The Secrets of Cryoprecipitate: A Blood Banking Process
Expires: October 7, 2022
James Stubbs, M.D., is the Division Chair of Transfusion Medicine in the Department of Laboratory Medicine and Pathology in Rochester, Minnesota. He holds the academic rank of Assistant Professor of Laboratory Medicine and Pathology.
Manish Gandhi, M.D., is a Consultant for Transfusion Medicine in the Department of Laboratory Medicine and Pathology in Rochester, Minnesota. He holds the academic rank of Assistant Professor of Laboratory Medicine and Pathology.
Jessie Swanson, MLS(ASCP), is a Clinical Laboratory Technician in the Component Laboratory for Transfusion Medicine in the Department of Laboratory Medicine and Pathology.
Michelle Soland is a Clinical Laboratory Technician in the Component Laboratory for Transfusion Medicine in the Department of Laboratory Medicine and Pathology.
Contact us: firstname.lastname@example.org.
Transcript and References
Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice in the Department of Laboratory Medicine and Pathology at Mayo Clinic. Transfusion of cryoprecipitate is used for patients who are in need of fibrinogen replacement and occasionally for inherited disorders. In this month’s “Hot Topic” my colleagues from the Component Laboratory, Division of Transfusion Medicine, will discuss the process improvement that helped decrease the standard cryoprecipitate dosage and the positive impact on patients. I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patient’s health care.
Hello, we are Jessie Swanson and Michelle Soland here to talk to you about “The Secrets of Cryoprecipitate: A Blood Banking Process Improvement.”
The objectives of this presentation are:
- What is cryoprecipitate?
- Discuss product storage and requirements for manufacturing
- What are some indications for use?
- Defining the standard dose
- Describe the process improvement of cryoprecipitate in detail.
What is Cryoprecipitate
What is cryoprecipitate? Cryoprecipitate often called “cryo” is a frozen blood product prepared from fresh frozen plasma. It is comprised of cold-insoluble proteins that precipitate when Fresh Frozen Plasma (FPP) is thawed.
It is rich in plasma proteins such factor 8, fibrinogen, factor 13, von Willebrand factor, and Fibronectin. One of those components, factor 8, is also called antihemophilic factor or AHF, which is why cryo is sometimes called cryoprecipitated antihemophilic factor or cryoprecipitated AHF.
Pictured here is a thawed cryoprecipitate pool ready for transfusion. They range in color from pale to dark yellow and can even appear as an even, thick, whitish liquid.
On a history note, current cryo use is far removed from that which it was originally intended.
How is it manufactured?
Let’s have a broad overview of how cryoprecipitate is manufactured:
Cryo is manufactured from Fresh frozen plasma (FFP) collected from whole blood donation. Fresh Frozen Plasma is plasma frozen within 8 hours of collection. Why is freezing plasma within 8 hours significant for making cryo? Research has shown the amount of liable factors degrades over time prior to freezing.
FFP of the same ABO is slowly thawed at 1-6°C until a slightly slushy consistency is achieved. This can be done by thawing in a refrigerator for 18-24 hours or a few hours in a circulating water bath. Here we have a FFP pulled from a water bath. Circled are precipitates that are visible prior to centrifugation. They are opaque white in appearance.
Once the plasma is thawed, the liquid plasma is separated from the cryoprecipitate by centrifugation, utilizing a “heavy” spin. This is done to concentrate the precipitates to the bottom of the bag. The precipitates will have a thick, white to opaqueish consistency. It can be mistaken as a fibrin clot.
After centrifugation, the supernatant is removed by gravity drain or plasma expressor. Approximately 10-15mL of supernatant plasma remains with the cryoprecipitate and is used to re-suspend the precipitates. At this point the cryo may be refrozen immediately or pooled. Pictured on the left is a single cryoprecipitate containing approximately 25-30 grams of supernatant and the precipitate pellet. Circled are the precipitated factors.
The supernatant plasma, pictured on the right, can be refrozen as a transfusable product called cryo-poor plasma or as a non-injectable product called recovered plasma. Both cryo-poor plasma and recovered plasma are deficient of those cryo factors. Cryo-poor plasma is an injectable product that is typically used for plasma exchange. Recovered plasma is an unlicensed product that can be shipped to manufactures of plasma products.
To create a pool, single cryo are welded onto a pooling harness bag set. The process of welding is sterile and allows the product to be a closed system. The single cryo are pooled to provide a single, combined therapeutically effective blood product, a cryoprecipitate pool. Pictured is a 5-unit pool.
Now, let’s discuss the storage and shelf life of cryo. Cryoprecipitate is stored at negative 18°C or colder, and has a shelf life of 1 year. For pooled cryo, the expiration date is determined to be 12 months from earliest date of FFP collection.
