2. Content
Antibody screening positive ( all cells)
Multiple antibodies
Antibody to high prevalence antigen
Warm autoantibodies
Drug induced immune hemolytic anemia
3. Antibody screening positive
( all cells)
Antibody screening Vs. Auto control
Manual polybrene ( MP) Vs. classic AHG method
Auto control Vs. DAT
6. MP Vs. classic AHG
Sensitivity MP>CAT> AHG
MP sensitive in cold reactive antibodies ( eg. Anti-E,
anti-P1, anti-Lea, anti-Leb, anti-M, anti-Mia……)
MP sensitive in IgM antibodies ( eg. Anti-I, anti-HI, anti-
E……)
Classic AHG more sensitive in Kell, kidd, Duffy, s
MPA is an alternate method with MP method.
7. Optimum formula of MP (LIP)
0.6mL or 1.0 mL LIM
0.05% polybrene , 0.25% polybrene, 0.5% polybrene
Agglutination chart
Enzyme treated RBC ? Eluent ? Suitable for MP
8. Autocontrol Vs. DAT
Auto control show relative to allo cells in deferent
condition. ( IS, 37, AHG, MP, prewarm )
MP AC Vs. MP DAT
Mix field DAT, AC
Transfusion within 3 months
9. Content
Antibody screening positive ( all cells)
Multiple antibodies
Antibody to high prevalence antigen
Warm autoantibodies
Drug induced immune hemolytic anemia
11. Evaluation of Initial Antibody
Identification Panel
When… And autocontrol is… Then antibody(ies) most likely
present is/are…
some panel cells are positive at any
phase of testing
negative single or multiple antibodies.
all panel cells are equally positive (2
to 4+) in the IAT
negative an antibody to a high-
prevalence antigen.
all panel cells are weakly positive
(2+) in the IAT with variable
reactivity
negative Knops antibody, anti-Yt
a
, anti-
JMH, or anti-Ch/Rg.
all panel cells are positive strongly positive (3 to 4+) warm autoantibody.
all panel cells are positive, or some
positive, some negative
weakly positive (2+) multiple antibodies, in a patient
experiencing a delayed
transfusion reaction.
all panel cells are negative negative an antibody directed against a
low-prevalence antigen.
all panel cells are equally positive (1
to 4+) at IS and negative or
weaker at 37 C and IAT
negative or positive cold-reactive autoantibody
(anti-I, -IH).
12. Differential of multiple
antibodies
Neutralization
Enzyme and chemical treated cells
Absorption & elution
Cord blood cells
pH & thermal
Dilution
15. TRY = trypsin-treated RBCs; CHY = chymotrypsin-treated RBCs; PAP = papain- or ficin-treated RBCs; PRO = pronase-
treated RBCs; NEU = neuraminidase-treated RBCs; DTT = RBCs treated with dithiothreitol or 2-
aminoethylisothiouronium bromide hydrobromide (AET); + = antibody reactive; 0 = antibody nonreactive; w =
reactions weakened (weak antibodies may be nonreactive); +/0 = some examples reactive, others nonreactive;
+/w = some examples reactive, others show weakened reactions.
