serological testing in blood transfusion services

4th year medical students

Serological Testing InBlood Transfusion Services

Salwa Hindawi

Medical Director of Blood Transfusion ServicesKAUH


Introduction

• Serology is the study of antigens and antibodies

• Immunohematology refers to testing done in blood banks on red blood cells

• All testing requires the use of Standard Precautions


Antigens and Antibodies

• Antigens are substances recognized as foreign by the body

• Antibodies are proteins produced by the body in response to specific antigens


ABO Group

• Four blood groups: A, B, O, or AB

• Almost all serum contains antibodies to ABO antigens it lacks

• Antibodies are crucial to safe transfusion


type A

type B type O

type AB

Red blood cell antigens

A antigen A and B antigen

B antigen H antigen(no A or B antigen)


Red cell Antigens: ABO type

• present on RBCs, GI tract and vascular endothelium

• three alleles A, B, O, the A and B alleles code for glycosyltransferases

• specificity of the antigen is in its terminal sugar– galactosamine for A– galactose for B


Molecular basis of ABO alleles

• fucose added to H-substance • precursor

• product of A allele adds • N-acetylgalactosamine

• product of B allele adds • galactose

• both molecules (A and B) can • be expressed on cell surface• at the same time

• in OO individuals H substance • is not modified


IAIA

IAIO

Blood GroupA 41% A Anti-B

IBIB

IBIO

Blood GroupB 10% B Anti-A

IOIO

Blood GroupO 45% Neither

Anti-AAnti-B

IAIB

Blood Group AB 4% Both Neither

Genotype Phenotype Frequency Antigen

Antibodyin

Serum

Note: Caucasian frequencies given. Frequencies differ with population.


Rh type

• Rh blood group antigens are present only on RBCs• Five important antigens of the Rh system are D, C, E,

c, and e• Rh positive means that the D antigen is present

(85% on the population)• Rh negative means that the D antigen is absent

(15% of the population)• the D antigen is highly immunogenic• More than 70-80% of D negative persons receiving D

positive blood are expected to develop anti-D


Rh Blood Group Alleles

• Weiner - One complex locus, 8 different alleles

• Fisher-Race - Three separate loci; simple alleles, D allele most

important; D = Rh+; dd = Rh-

• C and E loci may effect expression of Rh phenotype

Multiple AllelesWeiner

R1

R2

R0

Rz

rrl

rll

ry

Closely Linked LociFisher-Race

CDecDEcDeCDEcdeCdecdECdE

Rh+

Rh-


Other Blood Group Systems

• clinically significant blood group systems are Kell (K), Kidd (Jk), Duffy (Fy) and Rh (E,e,C,c) systems.

• antibodies are made by people who lack the antigen on their RBCs and have been exposed to RBCs containing the antigen


Steps in Blood Banking

• Type and Screen (T & S): • (Done for low probability of transfusion)

– ABO and Rh type– Antibody screen– Antibody identification

• Type and Crossmatch (T & C) • (Done for high probability of transfusion)• above steps plus Crossmatch


Pre-transfusion Testing: Purpose: To select blood components that will not cause harm to the recipient and will have acceptable survival when transfused.

When performed properly, pretransfusion tests will confirm ABO compatibility between the component and the recipient and will detect most clinically significant unexpected antibodies.


Guidelines for Pre-transfusion Testing:

• Positive identification of recipient and recipient sample• ABO group and Rh typing of recipient and donor blood.• Red cell antibody detection tests for clinically significant

antibodies using recipients serum or plasma.• Comparison of current findings of recipients sample with

record of previous patient results.• Selection of components of ABO group and Rh type

appropriate for the recipient.• Performance of a serologic or computer crossmatch.• Labeling of products with the recipients identifying

information.


