Module 6 Path

Front 1

Define immunohematology.

Back 1

body of knowledge concerning Ag on blood elements and their Ab

Front 2

Explain blood group antigens?

Back 2

The red cell mbrn contains over 300 different Ag classified into 29 blood groups systems.

BLOOD GROUP SYSTEM consists of:
-- a grp of Ag governed by either a single gene locus OR
-- by a complex of two or more very closely linked homologous genes w/ virtually no recombination btwn them

Front 3

Red cell Ag are determined by...?

Back 3

carbohydrates -- ABO, P, Lewis

proteins -- Rh, Duffy, Kidd

CHO/protein combos -- MNS

Front 4

What are the general principles of Ag detection techniques?

Back 4

1. Ag are detected by using known "reagent" antisera

2. Ab are detected and identified by using panels of "reagent cells," whose phenotypes are known

3. Two classes of Ab are signif in transfusion medicine: IgM and IgG

Front 5

How are red cell IgM Ab detected?

Back 5

1. Incubate the RBC at or below room temp with a serum containing IgM Ab to a specific Ag

2. Ab are detected by observation of agglutination or hemolysis

3. Because the IgM Ab are large and pentameric they can easily form a bridge btwn red cells. No specific agglutination enhancement media is needed.

**IgM Ab best react at lower temp and may often elute off the cells during a 37C incubation

Front 6

How are red cell IgG Ab detected?

Back 6

IgG are smaller and monomeric. They cannot form a bridge btwn two red cells w/o the help of an agglutination enhancer.

1. Antihuman globulin (Coombs') serum is an antibody directed toward human globulin, mostly IgG

2. The Coombs' can form a bridge btwn two red cells coated w/ IgG

Front 7

Compare direct vs. indirect antiglobulin testing.

Back 7

DIRECT ANTIGLOBULIN TEST (DAT)
-- tests patient’s RBCs directly
-- (+) cells clump; (-) cells stay free in soln
-- can be used to dx immune hemolytic anemia (using IgG or C3 immune reagent

INDIRECT ANTIGLOBULIN TEST (IAT)
-- tests patient’s serum for foreign Ag
ex: via transfusions or pregnancy
-- IgG Ab react best at 37oC and their detection in an IAT require lengthy (30min-1hr) incubation at that temp

-- reagent red cells + pt serum  incubate then wash (x3) to remove excess Ab  add antiglobulin, centrifuge  (+) clumping due to Ab in serum; (-) no clumping so no Ab in serum

Front 8

What kind of antigens do lymphocytes carry?

Back 8

Human leukocyte Ag (HLA) – very polymorphic group of Ag

Ag-Ab rxns in this Ag system are detected by a technique called microlymphocytotoxicity

Front 9

What kind of Ag do platelets carry?

Back 9

Platelet specific Ag – classified into 5 different systems of human platelet Ag, HPA 1-5

HLA class I Ag (products of the A and B locus)

ABO Ag

Platelet Ag-Ab rxns are detected by different technologies, most common being ELISA or a solid phase red cell adhesion assay (SPRCA).

Front 10

What kind of Ag do granulocytes carry?

Back 10

Granulocyte specific Ag classified into several humal ganulocytes Ag or HGA systems

HLA class I Ag

ABO Ag

Front 11

On which chromosome do we find the ABO Ag?

Back 11

ABO Ag are governed on a single locus on chromosome 9, which codes for glycosyltransferase.

Front 12

What does glycosyltransferase do?

Back 12

Adds an “immunodominant” sugar to the end of a CHO fhain linked to a lipid or a protein embedded in the red cell mbrn.

Front 13

What cells possess the ABO Ag?

Back 13

They are present on EVERY cell of the body, not just red cells.

Front 14

Compare A, B, and O alleles.

Back 14

O
- recessive
- codes for nonfxning protein and appears to be mutated form of A allele

A
- codominant with B
- transfers N-acetylgalactosamine

B
- codominant with A
- transfers D-galactose

Front 15

What is the unique feature of the ABO blood group?

Back 15

After 6 months of age, the corresponding Ab are always present in serum, when an Ag is not present on the cells (Landsteiner law)

Ab formation does not require previous exposure to foreign red cells – since CHOs containing these immunodominant sugars are widespread in nature, partic in bacteria and viruses

Front 16

What are the primary antibodies to A, B, and O?

