Advanced Transfusion Case Studies · AN patient with apparent Anti-C+D+E •ooking sample, 16 -17 weeks gestation • ZO neg [, with positive antibody screen •Previous pregnancy,

Advanced Transfusion Case Studies

1/31/2017

Case from NHSBT RCI

When is anti-D+C not AntiD+C?

1/31/2017 Julie Molloy, MSc CSci FIBMS [email protected]

AN patient with apparent Anti-C+D+E

•‘Booking’ sample, 16-17 weeks gestation

•‘O neg’, with positive antibody screen

•Previous pregnancy, delivered 18 months ago •transfusion episode, at end of last pregnancy

NBS Reagents Liverpool Antibody Investigation Worksheet

Patient‟s Name Requestor Hematos Ref No. Tested by

Date of Birth Hosp no Sample No. Date Tested

Product Lot No. Product Lot No. Product Lot No.

ID panel in Alsevers R1443119 ID panel Papainised in Alsevers

R1543119 ID Panel in LISP R1463119

ID panel in CellStab R1433119 ID panel Papainised in CellStab

R1533119 EXPIRY DATE : 2006.08.31

Rh M N S s P1 Lua K k Kp

a Le

a Le

b Fy

a Fy

b Jk

a Jk

b Other IAT ENZ

1 R1wR1 + + + 0 4 0 + + 0 0 + 0 + 0 + 4 5

2 R1R1 + + 0 + 3 + 0 + 0 + 0 + 0 + 0 4 5

3 R2R2 0 + 0 + 0 0 0 + 0 + 0 0 + + 0 3 5

4 r’r + 0 0 + 5 0 0 + 0 0 + 0 + 0 + 3 5

5 r’’r + 0 0 + 4 0 0 + + 0 + + 0 0 + Cob+ 0 0

6 rr + + + 0 0 0 0 + 0 + 0 + 0 + 0 0 0

7 rr + 0 0 + 4 0 + + 0 0 + 0 + + 0 0 0

8 rr + 0 + + 0 0 + 0 0 + 0 + 0 + + 0 0

9 rr 0 + 0 + 0 0 0 + 0 0 + 0 + 0 + 0 0

10 rr 0 + 0 + 5 0 0 + 0 0 + + 0 0 + Cob+Bg

b+ 0 0

Auto 0 0

Antibody Titre

Dilution: Cell Id

Group O D negative

Pheno D-C-c+E-e+K-

Anti-

Archive

Anti-

Archive

DAT Batch No

PS IgG IgA IgM C3c C3d Ctl

Conclusion

Entered into Hematos by:…………………………..Authorised by…………………….

Reagent

Batch No‟s

? anti-D + C








R1533119 EXPIRY DATE : 2006.08.31


a Le

a Le

b Fy

a Fy

b Jk

a Jk

b Other IAT ENZ

1 R1wR1 + + + 0 4 0 + + 0 0 + 0 + 0 + 4 5

2 R1R1 + + 0 + 3 + 0 + 0 + 0 + 0 + 0 4 5

3 R2R2 0 + 0 + 0 0 0 + 0 + 0 0 + + 0 3 5

4 r’r + 0 0 + 5 0 0 + 0 0 + 0 + 0 + 3 5

5 r’’r + 0 0 + 4 0 0 + + 0 + + 0 0 + Cob+ 0 0

6 rr + + + 0 0 0 0 + 0 + 0 + 0 + 0 0 0

7 rr + 0 0 + 4 0 + + 0 0 + 0 + + 0 0 0

8 rr + 0 + + 0 0 + 0 0 + 0 + 0 + + 0 0

9 rr 0 + 0 + 0 0 0 + 0 0 + 0 + 0 + 0 0

10 rr 0 + 0 + 5 0 0 + 0 0 + + 0 0 + Cob+Bg

b+ 0 0

Auto 0 0

Antibody Titre

Dilution: 2 4 8 16 32 64 128 256 512 1024 Cell Id

Group O D negative

Pheno D-C-c+E-e+K-

Anti-C 4 4 4 4 3 3 2 1 0 0 D-C+c+

Archive

Anti-

Archive

DAT Batch No


Conclusion

Additional; anti-D quantified at 5.8 iu/mL


Reagent

Batch No‟s

? anti-D + C

Suspicious result

• Anti-D of 5.8 iu/mL • Reactions with R2R2 cells should have been stronger

• Suggestions for further tests?