Direct Antiglobulin Test (DAT)

• also called the direct Coombs test• adding anti-IgG to detect IgG that is

attached to the RBCs• also detects C3 complement fragments on

the RBC surface• DAT is performed in the investigation of

immune hemolytic anemia and transfusion reactions


Indirect Antiglobulin Test (IAT)

• detects free antibodies in the serum

• the IAT test is performed during the antibody screen and antibody identification


ABO GROUPING

ABO grouping is the single most important serological test performed on pre-transfusion


ABO Grouping

Pt Cells Pt Serum

vs vs

anti -A anti-B A cells B cells

A + 0 0 + 40%

B 0 + + 0 11%

AB + + 0 0 4%

0 0 0 + + 45%


Type and Screen (T & S)

• an ABO and Rh type and an antibody screen and antibody identification are done when the patient is admitted

• only testing necessary if low probability of transfusion


Crossmatch to Transfusion ratio (C:T ratio)

• blood is used more efficiently when the number of units set aside for a particular patient (crossmatched) are actually transfused.

• when a patient does not need blood, it is good practice to get a T& S but not a T & C

• C:T ratio is less than 2:1


Maximum Surgical Blood Order Schedule (MSBOS)

• Is a guideline to order standard number of units of RBCs to be crossmatched for a specific surgical procedure, based on average use in the institution

• examples – angioplasty T&S– aortic dissection T&C 6 U


ANTIBODY SCREENING

that antibody screening should be performed in all pre-transfusion testing.

As a minimum the following antigens must be expressed within the screening cell set:

C, c, D, E, e, K, k, Fya, Fyb, Jka, Jkb, S, s, M, N, Lea


Antibody Screen (IAT)

• recipients serum is added to 3 test RBCs (in test tubes 1 to 3 ) which have all of the important RBC antigens on them

• therefore if one or more of the three screening cells is positive then RBC antibody is present in the serum

• then do an antibody panel to identify the antibody present


Antibody Identification (IAT)

• after the Ab screening are positive then do an antibody identification

• recipients’ serum is added to 10 test RBCs in a panel (test tubes 1 to 10) which contain all of the important antigens

• the antibody in the serum is identified


REAGENT RED CELL PANEL

NEGATIVE AUTO CONTROL

SOME CELLS POSITIVE ALL CELLS POSITIVE SOME CELLS POSITIVE

(SAME STRENGTH AND PHASES) (DIFFERENT STRENGTHS (DIFFERENT STRENGTHS AND/OR PHASES)

SOME CELLS NEGATIVE AND/OR PHASES) AND SOME CELLS NEGATIVE

Suspect Single Antibody Suspect Multiple Antibodies Suspect Multiple Antibodies

-Test other selected cells to -Test selected cells to confirm and

eliminate other specificities eliminate specificities

-Test patient’s cells to confirm -Additional techniques may be useful

they lack antigen (enzymes)

-Test patient’s cells to confirm

they lack antigens

ALL CELLS POSITIVE WEAK REACTIVITY SEEN WITH SOME CELLS ONLY ONE CELL POSITIVE

(SAME STRENGTH AND SAME PHASES) ALL SPECIFICITIES ELIMINATED (DONOR UNIT OR PANEL CELL)

Suspect Antibody to High-Incidence Antigen Suspect Weakly Reactive Antibody or Suspect Antibody to Low-Incidence Antigen or

(See also Multiple Antibodies) Antibody Showing Dosage Antibody to HLA Antigen

-Test cells negative for high-incidence -Enhancement techniques (enzyme panel, -Test cells positive for low-incidence

antigens increase amount of serum used, antigens or known strongly positive for

-May need help from Reference Laboratory increase incubation time) HLA antigens

for identification or confirmation -Test patient’s cells to confirm -May need to refer to Reference Laboratory

-Test patient’s cells to confirm they lack antigen for identification or confirmation

they lack antigen

Approaches for identifying antibodies


Major Crossmatch (Compatibility testing)

• donor RBCs (unit of blood) are tested with recipient serum

• to detect unexpected recipient antibodies

• this checks to see if the transfusion is compatible


CROSSMATCHING

Types of Cross Match:

-serological IAT crossmatch.

-abbreviated serological techniques immediate spin

-non-serological methods

electronic cross Match


PatientABO Type

Antibody(Serum)

RBCs orGranulocyt

es

Plasma-ContainingComponen

ts*

WholeBlood

O(45%)

Anti-A andAnti-B

O A, B, AB, O O

A(40%) Anti-B A, O A, AB A

B (11%)

Anti-A B, O B, AB B

AB(4%) None A, B, AB, O AB AB

ABO Selection of Blood Components

serological testing in blood transfusion services

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