The Cell Membrane 2007-2008.

The Cell Membrane

Overview Cell membrane separates living cell from nonliving surroundings thin barrier = 8nm thick Controls traffic in & out of the cell selectively permeable allows some substances to cross more easily than others hydrophobic vs hydrophilic Made of phospholipids, proteins & other macromolecules

Aaaah, one of those structure–function
Phospholipids Phosphate Fatty acid tails hydrophobic Phosphate group head hydrophilic Arranged as a bilayer Fatty acid Aaaah, one of those structure–function examples

Phospholipid bilayer polar hydrophilic heads nonpolar hydrophobic
tails polar hydrophilic heads

It’s like a fluid… It’s like a mosaic… It’s the Fluid Mosaic Model!
More than lipids… In 1972, S.J. Singer & G. Nicolson proposed that membrane proteins are inserted into the phospholipid bilayer It’s like a fluid… It’s like a mosaic… It’s the Fluid Mosaic Model!

Filaments of cytoskeleton
Membrane is a collage of proteins & other molecules embedded in the fluid matrix of the lipid bilayer Glycoprotein Extracellular fluid Glycolipid Transmembrane proteins The carbohydrates are not inserted into the membrane -- they are too hydrophilic for that. They are attached to embedded proteins -- glycoproteins. Phospholipids Filaments of cytoskeleton Cholesterol Peripheral protein Cytoplasm

Membrane fat composition varies
Fat composition affects flexibility membrane must be fluid & flexible about as fluid as thick salad oil % unsaturated fatty acids in phospholipids keep membrane less viscous cold-adapted organisms, like winter wheat increase % in autumn cholesterol in membrane

Membrane Proteins Proteins determine membrane’s specific functions
cell membrane & organelle membranes each have unique collections of proteins Membrane proteins: peripheral proteins loosely bound to surface of membrane cell surface identity marker (antigens) integral proteins penetrate lipid bilayer, usually across whole membrane transmembrane protein transport proteins channels, permeases (pumps)

Why are proteins the perfect molecule to build structures in the cell membrane?

nonpolar & hydrophobic
Classes of amino acids What do these amino acids have in common? nonpolar & hydrophobic

I like the polar ones the best!
Classes of amino acids What do these amino acids have in common? I like the polar ones the best! polar & hydrophilic

Proteins domains anchor molecule
Within membrane nonpolar amino acids hydrophobic anchors protein into membrane On outer surfaces of membrane polar amino acids hydrophilic extend into extracellular fluid & into cytosol Polar areas of protein Nonpolar areas of protein

Examples water channel in bacteria
NH2 H+ COOH Cytoplasm Retinal chromophore Nonpolar (hydrophobic) a-helices in the cell membrane Examples water channel in bacteria Porin monomer b-pleated sheets Bacterial outer membrane proton pump channel in photosynthetic bacteria function through conformational change = shape change

Many Functions of Membrane Proteins
Outside Plasma membrane Inside Transporter Enzyme activity Cell surface receptor Signal transduction - transmitting a signal from outside the cell to the cell nucleus, like receiving a hormone which triggers a receptor on the inside of the cell that then signals to the nucleus that a protein must be made. Cell surface identity marker Cell adhesion Attachment to the cytoskeleton

Membrane carbohydrates
Play a key role in cell-cell recognition ability of a cell to distinguish one cell from another antigens important in organ & tissue development basis for rejection of foreign cells by immune system The four human blood groups (A, B, AB, and O) differ in the external carbohydrates on red blood cells.

Any Questions??

Movement across the Cell Membrane

IMMUNOGENETICS IMMUNITY
The immune system in all its form is mankind’s defense mechanism, and in order to understand the inherited disorders of immunity, we must first understand the fundamentals of the genetic basis of immunity. Immune defense mechanism can be divided into two main types: innate immunity – includes a number of non-specific systems which do not require or involve prior contact with the infectious agent. specific acquired or adaptive immunity – involves a tailor-made immune response that occurs after exposure to an infectious agent. Both types can involve either humoral immunity, which combats extracellular infections, or cell-mediated immunity, which fights intracellular infections.

