Blood Bank Case Studies Case Studies from the reference laboratory Jackie Ensley, MLS(ASCP) CM SBB 1

Blood Bank Case StudiesCase Studies from the reference laboratory

Jackie Ensley, MLS(ASCP)CMSBB

• Present various case studies and describe the approach to serologic problem solving and antibody identification.

• Determine possible causes of pan-reactivity and steps to resolve complex antibody cases.

• Briefly review serologic and molecular characteristics of antibodies identified and their respective blood group system, including clinical significance.

• Explain the various techniques and methods used in the case studies for antibody identification.

Objectives

• Antibody detection and identification is a complex problem-solving process

• Many techs may have a “gut-feeling” about the antibody before testing completion and intuitively know what needs to be done for antibody identificationo Be prepared to reevaluate your hypothesis if testing results do

not fit with initial assessment

Antibody Identification

• Use the tools available to help you detect and then identify the antibody:o Gel/solid phaseo Tube testing: saline/PeG/LISS/albumin/Room temperature/4˚Co Enzymes such as ficin, papain, trypsino Chemicals such as 0.2M DTTo Adsorption/elutiono Reticulocyte/sickle cell separationo Phenotypically similar cellso Antisera/rare antigen negative cells

• Know phases of reactivityo Some antibodies react best at room temperature/4˚C (M, N, P1, Lewis,

etc)o Some antigens destroyed by enzymes/chemicals (Ficin destroys Fya,

Fyb, M, N, etc)o Enzyme treatment of red cells enhances reactivity of some antibodies

such as those in the Rh system, Jka, Jkb, Lea, Leb, P1

• Know strength/pattern of reactivityo Some antigens show variable antigen expression and some antibodies

show variable reactivity and may show dosage, such as -Jka/-Jkb and -M/-N

o Note: different strengths may also indicate more than one antibody is present

• Besides using the blood bank techniques available to detect the antibody, also keep in mind these tips to aid you in the identification process:

o Review patient’s records, including medication, age, gender, race, diagnosis and transfusion history

o Investigate/repeat any inconsistent or contradictory reactions in the patient’s workup

o Phenotype the patient to confirm they are antigen negative for the suspected or identified antibodies

Case Study 1PATIENT HISTORY

• Female, 51 years old • Caucasian• Diagnosis: Anemia and GI bleedThe patient was seen on 12/12/2013. She typed as A Positive and had a negative antibody screen. She was transfused at that time.Current H/H: 7.7/ 24.8The hospital reports on 2/14/2014 a positive antibody screen in tubes with LISS (3+) with a positive autocontrol. The DAT/IgG is positive (2+). 4 out of 4 units are crossmatch incompatible. Hospital decides to send to the reference laboratory.

Case Study 1

• Reference Lab testing:– ABO/Rh performed:

– DAT Performed:

Anti-A Anti-B Anti-D A1 Cell B Cell ABO/Rh4+ 0 4+ 0 4+ A Positive

Anti-IgG/ Gel Anti-C3/ Gel

Plasma

Rh System Kell Duffy Kidd Lewis P MNS Lutheran

Room Temp

AHG-PeG

D C c E e K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub

I + + 0 0 + 0 + 0 + 0 + 0 + + 0 + 0 0 0 + 0 + 0 + 0 2+II + 0 + + 0 0 + 0 + 0 + + 0 0 + + 0 + + + + + 0 + 0 1+III 0 0 + 0 + + + + + 0 + + + + 0 0 + + + 0 + 0 0 + 0 2+Auto 0 1+

Plasma

GELCell D C c E e K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub

1 + + 0 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 2+2 + + 0 0 + 0 + 0 + 0 + 0 + + + + 0 0 0 + 0 + + + 2+3 + 0 + + 0 + + 0 + 0 + 0 + + + 0 + 0 0 + 0 + 0 + 2+4 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 + + 0 + + + + + 2+5 0 + + 0 + 0 + 0 + 0 + + + + 0 0 + 0 + 0 0 + 0 + 2+6 0 0 + + + 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + 2+7 0 0 + 0 + + + 0 + 0 + 0 + + + 0 + + + 0 0 + 0 + 2+8 0 0 + 0 + 0 + 0 + 0 + + 0 0 + 0 0 + + + 0 + 0 + 2+9 0 0 + 0 + 0 + + + 0 + + 0 0 + 0 + 0 + + + + 0 + 2+10 + + 0 0 + + 0 0 + 0 + 0 + + 0 0 + + + + 0 + 0 + 2+11 + w+ 0 + 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + 2+Auto 2+