16. Chemical / physical treated
S : destroy with NaClO
Nform : enhance if patient on renal dialysis
HLA ( Bga) : test with chloroquine-treated RBC
( Gamma Quin)
M : enhance by acidification ( 0.2N HCl )
Kell / LW : 0.2M DTT destroy
17. Antigens Usually Denatured or Altered
by Proteolytic Enzymes
M, N, S, Fya, Fyb, Yta, Ch. Rg, Pr, Tn,
Mg, Mia/Vw, Cla, Jea, Nya, JMH,
some Ge, Inb
18. Antigens Usually Denatured or
Altered by DTT
Yta, JMH, Kna, McCa, Yka, LWa, LWb,
Ge, All Kell, Lutheran, Dombrock,
and Cromer blood group antigens
19. Anti- Approach Anti- Approach
Ch/Rg Destroy with proteases
Enhance with C4d-coated RBCs
Adsorb with C4-coated RBCs
Lu Destroy with trypsin/chymotrypsin and
AET
Do Enhance with proteases or PEG M Enhance by acidification
Destroy with proteases
Fy Destroy with proteases McC Destroy with AET/DTT
H Inhibit with H secretor saliva N Destroy with proteases
Enhance Nform if patient on renal
dialysis
Test for ‘N’ if patient is Black
HLA Test with chloroquine-treated RBCs P1
Inhibit with soluble P1 substance
Jk Enhance with proteases, LISS-Ficin, or
PEG, or
Enhance by two-stage EDTA-
antiglobulin test
Rg Destroy with proteases
Enhance with C4d-coated RBCs
Adsorb with C4-coated RBCs
JMH Destroy with proteases and AET S Destroy with proteases
Destroy with NaClO
KEL Destroy with AET/DTT Sda nhibit with Sd(a+) urine
Kn Destroy with AET/DTT Yka Destroy with AET/DTT
Le Inhibit with saliva containing Lea and/or
Leb
Yta Destroy with proteases
Destroy with AET
20. Content
Antibody screening positive ( all cells)
Multiple antibodies
Antibody to high prevalence antigen
Warm autoantibodies
Drug induced immune hemolytic anemia
21. Higt- titer, low-avidity HTLA
Anti-Ch ( Chido), -Csa ( Cost-Stirling), -JMH ( John-
Milton-Hagen), -Kna ( Knops), -McCa ( McCoy), -Rg
( Rodgers)
Such antibodies react at high serum dilutions but the
observed reactions are usually 2+ ( ± )with undiluted
serum. (titer≥ 64).
Titration tests are widely utilized to differentiate HTLA
antibodies from alloantibodies with a greater potential
to cause immune hemolysis.
22. Antibody to high prevalence
antigen
Anti-Jk3, anti-Hr0, anti-H, anti-Tja, anti-Dib, anti-Vel, anti-
KX, anti-KL, anti-Jra, anti-Wrb, anti-AnWj,
Ethnic high incidence antigen : s, Fya,
Anti-Lea+Leb ( LebH )
If transfusion, difficult to phenotype.
23.
24. Anti-Pr
Clinical no ( when antibody is inactive
at 37℃ )
Antibody characters IgM class; reactive
by RT; 4℃ ;37℃ phase
Technical tips sensitive for ficin/papain
Autoanti-Pr may cause autoimmune
hemolytic anemia.
Experts recommend to transfuse with
blood warmed to 37℃ in an approved
blood warmer.
25. Anti-P
Clinical no to severe ; antibodies
against P and/or Pk antigens are
addociated with a high rate of
spontaneous abortion in women .
Antibody characters
Reactive by RT; 37℃ ; IAT; complement
binding ; often hemolytic
Auto anti-P as a biphasic autohemolysin
in paroxysmal cold hemoglobinuria (PCH),
detected by the Donath-Landsteiner test.
