Coagulation – Lab Issues And Quality Control

Grouping and compatibility issues and their resolution 3rd Basic Haematopathology Course TMH Mumbai

ABO system  First human blood group system to be discovered  Only blood group system in which individuals have

predictably antibodies in their serum to antigens that are absent from their RBCs  4 major ABO phenotypes : A, B, O and AB - determined by the presence or absence of two antigens (A and B) on red cells

•Presence or absence of naturally occurring antibodies termed isohemaagglutinins, directed against missing A and B antigens. •Immunizing source- is gut and environmental bacteria which have been shown to possess ABO like structures on their lipopolysaccharide coats. • Predominantly IgM ,activate complement and react at room temperature or colder •Produce strong direct agglutination reactions during ABO testing

 ABO antibodies – Initiated at birth but detected only by 3 to

6 months of age ,peaks between 5-10 years and declines later in life  ABO antigens – develop by 37th day of fetal life and fully

developed by 2-4 years of age and remain constant for life

 Forward grouping or front typing:

Detection of antigens on an individual’s RBCs using known sources of commercial antisera  Reverse grouping or back typing:

Detection of ABO antibodies in the patient’s serum using known reagent RBCs namely A1 and B cells

Discrepancy – A conflict or variation …between things that ought to be the same Collins dictionary

In the blood bank  Reverse typing discrepant with forward grouping  Patients phenotype inconsistent with antibody profile  Group/type inconsistent with previously assigned group/type  Crossmatch results discrepant with prior or subsequent compatibility testing

Classification of grouping discrepancies  Group I – Unexpected reactions in serum grouping due to weakly

reacting or missing antibodies  Group II – Unexpected reaction in cell grouping due to weakly reacting or missing antigens  Group III – Unexpected reactions in cell and serum grouping caused by protein or plasma abnormalities  Group IV – Miscellaneous problems

Common sources of technical errors  Inadequate identification of blood specimens, test tubes or

• • • • •

slides A mix- up in samples Clerical error Cell suspension either too heavy or too light Failure to add reagent Failure to follow manufacturer’s instructions

 Contaminated poor quality reagents

• Missed observation of hemolysis • Uncalibrated centrifuge • Unclean/ contaminated glassware

General principles for resolution  Eliminate the chance of clerical error or misidentification by retesting

where possible on a fresh sample.

 Test with alternate platforms and reagents  Use washed cells and saline replacement/dilution to eliminate type III

reactions

 Use enhancement techniques to bring out weak or missing reactions

 Review and analyze the patient’s history for a possible role

Lab tests that aid in resolution The tools to him that has the ability to handle them. French proverb

 Multiple platforms and reagents  Antibody screening and identification  Non-routine serological tests (enhancement techniques,

secretor test, adsorption and elution, etc.)  Extended phenotyping  Genotyping

Case 1 - Where a discrepancy aided

diagnosis!

 A 55 year old lady slated to undergo an orthopaedic surgery

elsewhere, was referred to our blood bank. Results of grouping done elsewhere were:Anti A Anti B Anti AB

A cells B cells O cells

4+

2+

4+

4+

Forward group:AB

2+

2+

Reverse group:???

• Microscopy: Rouleaux

•Rouleaux – Stacking of erythrocytes in a coin like fashion (right) can be mistaken for agglutination (left) •Saline dilution/replacement will neutralise rouleaux formation

 With saline dilution reverse group consistent with AB.  In view of the clinical history of fracture, patient’s clinician

informed of need to exclude plasma cell dyscrasia.  On further investigation, patient diagnosed to have myeloma  The presence of a discrepancy may point to a significant clinical condition

Case 2 – Clinical history to the rescue!  Sample from a 1 year old child was sent for grouping  Grouping results

Anti A

Anti B

Anti AB

A cells B cells O cells

Neg

Neg

Neg

Neg

Forward group:O

Neg

Neg

Reverse group: AB?

 Sample retested after incubation at 4 degrees Celsius showed

similar results.  Clinical history enquired into:- history of recurrent infections; suspected case of severe combined immunodeficiency syndrome  Inference: Serum grouping negative because of hypogammaglobulinemia.  Clinical history can play an important role in resolving a discrepancy

Case 3 – A blood group makeover!  59 year old gentleman posted for surgery for carcinoma colon.  Previously documented A group  Repeat grouping:-

Anti A

Anti B

4+

2+

Anti AB 4+

Forward group: AB

A cells B cells O cells

Neg

4+

Neg

Reverse group: A?

