoperative donation
Blood dilution
Intraoperative blood salvage
Post operative blood collection

73. Experienced mild side effects by a donor
Stinging during insertion the needle
Upset stomach
Dizziness
A small amount of bruising
A donor may faint
Having muscle spasm
Suffering damage

75. No Whole blood BUT blood components

76. Plastic Blood Bags and Component Separation

79. Red blood cells For chronic anemia resulting from disorders
For acute blood loss
resulting from trauma or surgery
Shelf life of RBC = 42 days
Frozen for up to 10 years

80. Plasma Contain albumin – fibrinogen – globulins
Usually separated into specific products.
Fresh frozen plasma stored for 1 – 7 years.
Cryoprecipated AHF, rich in certain clotting factors.( factor VIII , fibrinogen, von Willbrand factor, factor XIII
AHF prevent or control bleeding in individuals with hemophilia and von Willbrand’s disease.

81. Platelets Prevent massive blood loss resulting from trauma.
Maybe obtained from donor by a process known as APHERESIS.
Stored at room temperature for up to 5 days.
used to treat thrombocytopenia.

82. White blood cells Transfused within 24 h after collection.
Used for infections that are unresponsive to antibiotic therapy.
The effectiveness is still being investigated.

85. Compatibility testing
ABO-Rh blood typing
Antibody screening
Cross-matching
Cross-matching is performed to determine if the patient has antibodies that react with the donor’s cells

86. The risk of infection from transfusion
About 1 in 600,000 units for hepatitis B
About 1 in 2 million units for HIV and hepatitis C

87. The greater concern is an ABO incompatibility and transfusion reactions. ABO incompatibility occurs when blood samples from two people with different ABO blood types are mixed.

88. Several types of transfusion reactions like allergic and febrile(characterized by fever)
Treatment will depend on type of reaction and patient’s symptoms.

89. Fully automated grouping and antibody screening

90. Play a game on Blood grouping for blood transfusion