ssg&sp ycreening & specificity of red blood cells …...ssg&sp ycreening &...

Screening & Specificity S g & Sp yof Red Blood Cells Allo-antibody(ies)

By:Ahmad Gharehbaghian MTD, PhD

Professor of Clinical ImmunohematologyShahid Beheshti University of Medical SciencesShahid Beheshti University of Medical Sciences

Head of Hematology & Blood banking Department

AlloAllo--antibody screening?antibody screening?Detection of broad range unexpected (usually clinicallysignificant) an antibody(s) against an antigen(s) (usually theminor blood group) in sample sera that he/she lacks, isg p) p ,called allo-Ab screening.

D t ti f t d tib d t ti iDetection of unexpected an antibody to an antigen insample sera that he/she possess, is called auto-antibodyscreening.

Uses patients plasma/serum against reagent red cells todetect unexpected antibodies that react only with allogeneicdetect unexpected antibodies that react only with allogeneicRBCs expressing the corresponding Ags.

Also called the indirect Coombs test or the indirectantiglobulin test (IAT).

Why Allo-antibody detection?The purpose of pre-transfusion compatibility testing is toprevent hemolytic transfusion reaction technical and clericalagainst blood components.g p

If the host or recipient recognizes the donor RBC surfacet e ost o ec p e t ecog es t e do o C su aceantigens as foreign, the host will mount an immune responseto the donor RBC’s.

Unexpected antibodies are a result of red cell stimulation (e.g.,transfusion, HDN, pregnancy, …)

Unexpected antibodies may be:Clinically significant (IgG)Not clinically significant (IgM)

Clinically significant antibodiesy gPositive in between 0.3-38% of samples depending on thepopulation study (blood donors or patients group) and thet t th d iti ittest method sensitivity.

Usually IgG and react best at 37° and AHG phase (IAT)

Clinically significant antibodies are associated:hemolytic transfusion reactions (HTR)hemolytic transfusion reactions (HTR)hemolytic disease of the fetus or newborn (HDFN)notable decrease in the survival of transfused RBCsspontaneous miscarriage!!!spontaneous miscarriage!!!

Although “naturally occurring allo-Abs” may be present.

Antibody screening are used for?

1. Patients needing a transfusion2. Pregnant women3 Cases of transfusion reactions3. Cases of transfusion reactions4. Blood and plasma donors

Pre-transfusion ScreeningScreeningScreening forfor antibodiesantibodies isis normallynormally performedperformedpriorprior toto bloodblood transfusiontransfusion toto detectdetect antibodiesantibodiesthatthat reactreact atat bodybody temperaturetemperature ((3737°°))thatthat reactreact atat bodybody temperaturetemperature ((3737 ))

ColderColder reactingreacting antibodiesantibodies (RT(RT andand below)below) areareColderColder reactingreacting antibodiesantibodies (RT(RT andand below)below) arearethereforetherefore consideredconsidered insignificantinsignificant andand justjust causecauseinterferenceinterference whenwhen performingperforming lablab testingtesting

TheThe onlyonly importantimportant thingthing toto rememberrememberconcerningconcerning coldcold antibodiesantibodies isis thatthat theythey maymay bindbindconcerningconcerning coldcold antibodiesantibodies isis thatthat theythey maymay bindbindcomplementcomplement ifif aa personspersons bodybody temperaturetemperaturebecomesbecomes lowlowo Open-heart surgeryo Hypothermia

Screening cellsS i ll f 2 3 t RBC lScreening cells come from 2 or 3 reagent RBC samples (not pooled) in 2-5% RBC suspension:

O cellsUsing screening cells that are homozygous for the clinically significant antigens including D, C, E, c, e, M, N, S, s, P1,Lea, Leb, K, k, Fya, Fyb, Jka, and JkbK, k, Fy , Fy , Jk , and JkHomozygous antigens will react strongerHeterozygous antigens will react weaker (Antigens may show dosage effect esp. for Rh, MNS, Duffy & Kidd systems; greater antigen density per cell increases sensitivity)antigen density per cell increases sensitivity)

Screening cells may also contain low-incidence antigens like Kpa but the presence of these antigens is notlike Kpa but the presence of these antigens is not required for screening cells

A reaction to one or more cells indicates the presence of an unexpected antibody

Interpretation:Very effective in detecting antibodies

If negative, then the cross-match should be compatible

Limitations:1) Dosage may weaken a reaction (variation in Ag. expression)) g y ( g p )

2) Dosage phenomenon (zygosity) that Abs react with “double-dose” Ag. Presentation

3) Antigen may not be present on screening cells (Low frequency antigens that found on < 10% of all human RBCs)