• Decided to perform ‘parallel’ adsorptions of patient plasma.

• Vs. R2R2 cells • Vs. r’r cells.

• Resulting adsorbed plasmas were then panelled by IAT

Why do this?

• Any time we see “anti-D+C” in AN patient we should be considering if it might in fact be Anti-G • (+anti-D, +anti-C?)

• R2R2 cells will ‘remove’ anti-D and anti-G, leaving behind anti-C

• r’r cells will ‘remove’ anti-C and anti-G, leaving behind anti-D

• The combination of results will give us a clue as to what antibodies are really present








R1533119 EXPIRY DATE : 2006.08.31


a Le

a Le

b Fy

a Fy

b Jk

a Jk

b Other IAT ENZ 1 2

1 R1wR1 + + + 0 4 0 + + 0 0 + 0 + 0 + 4 5 3 0

2 R1R1 + + 0 + 3 + 0 + 0 + 0 + 0 + 0 4 5 3 0

3 R2R2 0 + 0 + 0 0 0 + 0 + 0 0 + + 0 3 5 0 0

4 r’r + 0 0 + 5 0 0 + 0 0 + 0 + 0 + 3 5 3 0

5 r’’r + 0 0 + 4 0 0 + + 0 + + 0 0 + Cob+ 0 0 0 0

6 rr + + + 0 0 0 0 + 0 + 0 + 0 + 0 0 0 0 0

7 rr + 0 0 + 4 0 + + 0 0 + 0 + + 0 0 0 0 0

8 rr + 0 + + 0 0 + 0 0 + 0 + 0 + + 0 0 0 0

9 rr 0 + 0 + 0 0 0 + 0 0 + 0 + 0 + 0 0 0 0

10 rr 0 + 0 + 5 0 0 + 0 0 + + 0 0 + Cob+Bg

b+ 0 0 0 0

Auto 0 0 0 0

Antibody Titre

Dilution: 2 4 8 16 32 64 128 356 512 1024 Cell Id

Group O D negative

Pheno D-C-c+E-e+K-

Anti-C 4 4 4 4 3 3 2 1 0 0 D-C+c+

Archive

Anti-

Archive

DAT Batch No


Conclusion

Additional; anti-D quantified at 5.8 iu/mL


Reagent

Batch No‟s

Ads vs R2R2 remove’ anti-D and anti-G

Ads vs r’r remove anti-C and anti-G

? anti-D + C

After a „phone call...

•Previous pregnancy had delivered a group O D positive baby

•Sufficient prophylactic anti-D had been given to cover all recognised FMH

•One transfused unit was D-C-c+E-e+

•One transfused unit was D-C+c+E-e+

Conclusion

• Antibody specificity present • anti-G+C

• Against ‘standard’ panels anti-G (with or without anti-C) will appear to be anti-D+C

• Clue that anti-D might not be present • stronger reactivity with R1R1 (and often r’r) cells than with R2R2

cells

• If lab had known anti-D wasn’t present, would not have performed anti-D quantification

What advice was given to hospital?

•There is a risk of HDFN as the titre is 32 or greater

•Continue with anti-D prophylaxis

•Send samples for re-assessment at 28 weeks gestation

•Take the samples before prophylactic anti-D is given

Next …

•Sample taken between 31 & 32 weeks gestation

•500 iu of prophylactic anti-D had been administered (routinely, at 28 weeks)

