Opinion: Selecting a Pathogen Inactivation System

Transfusion-transmitted infectious disease are a continuing threat.  Despite donor infectious marker testing and new donor questioning, there are many threats which are not addressed by these measures.  Also new pathogens are being identified for which there are no tests or specific donor questions available.  How can we handle these new threats?

Pathogen inactivation can significantly reduce infectious agents in blood components, although the degree varies depends on the agent.  Theoretically any agent with nucleic acid—RNA or DNA is affected.  The only class of agents not affected at all are prions, which have NO nucleic acid at all.

In general, a photoactive agent is added to the blood component which binds to the nucleic acid.  Photoactive agents include riboflavin, psoralen dyes, and methylene blue.  Then the component is irradiated, the time proportional to the volume of the unit.

The component is then exposed to ultraviolet light to photoactivate it, which disrupts the DNA and RNA present, including in the white cells.  Thus, NO irradiation or bacterial culture is required.

Here are my questions to consider when selecting a pathogen inactivation system:

Targets?  Platelets vs plasma vs whole blood?

Methylene may be used for plasma, but riboflavin or psoralens may be used for platelets or plasma.  Whole blood inactivation with riboflavin is CE-approved.

Photoactive dye:  Is it riboflavin vs psoralen vs methylene based?

Riboflavin is vitamin B2—the amount used is small and does not need to be removed whereas the psoralen must be removed for clinical use.

Does the photoactive material need to be removed before transfusion?

You can immediately use the riboflavin-treated component but the psoralen must be removed before transfusion—this may take 6 or 20 hours depending on the licensing of the product.

What is the loss of platelets or coagulation factors after treatment?

With treatment by all methods, there is some loss of platelets and coagulation factors.  The platelet loss may be greater in psoralen-based methods and require additional components be added to the pool to reach the desired dose.  Likewise, plateletpheresis components treated with psoralen may require a recalibration of the donor apheresis equipment to collect more platelets per dose to compensate.  There may be some RBC loss additionally in whole blood pathogen inactivation.

What is the efficacy of pathogen reduction for the infectious agents, particularly the ones in your region?

Example:  How well does the treatment handle local agents like Hepatitis E?  Psoralen agents may be less effective than riboflavin for this agent.

Does it work with platelet additive solution PAS?

There are minimum and maximum volumes for pathogen inactivation set by the manufacturer.  Can you get sufficient yields within these volumes?

How good is the data management system?  Can it be integrated with your blood bank computer system?

Can the equipment be integrated with your system?  This is important to set rules and enforce good manufacturing processes GMP.

Does it work well with an automated blood component production system?

Such automated systems like the Reveos can free up personnel for pathogen inactivation.  Can the volumes produced be handled effectively by the pathogen-inactivation method?  Do the timings for separation of components work synergistically with the pathogen inactivation method?

Vendor issues:  how well will the local agent provide support?  Is someone else in your country or region using the system?

You need an experienced vendor to provide optimal support.

Automation and Synergism: Joint Use of Reveos and Mirasol

This is the virtual talk I gave at the Russian Transfusion Congress in Moscow on 13 May. It gives a brief description of automated component processing and riboflavin-based pathogen inactivation and then discusses the use of these technologies together to free up labor and enhance the quality of products, i.e. improved GMP, especially if a dedicated blood bank computer system is used to enforce production rules.

My Opinion: Issues in Transfusion Medicine Software and Component Production

I anticipate that there are several innovations coming or in the process of coming to mainstream blood component production and software.  Some of these I have already addressed in some of my previous posts:

Pathogen inactivation:  We have had this for over a decade.  However, with new emerging pathogens, this will become more important so I expect it will be adopted in many centers where it is not currently being used.  I expect we will close the loop and pathogen-inactivated RBCs will be available so all components will be treated.  Still, the first-generation pathogen-inactivated RBCs may have reduced shelf life compared to regular, untreated units.

Automated component production:  Although this is expensive, it does provide excellent GMP production.  It is fast and may provide higher yields, especially for platelets.  I expect more centers will adopt this technology, especially in combination with pathogen inactivation.

