immunohematologicalwork-up formakingsafe transfusion …kongre.kanver.org/files/19.pdf · 2019. 11....
TRANSCRIPT
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Immunohematological Work-Upfor making Safe Transfusion
available in Case ofAllo-/Autoantibodies
Christof [email protected]
4 November 2019
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Pathomechanism of immune haemolysis
1. Opsonisation
extravasal haemolysisSpleen, Liver
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Pathomechanism of immune haemolysis
2. complement activation + complement mediated cell lysis
intravasal haemolysis
free Haemoglobin
membrane-/cellfragments
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Die KomplementaktivierungComplement activation
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0 10° 20° 30° 37° Celsius0%
50%
activity100%
„Thermal-Amplitude“
ab-bindung Complement-activity
haemolysis
Under which conditionds would „cold reacting“ IgM ab´s lead to complement mediated
haemolysis?
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Complete / incomplete antibodies
„Zeta-Potential“
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RBC antibody terminology
RBC antibody
incomplete antibodies:
bind to RBC do not agglutinate RBC
complete antibodies:
bind to RBC and agglutinate RBC
IgM (e.g. anti-A, anti-B) IgG (e.g. anti-RhD-AK)
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RBC antibody terminology
RBC antibodies (ab)
Auto-antibodiesAllo-antibodies
regular ab irregular ab
naturaly preformed abz.B.: -Anti-A
-Anti-B-Anti-T
naturaly preformed abz.B. : -Anti-P1
-Anti-Lea/b
-Anti-M-Anti-N
immune abz.B. -Anti-D
-Anti-Fya
-Anti-Jka
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Institute for Transfusion Medicine German Red Cross Blood CenterUniversity Frankfurt / Main Germany
Primary immune reaction (i.e. Anti-Jka)
0 4 8 6 12weeks months
512256
12864
8
4
2
16
32
transfusion of a Jk a –positiv RBC unit
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Institute for Transfusion Medicine German Red Cross Blood CenterUniversity Frankfurt / Main Germany
Sekundary immune reaction
0 1 2 3 4 5 6 7 9days
delayed hemolytic Transfusion reaction
512256
12864
8
4
2
16
32transfusion of a Jk a –positiv RBC unit
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Institute for Transfusion Medicine German Red Cross Blood CenterUniversity Frankfurt / Main Germany
Delayed hemolytic transfusion reactionimmunisation and „Booster-Effect“
Titer
sekundaryreaction
Primary reaction
4 weeks 12 (months / jears)
IgMIgG
first contactwith an Antigen
first contactwith an Antigen
3 - 14 days
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Institut für Transfusionsmedizin DRK-BlutspendedienstBaden-Wuerttemberg-Hessen
Delayed transfusion reaction
Titer
Transfusion(Booster effect)
Detection limit
0 10 days (1 - 14 days)
Critical timepoint: DAT positivs, ab screening yet negative
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0
5
10
15
20
25
30
Rela
tiver
Ant
eil (
%)
Jka DJkb C E c e K k Kpa Fya Fyb Fy3 S M Lua
Häufigkeit von neu nachgewiesenen Alloantikörper-Spezifitäten bei verzögerten hämolytischen Transfusionsreaktionen (Auswertung der Angaben in den Jahresberichten 2006-2009 „Serious Hazards of Transfusion䇾 (SHOT) (www.shotuk.org).
DHTRDelayed Hemolytic Transfusion Reaction
RBC antibodies (Booster-Effect)1 : 1.000 - 1 : 4.000 Transfusionsfatal (< 1 auf 1.000.000 Transfusions)
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Elution II
Elution
Physico- / chemical „force䇾
(acid, heat, reagents)
centrifugation
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Elution III
Elution
Physico- / chemical „force䇾
(acid, heat, reagents)
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„Zeta-Potential“
charged ioniclayers
Supplement(e.g.. Albumin or LISS)-reduces Zeta-Potential
RBC reject each other
---
--
- ----
--
- -
---
--
- ----
--
- -
+ +++
+++
++++++++
++ + +
+ + + + ++++
+++++
++++
-----
- - ----
+++
+
+ +
+
+
+++
+-
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Antihumanglobuline test
YYY Y
Y
Y
Moreschi (1908): Anti-Protein-SeraCoombs (1945): Antihumanglobuline test for the detection of
incomplete ab
C. Moreschi
R. R. A. Coombs
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Antihumanglobuline test
RBC antibodies(human globuline)
Anti-Humanglobuline from rabbits (Coombsserum)
Coombs test (Antihumanglobuline test)
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crossmatch
Crossmatch is no substitute for antigen testing in antibody positive patients
Why: Antigendose
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• Expression depends on genotype in various blood group systems
Dose effect of blood group antigens
Fy (b)
14.0007.000
Fy (a)14.000
7.000
(a+b-)(a-b+)(a+b+)
Duffy (Fy)
s
170.00085.000
S250.000
125.000
SSssSs
Ss
Jk (b)
10.0005.000
Jk (a)10.000
5.000
(a+b-)(a-b+)(a+b+)
Kidd (Jk)
Antigens/RBCAntigens/RBCGenotype
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Crossmatch / antibody screening(3 stage – tube test)
1. NaCl 22° CIgM 2.
Suppl. 37° CIgM, IgG 3.
