Detection and Identification of Red Cell Antibodies

four types of unexpected antibodies

immune alloantibodies

naturally occurring alloantibodies

passively acquired antibodies

autoantibodies

immune alloantibodies are produced in responnse to

RBC stimulation

naturally occurring alloantibodies are caused by

exposure to environmental sources (ex: anti-D)

passively acquired antibodies

intravenous immunoglobulin

autoantibodies

antibodies directed against antigens expressed on one’s own RBCs

three main phase reactions occur in

immediate spin, 37C, and AHG

what is an antibody screen

pt. plasma + reagent screen cells

we use this to find unexpected antibodies in a patient’s plasma that may cause a transfusion reaction

antibody screens are preformed in which 3 areas of testing

donor blood testing

prenatal testing

compatibility testing for transplants (hematopoietic progenitor cells or hematopoietic stem cells, or bone marrow)

specimen requirements of blood bank

must be serum or plasma

need 5-10 mL aliquot of whole blood needed

when auto control is being tested in ab ID EDTA plasma is used

why is EDTA the preferred tube for bloodbank

it avoids the in vitro uptake of complement by red cells which can occur if clotted samples are used

three main methods of antibody testing

tube (traditional and gold standard)

gel

solid phase

what group type are the screener cells made of

group O

dosage meaning

you’ll have stronger reactions (3+ or 4+) when we have a homozygous antigen expression (considered double dose)

you’ll have weaker reactions (1+ or 2+) when we have hetereozygous antigen expression (considered single dose)

which antigens don’t show dosage

Kell, D, Lewis, I, and P

for the common blood group which antibodies show dosage

lutheran (Lu^a, Lu^b), kidd (Jk^a, Jk^b), duffy (Fy^a, Fy^b), Rh (C, E, c, e), MNS

Lutheran kids and duffy the rhesus monkey eat M and Ns

what is the purpose of enhancement reagents

it decreases incubation time and increases sensitivity (such as PEG)

3 possible enhancement reagents used in antibody detection

LISS

PEG (most common)

22% albumin (BSA, uncommon)

what are the differences between monospecific AHG and polyspecific AHG

monspecific contains only IgG, is expensive, and only works on one epitope/antigen

polyspecific contains IgG and C3d, cheaper, and works on multiple epitopes/antigens

check cells are also known as

coomb’s cells

why do we use check cells

to make sure there was adequate cell washing

to make sure AHG reagent was added

to make sure AHG reagent is functioning properly

limitation of antibody screen

the screen is designed to detect as many clinically significant antibodies as possible and avoid detecting as many clinically insignificant antibodies as possible

negative results should give great confidence that there were no clinically significant antibodies detected

what are check cells

type O Rh positive red blood cells coated with anti-D

what is auto control and its purpose

patient cells + patient plasma tested under the same conditions as reagent red cells are tested to looks for autoantibodies

should be tested as part of antiody ID but can be preformed with Absc

(do not confuse with DAT which tests for complement activation)

process of how to start the interpretation antibody screen results

negative and positive reactions are important in interpretation of panel

phase and strength of positive reactions helps suggest antibody specificity

negative reactions support the specificity suggested by positive reactions

single common alloantibody usually produces a clear pattern of positive and negative reactions

possible interpretation of antibody screen

single alloantibody

two alloantibodies, antigens only present on cell II

probably IgG alloantibody

possible interpretation of antibody screen

multiple allantibodies

single alloantibody (dosage)

probable IgG alloantibody

possible interpretation of antibody screen

single or multiple antibodies

probably IgM alloantibody

possible interpretation of antibody screen

multiple alloantibodies, warm and cold

potent cold alloantibody binding complement in AGT

possible interpretation of antibody screen

single warm alloantibody, antigen present on both cells

antibody to high prevelence antigen

complement binding by a cold alloantibody not detected at IS

possible interpretation of antibody screen

warm alloantibodies

transfusion reaction

probable IgG alloantibody

warm autoantibody

which antibodies tend to react at immediate spin

Le^a, Le^b, M, N, Lu^a, and P

which antibodies tend to react at 37C

cold IgM antibodies (I, P1, A, B, H) and warm antibodies (D, E, and K)

which antibodies tend to react at the antiglobulin phase (AHG)