Blood bank computer software:  This software must be considered as dynamically changing, and considerable resources are needed to keep in compliance with ever-changing international regulations and the latest epidemiologic data.  Production rules can be strictly and mercilessly enforced by a dedicated blood bank computer software.  It can also ensure that the final ISBT label is not applied unless all the production rules (registration, collection, processing, and testing) are met.  Manual processing is extremely risky nowadays with all the parameters to be monitored.

Patient Blood Management:  Current blood bank software does not adequately address the need for prospective review of component orders.  I expect that collaboration will occur between laboratory and blood bank software vendors to fill this gap.

Refrigerated platelets:  The pendulum swings back to this component which was used over 40 years ago.  Refrigerated platelets suspended in additive solution may be effective up to 14 days for hemostasis in the trauma setting.  These platelets are activated so standard 20-24C stored platelets may be preferred for prophylactic transfusions.

Low-titer group A universal plasma:  This is already available, but its use will increase because of the low numbers of group AB units available and increased demand.  This includes its production for COVID convalescent plasma.  Your transfusion medical director must decide what “low titer” means.  Also you need a robust way of performing anti-B titers, this may require use of an immunohematology analyzer with titration built-in.

Low titer group O whole blood:  Use of this product may reduce the need for components in massive transfusion settings but it requires performing anti-A and anti-B titers on large numbers of units.  Your transfusion medical director must decide what “low titer” means.  Also you need a robust way of performing anti-A and anti-B titers, this may require use of an immunohematology analyzer with titration built-in.  Also, you must decide whether to leukodeplete the whole blood units:  few whole blood filters are platelet-sparing.

Reveos Interface to Medinfo Hematos IIG

The Medinfo HIIG interface to the Reveos is a bidirectional interface, which was first developed by the Medinfo team for HMC in Qatar.  It is similar to the Atreus interface but there are 4 units processed simultaneously in each cycle of operation whereas the Atreus only processed 1 unit each cycle.  In Qatar it was used in conjunction with Mirasol riboflavin-based pathogen inactivation.

The process is:

  1. Medinfo controls registration, donor screening, and donor collection of whole blood and apheresis-derived (Trima) components.
  2. Medinfo will assign ISBT specimen labels for the whole blood collected with the Reveos blood kit.
  3. Medinfo will not allow processing of whole blood units not meeting donor criteria (donor screening, volumes, collection time, donor deferral database, etc.)
  4. Reveos will read ISBT specimen labels generated by Medinfo.
  5. Upon processing, Medinfo will receive from the Reveos machine the packed red blood cell, plasma, platelet, and buffy coat volumes for each bucket in the Reveos machine.
  6. If the volumes are within the specified ranges, platelet pooling and Mirasol pathogen inactivation of platelets and plasma may proceed.

For each component, the following information will be collected:

  1. All timestamps in the process
  2. Which Reveos machine used
  3. Which bucket in each machine used
  4. Volumes collected (packed RBCs, buffy coat platelet, plasma volume)
  5. Reveos collection set details
  6. Processing technologist ID

The key point is the complete TRACEABILITY of each component throughout its production.  Should there be a failure in production, we can trace exactly where the problem is and then quarantine this and any other affected units simply in the system.  Additionally, this information is part of the permanent record of the unit so it can retrieved subsequently at any time.

Reveos and Atreus Automated Blood Component Processing: My Experience

This post is about my over 10 years of experience with automated component processing using Terumo equipment, first Atreus and then Reveos at HMC Qatar.  The Reveos system is still in use at that institution.  There is also a previous post about Mirasol riboflavin-based pathogen inactivation.

We were the first place in the world to combine the automated component production Atreus with the Mirasol pathogen inactivation.  Their synergism was very important in the rapid throughput of component production for Qatar.

Terumo has two programs, 2C (C for components) to yield plasma and RBCs and 3C for yielding RBCs, plasma, and platelets.  The 2C program is faster but no platelets are separated.