 Patient’s serum tested against patient’s own cells gave a

negative reaction  Acquired B suspected in view of the above, and clinical history  Confirmed by secretor test: Showed only A substance  Inference: A group with acquired B

CH2OH

CH2OH O

OH OH

Bacterial deacetylating enzymes

O OH

O

OH

OH

NHCOA immunodminant sugar (N- acetyl D galactosamine)

O

OOH o

NH2 Acquired B (D galactosamine)

Anti B

CH2OH OH

O

OH OH OH B immunodominant sugar (D galactose)

Acquired B phenotype  Patients with gastrointestinal lesions, particularly carcinoma colon.  Always occur in group A patients, nearly always A1  Acquired B cells do not agglutinate with patient’s own anti B

 Secretors do not secrete B substance  Reversible on treatment with acetic anhydride

 Ph sensitive:- Anti B reagents with PH beyond the range of 6.0-8.5 do

not agglutinate acquired B cells

Case 4 – An argument in favour of different phases of compatibility testing!  A 19 year old youth posted for an elective surgery was grouped in

our blood bank.The patient had received 16 uneventful transfusion prior to this  Grouping results Anti A

Anti B

Anti AB A cells B cells O cells

4+ 4+ 4+ Forward group:AB

Neg Neg Neg Reverse group:AB

•Antibody screen - Negative

Crossmatch results Phase of testing

Donor 1 Group AB

Donor 2 Group A

Donor3 Group O

saline

Neg

Neg

Neg

37o C

3+

3+

Neg

Coombs

4+

4+

Neg

Patients cells tested with A1 lectin – Negative reaction signifying A2B group Incompatible units tested with lectin and found to be positive signifying A1 phenotype. Crossmatch performed with A2B unit was compatible Inference: A2 with anti A1

 Significance:- Anti A1 reactive at 37o C – clinically

significant!!  Would have been missed if immediate spin crossmatch alone was done eg. type and screen/electronic crossmatch scenario

Case – 5 Bombay group Anti A

Anti B

A1 cells

B cells

O cells

Auto control

Results

0

0

4+

4+

4+

0

Oh Bombay

•Reported by Bhende in 1952 in Bombay •Inheritance of double dose of h gene producing genotype hh •No expression of ABO genes and no H antigen and hence ABH antigens not formed •Serum contains anti A, Anti B, Anti A,B and Anti H •Anti H – potent and clinically significant at 370 C •IgM antibody-activates complement and RBC lysis •Transfuse blood only from another Bombay individual

Case 6 – Dilemma after resolution!  55 year old lady slated for elective surgery referred from

elsewhere. She had received 2 uneventful transfusions of O positive blood elsewhere one year previously.  Grouping performed elsewhere.

Anti A Anti B Anti AB Neg Neg Neg Forward group: O

A B O cells cells cells 4+ 4+ Neg Reverse group: O

Case 6 – Dilemma after resolution!  55 year old lady slated for elective surgery referred from

elsewhere. She had received 2 uneventful transfusions of O positive blood elsewhere one year previously.  Grouping performed elsewhere.

Anti A Anti B Anti AB Neg Neg Neg

Anti H Neg

A cells 4+

B cells 4+

O cells Neg

Our grouping Phas Anti e A

Anti B

Anti AB

Anti H

H A lectin cells

B cells

O cells poole d

O cells dono r1

O cells dono r2

O cells dono r3

IS/ LISS

Neg

Neg

Neg

Neg

Neg

4+

4+

Neg

Neg

Neg

Neg

4O inc

Neg

Neg

Neg

Neg

-

4+

4+

Neg

Neg

Neg

Neg

37O inc

Neg

Neg

Neg

Neg

-

4+

4+

Neg

Neg

Neg

Neg

Coo mbs

Neg

Neg

Neg

Neg

-

4+

4+

Neg

Neg

Neg

2+

Lewis typing: Le(a-b+) suggesting secretor positive status Inference: Parabombay (Oh secretor)

Para-bombay (Oh secretor)  H deficient secretor  Little or no A,B and H antigens

Ulex europaeus (common gorse)

 RBCs may be agglutinated by strong anti-H.  Usually not agglutinated by anti-A and anti-B but may occasionally be

agglutinated by potent antisera or anti-AB  Weak H like antibody (HI) reactive at low temperature almost always present in sera. This is not inhibited by secretor saliva and does not agglutinate cord cells.  Normal H levels in saliva because of secretor status

Bombay categories Classificatio n

Gene

Bombay (Red cell H deficient, non secretor)

Glycosyltransfe rase

Antigens

Secreti on

Antibodies

hh sese None or A A and/or B and/or B (depending on ABO genotype) in RBC or serum

None

None

Anti-A, antiB, anti-H

Parabombay (Red cell H partially deficient, non secretor)

hh (weak variant) sese

A and/or B (depending on genotype)

Weak A and/or B depending on genotype. Weak H in Oh.Residual H if A or B enzymatically removed in others

None

Anti H, anti A and/or anti B (depending on ABO genotype)

Parabombay (Red cell H deficient secretor)

hh Se

A and/or B(depending on genotype), H in serum

Weak A and/or B depending on genotype and weak H

H, A and/or B

Weak IH, anti-A and or anti-B

Bombay blood group not available! Can I transfuse O group cells in Parabombay phenotype?

