BL 02-27-14 10-11am Immunohematology - Cohen

Question 1

Transfusion - transplanting red cells

Answer 1

-Red cells DO NOT carry MHC antigens in humans
- The antigens they do carry are much less polymorphic in the population (many fewer
alleles)
- The white cells that come along with the white cells are recognized & destroyed (which is fine when you just are wanting the red cells)
- The ability to transfuse red cells from one person to another depends on avoiding immune responses
- When problems arise, they are often immunological

Question 2

Transfusion - transplating platelets

Answer 2

• Platelets DO bear HLA (Class I) antigens
• with repeated platelet transfusions, alloimmunization may be developed
• in that case, HLA typing becomes necessary (expensive, hard to find good matches)

Question 3

Blood group ANTIGENS

Answer 3

• glycolipids found on the surface of ALL BODY CELLS, including red cells of course
• The lipid backbone spans the plasma membrane
• The terminal sugars confer antigenic specificity, A, B or O

Question 4

Blood group SUBSTANCES

Answer 4

• glycoproteins with similar sugars, found in the body fluids of people who have the Secretor (Se) phenotype.
• ~80% of people are secretors
• their blood type can be determined from sweat stains, cigarette butts, etc.
• NO particular advantages to being a secretor

Question 5

O antigen - pros & cons

Answer 5

People who are O are…

• somewhat protected from pancreatic cancer
• much less likely to develop venous thromboembolic disorders

Question 6

Structure of Blood group antigens

Answer 6

• A set of glycosyl transferases assemble the
  basic “core” sugar chain which almost everybody has = called the “H” antigen
• Then a final glycosyl transferase, of which there
  are three alleles, can act and produce the different antigens

Question 7

Antigen differences in A, B, O, AB

Answer 7

• The O allele is an “amorph;” it does not
  code for a working transferase & so group O people have only the basic core, the H antigen.
• People who are group A have a glycosyl
  transferase allele which puts an additional sugar on the core chain
• People who are B have a different allelic form of this enzyme which adds a different sugar.
• Group AB individuals have both the A and B antigens on their red cells, because they got both the A and B transferases from their parents.

Question 8

Bombay phenotype

Answer 8

• Some people who lack the transferase gene that puts the final sugar on the “core”, and thus do not express even the H antigen
  = rare Bombay phenotype (Oh,)
• All blood, even type O, is foreign to such people (can only give Bombay blood)
• Appears as type O blood on normal typing tests

Question 9

Commonness of ABO antigens in environment

Answer 9

• ABO antigens are very simple carb structures, so it’s not surprising that they are found in many places in nature.
• Thus, one will inevitably come into contact w/ these carbs in the environment during infancy
• Those which are not the same as your own are, of course, foreign to you, & you BECOME IMMUNIZED to them (type A person will make Ab to B; type O person will make Ab to both A & B, etc.)
• These Abs are called “naturally-occurring” or ISOHEMAGGLUTININS

Question 10

Class of Isohemagglutinins

Answer 10

IgM

Question 11

Measure titers of isohemagglutinins

Answer 11

• Can be of use in Dx of B cell immunodeficiency, since they should begin to appear in blood between 3 & 6 mo of age, as antigen exposure occurs

Question 12

What kind of cells do Group A people have?

Answer 12

Cells with A carbohydrate antigens on them.

Question 13

What kind of cells do Group B people have?

Answer 13

Cells with B carbohydrate antigens on them.

Question 14

What kind of cells do Group O people have?

Answer 14

Cells with NO carbohydrate antigens on them.

Question 15

What kind of cells do Group AB people have?

Answer 15

Cells with both A & B carbohydrate antigens on them.

Question 16

What kind of antibodies do Group A people make?

Answer 16

anti-B

Question 17

What kind of antibodies do Group B people make?

Answer 17

anti-A

Question 18

What kind of antibodies do Group O people make?

Answer 18

anti-A & anti-B

Question 19

What kind of antibodies do Group AB people make?

Answer 19

None

Question 20

What are the possible genotype(s) for Group A people?

Answer 20

AA or AO

Question 21

What are the possible genotype(s) for Group B people?