Eluate

1 + + 0 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 3+2 + + 0 0 + 0 + 0 + 0 + 0 + + + + 0 0 0 + 0 + + + 3+3 + 0 + + 0 + + 0 + 0 + 0 + + + 0 + 0 0 + 0 + 0 + 3+4 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 + + 0 + + + + + 3+5 0 + + 0 + 0 + 0 + 0 + + + + 0 0 + 0 + 0 0 + 0 + 3+6 0 0 + + + 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + 3+7 0 0 + 0 + + + 0 + 0 + 0 + + + 0 + + + 0 0 + 0 + 3+8 0 0 + 0 + 0 + 0 + 0 + + 0 0 + 0 0 + + + 0 + 0 + 3+9 0 0 + 0 + 0 + + + 0 + + 0 0 + 0 + 0 + + + + 0 + 3+10 + + 0 0 + + 0 0 + 0 + 0 + + 0 0 + + + + 0 + 0 + 3+11 + w+ 0 + 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + 3+

The Eluate Last Wash is negative

• Let’s look at what we know:– Phase of reactivity: AHG– Strength/pattern of reactivity: pan-reactive, about

the same strength.– Patient history: recently transfused– DAT/autocontrol: positive/reactive– Other info: eluate is also pan-reactive with same

strength

Serologic Problem Solving

Question to ask yourself: So where do we go at this point?

• Narrowed down possibilities:1. Warm autoantibody 2. Multiple antibodies in plasma (and eluate)3. Antibody to a high incidence antigen

Next Step:

• Reference tech decides to perform adsorption on plasma only.

Why not an adsorption on the eluate?

• The patient has been transfused in the last 3 months…

Technical Manual States that “newly developed antibodies initially detectable only in the eluate are usually detectable in the serum after about 14 to 21 days”

Blood Bank Technique: Adsorption

What is an adsorption?

Blood bank technique where red cells and plasma (or eluate) are mixed, causing antibody to be adsorbed onto the red cell surface.

Types of Adsorption:

Autologous: Patient plasma is mixed with patient cells

PATIENT MUST NOT HAVE BEEN TRANSFUSED last 3 months

Differential/Allogeneic: Patient plasma is mixed with R1R1, R2R2, and rr donor cells of known phenotypes.

Antibodies to high incidence antigens may be adsorbed out

How is an alloadsorption performed?

• Alloadsorption: Patient has been transfused or transfusion is unknown.

Patient’s Plasma + Donor RBC’s

Incubate together to adsorb the antibodies onto the donor

red cells

Blood Bank Technique: Adsorption

Incubation allows any antibody to adsorb onto the

red cells (alloantibody or autoantibody)

lls- d

est R1R1

Centrifuge the tubes and separate the adsorbed plasma from the red cells for testing

R1R1(D+C+E-c-e+)

R2R2(D+C-E+c+e-)

rr (D-C-E-c+e+)

Run each adsorbed plasma with panel cells to identify any antibodies. Antibodies in adsorbed plasma will depend on the phenotype of the adsorbing cell.

Example: anti-E Example: anti-E

R1R1 Adsorption Rh System Kell Duffy Kidd Lewis P MNS Lutheran

X2 PeG- Plasma

Cell D C c E e K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub

R1R1 Cell Phenotype + + 0 0 + 0 + 0 + 0 + + 0 0 + + 0 0 0 + 0 + 0 +

1 + + 0 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 0√2 + + 0 0 + 0 + 0 + 0 + 0 + + + + 0 0 0 + 0 + + + 1+3 + 0 + + 0 + + 0 + 0 + 0 + + + 0 + 0 0 + 0 + 0 + 1+4 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 + + 0 + + + + + 2+5 0 + + 0 + 0 + 0 + 0 + + + + 0 0 + 0 + 0 0 + 0 + 2+6 0 0 + + + 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + 2+7 0 0 + 0 + + + 0 + 0 + 0 + + + 0 + + + 0 0 + 0 + 1+8 0 0 + 0 + 0 + 0 + 0 + + 0 0 + 0 0 + + + 0 + 0 + 0√9 0 0 + 0 + 0 + + + 0 + + 0 0 + 0 + 0 + + + + 0 + 0√10 + + 0 0 + + 0 0 + 0 + 0 + + 0 0 + + + + 0 + 0 + 2+11 + w+ 0 + 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + 2+