We used the Atreus since 2010 and later replaced it with the Reveos in 2016.  Both systems use a special blood bag set that collects the whole blood in European CPD.  The kit is carefully placed in the machine.  Atreus machines accepted one blood bag set, the Reveos can accept up to 4 sets.  In both cases, the whole blood is processed to yield packed RBCs, leukoreduced plasma (<1E6 residual WBCs), platelets, and a special WBC bag (i.e. the residual buffy coat, which is not for clinical use.

The Atreus took about 10 minutes to process the one bag set whereas the Reveos processes 4 bag sets in slightly more than 20 minutes.  Thus, the throughput from the Reveos is twice that of Atreus.

We had 4 Atreus and later 4 Reveos machines and these were handled by up to 4 technologists, depending on the number of the units.  While the machines were running, the staff were filtering the RBCs and platelet pools, pooling the platelets, and performing the PAS-Mirasol pathogen inactivation.  The workflow was not hectic and staff were not stressed out by the multiple tasks.  Normally 1 staff member ran the Reveos or Atreus machines at any one time.

When the processing was complete, the RBC bags were filtered with an integral leukodepletion filter designed to leave a residual of <1E6 WBCs in accordance with the CE Standard.  The platelets were combined to give a target yield of >= 2.4E11 absolute number of platelets.  Then the pool was leukodepleted by filtration to a residual of <1E6 WBCs.

Both Reveos and Atreus measured the RBC, platelet, and plasma volume yields.  Additionally, for platelets a Platelet Yield Index PYI was calculated as a relative measure of the platelet yield.  To reach a goal of 2.4E11 platelets, the PYI indices for the individual platelet bags were added so that the total exceeded 240.

When combined with the Mirasol system, the component volumes for the plasma and platelets needed to be within specified ranges.  Both systems could easily meet these requirements.

When we switched from Atreus to Reveos, our platelet yields increased.  The transition period was only two weeks.   When we adopted platelet additive solution PAS at the same time, the Reveos had a special program to make “dry” platelets with less volume so that the PAS could be added and still stay within the acceptable range for pathogen-inactivation.

Throughout these years, Terumo sent us special engineers to handle the Atreus then Reveos, Mirasol, and PAS processing.  All staff were trained by Terumo initially before we finalized their competency assessments. 

We had excellent local service:  we never had downtimes due to equipment failures.  During the COVID pandemic, all materials (kits, filters, Mirasol solution, and PAS) have been provided without interruption.

We went live with Medinfo Hematos IIG software for the entire blood donor center and hospital blood banks in 2013.  From the first day 30/6/13 we had bidirectional interfaces first to the Atreus and later to the Reveos—the world’s first.  Likewise, the Mirasol and PAS processing were fully integrated with Medinfo when they were activated.

The residual buffy coat was not used for patient care.  However, it has proven invaluable as a quality control material for the stem cell laboratory.  In addition, many researchers have used it to establish cell lines for investigational use.

Proper handling the collected whole blood units is critical to success:

  1. Maintain the temperature below 25C.
  2. Carefully stack the whole blood units in the blood containers—do not play “ring toss” and just throw them into the container.

In summary, I am very pleased with using this system for over 10 years.  In a few weeks, the production laboratory was fully GMP compliant using a diverse group of staff with varying technical backgrounds.

The following are some pictures of the Reveos and its prepared blood components.

Reveos Machine has 4 chambers to process the 4 whole blood units.
Reveos takes up little space: this crowded corner processed all whole blood for Qatar.
Buffy-coat platelets processed by the Reveos
Close-up of Reveos buffy coat platelets: notice there are NO streaks of RBCs.

International Perspective

When I first moved overseas from the United States, I brought the perspective of my American training and experience.  I saw everything in my new blood bank through those eyes.

Yet, most of my staff were not American or even North American.  Few were even native in English, and most of those  were not American.  They had different qualifications, many of which would not have been accepted by the American schemes.  Still, they functioned well.