Answer 21

BB or BO

Question 22

What are the possible genotype(s) for Group O people?

Answer 22

OO

Question 23

What are the possible genotype(s) for Group AB people?

Answer 23

AB

Question 24

What is the frequency of blood types in white & black Americans?

Answer 24

O most common - almost half of both whites & blacks
A almost as common as O in whites, with B much less common
A & B almost equal in blacks
AB is the rarest block type (3% in w, 4% in b)

Question 25

Difficult to type A & B antigens…

Answer 25

• There are variant A and B types (A2, A3, Ax, Bx, etc.) in which A or B antigen is expressed weakly
• Such people may be typed incorrectly or with difficulty in the blood bank.
• Suppression of ABO antigens seen in some diseases (leukemia)
• Titers of isohemagglutinin can be low in the elderly and in hypogammaglobulinemia.
• Any of these conditions can lead to an “ABO discrepancy” (lack of correlation between ABO phenotype as determined by cell & serum typing) which must be resolved.
• DNA typing is possible.

Question 26

Blood type & historically isolated populations

Answer 26

• Some historically isolated populations have very different distributions than we see in the US
• most South American natives are group O
• group O is rare in China
• group B is very common in Vietnam
• Used by anthropologists to estimate relatedness of peoples & times of migrations.
• Important if your donor & recipient populations are
  different; it can often be a problem for travelers.

Question 27

Rh factor

Answer 27

• 2nd most important blood group system (out of over 600 IDed blood group antigens!)

Rh antigens are on proteins coded for at two loci:

• One is for alleles d/D
• The other for c/C & e/E
• By far the most important allele is D, so that if you say you are “Rh positive” you really mean ►“Rh(D) positive”

Question 28

What does it mean when you say “Rh+”?

Answer 28

This truly means that you are Rh(D) positive, as the D allele is by far the most important Rh antigen.

Question 29

D allele of Rh

Answer 29

• D is dominant over d (the “d” allele, another amorph, is heavily mutated & does not make protein)
• People whose genotype is DD or Dd type as Rh+
• 92% of U.S. blacks are Rh+; 85% of whites.
• Rh(D) negative is rare in sub-Saharan Africa
• There are no “naturally occurring” isohemagglutinins for Rh
• It’s a protein, & not ubiquitous in nature, ►so you don’t make antibody to it unless you’re Rh(D) negative & become immunized w/ Rh(D)+ red cells.

Question 30

First process in the blood bank

Answer 30

“Type & Screen”
All donor units of blood are…
- typed for ABO & Rh
- tested for presence of “unexpected” antibodies
- tested for syphilis, Hep B & C, HIV, and West Nile Virus antibody
- In many blood banks, red cells are also typed for a long list of “minor” blood group antigens as well

Reverse typing is also performed

• makes sure isohemagglutinins in plasma are appropriate for determined red cell type
• screens for list of other RBC antibodies

Units are then banked

Question 31

Preparing the recipient for transfusion (screening, etc.)

Answer 31

Before transfusion, recipient is

• typed for ABO & Rh
• his plasma screened for expected & “unexpected” antibodies
• Computer can then tell which donor units are compatible with the recipient.

Question 32

Donors matching Recipients

Answer 32

• Must ordinarily be identical at ABO & Rh

Question 33

Crossmatch

Answer 33

• Despite ABO/Rh match, there may still Abs in recipient’s plasma which can react with Ags on donor’s RBCs… if so, this is called crossmatch.

Question 34

Consequence of crossmatch if blood transfused anyways…

Answer 34

• Generalized complement-mediated hemolysis
• Active complement-mediated inflammation
• Free hemoglobin deposited in kidneys leading to acute renal failure

Question 35

Testing for Crossmatch

Answer 35

• lab test in which plasma from prospective recipient is mixed w/ red cells from prospective donor
• The big question is, will this recipient’s plasma destroy the incoming red cells?
• If so, that could be catastrophic
• Cells are 1st suspended in saline & a drop of the recipient’s plasma added.
• If there is agglutination, it means there are lots of high-avidity antibodies in the serum (probably IgM) = most dangerous b/c of their potent complement-activating ability.
• Even if this test is negative there could still be lower-avidity antibodies that would shorten red cell survival, so the bank does some further tests to push the system.