Rh antigens, Kell, Duffy, Kidd, S, s, and Lu^b

what does exclusion rule out mean

the process of ruling out certain antibodies as the cause of reaction

it helps narrow down the possibilities and ID the specific antibody present in the patient’s blood

list specific techniques that can be used to further ID antibodies

enzyme (common)

neutralization

elution (common)

adsorption (common)

titration

enzyme ID method

used when multiple antibodies are suspected

certain antigens are destroyed or weakened, others are enhanced, some have no effect (antigens destroyed are more likely on RBC membrane)

never used for initial panel

enzyme ID method is used for

weakly reactive antibodies present which enhance the antibody reactivity

helps differentiate between multiple antibodies present, reaction strengths, and untreated vs treated

two ways you can preform enzyme ID

one step technique (dump everything in a tube (enzyme, plasma, rbc), incubate, absc

two step proccess (preferred): RBC + enzyme then wash then add plasma

which type of ezyme treatment is preferred and why

the two step process because in babies and the elderly it gives a cleaner sample because we want to make sure everything is 100% because of transfusions and RBCs can have antigens on them

blood groups enhanced by enzymes

ABO/H, Lewis, I, P, Rh, and Kidd

blood groups decreased by enzymes

MNS, Duffy, and Lutheran

blood groups unaffected by enzymes

Kell, Diego, and Colton

neutralization ID method

soluble forms of some blood group antigens exist in body fluids (ex; plasma, urine, saliva)

this inhibits the reactivity of corresponding antibody that could mask the prescence of undelrying clinically significant, nonuetralizable antibodies

most used substances for neutralization

Lewis’s substances (most common, saliva, plasma, urine, etc)

P1 substances

Sd^a substances

Chido and Rogers substances

technique for neutralization

neutralizing + pts plasma then incubate

the suspected antibody should be negative (or decrease in reactivity)

the control (pt plasma + saline, incubate) should be positive

substances used for neutralization

anti-P

anti-Lewis

anti-Chido, anti-Rodgers

anti-Sd^a

anti-I

anti-P neutralizing substances

hydatid cyst fluid, pigeon droppings, turtledoves’ egg whites

anti-Lewis neutralizing substances

plasma or serum, saliva

anti-chido/anti-rodgers neutralizing substances

serum containing complement

anti-Sd^a neutralziing substances

urine

anti-I neutralizing substances

human breast milk

elution ID method

used to identify one or more antibodies attached to RBC membrane by freeing them

antibody recovered/removed is called elute

useful for removing antibodies for RBC in HDN due to ABO antibodies

how does elution work

1. bound antibody us reaksed from RBC antigen

2. this disrupts the structure of the binding site because it reverses rhe forces of attraction keep the antigen-antibody complexes together

3. changes thermodynamics of antigen-antibody complex

two methods for elution

lansteiner and miller heat elution

wiener’s freeze-thaw method

landsteiner and miller heat elution method is best for

cold reactive antibodies or those that have a broad temperature rnage of reactivity

weiner’s freeze-thaw method

the sample is frozen causng ice crystals to form causing hemolysis of RBCs when the sample is thawed the antibodies are released

adsorption ID method

used to remove autoantibodies

two types of adsorption techniques

autologous adsorption (using pts own RBCs)

alladsorption (using other peoples RBCs paired with elution techniques)

when picking RBCs for adsorption, the antigens that stimulate the most clinicaly significant anitbodies are

Rh, K, Duffy, S, s, and Kidd

adsorption technique

used to remove unwanted antibodies from patient serum

patient plasma (containing antibodies) is mixed with RBCs that have known antigens

unwanted antibodies in the plasma bind to the antigens on the RBCs

the RBCs with the bound antibody are removed from the serum leaving behind the rest of the other antibodies making it easier to identify the remaining ones

antibody titration ID method

measures the strength of an antibody using serial dilutions of antibody containing plasma tested against selected RBCs to determine the highest dilution casuing a positive rxn

a 4+ titer means you have a titer score of

12

a 3+ titer means you have a titer score of

10

a 2+ titer means you have a titer score of

8

a 1+ titer means you have a titer score of

5

elution and adsorption are used to detect what kind of antigens

elution is used to indentify one or more antibodies attached to the RBC membrane specifically HDFN ad HTR antigens

adsorption is used to remove autoantibodies

significance of red cell phenotyping

confirms that the antibody indentified is the correct one (its the last step of identification)

what is the number one thing MLS need to do before starting testing

check patient history (blood type, known antibodies, history of transfusion)

why is antigen typing not really preformed on a patient that hsve been transfused within 3 months

because you’ll still have circulating donor RBCs so it would cause a mixed field reaction

which method of antibody detection is useful to detect cases of HTR and HDN

elution method

what is needed on a patient label

name (spelled correctly and with first, middle inital, and last name)

DOB

time

your intials

date of draw

a unit of blood can be returned if

it’s not open

the storage temperature was maintained

it’s been within 30 minutes of the unit leaving the window

ACOS 1:1:1 ratio includes

every unit of RBCs gets one unit of FFP and one unit of PLTs

if a patient has multiple myeloma what can happen to the RBCs

agglutination mixed with rouleux

drug given for multiple myeloma

DARA

what drug causes agglutination mixed with rouleux

DARA

when you treat a patient on DARA with DTT what does it do

it wipes out the Kell blood group so you’ll have to give Kell negative blood

dolichos biflorus lectin binds with

A₁ and A₁B

ulex europaeus binds

H and O cells

bandeiraea simplicifolia binds

B

vicia graminea binds

N

iberis amara binds

M