4) Variation in adult and infants (e.g. I, P1, Lea)

5) Patient may have a passive ABO antibody

6) Change with storage (e.g. Fya, Fyb, P1, M)

7) Patient medical history:

i. Collect some data such as diagnosis; age; gender; history of pregnancy, abortion or miscarriage; history of blood transfusion or surgery; transplantations; medications (e.g. IVIG, RhIG)/IV fluids etcetc

ii. Mixed red cell populations from a previous transfusion can remain for up to 3 months (transfusion history)

iii. Patient may have come from another hospital

i S di i t d ith tib di ( C ld l ti iiv. Some diseases are associated with antibodies (e.g. Cold agglutinin disease, Reynaud's phenomena, viral infections in children, PCH, SLE, Multiple myeloma, CLL, Lymphoma, Warm autoimmune hemolytic anemia, ……

v. Some antibodies occur at a higher frequency in some races (e.g. Oh or para-Bombay in Indians>any, Fy(a-b-) in Blacks>Arabs>Mediterranean>WhitesBlacks>Arabs>Mediterranean>Whites

Patients history with identified alloantibody

operation, 15 (50%)

abortion, 11 (36.7)

D&C, 1 (3.3%)

transfusion, 18 (60%)

pregnancy, 15 (50%)

Cross match limitations1 Varying age of donor cells

Cross-match limitations1. Varying age of donor cells2. Varying haematocrite by stored at in a

ll l ismall plastic tag 3. Diluted by eye to roughly the right cell

concentration4. Performed once only y5. Without a positive control

Laboratory EvaluationCheck pre & post Transfusion Hemoglobin

Antibody Detection :- Direct Coomb’s Test ( DAT) : Detect RBC covered

with Antibody ; IgGwith Antibody ; IgG- Indirect Coomb’s Test (IAT): Detect Allo-Ab in

plasma (screening antibody)p ( g y)

Antibody Identification :- Panel Cell If screening ab were positive

Auto Control: Serum Pt + RBC Pt for Auto AbAuto Control: Serum Pt + RBC Pt for Auto-AbDetection

Results in Tube method

Results in Tube method

Grading of Reactionsg

Negative

Gel Method

0

2+

0

00

0

0

2+

0

0

2+

0

0

Anti-K

0

2+

0

00

0

0

2+

0

0

2+

0

0

Anti-e, Fya; Anti-K?

3+

3+

3+

3+3+

3+

3+

3+

3+

3+

3+

3+

0

AIHA; Anti-K?

3+

3+

3+

3+3+

3+

3+

3+

3+

3+

3+

3+

3+

Compatible blood unit selecting for patient with antibody screening positive:

Compatible blood unit selecting for patient with antibody screening positive:antibody screening positive:antibody screening positive:

If patient has an allo-antibody, he/she needs units that are negative for that antigen

Select random ABO/Rh compatible units, perform compatibility test and, if negative, check units for antigen of interest using known antiserum

How many random donor units will it take to find needed units? Use known antigen frequencies to determine

Divide number of units needed by the frequency of population that is negative for the antigen ; screen that many random units

What if the patient has multiple (clinically significant) allo-antibodies?

Multiply the population frequency of those negative for one with theMultiply the population frequency of those negative for one with the frequency of those negative for the otherDivide # units needed by that number

Cross-match advantages paralleling with Ab screening

I. It acts as a double check, when the screen have been performed badly or misread

Ab-screening

have been performed badly or misread

II It might pick up antibodies to antigens present II. It might pick up antibodies to antigens present on the donor red blood cells but not present on the screening cells

III. As serologists became confident in the atypical antibody screen, it was possible to yp y , prevert to the use of the cross-match for the detection of ABO mismatch alone, the so called “Quick spin cross-match”called Quick spin cross match

Autocontrol(AC)Tests patient serum with their own red cells

H th AC h ld b ith thHowever, the AC should be run with the antibody panel

AC is incubated with the antibody screen (or antibody panel)y p )

When positive, indicates that unknown RBCs p ,have:

An unexpected auto-antibody (eg., AIHA)A positive DAT (eg., recently transfused with incompatible blood)

Study designAnalysis of 8000 non-chronically transfused patients who were

candidate for elective surgery, was conducted in Tehran, Shiraz, Ardebil.

The patients' questioner included: sex, date of birth, transfusion history, previous alloantibody specify, surgery history and pregnancy, abortion or miscarriage history.

71 out of 8000 patients had alloantibody (prevalence rate of 0.89%), in other hand 43 out of 71 (PR of 60 6% or PR of 0 54%) positivein other hand 43 out of 71 (PR of 60.6% or PR of 0.54%) positive samples for Allo-Abs were clinically significant.