Back 16

Ab to the A and B Ag are mostly IgM, thus an ABO blood type is determined by DIRECT agglutination at room temp incubation, w/o use of antihuman globulin

Indiv w/ type O blood will also have an IgG component to their anti-A and anti-B

Front 17

Describe ABO incompatibility.

Back 17

-- causes severe, often fatal hemolytic transfusion rxn w/ RBC transfusion
-- causes hyperacute rejection in organ transplantation
-- is most common cause of hemolytic disease of the newborn (HDN); however, hemolysis is usually mild
-- is assoc w/ hemolysis and/or delayed engraftment in allogeneic BM transplantation

Front 18

Which chromosome controls Rhesus blood group system Ag?

Back 18

Controlled by two very closely lilnked and homologous loci on Chr 1
-- one locus codes for presence or absence of D Ag
-- second locus codes for four other common Ag: C, c, E, e

Front 19

Rh Ag are restricted to…?

Back 19

Red cells
-- carried by a multipass transmbrn protein.

Front 20

What determines C/c and E/e polymorphisms?

Back 20

A few single point mutations control the aa changes that determine the C/c and E/e polymorphisms

Front 21

Compare Rh (+) and Rh (-).

Back 21

Rh (+) = presence of D Ag
Rh (-) = absence of D Ag, often referred to as the “d” phenotype; however, there is no “d” Ag, consists of deleted D gene
-- most (-) phenotypes are dce phenotype (no C or E Ag)

Front 22

Rh (-) individuals are as risk of making..?..when exposed to Rg (+) blood.

Back 22

AntiD – 90% chance if through transfusion; 30% chance if through pregnancy
-- immunization to the D Ag after exposure to small amt Rh(+) RBCs can be prevented w/ injection of RhIg w/in 72 hours

Front 23

Describe Rh incompatibility.

Back 23

-- causes morbidity ONLY if pt has already formed antiD
-- assoc w/ hemolytic transfusion rxns (not as severe as ABO incompatibility)
-- is still the most common cause of severe HDN (erythroblastosis fetalis)
-- has very little, if any, impact in organ or BM transplantation

Front 24

List other erythrocytic blood grp systems and their impact on transfusions.

Back 24

Kell, Kidd, Duffy, MNS, Diego, Lewis, Lutheran, P, etc.
-- Ab to these Ag present in <1% of the pt population and usually require previous transfusion or pregnancy
-- these Ag are not very immunogenic
-- Are occasionally involved in hemolytic transfusion rxn if pretransfusion testing is incomplete/inaccurate
-- incompatibility has little, if any, implication in organ or BM transplantation (EXCEPT Lewis group in renal transplantation)

Front 25

Which of the “other” blood grp systems are the most clinically significant?

Back 25

AntiK to the K Ag in the Kell system is the most common and clinically signif Ab
-- most immunogenic after the D Ag
-- often complement fixing and can cause severe intravascular hemolytic rxns
-- AntiK usually formed as result of transfusion of one or more units of K(+) RBCs.
-- K Ag frequency is only 10% in pops of European orgin and 5% or less in pops from other geograph origin

Front 26

What is the clinical significance of a positive DAT?

Back 26

DAT done on pt’s RBCs
(+) DAT reflects presence of IgG and/or complement components (C3b and C3d) on these red cells
-- (+) is ABNORMAL
-- should be ordered when one suspects or wants to r/o an immune mediated hemolysis

1. Autoimmune disorder
2. HDN
3. Hemolytic transfusion rxn
4. Drug-induced
5. Non-specific binding

Front 27

Once an initial DAT is determined (+), what is the next step?

Back 27

Done initially w/ polyspecific antihuman globulin, which will detect both IgG and C3.

Next step is to use monospecific globulin to determine whether it is IgG, C3, or both.

If IgG present: it is eluted off the RBCs and reacted w/ reagent cells to determine whether it is directed toward RBC Ag (positive eluate), or bound to the RBCs in a less specific fashion (negative eluate)

Front 28

A positive DAT is always present…?