X3 GEL

-Plasma

R2R2 Cell Phenotype

+ 0 + + 0 0 + 0 + 0 + + 0 + 0 0 + + + 0 + 0 0 +

1 + + 0 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 0√2 + + 0 0 + 0 + 0 + 0 + 0 + + + + 0 0 0 + 0 + + + 0√3 + 0 + + 0 + + 0 + 0 + 0 + + + 0 + 0 0 + 0 + 0 + 0√4 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 + + 0 + + + + + 0√5 0 + + 0 + 0 + 0 + 0 + + + + 0 0 + 0 + 0 0 + 0 + 0√6 0 0 + + + 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + 0√7 0 0 + 0 + + + 0 + 0 + 0 + + + 0 + + + 0 0 + 0 + 0√8 0 0 + 0 + 0 + 0 + 0 + + 0 0 + 0 0 + + + 0 + 0 + 0√9 0 0 + 0 + 0 + + + 0 + + 0 0 + 0 + 0 + + + + 0 + 0√10 + + 0 0 + + 0 0 + 0 + 0 + + 0 0 + + + + 0 + 0 + 0√11 + w+ 0 + 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + 0√

rr Adsorption Rh System Kell Duffy Kidd Lewis P MNS Lutheran

X3 GEL

-Plasma

r r Cell Phenotype

0 0 + 0 + 0 + 0 + 0 + 0 + + 0 0 + + 0 + + + 0 +

1 + + 0 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 0√2 + + 0 0 + 0 + 0 + 0 + 0 + + + + 0 0 0 + 0 + + + 0√3 + 0 + + 0 + + 0 + 0 + 0 + + + 0 + 0 0 + 0 + 0 + 0√4 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 + + 0 + + + + + 0√5 0 + + 0 + 0 + 0 + 0 + + + + 0 0 + 0 + 0 0 + 0 + 0√6 0 0 + + + 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + 0√7 0 0 + 0 + + + 0 + 0 + 0 + + + 0 + + + 0 0 + 0 + 0√8 0 0 + 0 + 0 + 0 + 0 + + 0 0 + 0 0 + + + 0 + 0 + 0√9 0 0 + 0 + 0 + + + 0 + + 0 0 + 0 + 0 + + + + 0 + 0√10 + + 0 0 + + 0 0 + 0 + 0 + + 0 0 + + + + 0 + 0 + 0√11 + w+ 0 + 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + 0√

X2 PeG- Plasma

R1R1 Cell Phenotype + + 0 0 + 0 + 0 + 0 + + 0 0 + + 0 0 0 + 0 + 0 +

1 + + 0 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + 0 + 0 0 + 0√2 + + 0 0 + 0 + 0 + 0 + 0 + + + + 0 0 0 + 0 + + + 1+3 + 0 + + 0 + + 0 + 0 + 0 + + + 0 + 0 0 + 0 + 0 + 1+4 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 + + 0 + + + + + 2+5 0 + + 0 + 0 + 0 + 0 + + + + 0 0 + 0 + 0 0 + 0 + 2+6 0 0 + + + 0 + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + 2+7 0 0 + 0 + + + 0 + 0 + 0 + + + 0 + + + 0 0 + 0 + 1+8 0 0 + 0 + 0 + 0 + 0 + + 0 0 + 0 0 + + + 0 + 0 + 0√9 0 0 + 0 + 0 + + + 0 + + 0 0 + 0 + 0 + + + + 0 + 0√10 + + 0 0 + + 0 0 + 0 + 0 + + 0 0 + + + + 0 + 0 + 2+11 + w+ 0 + 0 + 0 + 0 + 0 0 + 0 0 0 + + + 0 + 0 + 2+

Autoantibody Confirmation Testing

• Patient had been transfused in the last 3 months so need to perform reticulocyte separation– Want to be testing patient cells and not donor

How is a Reticulocyte Cell Separation Performed?

• Patient has been transfused so need to separate patient cells from donor red cells

Spin the sample down and fill microhematocrit tubes with the red cells.

Stopper one end of the hematocrit tube with clay.

Blood Bank Technique: Reticulocyte Cell Separation

How is a Reticulocyte Cell Separation Performed?

Clay Plug

Newer Red Cells

Older Red Cells

Excess saline/plasmaBuffy Coat

Spin the microhematocrit tubes and then cut the tubes to get the reticulocytes

Blood Bank Technique: Reticulocyte Cell Separation

Now that we have the retics:• DAT/IgG had been positive so perform DAT/IgG

on retics:

Retics

Anti-IgG/ tube

Can not proceed with testing to identify warm autoantibody until the DAT is negative

How do we get the DAT/IgG negative?

EGA Treatment• What is EGA? – EDTA glycine acid dissociates IgG from red blood

cells so the treated red cells can be used for further testing or antigen typing using the AHG phase.