CoombsphaseIgG/Komplement
Compatibility testing
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Compatibility testing
Relevance of antibodies according to reaction pattern
1.StageNaCl; 22° C
2. Stage Supplement; 37° C
3. StageCoombsphase
Cold reactive = mostly irrelevant.
Warm reactive = potentially relevant
Warm reactive = relevant
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Clinically relevant:Anti- …
Potentially relevant: Anti- …
Mostly irrelevant: Anti- …
C, c, D, E, e, Cw A1, H, HI Kälte-Ak (bei 37°CK, k, Kpa, Kpb, Jsa, Jsb M, N nicht wirksam)Jka, Jkb Lea, Leb NFya, Fyb P(1) P(1)S, s, U Xga Sda
Lub, Yta Cob Bga, Bgb, Bgc (HLA)Vel, P, Tja3 Ytb HTLA: Chido, Rogers, Coa, Dia, Dib, Doa, Dob, Lanund viele weitere …
Irregular RBC antibodies
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Aus: DiaMedArbeitsanleitungenImmunhämatologie
Characteristic of irregular RBC antibodies
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Compatibility testing
today:
• crossmatch often performed in column centrifugation test (equals 3. stage of tube testing)
• (difficult differentiation of warm-/cold reactive antibodies)
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Antibody identificationPatient: Case 1 Sample taken: Method: T = TubeBloodgr.: A1 Rh neg (ccddee), Kell neg.Techn.: Invest.-Date: M = Micro column (Diamed)
Sample: S = Serum, E = Eluate
22°NaCl
37°Sup. CT
Test- St. 1
St. 2
St. 3
Co.
-Co.
Zellen Rhesus MNSs P Lewis Lutheran Kell Duffy KiddD C E c e Cw V M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xg
I + + o o + + NT + + o + + o + o + o + o o + o o + NT I o 2 3II + o + + o o o o + o + 0 + o o + o + o o o + + o NT II o 2 3
III o o o + + o o + o + o +s o + + + + + o o o + + o NT III o o o1 o o o + + o o o + + + +w + o + + o + o o + o o + + 1 o o o2 o o o + + o o + + o + + o o o + + + o o o + + o o 2 o o o3 o + o + + o o + o + o +s o + o + o + o o + + o + + 3 o o o4 o o + + + o o + o o + + o o o + o + o + o o + + + 4 o o o5 o o o + + o o + o + o + o + o + o + + o o + + + + 5 o o o6 + o o + + o o + o + + +s o o o + o + o o o o + o + 6 o 2 37 + + + o + o o + + + + +w + o o + o + o o o + + o o 7 o 2 38 + + o o + o o o + o + o o + o + + + o o o + + o + 8 o 2 39 + + o o + + o + o o + + o + o + + + o o + o + + o 9 o 2 3
10 + o + + o o o + o o + + o + o + o + o o + + o + + 10 o 2 311 + + o + + o o + o o + + o + o + + + o o + + o + + 11 o 2 3
autolog AC o o oA1 o 2 3A2 o o o +B 4 3 3
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Antibody identification-Checklist-
• Auto- or Allo-ab? 1. Autolog. controll (AC) neg -> Allo-ab2. Patient is antigen negative
• Cold or warm reactive? (Stage 1, 2 oder 3)weak in 2., strong in 3. Stage -> warmreactice ab
• complete or incomplete reacting? (Stage 1 or 3)only in 3. Stage -> „incomplete“ ab
Conclusion:
Warm reactive, incomplete Allo-ab of the speficity XY
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Antibody identificationPatient: Fall 1 Sample taken: Method: T = TubeBloodgr.: A1 Rh neg (ccddee), Kell neg.Techn.: Invest.-Date: M = Micro column (Diamed)
Sample: S = Serum, E = Eluate
22°NaCl
37°Sup. CT
Test- Stuf
e 1
Stuf
e 2
Stuf
e 3
Co.
-Co.
