Indirect and Direct Antiglobulin (Coombs)

Testing and the Crossmatch

Darrell J. Triulzi, M.D.Medical Director

INTRODUCTION
The antiglobulin test was first introduced into clinical medicine in 1945
by R.R. Coombs who showed that it could be used to detect nonagglutinating red cell antibodies (indirect antiglobulin test, IAT) or
sensitized red cells (direct antiglobulin test, DAT). Most nonagglutinating (incomplete) antibodies are IgG, although some antibodies
are IgM. These antibodies do not spontaneously cause agglutination
due to a strong electronegative charge on the red cell surface that
prevents the cells from coming into close proximity. The antiglobulin
reagent is able to bridge these negative forces. Current antiglobulin
reagent (Coombs reagent) preparations contain a "cocktail" of
monoclonal antibodies directed against human IgG and C3. The latter
is more effective than an anti-IgM reagent for detecting IgM antibodies
because a single IgM molecule will bind numerous complement
molecules to the red cell surface and IgM antibodies tend to
spontaneously dissociate from the red cell membrane.

DIRECT ANTIGLOBULIN TEST (DAT)

The DAT is used to detect IgG or C3 bound to the surface of the red
cell. In patients with hemolysis, the DAT is useful in determining
whether there is an immune etiology. Non-immune causes of hemolysis
such as DIC, thrombotic thrombocytopenic purpura, mechanical
hemolysis such as those due to artificial valves or burns,
hemoglobinopathies (sickle cell, thalassemia), red cell enzyme
deficiencies (G6PDP, pyruvate kinase), and red cell membrane defects
(hereditary spherocytosis, PNH) will have a negative DAT. Immune
causes of hemolysis including autoimmune hemolytic anemias, drug
induced hemolysis, and delayed or acute hemolytic transfusion
reactions are characterized by a positive DAT. A positive DAT can occur
without hemolysis. A small proportion of normal individuals have a
positive DAT without evidence of decreased red cell survival. Thus, a
positive DAT, by itself, does not mean that the patient has an immune
hemolytic anemia.

The DAT is a 5-10 minute procedure performed by incubating patient

red cells with the antiglobulin (Coombs) reagent. A positive DAT due to
IgG is seen most frequently in patients with warm autoantibodies.
Approximately 1/3 of these patients also have C3 on the red cell
membrane. IgG coated red cells may also be seen in patients who
have received an incompatible transfusion. Thus, the DAT is routinely
performed as part of all transfusion reaction investigations. IgG bound
to the red cell surface can be eluted and its specificity determined.
Eluted autoantibodies usually bind to all red cells (panagglutinin) but
occasionally have specificity within the Rh system. Eluted
alloantibodies can usually be given a distinct specificity.(eg. anti-D, antiK) Red cells sensitized with IgG may be destroyed by extravascular
hemolysis. The primary site of removal is the spleen via Fc receptors
on phagocytic cells. Factors that determine whether hemolysis will
occur include: antibody titer, number of IgG molecules on the red cell,
number of antigen sites on the red cell, IgG subclass, and splenic
function.
A positive DAT due to complement (C3) alone is seen in patients with
cold autoantibodies, paroxysmal cold hemoglobinuria, and in some drug
induced hemolytic anemias. The offending antibodies are typically of
the IgM isotypes that efficiently bind complement. IgM antibodies are
not directly detected by the DAT, but are detected indirectly by the
presence of C3 on the red cell surface. Cold autoimmune hemolytic
anemias may be associated with intravascular hemolysis due to
complement mediated lysis. Extravascular removal of C3 coated cells
can also occur via complement receptors on phagocytes in the liver.
Rarely, patients with immune hemolysis may have a negative DAT.
Many of these patients have IgG, IgM, or IgA antibodies detectable on
the red cell only with more sensitive techniques. (MicroCoombs test)

INDIRECT ANTIGLOBULIN TEST (IAT)

The IAT is used to detect red cell antibodies in patient serum. In clinical
practice this is referred to as the "antibody screen" and is part of the
type and screen procedure. Approximately 5% of patients have a
positive IAT due to IgG antibodies, IgM antibodies, or both. Most
clinically significant alloantibodies are IgG antibodies that react best at
37C and are formed as a result of previous exposure via transfusion or
pregnancy. Examples include antibodies to Rh, Kell, Kidd, and Duffy
red cell antigens. IgM antibodies are usually not clinically significant
(except for ABO antibodies) but are a source of in-vitro serologic
difficulty that may delay transfusion. Examples include antibodies to the
Lewis, I, P, M, and N red cell antigens. IgM antibodies react best at cold
temperatures (4C) and are usually naturally occurring in that they do

not require a sensitizing event.