– Use when direct antiglobulin phase (DAT) is positive

– Does not impair red cell surface antigens

Blood Bank Technique: EGA Treatment

EGA Treatment• The Process– Wash IgG coated red cells thoroughly– Suspend cells briefly in EGA solution to dissociate

bound IgG antibody– Bring mixture to neutral pH– Centrifuge and wash cells with saline

• Test treated cells by performing a DAT• Limitation: destroys Kell, Era, Bg antigens

Blood Bank Technique: EGA Treatment

Autoantibody investigation

• EGA testing performed and DAT negative retics obtained

• To confirm the antibody is warm autoantibody the DAT negative retics are tested against the plasma and eluate:

Retics-Plasma Retics-Eluate

Gel Gel

This is what was expected if the antibody was autoantibody! Further testing is not required, the warm autoantibody has been confirmed.

Antibody Confirmation

• Lastly need to confirm anti-Jka (JK1) by antigen typing

• Use retics so that typing patient cells and not donor cells

Anti-Jk

Patient types Jka negative

Results

• Patient has warm autoantibody and anti-Jka (JK1).

• Transfusion recommendations:Transfuse Jka- (JK1), AHG crossmatch least

incompatible, red blood cell products.

Kidd Blood Group System

·Daniels, G. (2013) Kidd Blood Group System, in Human Blood Groups, 3rd edition, Wiley-Blackwell, Oxford, UK.

• Located Chromosome 18

• Glycoprotein with 10 membrane spanning domains

Kidd antibodies are often difficult to work with and are a common cause of delayed hemolytic reactions

Jka (JK1) Antibody & AntigenJka Antibody Characteristics

History 1951

Clinical Significance Yes! Clinically significant·Transfusion Reactions possible, immediate or delayed hemolytic·HDN possible, mild to moderate

Antibody IgG/IgM

Other facts ·Jka has been demonstrated on fetal cells as early as 11 weeks·Antibody fades in vitro and in vivo·Can show dosage

Jka Antigen Characteristics

Occurrence Caucasians 77%Blacks 92%

Reid, Marion and Christine Lomas-Francis (2012). The Blood Group Antigen FactsBook, 3rd Edition, Elsevier.

• Female, 38 years old • African American• DIAGNOSIS:-Severe Sepsis--blood cultures showed Finegoldia magna (normal flora of the gastrointestinal and genitourinary tract, and can be isolated from skin and the oral; often regarded as a contaminant in cultures) with subsequent cultures after that date with no growth. -Probable pneumonia -Cardiac arrest -Hypertensive-Acute respiratory failure

-Acute renal failure -Positive for influenza A

• The patient arrived as in-patient on 1/15/2014 and was typed as B Positive with negative antibody screen. Patient was transfused 2 B Positive RBCs at that time.

• Patient was monitored and was still very ill

• On 1/24/2014 patient required another transfusion and sample was sent to hospital blood bank.

Case Study 2

• The 2nd sample was collected on 1/24/2014, 9 days after transfusion.

• Sample was sent to the reference laboratory

Hospital Results on 1/24/2014:B PositiveAll cells reactive 2+ in gelAutocontrol positive

Case Study 2

– DAT Performed:

Anti-A Anti-B Anti-D A1 Cell B Cell ABO/Rh0 4+ 4+ 4+ 0 B Positive

Anti-IgG/ Gel Anti-C3/ tube

W+ 0√

Plasma

Room Temp

AHG-PeG

I + + 0 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + + + + 0 + 0 1+II + 0 + + 0 + + 0 + 0 + + + + + + 0 0 + 0 + 0 0 + 0 2+III 0 0 + 0 + 0 + 0 + 0 + + 0 + 0 0 + + 0 + 0 + 0 + 0 2+Auto 0 0√

Plasma

1 + + 0 0 + + + + + 0 + + + + 0 + 0 W0 + + 0 0 + 2+2 + + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 2+3 + 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 2+4 + 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 2+5 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 2+6 0 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 2+7 0 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 2+8 0 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 2+9 0 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 2+10 0 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 2+11 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 2+Auto W+

Eluate

1 + + 0 0 + + + + + 0 + + + + 0 + 0 W 0 + + 0 0 + 4+2 + + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 4+3 + 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 4+4 + 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 4+5 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 4+6 0 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 4+7 0 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 4+8 0 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 4+9 0 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 4+10 0 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 4+11 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 4+

The Eluate Last Wash is negative

• Let’s look at what we know:– Phase of reactivity: AHG– Strength/pattern of reactivity: pan-reactive, same

strength.– Patient history: recently transfused– DAT/autocontrol: positive/reactive– Other info: eluate is also pan-reactive with same

strength

• Narrowed down possibilities:1. Warm autoantibody 2. Multiple antibodies in plasma and eluate3. Antibody to a high incidence antigen

Next Step:

• Reference tech decides to perform adsorptions on plasma & eluate.