INNATE IMMUNITY The first simple type of defense against infection is a mechanical barrier. This barrier is represented by skin and by membranes lining the respiratory and gastrointestinal tracts. If an organism succeeds in invading the body, phagocytosis and bactericidal agents come into effect. HUMORAL INNATE IMMUNITY A number of factors are involved in innate immunity by helping to minimize tissue injury by limiting the spread of infectious microorganisms. These are often called the acute phase proteins and include C-reactive protein, mannose binding protein and serum amyloid P component. In addition, cells when infected by a virus synthesize and secrete interferon , which interferes with viral replication through reducing mRNA stability and interfering with translation.

INNATE IMMUNITY COMPLEMENT
Is a complex series of 20 or so interacting plasma proteins that can be activated by the cell membranes of invading microorganisms, in what is termed the alternative pathway. The various components of complement interact in a specific cascade sequence, resulting in a localized acute inflammatory response through the action of mediators released from mast cells and tissue macrophages. These result in increased vascular permeability and the attraction of phagocytes in the process known as chemotaxis. In addition, the latter components of the complement cascade generate a membrane attack complex that induces defects in the cell membrane, resulting in the lysis of microorganisms. Complement can also be activated through the classic pathway, by the binding of antibody with antigen.

Classic and alternative pathways of complement activation

INNATE IMMUNITY CELL MEDIATED INNATE IMMUNITY PHAGOCYTOSIS
Microorganism are engulfed and digested by two major types of cells., either polymorphonuclear neutrophils or macrophages. Polymorphonuclear neutrophils are found mainly in the bloodstream, while macrophages occur primarily in tissue around the basement membrane of blood vessels in connective tissue, lung, liver and in the lining of sinusoids of the spleen and the meddullary sinuses of the lymph nodes. Surface antigen (Ag) on microorganisms result in their being engulfed and fusing with the granules of the phagocytes-which leads to their destruction. EXTRACELLULAR KILLING Virally infected cells can be killed by large granular lymphocytes, known as natural killer cells. Attachment of the natural killer cells to the infected cells results in the release of a number of agents, which in turn results in damage to the membrane of infected cell, leading to cell death.

SPECIFIC ACQUIRED IMMUNITY
Many infective microorganisms have, through mutation and selective pressures, developed strategies to overcome or evade the mechanisms associated with innate immunity. There is the need, therefore, to be able to generate specific acquired or adaptive immunity. This can, like innate immunity, be separated into both humoral and cell-mediated processes. HUMORAL SPECIFIC ACQUIRED IMMUNITY The main mediators of this immunity are immunoglobulins or antibodies. Antibodies are able to recognize and bind to antigens of infecting microorganisms. Exposure to a specific antigen results in the clonal proliferation of a small lymphocyte derived from the bone marrow, hence “B” lymphocytes, resulting in mature antibody-producing cells or plasma cells.

HUMORAL SPECIFIC ACQUIRED IMMUNITY
Immunoglobulins The immunoglobulins (Ig), or antibodies, are one of the major classes of serum protein. Their function, both in the recognition of antigenic variability and in effector activities, was initially revealed by protein, and more recently by DNA, studies of their structure. Ig structure – papain (a proteolytic enzyme), splits the Ig molecule into three fragments. Two fragments are similar, each containing an antibody site capable of combining with a specific antigen and therefore referred to as the antigen binding fragment or Fab. The third fragment can be crystallized and was therefore called Fc. The Ig molecule is made up of four polypeptide chains: two ‘light’ (L)-220 amino acid, and two ‘heavy’ (H)-440 amino acid length. They are held together in Y-shape by disulfide bonds and non-covalent interactions. There are five different types of H chain, designated respectively as α,γ,µ,δ, and ε, one each respectively for the five major antibody classes, or what are known as isotypes, IgA, IgG, IgM, IgD and IgE. The L chains are one of two types, either kappa(κ) or lambda (λ), occurring in all five classes of antibody, but with only one type of L chain occurring in each individual antibody. In addition there are four IgG subclasses(IgG1-IgG4), and two IgA subclasses (IgA1,IgA2).