The IAT (antibody screen) is performed by incubating patient serum with
reagent screening red cells for approximately 20 minutes and then
observing for agglutination. If the antibody screen is positive, additional
testing is required to determine the specificity of the antibody.
If transfusion is necessary, patients with clinically significant red cell
alloantibodies identified in the antibody screen should receive antigen
negative red cells. Compatible blood may be difficult to find if antigen
negative blood is rare or if multiple antibodies are present. Consultation
with the transfusion service is helpful in developing a transfusion
strategy in these cases.
CROSSMATCH
The crossmatch (CM) represents a special form of the IAT in which the
red cells used for testing are from the unit intended for transfusion. The
purpose is to establish in vitro compatibility in the expectation that the
transfused cells will exhibit normal in-vivo survival. Historically, a major
CM involving patient serum and donor red cells was performed on every
unit intended for transfusion. The major crossmatch takes 20-30
minutes and is now reserved only for patients with clinically significant
red cell antibodies. The minor CM involving patient red cells and donor
plasma has not been performed for more than 20 years because
packed red cells have <70 ml of plasma. More recently it has been
recognized that patients with a negative antibody screen and no history
of red cell antibodies do not require a complete 20-30 minute
crossmatch. The chances of a clinically significant red cell antibody
being missed in a patient with a negative antibody screen (false
negative) is 1-4/10,000. Approximately 95% of transfusions occur in
patients with a negative antibody screen. Such patients can undergo
abbreviated CM testing in which only ABO compatibility of the unit need
be established. There are two methods for abbreviated CM testing.
The immediate spin crossmatch (ISCM) requires only a 5 minute
incubation at room temperature with patient serum and donor red cells.
For the last 4 years the American Association of Blood Banks has also
allowed an abbreviated CM to be performed by computer confirmation
of ABO compatibility without any serologic testing: computer
crossmatch. The patient must have two determinations of their ABO
group on record and a negative antibody screen. Using a validated
computer system the bar code on unit of blood can be wanded and the
computer will compare the ABO of the unit to that of the patient and
indicate whether it is compatible. Advantages of the computer
crossmatch includes: faster turn-around, computer prevents release of
ABO incompatible units, lower reagent costs, and improved quality

control.

SUMMARY
The IAT and DAT are used to detect red cell antibodies in the serum
and on the red cell, respectively. The DAT is used to determine whether
patients with hemolysis have an immune etiology. The IAT is used to
identify clinically significant red cell alloantibodies that are important in
choosing compatible blood products.

1.

Felson D, Lavalie MP, et al. The Prosorba column for treatment of

refractory rheumatoid

2.

Snyder HW, Cochran SK, et al. Experience with protein Aimmunoadsorption in treatment-resistant adult
immunothrombocytopenic purpura. Blood 1992; 79: 2237-45.

3.

George JN, Woolf SH, et al. Idiopathic thrombocytopenic purpura: a

practice guideline developed by explicit methods for the American
Society of Hematology. Blood 1996; 88: 3-40.

CARA KERJA CROSS MATCH DENGAN

BIORAD/DIAMED GEL TES
I. BUAT SUSPENSI SEL PASIEN & DONOR 0.8 - 1%.
CARA : 1. Masukkan 0,5 ml Dil 2 dengan Dispenser ke
dalam tabung
2. Ambil 5 ul (mikroliter) PRC atau 10 ul WB, masukkan
tabung
3. Campur dan homogenkan Suspensi 0,8 1%
II. Ambil Liss / Coombs Card, tandai dengan identitas Pasien /
Donor, buka
penutup alumunium. Dengan bantuan mikropipet,

masukkan :