Why perform an adsorption?• To adsorb out suspected warm autoantibody

and determine if there are any alloantibodies hiding under the pan-reactivity.

Plasma

Eluate

R1R1 Cell Phenotype

+ + 0 0 + 0 + 0 + 0 + + 0 + 0 0 + + + + + + 0 +

1+ + 0 0 + + + + + 0 + + + + 0 + 0 W 0 + + 0 0 + 0 0

2+ + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 0 0

3+ 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 0 0

4+ 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 0 0

50 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 0 0

60 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 0 0

70 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 0 0

80 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 0 0

90 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 0 0

100 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 0 0

11+ 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 0 0

GEL-Plasma

GEL-Eluate

R2R2 Cell Phenotype

+ 0 + + 0 0 + 0 + 0 + 0 + + + 0 + + 0 + 0 + 0 +

1+ + 0 0 + + + + + 0 + + + + 0 + 0 W 0 + + 0 0 + 0 0

2+ + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 0 0

3+ 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 0 0

4+ 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 0 0

50 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 0 0

60 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 0 0

70 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 0 0

80 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 0 0

90 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 0 0

100 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 0 0

11+ 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 0 0

rr Adsorption Rh System Kell Duffy Kidd Lewis P MNS Lutheran

GEL-Plasma

GEL-Eluate

r r Cell Phenotype

0 0 + 0 + 0 + 0 + 0 + + + 0 + + 0 0 + 0 + 0 0 +

1+ + 0 0 + + + + + 0 + + + + 0 + 0 W 0 + + 0 0 + 0 0

2+ + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 0 0

3+ 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 0 0

4+ 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 0 0

50 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 0 0

60 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 0 0

70 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 0 0

80 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 0 0

90 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 0 0

100 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 0 0

11+ 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 0 0

Results

• Appears to be warm autoantibody• No alloantibodies were detected in the

alloadsorbed plasma or eluate

Need to confirm warm autoantibody

• Patient had been transfused 9 days ago so perform reticulocyte separation.

• DAT/IgG had been positive so perform DAT/IgG on retics:

Retics

Anti-IgG/ Gel

Proceed with further testing to identify warm autoantibody

• To confirm the antibody is warm autoantibody the retics are tested against the plasma and eluate:

Retics-Plasma Retics-Eluate

Gel Gel

This is NOT what was expected if the antibody was autoantibody! Further testing is required and now antibody to a high incidence antigen is suspected

• Narrowed down possibilities:1. Warm autoantibody 2. Multiple antibodies in plasma and eluate3. Antibody to a high incidence antigen

Next Step:

• Use blood bank techniques, reagents and cells to try and determine the antibody

SOME TECHNIQUES/OPTIONS AVAILABLE:

• ENZYMES (FICIN, PAPAIN, TRYPSIN, ETC)

• CHEMICALS SUCH AS DTT

• PHENOTYPE PATIENT

• RARE ANTISERA

• RARE CELLS

Ficin Panel Rh System Kell Duffy Kidd Lewis P MNS Lutheran

GEL-Ficin

1 + + 0 0 + + + + + 0 + + + + 0 + 0 W 0 + + 0 0 + 2+ 3+2 + + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 2+ 3+3 + 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 2+ 3+4 + 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 2+ 3+5 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 2+ 3+6 0 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 2+ 3+7 0 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 2+ 3+8 0 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 2+ 3+9 0 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 2+ 3+10 0 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 2+ 3+11 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 2+ 3+Auto

0.2 M DTT Panel Rh System Kell Duffy Kidd Lewis P MNS Lutheran

GEL-0.2M DTT

1+ + 0 0 + + + + + 0 + + + + 0 + 0 W 0 + + 0 0 + 2+ 2+

2+ + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 2+ 2+

3+ 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 2+ 2+

4+ 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 2+ 2+

50 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 2+ 2+

60 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 2+ 2+

70 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 2+ 2+

80 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 2+ 2+

90 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 2+ 2+

100 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 2+ 2+

11+ 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 2+ 2+

Auto W+

Case Study 2• Ficin and DTT testing has helped narrow down

the possibilities. Some high incidence antigens resistant to Ficin and 0.2M DTT Treatment:

Lan ABTI PEL U Fy3

Ata MAM Dib Ge3 Fy5

Emm Oka Wrb EnaFR Era

Sda (Ficin enhanced0

Coa CO3 Vel (Ficin enhanced)

Jra (Ficin enhanced)

Consider the race of patient and start with the easiest to test for

The list is not all-inclusive. Refer to The Blood Group Antigen FactsBook by Marion E Reid and Christine Lomas-Francis for support regarding antigen/antibody reactivity.