Technical tips: ficin/papain enhanced
biphasic D-L test (+)
26. Anti-Dib
Clinical no to severe / delayed HTR, mild to severe
HDN
Antibody characters
Anti-Dib屬於高頻抗原抗體，台灣彰基醫院第一次發現
這抗體，但陸續台灣地區也發現多例anti-Dib抗體。
台北捐中Dia(a+b-)冷凍紅血球庫存A 3U, O 12U ，高雄
捐中O 6U ( 2012.6)
Technical tip
Anti-Dib 因dosage effect對於Di(a+b+)的反應強度會弱
於Di(a-b+)
Enzyme treated cell enhanced anti-Dib antigen
27. Anti-Ku
Clinical mild to severe
Antigen characters
only RBCs with the K0 phenotype lack the Ku
antigen
Sensitive to 0.2M DTT( thus sensitive to WARM
and ZZAP)
Sensitive to acid ( thus sensitive to EGA)
Technical tips 借馬階血庫咨詢實驗室K0
(K-k-)cells
Blood component transfusion 台北捐中
A 8U 高雄捐中A U 冷凍紅血球(2012.7)
28. Anti-KL
Anti-KL= anti-Kx + anti-Km, is make by males with the
McLeod phenotype and CGD
Clinical mild / delayed transfusion reaction,
Technical tips
Antigen is enhanced by 0.2M DTT( also with W.A.R.M. and
ZZAP)
對K0 cell reactive ( anti-Kx) weak k Kpb expression
Anti-Kx can be prepared by adsorption of anti-Kx +anti-
Km onto and elution from K0 RBCs
Blood component transfusion brothers of patients
with anti-Kx should be tested for compatibility and
the patient urged to donate blood for cryogenic
storage when his clinical state permits
33. Least incompatible
“least incompatible” units for autoimmune hemolytic
anemia: should we eliminate this meaningless term? .
“least incompatible” is not an acceptable alternative
for selecting red cells units for transfusion
Use of the term should be strictly discouraged.
( Transfusion 2003 43,
1503-1507)
34. Serotyping in chronic
transfusion
But most importantly, even the results obtained in the
reference laboratory were often incorrect: if the
strictest possible criteria were used, 28% of antigen
determinations had to be considered invalid, and 4%
of the antigen determinations considered correct
were proven to be wrong by molecular typing.
There is only one possible conclusion regarding
serological antigen typing in chronically transfused
patients: don't do it. Or if you do it, verify the results by
molecular techniques as soon as possible.
Blood Transfus DOI
10.2450/2013.0186-13
35.
36. 1.The Transfusion Service / Blood Bank cannot ensure
that the patient does not have an allogeneic
antibody (alloantibody) that may react with this blood,
leading to a type of transfusion reaction.
2.Although all major red blood cell antibodies have
been excluded, the patient is exhibiting an
autoantibody.
3.Accrediting/Regulatory Agencies require that we
notify you that we have been unable to find blood
which is matched to the patient, without evidence of
unwanted antibodies.
37.
38. Content
Antibody screening positive ( all cells)
Multiple antibodies
Antibody to high prevalence antigen
Warm autoantibodies
Drug induced immune hemolytic anemia
39. DIIHA
42% were antimicrobials
15% were anti-inflammatory
11% were anti-neoplastics
Several groups reported that about 20% (up to 35%) of
patients with chronic lymphocytic leukemia (CLL)
treated with fludarabine developed AIHA
40. Two types of DIIHA
Drug independent antibodies are those antibodies
that can be detected in vitro without adding any drug;
thus, in vitro and in vivo characteristics are identical to
cell red blood cell (RBC) autoantibodies.
Drug-dependent antibodies are those antibodies that
will only react in vitro in the presence of drug (eg,
bound to RBCs or added to the patient’s serum in test
systems to detect drug antibodies)
41. Mechanisms of drug-
induced immune hemolysis
Induction of autoimmunity (IA) : alfa-methyldopa,
modify immune system.
Drug adsorption (DA): penicillins, bind to RBC
membranes. IgG, C3 may present .
Immune complex formation (IC): plenacetin, drug-
anti-drug interaction activates complement.
Membrane modification (MM): cephalosporin, RBC
membranes are modified by the drug
42. Investigation of DIIHA
Certain biological materials may also account for a
positive DAT ( eg. plasma Hemoglobin; IgG1, IgG3
subclass and titration)
Eluate from DAT positive cells. Control serum.
Get drug information ( serum level )
RBCs: group O rr cells, barbital buffer at pH 9.6
43. IgG titration to assess the risk
of haemolysis
A titer of 1:30 or low is not relevant therefore no risk of
hemolysis
A differentiation of subclass is not necessary.