Model of antibody molecule structure.
© 2005 Elsevier

Estimated number of the various DNA segments coding
for the κ, λ and various heavy chains. Downloaded from: StudentConsult (on 6 October :59 AM) © 2005 Elsevier

Classes of human immunoglobulin
Class Mol. Wt. Serum Antibody activity concentration (mg/ml) IgG – Binds to microorganisms, neutralizes bacterial toxins IgM Produced in early immune response IgA Guards mucosal surfaces IgD – On lymphocyte cell surface, involved in control of activation and suppression IgE trace In parasitic and allergic reactions

THE MAJOR HISTOCOMPATIBILITY COMPLEX
The major histocompatibility complex (MHC) plays a central role in the immune system. Association of an antigen with the MHC molecule on the surface of the cells is required for recognition of the antigen by the T-cell receptor that, in conjunction with the closely associated protein β2-microglobulin, results in the recruitment of cytotoxic and helper T cells in the immune response. MHC molecules occur in three classes: class I molecules occurring on virtually all cells and which are responsible for recruiting cytotoxic T cells; class II molecules that occur on B cells and macrophages and are involved in signaling T helper cells to recruit further B cells and macrophages; the non-classical class III molecules that include a number of other proteins with a variety of other immunological functions. Structural analysis of the class I and II MHC molecules reveals them to heterodimeres with homology to immunoglobulin. The genes coding for the class I (A,B,C,E,F and G), class II (DR, DQ and DP) and class III MHC molecules, or what is also known as the human leucocyte antigen (HLA) system, are located on chromosome 6.

Transplantation genetics
Replacement of diseased organs by transplantation has become routine in clinical medicine. Except for corneal and boner grafts, the success of such transplants depends on the degree of antigenic similarity between the donor and recipient. Rejection of the transplanted organ or tissue does not occur between identical twins, or between non-identical twins where there has been mixing of the placental circulations before birth. In all other instances, the antigenic similarity of donor and recipient has to be assessed by testing them with suitable antisera or monoclonal antibodies for antigens on donor and recipient tissue. As a general rule, a recipient will reject a graft from any person who has antigens the recipient lacks. The HLA system is highly polymorphic, two unrelated individuals are therefore very unlikely to have identical HLA phenotypes. The close linkage of the HLA loci means that they tend to be inherited en block, the term haplotype being used to indicate the particular HLA alleles that an individual carries on each of his two number 6 chromosome. Although crossing over does occur within the HLA region, certain alleles tend to occur together more frequently than would be expected by chance., i.e. they tend to exhibit linkage disequilibrium. An example is the association of the HLA antigens A1 and B8 in populations of Western European origin.

HLA polymorphism and disease associations
A finding which helps to throw light on the pathogenesis of certain diseases is the demonstration of their association with certain HLA types. The best documented is that between ankylosing spondylitis and HLA B27. In the case of narcolepsy, a condition of unknown etiology characterized by a periodic uncontrollable tendency to fall asleep, almost all affected individuals are HLA DR2. The possession of a particular HLA antigen does not mean that an individual will necessarily develop the associated disease, merely that he or she has a greater relative risk of being affected than the general population. In family, the risks to first-degree relatives of those affected is low, usually no more than 5%. In general, the mechanisms involved in most HLA disease associations are not well understood.