Selected Cells Run

Rh System Kell Duffy Kidd Lewis P MNS LutheranGEL

D C c E e K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub U

+ 0 + 0 + 0+ 0 + 0 + + 0 + + 0 + + 0 + 0 0 + + 0 0N1727

0 0 + 0 + 0+ 0 + 0 + 0 0 + + 0 + + + + 0 0 0 + 0 0

Rh System Kell Duffy Kidd Lewis P MNS LutheranGEL

D C c E e K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub U

+ 0 + 0 + 0+ 0 + 0 + + 0 + + 0 + + 0 + 0 0 + + 0 0N1727

0 0 + 0 + 0+ 0 + 0 + 0 0 + + 0 + + + + 0 0 0 + 0 0

Eluate Testing

Plasma Testing

Patient is antigen typed with the retics and is U-

BioArray Molecular ResultsRh c +

Duffy Fya +

Dombrock Doa 0

C 0 Fyb 0 Dob +

e + MNS M + Joa +

E 0 N 0 Hy +

Kell K 0 S LS LW Lwa +

k + s LS Lwb 0

Kpa 0 Lutheran Lua 0 Scianna Sc1 +

Kpb + Lub + Sc2 0

Jsa 0 Diego Dia 0 Hemoglobin S HbS 0

Jsb + Dib + U (-)

Kidd Jka + Colton Coa +

Jkb + Cob 0

Results

• The antibody is anti-U (MNS5), not a warm autoantibody as was suspected at first.

Antigen Negative Units Requested:Two U- (MNS5) units were deglycerolized and sent to hospital

Deglycerolization

Red cells are frozen with glycerol, a cryoprotective agent that prevents cellular damage and hemolysis as well as allows them to be frozen at < -65°C for 10 years.

To deglycerolize, the red cells are warmed and then washed with decreasing % NaCl to remove the glycerol and then suspended for transfusion. Once thawed they have a shelf life of 24 hours (if an open system was used).

U (MNS5) AntibodyU Antibody Characteristics

History Anti-U was first described by Wiener et al in 1953. It was called “U” for the universal distribution of the antigen. Not ‘naturally occuring’

Clinical Significance ·Yes! Clinically significant·Transfusion Reactions possible, mild to severe·HDN possible, mild to severe

Antibody ·IgG, reacts best at 37°C/AHG·Autoanti-U is possible

Other facts Some examples of anti-U are not compatible with all U- red cells. This is because some U- red cells are actually U variant and so have small quantities of U antigen.

U (MNS5) AntigenU Antigen Characteristics

Occurrence Caucasians 99.9%Blacks 99%Well developed at birth

Other Facts ·All U- individuals are S-s- but not all S-s- individuals are U-. ·The S-s- phenotype not common in the Caucasian population·U negative phenotype is associated with absence of Glycophorin B (GPB)

Variants U variant is possible

Sources for further reading: ·Reid, Marion and Christine Lomas-Francis (2012). The Blood Group Antigen FactsBook, 3rd Edition, Elsevier.·Daniels, G. (2013) MNS Blood Group System, in Human Blood Groups, 3rd edition, Wiley-Blackwell, Oxford, UK.

Genetics and Biochemistry

• Genes encoding MNS system antigens reside on chromosome 4 – Responsible for the

production of glycophorin A (GPA) and glycophorin B (GPB) on red cells

Genetics and BiochemistryGlycophorin A (GPA)

M and N antigens

Glycophorin B (GPB)S, s and U antigens

GPA and GPB are the major sialic acid containing structures of the red cell

membrane.

Photo Source: http://classconnection.s3.amazonaws.com/414/flashcards/1065414/jpg/mns_biochem1330653387883.jpg

U variants

• S-s-U+ or S-s-U+var

• Almost exclusively in those of African Origin• About 50% of S-s- are U+var

• Strength of expression is variable; adsorption/elution tests may be needed to detect the U antigen

• Strong correlation of U variant antigen cells being He+ (low frequency MNSs antigen).

• Reactivity– The anti-U of S-s-U- will react with S-s-U+var

– The anti-U of U variants will not react with S-s-U- cells.

• GPB of the cell– U- cells are totally GPB-deficient– U variants have a variant GPB molecule that

doesn’t express S or s

U vs U variants

• Female, 65 years old • African American• DIAGNOSIS: strokePatient had 45 minute seizure at nursing home before being transported to hospital. Speech was slurred upon arrival to emergency department with facial drooping.Patient has history of seizures, hypothyroidism, GERD, severe anemia, hypertension, congestive heart failure, etc.H/H: 9.9/ 32.1 Last transfusion was 10/27/2012 (>3 months)

Case Study 3• The sample was collected on 01/30/2013

• Sample was sent to the reference laboratory

Hospital Results on 1/30/2013:O PositiveAll cells reactive 2+ in gelAutocontrol not testedAdditional history includes anti-Chido and antibody in Knops system from another facility.