I also worked with the US military technologist staff during Gulf War One.  Some did not even have a Bachelor’s degree;  yet, they performed the work well.

I used many technologies that were not yet (or never) US FDA approved such as gel or glass bead typings and pooled buffy coat platelet production.  There were rare reagents I could buy off the shelf (e.g. anti-Tja/PP1Pk).

Later, I adopted pathogen-reduction technology (Mirasol), automated component production (Atreus then Reveos), and platelet additive solution.  I achieve a level of good manufacturing practice that would have been difficult to achieve by the FDA-approved methods.

My perspective had changed.  In the Middle East, I studied many frameworks and came to the conclusion that the best approach was to customize them to our local needs.  My particular experience was to start with one framework, i.e. Council of Europe CE, and then localize it.

To do this, I could not use an American turnkey blood bank software for either the donor or patient operations.  I needed a flexible system that could be customized to my needs.  Again, I chose a CE-marked system, Medinfo Hematos IIG that had already been adapted to many frameworks.

It is much easier to work solely within one system such as FDA.  However, if I had done that, I would have lost so much flexibility and not had a system optimized for local conditions.  I would not have used Mirasol, Reveos, Diamed, and many other reagents.

One big disappointment at such international meetings is the perspective by one country’s regulatory agency that they feel its regulations and framework will work well overseas.  I would wager that those people were not well acquainted with international conditions.

Another frustration was attending another international meeting in which the presenters apologized for the source of information since it came from a foreign country (France) and not their own (United States).

No country has a monopoly on what is best for everyone.  To share our experiences and compare is so valuable.  No one assume his way is the best.  In my career, I have had the richest experiences studying other perspectives and my organizations have benefited greatly from the exchange.  We can all learn from each other.  We are citizens of the world.

Reposted: Granulocyte Concentrates Prepared from Reveos Buffy Coats

The Reveos buffy coat is not approved for clinical use.  In my laboratory, I have offered this discard product to the stem cell laboratory and researchers as a quality control QC and a substrate to extract CD34+ cells for expansion and modification.

In this article (abstract attached) from Transfusion and Apheresis Science 59 (2020) 102682, the authors study pools of ABO-identical Reveos buffy coats for their granulocyte functionality and as a possible emergency replacement for granulocyte concentrates when the latter are not available.

I want to thank Terumo BCT (Brussels, Belgium) for referring me to this article and to the authors for a very interesting paper.

1/1/21

Therapeutic Apheresis Volume Calculations

You can get the values off the therapeutic apheresis machine, but in the middle of the night when you have to write orders, it is convenient to estimate the volumes (whole blood, plasma, RBCs).  These are the values from my lectures to hematology fellows while I was at HMC Doha:

Whole Blood:

Weight in kg X 70 ml/kg = whole (whole) blood volume adult

Weight in kg X 85 ml/kg =whole blood volume for child (prepubertal)

Weight in kg X 100 ml/kg = whole blood volume for neonates/premature

Example:  70 kg adult has 4900 ml blood volume (I round up to 5 liters)

Plasmacrit + hematocrit = 1.00 in fractions (100%), ignore buffy coat volume

Plasmacrit = 1- hematocrit

Plasma volume:

Plasma volume = whole blood volume x plasmacrit = whole blood volume X (1-hematocrit)

RBC volume:

RBC volume = whole blood volume x hematocrit

Estimates for blood components:

The volumes will depend on the original amount collected (e.g. 450 vs 500 ml),  original preservative solution used (e.g. CPD), use of automated component production such as Terumo Atreus or Reveos, use of RBC additive solution (e.g. SAGM), leukodepletion, platelet additive solution, pathogen inactivation.

At HMC Doha, the average values were:

300 ml for leukodepleted RBCs in SAGM prepared by Reveos

300 ml for platelet pools in Mirasol and platelet additive solution (residual WBC < 1E6)

300 ml for plateletpheresis concentrate (2.4E11) in Mirasol and platelet additive solution

250 ml for leukodepleted, pathogen inactivated plasma