Zellen Rhesus MNSs P Lewis Lutheran Kell Duffy KiddD C E c e Cw V M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xg
I + + o o + + NT + + o + + o + o + o + o o + o o + NT I o 2 3II + o + + o o o o + o + 0 + o o + o + o o o + + o NT II o 2 3
III o o o + + o o + o + o +s o + + + + + o o o + + o NT III o o o1 o o o + + o o o + + + +w + o + + o + o o + o o + + 1 o o o2 o o o + + o o + + o + + o o o + + + o o o + + o o 2 o o o3 o + o + + o o + o + o +s o + o + o + o o + + o + + 3 o o o4 o o + + + o o + o o + + o o o + o + o + o o + + + 4 o o o5 o o o + + o o + o + o + o + o + o + + o o + + + + 5 o o o6 + o o + + o o + o + + +s o o o + o + o o o o + o + 6 o 2 37 + + + o + o o + + + + +w + o o + o + o o o + + o o 7 o 2 38 + + o o + o o o + o + o o + o + + + o o o + + o + 8 o 2 39 + + o o + + o + o o + + o + o + + + o o + o + + o 9 o 2 3
10 + o + + o o o + o o + + o + o + o + o o + + o + + 10 o 2 311 + + o + + o o + o o + + o + o + + + o o + + o + + 11 o 2 3
autolog AC o o oA1 o 2 3A2 o o o +B 4 3 3
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Antibody identificationPatient: Fall 1 Sample taken: Method: T = TubeBloodgr.: A1 Rh neg (ccddee), Kell neg.Techn.: Invest.-Date: M = Micro column (Diamed)
Sample: S = Serum, E = Eluate
22°NaCl
37°Sup. CT
Test- Stuf
e 1
Stuf
e 2
Stuf
e 3
Co.
-Co.
Zellen Rhesus MNSs P Lewis Lutheran Kell Duffy KiddD C E c e Cw V M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xg
I + + o o + + NT + + o + + o + o + o + o o + o o + NT I o 2 3II + o + + o o o o + o + 0 + o o + o + o o o + + o NT II o 2 3
III o o o + + o o + o + o +s o + + + + + o o o + + o NT III o o o1 o o o + + o o o + + + +w + o + + o + o o + o o + + 1 o o o2 o o o + + o o + + o + + o o o + + + o o o + + o o 2 o o o3 o + o + + o o + o + o +s o + o + o + o o + + o + + 3 o o o4 o o + + + o o + o o + + o o o + o + o + o o + + + 4 o o o5 o o o + + o o + o + o + o + o + o + + o o + + + + 5 o o o6 + o o + + o o + o + + +s o o o + o + o o o o + o + 6 o 2 37 + + + o + o o + + + + +w + o o + o + o o o + + o o 7 o 2 38 + + o o + o o o + o + o o + o + + + o o o + + o + 8 o 2 39 + + o o + + o + o o + + o + o + + + o o + o + + o 9 o 2 3
10 + o + + o o o + o o + + o + o + o + o o + + o + + 10 o 2 311 + + o + + o o + o o + + o + o + + + o o + + o + + 11 o 2 3
autolog EP o o oA1 o 2 3A2 o o oB 4 3 3
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Antibody identification-checklist-
• Auto- or Allo-ab? (AC, DAT? Antigenstatus of the Pt.?)
• cold or warm reactive? (Stage 1, 2 or 3)
• complete or incomplete reacting? (Stage 1 or 3)
• identification of the specificity of the antibody
• exclusion of antibodies against other antigens
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Antibody identification:further methods
• prewarming technique (“strict wamth“): IAT with prewarmed (37 °C) pt. Plasma and test cells (sometimes also gel cards):
Principle: prewarming avoids reacting of cold reacting antibodies with increased temperature altitude
Differentiation between “normal” warm reacting allo-ab (IgG) und “cold reacting ab (IgM) with increased temperature amplitude
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page 33
Specificity Ficin/Papain Trypsin g-Chymo-trypsin
0.2 M DTT/AET*
Lu pos neg neg Weak/negYt neg pos neg Weak/negM,N Ge2, 3, 4 neg (Ge3 +) neg pos posS, s, Fy(a), Fy(b) neg pos neg posKel / LW pos pos pos negKn/Yk ? neg neg negCh/Rg neg neg neg posJMH neg neg/± neg negCs(a) pos pos pos posJr(a) mf pos pos posDo pos neg weak negABO, Rh, U, pos pos pos posFy3, P, Vel, Di pos pos pos pos
Methods: Enzyme – treatment of testcells:
IGLD 2017* Aminoethylisothiouronium bromide. Immunohematology 1992 vol.8 nr.3 / Factsbook M. Reid
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Antibody identification:further methods
• „Super-Coombs“: IAT with enzym treated Cells
Suspicion of weal allo-ab, e.g. Kidd-ab
Advantage: very high sensitivity
Disadvantage: unspecific positivity due to cold reactingautoantibodies
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Antibody identification:further methods
• REST (rabbit erythrocyte stroma)-Absorption
In high titer cold reacting auto antibodies
principle: Incubation of Plasma with REST, cold reacting auto ab are adsorbedwarm reacting ab remaincentrifugation: use of supernatant for further ab identification
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Antibody identification:further methods
• HPC (human platelet concentrate)-Absorption
In accompanying HLA-ab (Bennet – Goodspeed ab)
principle: Incubation v. Plasma with HPC, HLA-ab are absorbed, anti-RBC-ab remain centrifugation: use of supernatant for further ab identification
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Antibody identification:further methods
• AB-Serum-Inhibition
In suspicion of HTLA-AK (Rogers, Chido)with characteristic raction pattern (high titer, low avidity)
principle: Mixing of Pt.-Plasma with AB-Serum, HTLA-ab will be inhibited (bound to soluble antigen) (Controll: Mixing with NaCl)
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