Question 36

RBCs & Zeta potential

Answer 36

Zeta potential = net negative electrical change

• keeps RBCs apart from each other
• -> don’t clump in capillaries
• -> makes harder to agglutinate in blood bank test
• So, tests are done in a medium that neutralizes zeta potential

Question 37

Agglutination & crossmatch test results

Answer 37

• NO agglutination = blood & recipient are “compatible”
• Group O RBCs can be given to recipients of almost any phenotype (universal donor).
• Type AB people can be universal recipients.

Question 38

Antiglobulin (Coombs) Test

Answer 38

= a test which uses antibody against human Ig to

detect human Ig on surface of RBCs (direct test) or in plasma (indirect)

Question 39

Example/Scenario of Direct Antiglobin (Coombs) Test

Answer 39

What to know if pt has Ab against own RBCs… Direct test

Ab wouldn’t be enough (or of high enough avidity) to agglutinate her cells in vivo, b/c she’d be dead
- But, there could be enough to decrease RBC survival from 120 to 30 days, say, and that would make her anemic.

1. Take some of her RBCs, wash them, and then add Ab against human IgG to them.
   - -> If they had some human IgG sticking to their surface, this “antiglobulin” could cross-link it, and the cells would agglutinate.

Question 40

Example/Scenario of Indirect Antiglobin (Coombs) Test

Answer 40

Want to know if preg. woman has antibody in her system to Rh(D)+ [in that case, can’t use RhoGAM…already immunized]

1. Take donor red cells & add recipient plasma, and then wash off any unbound proteins.
   – If there was Ab against the cells, it might bind but not agglutinate them.
   2 Now we added the antiglobulin, it could cross-link bound antibodies & agglutinate cells.
   - This is an indirect antiglobulin test.

Question 41

Direct Antiglobulin Test (DAT) asks…

Answer 41

Is there antibody already on these cells I am interested in?

You rinse off the cells & add antiglobulin to find out.

DAT detects cells that were coated w/ Ab in vivo.

Question 42

Indirect Antiglobulin Test asks…

Answer 42

Is there unexpected antibody to red cell antigens in the plasma of this potential recipient?

You take red cells, add plasma, rinse cells (we assume they haven’t agglutinated,) and then add antiglobulin.
- If cells now agglutinate, there must have been Ab to them in the plasma, b/c antiglobulin alone won’t react with red cells.

[Memory aid: There’s always one more step in an indirect test.]

Question 43

Hemolytic Disease of the Newborn - basics (aka, when & to whom occurs)

Answer 43

Aka erythroblastosis fetalis
- occurs in Rh(D)+ babies of Rh(D)- mothers
- In last trimester & especially at time of delivery, some red cells from baby enter mother’s circulation
- If she is Rh(D)- & baby is Rh(D)+, she may make anti-Rh(D)
= No problem for the baby, who isn’t there anymore.
- But in subsequent pregnancy w/ another Rh(D)+ fetus, mother’s antibodies, formed after the first pregnancy & boosted by the second, can cross the placenta and destroy fetus’ RBCs
- In addition, each subsequent pregnancy with an Rh(D) fetus boosts her response.

Question 44

Consequences of Hemolytic Disease of the Newborn

Answer 44

Fetus will be born jaundiced…

• Can be dangerous
• High levels of bilirubin (a breakdown product of Hgb) can cross BBB & damage basal ganglia
• Results in cerebral palsy or, if there is very severe damage, fetal death

Question 45

Prevention of Hemolytic Disease of the Newborn

Answer 45

• The disease is preventable if, at the time that the mother delivers her FIRST Rh(D)+ baby, she is
  given IgG antibody to Rh(D) (Rh-immune globulin)
• Most familiar brand being Ortho’s RhoGAM®.
• These antibodies combine w/ fetal red cells, opsonizing them, and they are destroyed before they get a chance to immunize her.
• Note: she is not made tolerant– just not immunized
• She must receive Rh immune globulin each time there is a chance of being immunized by Rh(D)+ cells, including all subsequent normal deliveries, abortions, fetal manipulations, amniocenteses, etc.

