Immunohematology

workshop

(all cells reactive)張志昇

2014.4.30

Content

Antibody screening positive ( all cells)

Multiple antibodies

Antibody to high prevalence antigen

Warm autoantibodies

Drug induced immune hemolytic anemia

Antibody screening positive

( all cells)

Antibody screening Vs. Auto control

Manual polybrene ( MP) Vs. classic AHG method

Auto control Vs. DAT

Antibody screening Vs. AC

negative

Antibody screening Vs. AC

positive

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.

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

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

Content

Antibody screening positive ( all cells)

Multiple antibodies

Antibody to high prevalence antigen

Warm autoantibodies

Drug induced immune hemolytic anemia

Classification of Immune

Hemolytic Anemias

Autoimmune Hemolytic Anemia (AIHA)

Warm AIHA

Cold agglutinin syndrome

Mixed-type AIHA

Paroxysmal cold hemoglobinuria

Alloimmune Hemolytic Anemia

Hemolytic transfusion reaction

Hemolytic disease of the fetus and newborn

Drug-Induced Immune Hemolytic Anemia

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-Yta, 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).

Differential of multiple

antibodies

Neutralization

Enzyme and chemical treated cells

Absorption & elution

Cord blood cells

pH & thermal

Dilution

Neutralization

Maternal milk ( inhibits natural anti-ID more readily than

pathological anti-IF )

Rabbit erythrocyte stroma (RESt Immucor )

note: anti-Vel reactivity diminished by RESt

Earth worm , pigeon egg (P1 substances)

Guinea-pig urine ( Sda )

Commercial group substance ( Immucor Lewis, P1

substances )

Examples of effect of

enzymes /chemical

modificationPapain/

Ficin

Trypsin Chymotrypsin Pronase AET 200mM

DTT

Dib/Vel + + + + + +

JMH/ lnb – – – – – –

Sc + + + – + –

LW + + + – – –

Cromer + + – – +/– +/–

Knops +/– – – + – +

Ch/Rg – – – – + +

Kell + + +/– + – –

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.

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

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

Antigens Usually Denatured or

Altered by DTT

Yta, JMH, Kna, McCa, Yka, LWa, LWb,

Ge, All Kell, Lutheran, Dombrock,

and Cromer blood group antigens

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 P1Inhibit 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

Content

Antibody screening positive ( all cells)

Multiple antibodies

Antibody to high prevalence antigen

Warm autoantibodies

Drug induced immune hemolytic anemia

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.

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.

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.

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 (+)

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

Anti-KuClinical \ 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)

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

Anti-Jra

Clinical \ Anti-Jra大多數是經由輸血或懷孕免疫刺激產生IgG (subclass 是IgG1)的Anti-Jra，但也曾發現自然產生IgM的Anti-Jra

Antibody characters \

Junior(Jr)血型抗原原歸納在ISBT 901series(901005), 抗原頻率大於99%

。

抗原表現在日本人最多。根據日本大阪紅十字會血液中心資料，在日本Jr(a-)的比例約是1/3000

Technical tips \

Jra抗原可抵抗Enzyme與0.2 M DTT的處理。

疑似HTLA抗體特性及papain two stage 會有double cell population現象

台北榮總曾發現一例，高雄捐中最近也發現有兩例Jr(a-)血型血清中有anti-Jra

之案例。

Blood component transfusion \文獻上報告患者輸注Jr(a+)血球會引起輕微的遲發性溶血輸血反應，建議輸Jr(a-)血球，目前北捐、高捐已有Jr(a-)O型血冷凍庫存。

Anti-AnWj

AnWj抗原可抵抗Enzyme與Acid處理，但經0.2 M DTT處理會變弱。

Anti-AnWj是臨床有意義的IgG抗體，因為它曾引起嚴重溶血性輸血反應；但它不會引起新生兒溶血症，因為AnWj抗原不表現在Cord

RBCs上。

高雄捐中曾鑑定一例anti-AnWj抗體。

Tip /抗體在Papain/Ficin與EGA處理後RBCs未減弱或改變反應，而0.2 M DTT處理後RBCs有稍微變弱，利用Cord RBCs與i adult RBCs

測試，發現抗體與Cord RBCs為陰性反應，而i adult RBCs仍為陽性反應

由於Anti-AnWj是臨床有意義的抗體，原則上是需要提供AnWj抗原陰性RBCs的輸血，但AnWj抗原陰性表現非常罕見，所以文獻上建議可以提供Lu(a-b-)抗原表現的RBCs輸血，這是由於AnWj抗原在Lu(a-b-)

抗原表現的RBCs是非常弱的。

目前台北捐血中心已發現一位O型Lu(a-b-)抗原捐血人，也作成冷凍紅血球庫存。

Content

Antibody screening positive ( all cells)

Multiple antibodies

Antibody to high prevalence antigen

Warm autoantibodies

Drug induced immune hemolytic anemia

Serologic findings in WAIHA

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)

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

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.

Content

Antibody screening positive ( all cells)

Multiple antibodies

Antibody to high prevalence antigen

Warm autoantibodies

Drug induced immune hemolytic anemia

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

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)

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

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

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.

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%).

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.

Other procedures involve adsorption; two widely used

approaches are discussed below.

Adsorption with Autologous Red Cells

Adsorption with Allogeneic Red Cells

Antibody titration

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

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.

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).

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.

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?

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

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.

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