•Same work-up as before, including absorptions with R2R2 and r’r cells








R1533119 EXPIRY DATE : 2006.08.31


a Le

a Le

b Fy

a Fy

b Jk

a Jk

b Other IAT ENZ 1 2

1 R1wR1 + + + 0 4 0 + + 0 0 + 0 + 0 + 4 5 3 3

2 R1R1 + + 0 + 3 + 0 + 0 + 0 + 0 + 0 4 5 3 3

3 R2R2 0 + 0 + 0 0 0 + 0 + 0 0 + + 0 4 5 0 3

4 r’r + 0 0 + 5 0 0 + 0 0 + 0 + 0 + 3 5 3 0

5 r’’r + 0 0 + 4 0 0 + + 0 + + 0 0 + Cob+ 0 0 0 0

6 rr + + + 0 0 0 0 + 0 + 0 + 0 + 0 0 0 0 0

7 rr + 0 0 + 4 0 + + 0 0 + 0 + + 0 0 0 0 0

8 rr + 0 + + 0 0 + 0 0 + 0 + 0 + + 0 0 0 0

9 rr 0 + 0 + 0 0 0 + 0 0 + 0 + 0 + 0 0 0 0

10 rr 0 + 0 + 5 0 0 + 0 0 + + 0 0 + Cob+Bg

b+ 0 0 0 0

Auto 0 0 0 0

Antibody Titre

Dilution: 64 128 256 512 1024 Cell Id

Group O D negative

Pheno D-C-c+E-e+K-

Anti- 3 2 1 0 0

Archive 3 2 1 0 0

Anti-

Archive

DAT Batch No


Ads vs R2R2 remove’ anti-D and anti-G

Ads vs r’r remove anti-C and anti-G ? anti-D + C

Interpretation?

• Anti-D and anti-C are present • From these results you can’t tell if anti-G is present

• As anti-G previously ident, it seemed reasonable to assume it was still present

• Titration results suggest level of anti-G+C was unchanged

• Was the anti-D immune or prophylactic?

Next steps?

• Performed anti-D quantification. • If immune anti-D was present the level might be higher

than it was before. After all, the level of anti-G+C had not changed

• 17.1 iu, so suggested immune anti-D is present?

• In addition quantified the antibody in the plasma adsorped with r’r cells (contains only anti-D)

• 0.11 iu, so only prophylactic anti-D is present?

What does this mean?

• Anti-D quantification results can be affected by the presence other antibodies, e.g., anti-C

• The results from the first sample show this. • There was no anti-D but we recorded a level of 5.8 iu

• Must have been due to the presence of anti-G+C

• Titres against r’r cells for the first and second samples were the same, so the level of anti-G+C was unchanged

• Why had the quant result risen to 17.1 when the concentration of anti-D was actually 0.11iu/mL?

Advice given

• Anti-G+C present, no significant change in antibody level

• Anti-D present likely, but not certain, of prophylactic origin

• Refer patient to a Fetal Medicine Centre

• Send more maternal samples

• Send a paternal sample

• If RCI Lab hadn’t detected the anti-D, would not have asked for more samples

What came next


•No anti-D given, since last investigation

•Same work-up as before, including absorptions with R2R2 and r’r cells and quantification against R1R1 cells

•No paternal sample received

What did we find?

•Anti-D and anti-C still present after absorptions •Anti-G presumed present

• Titred to 128 against r’r cells

• ‘Raw’ plasma (anti-D) quantified at 15.6 • ‘Pure’ anti-D quantified at 0.08 •Nothing else done – at this point ran out of ideas as to how to proceed!

Advice given

• Anti-G+C present, no significant change in antibody level


• Refer patient to a Fetal Medicine Centre • Send more maternal samples • Send a paternal sample • “De-ja vu”

Next


•No anti-D given, since last investigation •No paternal sample was going to be received

•This time RCI Lab used a little more imagination …

What was done?

• Absorptions with r’r and R2R2 cells • leaving behind ‘pure’ anti-D and ‘pure’ anti-C

• Perform an elution on the r’r cells • elution contained a mixture of anti-C and anti-G

• adsorb this elution with R2R2 cells then perform an elution on these cells

• elution contained ‘pure’ anti-G

• Quantified anti-D from r’r adsorption (0.17)

• and ‘raw’ plasma (16.4)

Something to think about

• R1R1 cells • For every anti-D binding

site … • one site for anti-C binding • two sites for anti-G

binding • R2R2 cells

• For every anti-D binding site …

• one site for anti-G binding • no anti-C binding site

• r’r cells • For every anti-C binding

site … • one site for anti-G

binding • no anti-D binding site

Theoretical ratio of „total antibody binding capacity‟ (assuming equal antigen density)

R1R1 R2R2 r’r

anti-G+C 6 : 2 : 2

anti-G+C+D 8 : 4 : 2

anti-G 4 : 2 : 1

anti-C 2 : 0 : 1

anti-D 2 : 2 : 0


If the different cells are able to bind differing amounts of Ab, then interpreting titres quite ‘difficult’