Question 46

Indirect Antiglobulin (Coombs) Tests to see if mother has been immunized by Rh(D)+

Answer 46

• Rh(D)- woman is pregnant
• She had two previous miscarriages.
• Did they immunize her against Rh(D)?
• Take some of her plasma & add to ABO-compatible, Rh(D)+ cells.
• We see nothing, but did it bind?
• So we now wash the cells and add
  antiglobulin; they agglutinate.
• She is, in fact, already immunized.

Question 47

Rh(D)+ Newborn w/high Hgb, and with RH(D)- mother…testing for hemolytic disease of the newborn among other possible explanations

Answer 47

• Hemolytic disease of the newborn is possible, but there are other causes of hyperbilirubinemia.
• We take some of his red cells, wash then, and add antiglobulin.
• They are agglutinated

Question 48

“Prophylactic” RhoGAM shots

Answer 48

• A later development in prevention of HDN is the practice of giving a shot of RhoGAM to
  Rh negative women at 28 weeks gestation
  = to prevent immunization by small transplacental
  bleeds during 3rd trimester
• The Rh-immune globulin would also be given at or shortly after delivery, if the child turns out to be Rh(D)+

Question 49

Why are there not hemolytic diseases of newborns as a result of blood cell antigens other than Rh (such as ABO)?

Answer 49

• Since a type A mother would be expected to ahve anti-B antibodies, why don’t these anti-B’s not destrol a fetus with type B blood?
• Normally, isohemagglutinins are IgM, probably b/c the ubiquitous antigens that stimulate them are T-independent (carbs often are)
• So they don’t cross the placenta.
• Anti-Rh antibodies are usually IgG (being anti-proteins), and do cross the placenta.

Note:

• Occasional people do make IgG isohemagglutinins, esp. in group O people.
• So A or B fetuses of these women are at some risk of ABO hemolytic disease.
• But, there is no “RhoGAM” for this— these mothers are already immunized so there would be no point of a shot (?)

Question 50

Heterophile antibodies

Answer 50

= antibodies to one antigen which bind, fortuitously,
to another
= a fancy name for cross-reactive antibodies

Question 51

Example of Heterophile Antibodies– Infectious Mono

Answer 51

Best example of a heterophile antibody:
= Ab that appears in serum of pt w/ infectious mono
= Ab is in response to a viral antigen
- BUT, it happens to react also w/ sheep RBCs
–> Gives us a quick & cheap presumptive test (the Monospot) for mono

Question 52

Example of Heterophile Antibodies– Syphilis

Answer 52

Ab made by people w/ syphilis
= similar phosphodiester group in bacterium Treponema pallidum & in phospholipids extracted from beef heart (cardiolipin)
- Simple test can be performed that doesn’t require Treponema

Question 53

ASK YOURSELF: All normal people except Group AB have isohemagglutinins (Abs to A or B antigens), so a Group A mother has anti-B. If father is BB, fetus will be AB or B; why does her anti-B not destroy the fetus’s red cells?

Answer 53

b/c the antibody is IgM

• b/c its antibody to carbohydrate (–> IgM with no switching)
• so, can’t cross placenta

HOWEVER: Some O mothers can make some IgG to the ABO carb antigens –> can’t get newborn hemolytic disease

• normally not as serious as Rh disease
• no RhoGAM b/c the woman has been making the Ab since childhood

Question 54

A man is group A, his wife group O. They have a baby who turns out to be AB. The baby looks so much like him that he accuses her of non-maternity! But she really is the mother, and he really is the father. How could this be?

Answer 54

Mother is Bombay
Enzyme must see entire structure to put that sugar on.
In mother, she is genetically B (has enzyme to put that B sugar on) but doesn’t have last red triangle b/c she’s bombay …the full sugar is the substrate to put the B sugar on, so the mom can’t
The baby mades the full substrate b/c of father & got B from mother