MAYOR : 50 ul Suspensi Sel Donor + 25 ul

Serum Pasien
MINOR : 50 ul Suspensi Sel Os + 25 ul
Serum Donor
A.C
: 50 ul Suspensi Sel Os + 25 ul
Serum Pasien
III. Masukkan kartu ke Inkubator.
Inkubasi 37 C, 15 menit ( tekan tombol timer 1 / 2 / 3 )
IV. Pindahkan kartu ke Centrifuge
Tekan tombol Start ( Centrifuge selama 10 menit )
V. Baca Reaksi secara makroskopis
CARA KERJA DIRECT COOMBS TES
1. Buat Suspensi Os 0,8 1% ( cara sama seperti diatas )
2. Ambil Liss / Coombs Card, tandai dengan identitas
Pasien.
3. Masukkan 50 ul Suspensi Sel Pasien.
4. Putar di Centrifuge ( tekan tombol Start )
5. Baca Reaksi
CARA POOLING UNTUK INTER CROSS DONOR ( AUTO
POOL )
Maksimum pooling untuk 3 kantong darah
Cara Pooling :
a. Potong selang pada kantong donor yang akan di PoolinG
b. Teteskan pada 2 tabung kosong masing-masing sel
darah merah
donor yang akan di-pool dan serum/plasma donor yang
akan
di-pool dengan jumlah yang sama .

c. Homogenkan sel darah merah pada tabung yang berisi

pooling sel
darah merah donor, buat suspensi 1% dengan Diluent 2
dengan cara seperti di atas.
d. Lakukan Cross Match seperti biasa
INTER CROSS : 50 ul pool Suspensi Sel Donor +
25 ul pool serum Donor
INTEPRETASI HASIL CROSS MATCH
No
.

MAY
OR

MIN
OR

AC/D
CT

Kesimpulan

Darah keluar

Ganti darah donor

Ganti darah Donor

Darah keluar bila minor lebih kecil

atau sama dg AC/DCT inform
concent

Lihat ket. No.5

Keterangan :
1. Crossmatch Mayor, Minor dan AC = negatif
Darah pasien kompatibel dengan darah donor
Darah boleh dikeluarkan
2. Crossmtacth Mayor = positif, Minor = negatif, AC = negatif
Periksa sekali lagi Golongan darah Os apakah sudah sama
dengan
donor, apabila gol. Darah sudah sama :
Artinya ada Irregular Antibody pada Serum Os
Ganti darah donor, lakukan crossmatch lagi sampai didapat
hasil cross negatif pada mayor dan minor
Apabila tidak ditemukan hasil crossmatch yang kompatibel
meskipun darah donor telah diganti maka harus dilakukan
Screening dan Identifikasi Antibody pada Serum Os, dalam
hal ini sampel darah dikirim ke UTD Pembina terdekat
3. Crossmatch Mayor = negatif, Minor = positif, AC = Negatif

Artinya ada Irregular Anti Body pada Serum / Plasma Donor.

Solusi : Ganti dengan darah donor yang lain, lakukan
crossmatch lagi
4. Crossmatch Mayor = negatif, Minor = positif, AC = Positif
Lakukan Direct Coombs Test pada OS
Apabila DCT = positif, hasil positif pada crossmatch Minor dan
AC berasal dari autoantibody
Apabila derajad positif pada Minor sama atau lebih kecil
dibandingkan derajad positif pada AC / DCT, darah boleh
dikeluarkan
Apabila derajad positif pada Minor lebih besar dibandingkan
derajad positif pada AC / DCT, darah tidak boleh dikeluarkan.
Ganti darah donor, lakukan crossmatch lagi sampai ditemukan
positif pada Minor sama atau lebih kecil dibanding AC / DCT
5. Mayor, Minor, AC = positif :
Periksa ulang golongan darah Os maupun donor, baik dengan
cell grouping maupun back typing, pastikan tidak ada
kesalahan gol. Darah
Lakukan DCT pada Os, apabila positif, bandingkan derajat
positif DCT dg Minor, apabila derajat positif Minor sama atau
lebih rendah dari DCT, maka positif pada Minor dapat
diabaikan, artinya positif tersebut berasal dari autoantibody.
Sedangkan positif pada Mayor, disebabkan adanya Irregular
Anti Body pada Serum Os, ganti dengan darah donor baru
sampai ditemukan hasil Mayor negatif