Ab Titres compared

‘Raw’ Anti-D* Anti-C

R1R1 1024 4 4

R2R2 512 4 0

r’r 256 0 4


* NB Anti-D expressed as reciprocal of diltution NOT IU/mL

IgG Subclasses determined Various Ab/dilutions incubated Vs R1R1 cells by IAT

Anti-IgG1 Anti-IgG3 Anti-IgG Ctrl

1:1 1:100 1:1 1:100 1:10

G+C 5 3 1 0 5 0

G 4 0 0 0 5 0

C 0 0 0 0 3 0

D 0 0 0 0 3 0


This is of concern …

• IgG1 is more efficiently transported across the placenta • Close to term IgG1 concentrations are often,

relatively, higher in the fetus than in the mother

•Results in this case indicate a ‘lytic’ anti-G

What was advised • Anti-G+C present, no significant change in antibody

level


• Refer patient to a Fetal Medicine Centre

• Refer maternal samples for molecular typing of fetal DNA

• Send maternal samples every 2 weeks

What came next

•Sample from baby boy, delivered at about 39 weeks (on a Sunday)

•No request form received

•Request form later faxed stating ‘pre-exchange samples’ requesting ‘DAT+’

•Nothing from mother

Next steps? • Visual examination of the sample showed lysis • Rh phenotype on baby

• C-c+D+E+e+ • Elution of baby’s cells

• looked like anti-D+C, • however as baby C-, must be either anti-G or anti-G+D

• adsorbed eluate with r’r cells • Anti-D left behind

• Elution of r’r cells (used in adsorption) • Anti-G eluted (i.e. looks like anti-D+C)

Baby DAT


1:1 1:100 1:1 1:100 1:10

5 2 0 0 5 0


Confirming the culprit


1:1 1:100 1:1 1:100 1:10

G+D 0 0 0 0 3 0

G 4 0 0 0 5 0

D 0 0 0 0 3 0


The various specificities incubated with R2R2 cells and tested with AHG

What about the baby?

• At delivery • 4.07 kilos • Hb 15.5! • bilirubin’ 349 umol/L • Given ‘double dose’ phototherapy

• Hb derived from colorimetric method, so don’t know what it actually was

• ‘Bilirubin’ level is sufficient to cause kernicterus

Following on... • Next day

• Hb 18.1 • ‘bilirubin’ 339 • triple exchange transfusion performed

• Next day • Hb 19.1 • ‘bilirubin’ 301 • ‘single dose’ phototherapy performed

• Four days after delivery the ‘bilirubin’ had fallen to 232

• Eight days after delivery the FBC derived Hb was 19

• Ten days after delivery the baby was ‘thriving’

Could the investigation have proceeded differently?

• If Ab had been mistaken this for anti-D+C would the pregnancy have been managed differently?

• Did the need to ensure the patient would be given prophylactic anti-D lead to underestimation of the seriousness of the case?

• Later became apparent that the woman had suffered a number sensitising events, after Ab had been identified as anti-G+C.

• If prophylactic anti-D hadn’t been given the outcome might have been much worse!

Cold Hemagglutinin Disease (CHAD) Case


• Patient PW

• Male, DOB 1938

• CHAD

• Requested investigation - Antibody ID

• Previous Tx 11 days

• Manual ABO/D using x 6 warm washed cells – Gp O D Positive

• Known case - unable to group on Bio-Rad IH-1000 so manual groups needed - pre-warm/warm washed tube done



CHAD

Group O Rh D Positive

DAT Positive IgM 2+. No alloantibodies detected

using prewarmed and warm washed anti-IgG

LISP tube IAT

High risk patient Known case, Minimal investigation due to

risk of patient


High Risk

• Patient ST

• Male, DOB 1944

• Aplastic Anaemia

• Requested investigation - XM 2 units RC

• Previous Tx 4/52

• Previous results -

• Gp A D Positive, C-c+E+e-K+k+Kpa-,

• Anti-C+e+Kpa+Auto-pan



HR Pt

A Positive R2R2K+k+Kp(a-)

Anti-Kpa detected by IAT.

Previous anti-C not apparent.