•(In red cell H deficient secretors) ‘Anti-HI is unlikely to be active at 37oC. ABO-identical blood, compatible by ICT at 37oC, can be used for transfusion.’ •(In H partially deficient non secretors) ‘little information exists on the clinical significance of anti H. Ideally Oh (Bombay) phenotype should be selected, but if not available red cells of the appropriate ABO group (A for Ah, B for Bh) compatible by ICT at 37oC, may be used.’ Source: The clinical significance of blood group alloantibodies and the supply of blood for transfusion. (NHSBT specification SPN214/1.1)

Case 7- ABO HDN

ABO - HDN  Most common cause of HDN  Maternal ABO antibodies that are IgG can cross the placenta and

attach to the ABO incompatible antigens of the fetal RBCs.  Occur more frequently as high-titered IgG antibodies in group O individuals than in group A or B individuals.  ABO HDN is nearly always limited to A or B infants of group O mothers with potent anti-A,B.

 The mother’s history of prior transfusions or pregnancies -

unrelated to the occurrence and severity of the disease.  ABO HDN may occur in the first pregnancy and in any, subsequent pregnancies •Disease is manifested by the onset of hyperbilirubinemia and jaundice within 12 to 48 hours of birth. • Microspherocytes and increased RBC fragility in the infant are characteristic of ABO HDN

 The DAT result is positive in 90 percent of the cases complicated

by jaundice  The bilirubin peak is at 1 to 3 days  Phototherapy is usually sufficient for slowly rising bilirubin levels  With rapidly increasing bilirubin levels - exchange transfusion with group O RBCs may be required

Case 8 – Transfusion and transplantation     

39 year old male with Chronic Myeloid Leukaemia Underwent bone marrow transplant using stem cells from HLA identical sibling Blood group of patient – AB positive Blood group of donor – O positive Received 2 units of compatible O+ve red cells in CMC, 6 days and 3 days prior to transplant

 1 day after transplant blood request for 1 unit packed cells  

 

received. Forward typing of patient sample – AB+ve Backward typing of patient sample –AB+ve Antibody screen – Negative Issued 1 unit of O positive blood crossmatched and compatible till Coombs

 1 day after transfusion haemoglobinuria reported.

DD of haemolysis following haematopoietic stem cell transplantation  Major ABO incompatibility between donor and recipient  Minor ABO incompatibility between donor and recipient  Major incompatibility for other blood group antigens  Minor incompatibility for other blood group antigens  Transfusion of erythrocytes incompatible with donor or patient  Other causes of immune haemolysis eg. auto immune

haemolytic anaemia, drug induced  Non immune haemolysis - TTP

Major incompatibility between donor and recipient  Due to recipient-derived antierythrocyte antibodies  Haemolysis immediately after transplant

 Should be suspected when DCT positive  Eluate must be tested for anti A and anti B to identify

haemolysis due to ABO mismatch

Pure red cell aplasia  Occurs in major mismatch

 Recipient lymphocytes may remain and later produce antibodies  

  

to transplanted donor derived erythrocytes Can occur early or late (>100 days) Suppress erythropoiesis by destroying erythroid progenitors Diagnosis – Based on reticulocytopenia persisting for more than 60 days with absence of erythroids in marrow Delayed erythrocyte engraftment occurs in 20% of ABO mismatched transplants ?related to haemagglutinins

Minor incompatibility  Usually delayed haemolysis  May be due to plasma or ‘passenger lymphocytes’  Plasma in a bone marrow product to be depleted depending

on agglutinin tire.  Diagnosis - DCT positive with eluate showing anti A or anti B

Passenger lymphocyte syndrome  Donor derived lymphocytes in the HPC graft form blood group specific

antibodies to patient erythrocytes

 Usually ABO antibodies but can involve other blood group antibodies.

(Kell, Kidd, Duffy reported)

 Risk factors - Treatment with cyclosporine alone without an

antproliferative agent like methotrexate, low intensity conditioning and ?PBSCT (theoretically, since it contains more B lymphocytes)

 Usually begins 1-2 weeks after transplant, persists for 5-10 days, then

subsides

 Hemolysis can be severe

 Can involve transfused compatible erythrocytes (bystander

immune hemolysis)

 Management - Transfusion of compatible erythrocytes at a pace

that matches hemolysis

 In massive hemolysis - Exchange transfusion may be considered

Incompatibility due to other blood group antibodies  Supected when DCT is positive and antibody screen positive

for donor or recipient  The eluate shows an irregular antibody

Investigations in our patient  Grouping of the patient: AB  Grouping of the donor: O  DCT +ve (2+)  Post transfusion sample – ICT +ve; antibody screen positive

(1+)  Antibody ID – anti Jkb (3+)  Transfused red cells – Jkb positive

• Phenotype of stem cell donor- D+; C4+; E-; c4+; e4+; K-; Fya3+; Fyb -; Jka3+; Jkb3+ • Phenotype of patient – D+; C4+; E-; c4+; e4+; K-; Fya3+mf; Fyb 3+mf; Jka3+mf; Jkb3+mf

 Inference: Delayed haemolytic transfusion

reaction due to anti-Jk(b) formed by patient reacting with transfused cells.  Subsequent transfusions with Jkb negative fully compatible units.  However, haemoglobinuria persisted until 54 days after transplant, when antibody screen turned negative.

