Questions and Answers on Proteolytic Enzymes Used in Blood Group Serology

A Blog from Eric Ching:

Questions

• What are the common proteolytic enzymes used in antibody identification?
• What are the uncommon proteolytic enzyme used in reference laboratories?
• What blood group antigens are sensitive to enzyme treatment?
• What antibodies are usually enhanced by enzyme?
• How do they enhance antibody uptake?
• How does an enzyme abolish polyagglutination?
• Why are enzyme treated red cells no longer used in routine antibody screen?
• What is the difference between a one-stage and two-stage technique?
• Why would over treatment of red cells by proteolytic enzymes cause self agglutination?
• Other than enhancing antibody reactivity and denaturing blood group antigents, are there any other applications for enzymes?
• Should we prepare proteolytic enzymes “in house”?
• Must enzyme IAT be read macroscopically?

Answers

What are the common proteolytic enzymes used in antibody identification?
Ficin, papain and bromelin are commonly used. Ficin and papain are extracted from full grown but immature green fig, papaya latex while bromelin is prepared from crushed pineapple cores.  The raw material is dried and marketed in powder forms and may contain other enzymes.

What are the uncommon proteolytic enzyme used in reference laboratories?
Neuraminidase aka sialidase (Vibrio cholerae), pronase (Streptomyces griseus), trypsin (porcine stomach), chymotrypsin (bovine/porcine pancreas) and others.

What blood group antigens are sensitive to papain/ficin treatment?
M,N,S,Fy^a,Fy^b
En^a(TS, FS), Pr, Ch^a, Rg, JMH, Yt^a, T, Tn, Mg, Mi^a/Vw, Cl^a, Je^a, Ny^a, Lu8,Lu14, In^a, In^b, Xg^a and others

What antibodies are usually enhanced by papain and ficin?
Detection of antibodies to the following blood group systems are usually enhanced by papain and ficin:
ABO, Hh, Rh, Lewis, Kidd, Ii, P, Globoside, Colton and Dombrock
 
How do they enhance antibody uptake?
Proteolytic enzymes are “serological lumberjacks” that cleave specific peptide bonds of transmembrane glycoproteins where some blood group antigens reside. Notably the glycophorins (I refer them as trees) that house the MNS blood group antigens. These hydrophilic glycoproteins attract water molecules to form a hydration shield to keep the antibody from interacting with the hydrophobic antigens closer to the red cell surface in the first stage of antibody, antigen interactions- sensitization.  In addition, the sialic acid residues aka N-acetyl neuraminic acid (NANA) (I refer to them as leaves) attached to the glycophorins are also removed to allow the red cells to come closer in the second stage of cell-cell interaction. This phenomenon can be demonstrated by the use of pure IgG anti-D can directly agglutinate enzyme-treated Rh+ red cells suspended in saline.

Another point of interest: the thermal range of cold agglutinin can be widened by enzyme-treated red cell owing to the increased antibody uptake and enhanced agglutinability.

How does an enzyme abolish polyagglutination?
Polyagglutination is a phenomenon of patient’s red cells agglutinated by almost all normal human sera. It is a result of a viral or bacterial enzyme neuraminidase (sialidase) to expose the hidden antigen (T being the most common) which is readily recognized by antibodies found in normal sera. These crypt or hidden antigens ( tetra saccharides or four sugar residues composed of 2 sialic acids, galactose and N-acetyl galactosamine which I refer them as leaves, are attached to the glycophorin trees), enzymes such as ficin or papain can chop off the glycophorins thereby abolishing polyagglutination. The use of neuraminidase (sialidase) can do the same job itself as they are able to nip just the leaves off.

Why are enzyme-treated red cells no longer used in antibody screen?
Before the mid-70's, many blood banks used enzyme treated red cells as an adjunct with untreated red cells in routine antibody screen to increase sensitivity. However, with decreased specificity due to cold and warm autoantibody and the enhanced sensitivity offered by LISS and later PEG, “enzyme screen” were eventually dropped. However, enzyme technique is still very useful in resolving complicated antibody combination cases.

What is the difference between a one-stage and two-stage technique?
One stage enzyme is the sequential addition of serum, cell and enzyme before incubation.
Two stage method is to enzyme treat red cells, wash off enzyme before adding serum or plasma. This is a preferred method as there is a chance that an antibody has already attached to the antigen before enzymatic reaction begins. This may theoretically, reduce effectiveness. Commercial ficinized panels belong to the two stage technique. 

Why would over treatment of red cells by proteolytic enzymes cause self agglutination?
Over treatment of proteolytic enzyme cleaves sialic acid residues found on the membrane glycoprotein and glycolipids causing self agglutination. Under electronic microscope , papain treated red cells found to be deformed and the surface appeared “stringy and corrugated”.

In general, proteolytic enzymes can modify red cell membrane to different levels of intensity:

1. Enhance reactivities of some antibodies
2. Denaturation of some antigens
3. Self agglutination with inert serum
4. Self agglutination in saline
5. Lysis

Other than enhancing antibody reactivity and denature blood group antigens, are there any other enzyme applications ?
Enzyme treated red cell are useful in the following situations:

1. Enzyme treated red cells are more likely to be hemolyzed by pathological auto anti-I at 20⁰C
2. Low level warm reactive autoantibodies may mimic alloantibodies in IAT, panagglutination in an enzyme panel can reveal their interference.
3. Enzyme treated autologous red cells are more efficient to remove both cold and warm reacting autoantibody.
4. Diagnosing polyagglutination.

 Note:

• Possible association of auto papain among children with scoliosis- just personal unpublished observation, no useful application.
• In the era of using both papainized and untreated red cells routinely for antibody screen in the 1970’s, most “auto pap”  identified were taken from pre-op samples from the local children’s hospital. I wonder if colleagues from my generation had experienced similarly?!  

 Should we prepare proteolytic enzymes “in house”?
In light of the cGMP environment, clinical laboratories should avoid preparing home made reagents as much as possible. Ficin and papain panels are available commercially. However, if money is tight, one can purchase freeze dried papain, reconstitute to 2 ml at a 1%  solution, qs to 20 ml with PBS @ pH7.3  and prepare 10 2 ml aliquots. They can be stored at -30^oC for up to 12 months. Internal QC and validation must be documented for this money saving deviation.

Must enzyme IAT be read macroscopically?
Although it is advisable to read enzyme IAT macroscopically for routine testing, it may be useful, especially in experienced hands, to decipher valuable information when macroscopic reading is negative but microscopic is positive with a SPECIFIC PATTERN. For example, if only Jk(a+b-) panel cells are positive, this may indicate an early phase of a delayed hemolytic transfusion reaction in which the antibody level has just begun to rise. A fresh sample taken in the following morning may give a straight forward specificity.  Another “trick” to enhance a possible weak anti-Jk^a is the use of 4:1 papain or ficin IAT with polyspecific AHG in hope the anti-complement may help. Fresh serum sample must be used in this case.
 
Suggested Further Reading

• Use of uncommon enzymes to differentiate rare alloantibodies: The Blood Group Antigen Facts Book by Marion Reid and Christine Lomas-Francis
• Standardizing Enzyme Procedures: AABB Technical Manual
• Evaluating enzyme-treated red cells: AABB Technical Manual

 
 Your comments are encouraged!!