At titer of 1:300 and higher is clinical relevant therefore
high risk of hemolysis
A differentiation of subclass is necessary.
44. WAIHA
There are multiple reports in the literature
demonstrating that patients who have warm
autoantibodies in their sera have a higher rate of
alloimmunization (eg, 12% to 40%, with a mean of 32%).
45. Auto + allo antibodies
Methods to detect alloantibodies in the presence of
warm-reactive autoantibodies attempt to remove,
reduce, or circumvent the autoantibody.
Antibody detection methods that use PEG, enzymes,
column agglutination, or solid-phase red cell
adherence generally enhance autoantibodies.
Antibody detection tests using LISS or saline tube
methods may not detect autoantibodies, but most
significant alloantibodies will be detected.
46. Other procedures involve adsorption; two widely used
approaches are discussed below.
Adsorption with Autologous Red Cells
Adsorption with Allogeneic Red Cells
Antibody titration
47. Auto adsorption
A gentle heat elution at 56 C for 5 minutes can
dissociate some of the bound IgG.
Treatment of the red cells with ZZAP, a mixture of
papain or ficin and DTT.
Warm Autoantibody Remove Medium ( W.A.R.M.)
Autologous adsorption is not recommended for
patients who have been transfused within the last 3
months because a blood sample may contain some
of the transfused red cells that might adsorb
alloantibody
48. Adsorption with Allogeneic
Red Cells
When the patient’s phenotype is not known, group O
red cell samples of three different Rh phenotypes
(R1R1, R2R2, and rr) should be selected
The adsorbing red cells must include, at a minimum, at
least one negative for the S, s, Fya, Fyb, and K antigens
in addition to the Rh and Kidd requirements stated
above.
If the patient’s phenotype is known or can be
determined, adsorption with a single sample of red
cells may be possible.
49.
50. Characterizers of WAIHA
The majority of AIHA cases are caused by warm-
reactive autoantibodies, optimally reactive with red
cells at 37 C. The autoantibody is usually IgG (but can
be IgM or IgA).
51. Mix type WAIHA
Approximately 60% of patients with WAIHA have serum
antibodies that react with untreated saline-suspended
red cells. When testing with PEG, enzyme-treated red
cells, or solid-phase methods, over 90% of these sera
can be shown to contain autoantibody.
Agglutination at room temperature can be seen in
about one-third of patients with WAIHA, but the cold
agglutinins have normal titers at 4 C and are
nonreactive at 30 C and 37 C.
52. Crossmatch for patients with
warm auto antibodies ( old
sense)
Determine Pt’s Rh and Kidd phenotypes if no
transfusion in previous 3 months.
Warm auto without alloab : select packed RBC of the
same Rh and Kidd phenotype. Perform complete
crossmatches. Choose the least incompatible unit.
Warm auto + alloab : selected packed RBC of the
same Rh and Kidd phenotype and antigen-neg resp
to allo antibodies, if alloantibodies are clinically
significant. Perform complete crossmatches. Choose
the least incompatible one.
Auto specific self antigen?
53. Selection blood for
transfusion of AIHA
Red cell transfusions in patients with AIHA should be
undertaken carefully although they should never be
denied blood transfusions because of inability to find
compatible units.
As far as possible, phenotype matched red cells, cross-
match compatible with the patient’s autoadsorbed
serum should be transfused.
If coincident alloantibodies are identified, antigen-
negative red cells will need to be selected Red cell
transfusions in patients with AIHA should be
undertaken carefully although
54. Selection blood for
transfusion
If no alloantibodies are detected in adsorbed serum,
random units of the appropriate ABO group and Rh
type may be selected for transfusion.
If clinically significant alloantibodies are present, the
transfused cells should lack the corresponding
antigen(s).
If the autoantibody has clear-cut specificity for a
single antigen (eg, anti-e) and there is active ongoing
hemolysis, blood lacking that antigen may be
selected.