Case Study

– DAT Performed:

Anti-A Anti-B Anti-D A1 Cell B Cell ABO/Rh0 0 4+ 4+ 4+ 0 Positive

Anti-IgG/ tube Anti-C3/ tube

0√ 0√

PlasmaRh System Kell Duffy Kidd Lewis P MNS Lutheran

Room Temp

AHG-PeG

+ + 0 0 + + + 0 + 0 + + 0 + + + 0 + + 0 + + 0 + 0 1+

+ 0 + + 0 0 + 0 + 0 + 0 + + 0 0 + 0 + 0 + 0 0 + 0 w+

0 0 + 0 + 0 + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + 0 w+

0 0√

Plasma

1 + + 0 0 + + + + + 0 + + + + 0 0 0 + 0 + + 0 0 + 1+2 + + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 1+3 + 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 1+4 + 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 2+5 0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 1+6 0 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 1+7 0 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 2+8 0 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 1+9 0 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 2+

10 0 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 2+11 + 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 2+

Auto 0

• Let’s look at what we know:– Phase of reactivity: AHG– Strength/pattern of reactivity: pan-reactive,

different strengths.– Patient history: not recently transfused– DAT/autocontrol: negative

• Narrowed down possibilities:1. One antibody with different strengths2. Multiple antibodies in plasma • Keep in mind that patient has history of anti-

Chido or antibody in Knops system

Next Step:

• Use blood bank techniques, reagents and cells to try and determine the antibody

SOME TECHNIQUES/OPTIONS AVAILABLE:

• ENZYMES (FICIN, PAPAIN, TRYPSIN, ETC)

• CHEMICALS SUCH AS DTT

• PHENOTYPE PATIENT

• RARE ANTISERA

• RARE CELLS

Patient phenotypeRh c + Duffy Fya 0

C 0 Fyb 0

e + MNS M +

E + N 0

Kell K 0 S 0

Kidd Jka + s +

Jkb 0 Lewis Lea 0

Some of the Selected CellsRh System Kell Duffy Kidd Lewis P MNS Lutheran

GELD C c E e K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub

+ + 0 0 + + + 0 + 0 + + + + 0 + 0 + + 0 + 0 0 + 1++ + 0 + + + + 0 + 0 + + 0 + 0 0 + + 0 + 0 + 0 + Co(b+) 2++ 0 + + 0 0 + 0 + 0 + 0 + 0 + 0 + 0 + + 0 + + + 2+0 0 + 0 + + + 0 + 0 + 0 + 0 + 0 + + + 0 + 0 0 + 0+ + 0 0 + + 0 0 + 0 + + + + 0 0 + + + + 0 + 0 + w+0 0 + 0 + 0 + 0 + 0 + + + 0 + + 0 0 + + + + 0 + w++ 0 + + 0 + + 0 + 0 + + 0 + 0 0 + + + + + + 0 + 1++ + 0 0 + + + + + 0 + 0 0 + 0 + 0 + 0 + + 0 0 + 0+ + 0 0 + + + + + 0 + 0 0 + 0 + 0 0 0 + 0 + 0 + 00 0 + 0 + + + 0 + 0 + + 0 + + 0 + 0 + 0 + 0 0 + 00 0 + 0 + + + 0 + 0 + 0 + + + 0 + 0 + + 0 + 0 + Co(b+) 1+

Ficin Panel

+ + 0 0 + + + + + 0 + + + + 0 0 0 + 0 + + 0 0 + 1+ 1++ + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 1+ 1++ 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 1+ 1++ 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 2+ 1+0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 1+ 1+0 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 1+ 1+0 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 2+ 1+0 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 1+ w+0 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 2+ 1+0 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 2+ 1++ 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 2+ 1+

Auto 0 0

0.2M DTT Panel

GEL-Ficin

GEL-DTT

+ + 0 0 + + + + + 0 + + + + 0 0 0 + 0 + + 0 0 + 1+ 1+ 0

+ + 0 0 + 0 + 0 + 0 + + + + 0 0 + + + 0 + + 0 + 1+ 1+ 0

+ 0 + + 0 0 + 0 + 0 + + + + 0 0 + 0 + 0 + + + + 1+ 1+ 0

+ 0 + 0 + 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + 0 0 + 2+ 1+ 0

0 + + 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + 1+ 1+ 0

0 0 + + + 0 + 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 1+ 1+ 0

0 0 + 0 + + + 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 2+ 1+ 0

0 0 + 0 + 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + 1+ w+ 0

0 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 0 + + + + + 0 + 2+ 1+ 0

0 0 + + + + + 0 + 0 + 0 + 0 + + 0 + + + + + + + 2+ 1+ 0

+ 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 + 0 + + + 0 + 2+ 1+ 0Auto 0 0