The most common clinically significant Allo-Abs were anti-KThe most common clinically significant Allo-Abs were anti-K (25.35%), anti- E (18.3%), anti-c (11.27%), anti-C (7.04%) and anti-D (2.82%).

The non-significant Allo-Abs were Lea , Leb , Lua , Fya

Conclusion:1. Educating and training program for those people in

charge of blood banking in hospital.

2. Extended antigen matching (esp.: C, c, E, and K) preventing the formation irregular antibodies in patients especially in multi transfused patientsespecially in multi- transfused patients

3. There is still concern problem in blood grouping, indirect C b t t d t hCoombs test and cross-match.

4. Electronic X-match must be substituted after universal ab-screening by blood transfusion service and hospital.

5. Documentation of adverse effect due to blood components usage ( implementation of heamovigilancesystem)

“To give safe blood is “To give safe blood is “To give safe blood is “To give safe blood is a privilege”a privilege”

BUTBUTBUTBUT“To receive safe blood “To receive safe blood

Is a right”Is a right”gg

Reaction media (potentiator)Reaction media (potentiator)Decreases zeta potential by bufferingDecreases zeta potential by buffering

2222% Alb i% Alb i

Decreases zeta potential by bufferingDecreases zeta potential by bufferingAllows AbAllows Ab--coated cells to come closer coated cells to come closer

together together 2222% Albumin % Albumin Serum/cell mixture should incubate at Serum/cell mixture should incubate at

least least 2020--30 30 minutes minutes Doesn’t enhance warm autoDoesn’t enhance warm auto antibodiesantibodiesDoesn t enhance warm autoDoesn t enhance warm auto--antibodiesantibodies

LISSLISSIncubation time of Incubation time of 10 10 minutes minutes Decreases zeta potentialDecreases zeta potentialLISSLISS Decreases zeta potentialDecreases zeta potentialLowers ionic strength allowing better Lowers ionic strength allowing better

reaction sensitive and quickreaction sensitive and quick

PEGPEGRemoves water, concentrating Ab Removes water, concentrating Ab Enhances warm autoEnhances warm auto--antibodiesantibodiesDoes not react well with insignificant Does not react well with insignificant

antibodies (IgM)antibodies (IgM)

ResultsResultsII Cli i ll i ifi t d bl d llCli i ll i ifi t d bl d ll llll tib di f d itib di f d i 2020I.I. Clinically significant red blood cell Clinically significant red blood cell alloallo--antibodies were found in antibodies were found in 20 20

patients.patients.

II.II. Therefore prevalence of positive antibody screen in nonTherefore prevalence of positive antibody screen in non--chronically chronically t f d ti t h d t l ti dt f d ti t h d t l ti d t ht htransfused patients, who underwent elective surgery and crosstransfused patients, who underwent elective surgery and cross--match match had been done for them, was had been done for them, was 00..6767% .% .

III.III. Besides there were Besides there were 10 10 patients with patients with alloallo--antibodies that reacted in room antibodies that reacted in room t t h (RT)t t h (RT)temperature phase (RT). temperature phase (RT).

IV.IV. Frequency of Frequency of alloallo--immunization was greater among female than male immunization was greater among female than male patients (patients (1313F to F to 77M or M or 11..8686F to F to 11M). M).

V.V. All of them had surgery history or transfusion during less than two years All of them had surgery history or transfusion during less than two years ago. ago.

VI.VI. Two of them are Two of them are RhRh-- and positive for antiand positive for anti--D and had abortion in their D and had abortion in their histories but there aren’t any antihistories but there aren’t any anti--D in their sample now. D in their sample now.

VIIVII The four most frequentThe four most frequent alloallo antibodies were antiantibodies were anti--K (K (00 44%) anti%) anti--c (c (00 33%)%)VII.VII. The four most frequent The four most frequent alloallo antibodies were antiantibodies were anti--K (K (00..44%), anti%), anti--c (c (00..33%), %), antianti--E (E (00..22%), anti%), anti--C (C (00..0505%). %).

Antigen male female Total

Frequency of identified allo-antibodiesAntigen male female Total

K 4 (33.33%) 4 (22.22%) 8 (26.67%)

E 1 (8.33%) 6 (33.33%) 7 (23.33%)

c 2 (16.67%) 4 (22.22%) 6 (20%)

Leb 1 (8.33%) 3 (16.67%) 4 (13%)

Lua 2 (16.67%) 0 2 (6.5%)

D 1 (8.33%) 0 1 (3.33%)

Fya 0 1(5.56) 1 (3.33%)

C 1(8.33%) 0 1 (3.33%)

Total 12 (40%) 18 (60%) 30 (100%)