Transfusion support in mismatched transplants  Must be compatible with recipient and donor  Such compatible blood must be selected prior to

transplantation (from the start of myeloablation) as red cells may persist for weeks

3 phases of transplantation RBC tx:DCT negative and recipient isoagglutinins not detectable

Beginning of preparation

Phase 1

Phase 2

Transplant Recipient group Compatible with both donor and recipient

Donor group

Phase 3

Plasma and platelets tx: Recipient red cells not detectable

Selection of blood products in phase 2 Recipient RBC group

Donor's RBC group

Category of ABO mismatch†

Erythrocyte transfusion

Platelet or plasma transfusion

A

O

Minor

O

A, AB

B

O

Minor

O

B, AB

AB

O

Minor

O

AB

AB

A

Minor

A, O

AB

AB

B

Minor

B, O

AB

O

A

Major

O

A, AB

O

B

Major

O

B, AB

O

AB

Major

O

AB

A

AB

Major

A, O

AB

B

AB

Major

B, O

AB

A

B

Minor and major

O

AB

B

A

Minor and major

O

AB

A

A

None

A, O

A, AB

B

B

None

B, O

B, AB

AB

AB

None

AB, A, B, O

AB

O

O

None

O

O, A, B, AB

Blood group chimerism  Forward or backward grouping discrepancies and mixed field

agglutination  Intrinsic characteristic of mismatched transplantation eg. A group recipient transplanted with O group donor Anti Anti Anti A -A -B -AB cell s MF

Neg MF

B cell s

Neg 4+

Neg Neg Neg 2+

4+

O cell s

Inference

Neg Mixed field on front type may be due to transfused O cells/ conversion. Recipient red cells still seen. Reverse type is recipient group Neg Front and reverse type donor group, indicating full donor engraftment

Case 9 – ABO mismatch in kidney transplant  A 21 year old gentleman of blood group O-positive, with end

stage kidney disease  Only available related donor was mother whose blood group was A-positive.  HLA-crossmatch by Complement Dependent Cytotoxicity (CDC) was negative  Anti-A titre at presentation was 1:512.  Desensitization protocol involving Rituximab, plasmapheresis and Anti-thymocyte globulin initiated

 Column agglutination test used for monitoring  Schedule of monitoring: Pre-pheresis, immediately post pheresis    

and 24 hours later to detect rebound A group FFP used for replacement in order to quench anti A in recipient. Rituximab interfered with HLA CDC crossmatch. HLA antibody monitoring supplemented with solid phase assays Anti A titre reduced to 1:2 against which transplant performed successfully. 3 years post transplant - graft functional, patient well

Principles of ABOi KT  Removal of existing antibody – plasma exchange, double

filtration plasmapheresis, immunoadsorption  Preventing recurrence of antibody – IVIg, immunosuppression, splenectomy/ Rituximab  Waiting until titres achieve safe levels for transplantation

Range of Plasmapheresis Treatments required Before and After ABOiKT based on initial antibody titre

Lipshutz GS et al. Arch Surg. 2011;146(4):453-458

Monitoring of antibody titre  Baseline antibody levels (> 256) predict severity of

antibody mediated graft injury and graft survival. Reports suggest this is no longer true in patients that received tacrolimus or mycophenolate mofetil for immunosuppression*  Titre cutoff for transplant 16– empirically chosen  Titre endpoint based on IgG or IgG and IgM. *

H. Chung, J.Y. Lee, S. H. Kang et al., “Comparison of clinical outcome between high and low baseline anti-ABO antibody titers in ABO-incompatible kidney transplantation,” Renal Failure, vol. 33, no. 2, pp. 150–158, 2011.

Summary  ABO discrepancy recognition AND resolution is

imperative in the blood bank.  ABO discrepancies can present themselves as a front type

problem, reverse type problem, or combination of both  If ABO discrepancy resolution cannot be performed, and

blood is needed immediately, transfuse with O red cells

If we cannot end our differences, at least we can make the world safe for diversity John F. Kennedy Acknowledgement : Dr. Mary Purna Chacko Asst. Prof: Dept of Transfusion Medicine