H-Y antigen In number of different animal species it was noted that tissue grafts from males were rejected by females of the same inbred strains. These incompatibilities were found to be due to a histocompatibility antigen, known as H-Y antigen. The H-Y antigen seems, however, to play little part in transplantation in humans. Although the H-Y antigen seems to be important for testicular differentiation and function, its expression does not necessarily correlate with the presence or absence of testicular tissue. A separate sex-determining region of the Y chromosome (SRY) has been isolated which is now known to be the testis-determining gene.

The Basics Of Blood W.B.C. & Platelet R.B.C. Plasma ANTIBODY ANTIGEN
Natural & Immune Agglutinins/ Isoantibodies ANTIGEN >400 Agglutinogens on the cell membrane Antigen-Antobody reaction on the cell surface  Hemolysis

The Basics Of Blood Antigens: -
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.

The Basics Of Blood Antigens: -
> 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%

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

The Basics Of Blood Natural Antibodies: -
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.

The Basics Of Blood Immune Antibodies: -
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.

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

ABO Blood Grouping

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.

ABO blood groups found on outside of cell
Introduction to Blood ABO blood groups found on outside of cell

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

Hemoglobin picks up and drops off oxygen
Introduction to Blood Hemoglobin picks up and drops off oxygen

This diagram illustrates the terminal sugar for each blood group.

ABO Type Frequencies In U.S.
Per Cent O 45% A 40% B 11% AB 4%

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.

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.

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.

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.

Example of Determining Genotype
Mom’s phenotype is group A, genotype AO Dad’s phenotype is group B, genotype BO B O A AB 25% AO 25% (Group A) BO 25% (Group B) OO 25% (Group O)

Other Examples Mom Dad Offspring Blood Group AA BB 100% AB BO OO
50% each of B or O 100% O AO 50% each of A or O

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.

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.

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.

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.

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.

Summary A Anti-B A or O B Anti-A B or O AB A and B none AB, A, B or O
Blood Group Antigens on cell Antibodies in plasma Transfuse with group A Anti-B A or O B Anti-A B or O AB A and B none AB, A, B or O O None Anti-A & B

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 ABO & Rh(D)

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)

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

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.

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.

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.

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

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 ABO & Rh(D)

Distribution of Rh(D) Types
Population Rh(D) pos Rh(D) neg Caucasian 86% 14% African-American 95% 5% Oriental >99% <1% ABO & Rh(D)

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 ABO & Rh(D)

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 ABO & Rh(D)

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 ABO & Rh(D)

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

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

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

Pathology Of Iso-immunisation
AFTER BIRTH  HAEMOLYSIS  IN UTERO    Jaundice Kernicterus Hepatic Failure ANAEMIA BILLIRUBIN    HEPATIC ERYTHROPOESIS & DYSFUNCTION MAT. LIV NO EFFECT   DEATH IUD       PORTAL & UMBILICAL VEIN HYPERTNSION, HEART FAILURE ERYTHROBLASTOSIS FETALIS         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.

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.

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

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

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.

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

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

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 .

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.

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.

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

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.

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.

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.

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.

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.

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.

Summary of Slide Typing
Anti-A Anti-B Blood Group NEG O POS A B AB

The ABO Antigens Added to Proteins or Lipids in Red Cells
ABO & Rh(D) 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 ABO & Rh(D)

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 ABO & Rh(D)

Distribution of ABO Groups
Population O A B AB Aborigines 61 39 Basques 51 44 4 1 Blackfoot (N. Am. Indian) 17 82 Bororo 100 Chinese-Canton 46 23 25 6 Chinese-Peking 29 27 32 13 English 47 42 8 3 Hawaiians 37 2 Irish 52 35 10 Mayas 98 Navajo (N. Am. Indian) 73 Peru (Indians) United Kingdom (GB) USA (blacks) 49 20 USA (whites) 45 40 11 ABO & Rh(D)

Distribution of the A allele
ABO & Rh(D)

Distribution of the B Allele
ABO & Rh(D)

Distribution of the O Allele
ABO & Rh(D)

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