Patient History

• anti-Chido

• Knops

Reactivity

Ficin DTT

Negative Reactive

Reactivity

Ficin DTT

Weakened Negative

Knops SystemAntigen Occurrence

CaucasianOccurrence

Blacks

Kna 98% 99%

Knb 4.5% <.01%

McCa 98% 94%

McCb 0% 45%

Sla (Sl1) 98% 50-60% (30% West Africans)

Yka 92% 98%

Vil (Sl2) 0% 80%

Sl3 100% 100%

Available Selected Knops CellsRh System Kell Duffy Kidd Lewis P MNS Lutheran

GELD C c E e K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub

0 0 + 0 + + + 0 + 0 + 0 + 0 + 0 + 0 + + + + 0 + Yk(a-) 1+0 0 + 0 + 0 + 0 + + + 0 0 + 0 0 0 0 + 0 0 + 0 + Sl(a-) 0+ 0 + + + 0 + 0 + 0 + + 0 + + 0 + + + + 0 + 0 + Sl(a-) 0

Appears to be anti-Sla but due to known weak reactivity of the antibody do molecular to confirm

Molecular TestingRh c +

MNS M +

Dombrock Doa 0

C 0 N 0 Dob +

e + S 0 Joa +

E + s + Hy +

Kell K 0 Lutheran Lua 0 LW Lwa +

k + Lub + Lwb 0

Kpa 0 Diego Dia 0 Scianna Sc1 +

Kpb + Dib + Sc2 0

Jsa 0 Cromer Cra +

Jsb + Colton Coa + Knops Kna +Knb 0

Kidd Jka + Cob 0McCa +

Jkb 0 Cartwright Yta + McCb 0Duffy Fya 0 Ytb 0 Sl1 0 Fyb 0 Hemoglobin S HbS 0 Sl2 +

Knops System• Knops antigens are

located on complement receptor 1 (CR1)

• CR1 gene resides on chromosome 1

Complement Receptors

What is CR1 (CD35)?CR1 is a glycoprotein on cells that binds particles coated with C3b and C4b

Neutophils and monocytes then phagocytize those particles and processes the immune complexes.

These are transported to the liver/spleen for removal from circulation.

• Has inhibitory effect on complement activities by classical and alternative pathways so it protects the red cells from autohemolysis

What is CR1 (CD35)?

https://www.inkling.com/read/the-immune-system-peter-parham-3rd/chapter-9/antibody-production-by-b

Knops system

Structure of CR1 glycoprotein (CD35)

Knops system characteristics• Variation in antigen strength, related to CR1 red cell levels

• Generally, a reduction in antigen strength with storage of red cells as the CR1 copy per RBC may be decreased in stored samples

• High titer low avidity (HTLA) has been used to describe the antibodies

• Difficult to adsorb out antibodies

• Can be hard to distinguish antigen negative from weakly positive cells

• Clinically benign but can mask other significant antibodies

Knops System

Null phenotype: Kn(a-b-), McC(a-), Sl(a-), Yk(a-)aka Helgeson type

Knops antigens can be depressed in

cutaneous lupus erythematosus (CLE)

Cold Hemagglutinin Disease (CHAD)

Paroxysmal nocturnal hemoglobinuria (PNH)

hemolytic anemia insulin-dependent diabetes

some malignant tumors

any condition with increased clearance of immune complexes

Sla antibody

Sla Antibody Characteristics

History Reported in 1980 and named after Swain and Langely, the first two antibody producers.

Clinical Significance No! Clinically insignificant·No Transfusion Reactions·No HDN

Antibody ·IgG, reacts best at 37°C/AHG

Other facts May be confused with anti-Fy3 because most Fy(a-b-) red cells are likely to be Sl(a-). Common antibody made by blacks.

Sla antigenSla Antigen Characteristics

Occurrence 98% 50-60% (30% West Africans)

Other Facts Also known as Sl1

Disease processes causing red cell CR1 deficiency can lead to false negative antigen typing. Also, variability in antigen strength has been described.

Sources for further reading: ·Reid, Marion and Christine Lomas-Francis (2012). The Blood Group Antigen FactsBook, 3rd Edition, Elsevier.·Daniels, G. (2013) MNS Blood Group System, in Human Blood Groups, 3rd edition, Wiley-Blackwell, Oxford, UK.

Blood Bank Case Studies Case Studies from the reference laboratory Jackie Ensley, MLS(ASCP) CM SBB 1

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