Known anti-e

Pan Reactive Ab


Pre-op

• Patient KM

• Male, DOB 1945

• Pre-op splenectomy

• Requested investigation - XM 4 units RC

• Ab Screen Positive, Pan reactive

• No previous Tx



Pre-op PAN

A D Positive C+c-E-e+K-

Solution dependant antibody, reacting to

preservative. NAD in preservative free cells by

IAT


Pre-op PAN

Neonate

1/31/2017 Julie Molloy, MSc CSci FIBMS [email protected]; [email protected]

mailto:[email protected]


Neonate

• Patient MJ

• Male, DOB 3 days ago!

• Mother has anti-c+E

• Investigation requested – Group

• DAT 3+

• No record of IUT

• Group O D Positive, no reverse Gp

• C+c+E-e+K-





Neonate

Anti-c in eluate



Pt with Myelodysplastic Syndrome (MDS)




• Patient NT

• Male, DOB 1935

• Myelodysplastic Syndrome (MDS)

• Investigation requested Antibody ID, Urgent XM 4 units RC

• DAT + IgG

• Positive Ab Sc (capture) Ab NOT removed by x 5 Auto Adsorption!

• No history of Tx, pt not seen before by RCI





Auto Pan by gel, Enz and tube IAT.

Auto anti-D by gel IAT in plasma AA x 4 Vs rr

NAD by gel IAT in plasma AA x 4 Vs R1R1

MDS



Following discussion with NHSBT MO

• Gp O Pos C-K- issued as suitable

• Summary of results • Pt Gp O R2r K-

• DAT 5+ IgG

• Hb 64 g/l

• Strong Pan Agg

• AA x 4, auto anti-D detected.

• Units 3+ (gel IAT) Vs. rr ads plasma,

• NAD/1+ Vs. R1R1 ads plasma

• 1/31/2017 Julie Molloy, MSc CSci FIBMS [email protected]; [email protected]



SCD Patient Antibody ID & XM




• Patient MB

• Male, DOB 2000

• Sickle Cell disease

• Ab ID & XM – 12 units RC • (Ro or rr, K- HbS neg, CMV neg)

• Known pt • Knopps/McCoy with known -C and –K

• Investigated using inhibition agent





SCD

O Rh D ??, Phenotype Ro but recorded as ?? Due

to recent Tx with Orr

Anti-C, anti-K, anti-Kna/McCa & Pan previously

detected by IAT.



Able to supply Gp O (rr) D-C-E-K-CMV neg HbS neg units following XM Vs. inhibited plasma




Iron Def Patient Antibody ID & XM




• Patient LF

• Female, DOB 1934

• Iron Deficiency, Hb 53 g/l

• Ab ID & XM 2 unit RC

• Referring lab Ab Sc pos

• PAN Ab Capture & IAT


A Rh D Pos R1rK-

DAT 5+ Insufficient sample for auto adsorptions,

strong pan reactive autoantibody removed

following 3 x allo adsorption

IDA



“Low Hb” Ab ID




• Patient LR

• Female, DOB 1954

• Low Hb, level not stated

• Requested investigation Ab ID

• DAT 3+ IgG


LR Low Hb

O Rh D Neg C-c+E-e+K-M-N+S-s+Jk(a+b+)

DAT 3+ IgG

Previously detected anti-D+K+Pan




LR Low Hb

Additionally this time anti-Fya detected O Rh D Neg C-c+E-e+K-M-N+S-s+Jk(a+b+)

DAT 3+ IgG

Previously detected anti-D+K+Pan

Additionally on this occasion anti-Fya detected



Summary of results


Historical Results

Gp O Rh D Negative

C-c+E-e+K-M-N+S-s+Jk(a+b+)

Allo Anti-D+K+

Anti-Lua, anti-Fya not detected

Auto anti-Pan reactive Ab

Current sample

Gp O Rh D Negative

C-c+E-e+K-M-N+S-s+Jk(a+b+)

Allo Anti-D+K+Fya detected by IAT Tube

Auto anti-Pan reactive Ab



Documents

Advanced Transfusion Case Studies · AN patient with apparent Anti-C+D+E •ooking sample, 16 -17 weeks gestation • ZO neg [, with positive antibody screen •Previous pregnancy,