Back 28

In any of the autoimmune hemolytic anemias

WARM – will demonstrate IgG on the RBCs and sometomes C3 and the eluate will be (+)

COLD and PAROXYSMAL COLD – will demonstrate C3 only on the RBCs

Front 29

In hemolytic disease of the newborn (HDN), the DAT will be…?

Back 29

POSITIVE with IgG only and

The ELUATE will be (+) demonstrating the specificity of the maternal Ab that crossed the placenta

Front 30

In a hemolytic transfusion rxn, the DAT will be…?

Back 30

POSITIVE – the incompatible RBCs will be coated w/ an Ab causing a (+) DAT w/ a characteristic mixed field pattern.

The ELUATE will be (+) demonstrating the specificity of the Ab causing the transfusion rxn

Front 31

What are the three mechanisms that can result in immune-mediated drug induced hemolysis?

Back 31

1. hapten mechanism

2. immune complex mechanism

3. autoimmune phenomenon

Front 32

Describe the hapten mechanism.

Back 32

Mostly assoc w/ PCNs or cephalosporins
-- drug binds tightly to RBC mbrn and Ab binds to drug
-- DAT demonstrates IgG
-- ELUATE is (-)

Front 33

Describe the immune cmplx mechanism.

Back 33

Numerous drugs, with quinidine as the prototype
-- immune cmplx formed to the drug
-- complement activated
-- DAT only demonstrates C3

Front 34

Describe the autoimmune phenomenon.

Back 34

Only four drugs assoc: procainamide, L-dopa, alpha-methyldopa, and mefenamic acid
-- drugs elicit an autoimmune response w/ true autoAb to RBCs
-- DAT indistinguishable from warm autoimmune hemolytic anemia

Front 35

What is the clinical significance of a positive IAT?

Back 35

Positive IAT done on pt’s serum and reflects presence of an Ab to Ag in one of the blood grp systems except for ABO.
-- (+) IAT found in 1-3% of pts and does not mean that disease process is present

Usually the result of alloimmunization following exposure to foreign RBC Ag from prior transfusion or pregnancy
-- puts pt at risk for incompatible blood transfusion resulting in a hemolytic transfusion rxn
-- puts pregnant woman at risk for HDN

Front 36

What is the immunologic mechanism of HDN?

Back 36

1. The mother’s plasma contains RBC alloantibodies
-- mother (+) IAT or
-- mother is ABO incompatible w/ fetus (O mother w/ A or B child)

2. Placenta actively transfers IgG Ab into the fetal circulation

3. If the fetal RBCs have the corresponding Ag, the Ab will bind to the RBCs and cause extravascular hemolysis

Front 37

Clinical findings in fetus w/ HDN?

Back 37

1. anemia secondary to RBC destruction

2. hepatosplenomegaly secondary to extramedullary hematopoiesis

3. if hemolysis is severe and not sufficiently compensated by erythropoietic hyperplasia, hydrops fetalis occurs which may result in fetal death in the third trimester

Front 38

Clinical findings in neonate w/ HDN?

Back 38

1. anemia w/ reticulocytosis and very large number of nucleated RBCs in peripheral blood

2. positive DAT

3. brisk rise of indirect bilirubin w/in 24hrs of birth, which may cause kernicterus

Front 39

HDN therapy?

Back 39

1. intrauterine transfusion w/ RBC compatible w/ the mother’s blood is performed if fetal anemia is severe and fetal demise is a risk

2. exchange transfusion is performed after birth if indirect bilirubin level is in a range that could result in kernicterus

Front 40

Clinical significance of HLA incompatibility?

Back 40

1. severe GVHD in allogeneic BM transplant

2. acute or chronic rejection in organ transplantation

3. febrile non-hemolytic (“white cell”) rxn during transfusion of cellular blood components (RBCs or platelets)

4. refractoriness to platelet therapy

5. transfusion assoc acute lung injury (TRALI) after transfusion of plasma-containing blood components

Front 41

HPA incompatibility is assoc with…?

Back 41

1. neonatal alloimmune thrombocytopenia (attracting platelets)

2. refractoriness to platelet therapy

3. post-transfusion purpura

Front 42

HGA incompatibility is assoc with…?

Back 42

1. febrile non-hemolytic (“white cell”) rxn during transfusion of cellular blood components (RBCs or platelets)

2. TRALI after transfusion of plasma-containing blood components

3. neonatal alloimmune neutropenia