To prepare cryoprecipitate for transfusion, it is thawed quickly at 30-37°C and then stored at room temperature; 20-24°C. Thawed single cryo and pooled cryo which are sterilely manufactured have a shelf life of 6 hours. Currently thawed cryo cannot be refrozen or refrigerated, due to regulatory standards. Fun fact: refrigeration causes the factors to re-precipitate making it unsuitable for transfusion.
Each cryo product has to meet regulatory agency requirements. The Food and Drug Administration (FDA) requires cryo to be quality control tested “on at least four representative containers each month.” Each unit must contain a minimum of 80 international units of Factor 8 and a minimum of 150 mg of fibrinogen times the number of units in the pool per AABB standards.
For example, Mayo Clinic Rochester manufactures pools of 5 units, therefore, the QC values we need to pass are greater than or equal to 400 international units per pool of Factor 8 and greater than or equal to 750mg per pool of fibrinogen.
Indications for Use
Cryoprecipitate was originally prepared as a source of Factor 8 for hemophilia A patients and to treat patients with von Willebrand’s disease. It is no longer the preferred product for treatment of hemophilia A and von Willebrand’s disease as coagulation factor concentrates are readily available.
Patients that benefit from cryoprecipitate are typically patients needing fibrinogen replacement when clinical bleeding has occurred and individuals with disseminated intravascular coagulation (DIC). Occasionally, cryo is transfused for inherited disorders of fibrinogen and uremic bleeding. Another indication for cryo is during a massive transfusion, in which it helps maintain hemostasis due to post-partum hemorrhage, and surgical cases such as cardiac, gastrointestinal, transplants, or trauma incidents.
Just like other blood products, it is subject to misuse. Common misuse of cryo is reversal of warfarin therapy, replacement therapy in patients with normal fibrinogen levels, and treatment of bleeding without evidence of fibrinogen deficiency.
It can also be underutilized in massive transfusions with dilution coagulopathy and bleeding.
Selection and Administration
When choosing cryoprecipitate for transfusion, cryo should be selected as ABO-compatible. In some instances, large volumes of cryo may cause a positive direct antiglobulin test and mild hemolytic transfusion reactions. This can be caused by the isohemagglutinins, anti-A and anti-B.
However, cryo can be administered regardless of Rh. This is due to the fact neither plasma nor cryoprecipitate contain red cells. Compatibility testing is also not required.
Transfusion of cryoprecipitate carries the same risks as plasma transfusion. Potential risks and adverse events such as infection- viral or bacterial, volume overload, and transfusion reactions. Cryoprecipitate has a lower risk of causing hemolytic transfusion reactions than plasma, because of the smaller volume administered.
Cryo is administered utilizing aseptic technique, through a filter designed to remove clots and aggregates. Transfusion is started before cryo expires and completed within 4 hours.
The standard adult dose of cryoprecipitate at Mayo Clinic Rochester is 10 units or 2 pools with each pool containing on average 277mg/unit or 1389mg/pool of fibrinogen.
External suppliers, such as the American Red Cross, provide blood products to other Mayo campuses. These sites are producing cryo pools of 5 units at dosages >300 mg/unit or >1500 mg/pool.
This allows for other Mayo campuses to drop their standard adult dose from 10 units to 5 units.
Mayo Clinic Rochester recognized the need to standardize and improve their concentrations to decrease the patient dose. The goal was to drop the adult dose from 10 units to 5 allowing ordering consistency across all Mayo sites.
Now that you some background information of cryoprecipitate requirements and indications, let’s learn about a case study presented by Michelle.
Thank you Jessie. A middle aged man underwent a re-operative aorta surgery. This surgery required a long cardiopulmonary bypass run and cooling of the patient to 18°C for a good duration. The patient started to bleed a lot, and like most, their new graft caused depletion of fibrinogen levels significantly. In the end, the patient was transfused 40 units of cryo.
That’s 5 donors in each pooled cryo. 200 FFP were manufactured into 200 single cryo, and were used to create 40 pools. That means this patient was exposed to 200 donors!
Therefore, if we benchmarked a process improvement, then this patient could have had fewer products, less donor exposure, and less inventory used on one individual.
Now, let’s talk about this process improvement project that took place in the Component Laboratory at Mayo Clinic Rochester.
Process Improvement: Thawing
We experimented 12 different process changes including, but not limited to centrifuge settings, thaw rates, and differing product manipulation. Our group also toured other product manufacturers to see their processes.
We re-evaluated multiple steps in the process, starting with implementing new thawing techniques.
Fresh Frozen Plasma varies in volume due to various donor factors, it is important to thaw them evenly. Even thawing helps prevent some FFP from being completely thawed while another FFP could be icy in the batch. Too slushy or icy of a product is difficult to manipulate. To increase this chance that your products will thaw evenly, the process is now defined to choose FFP that are within 20g of each other.
We defined a “desired consistency” of thawed FFP. For best recovery, they should be thawed to a slushy or slightly slushy state. Therefore, impacting Factor 8, as it is a temperature sensitive product.
We also defined thaw time in the water bath. As a reminder, cryo precipitates are cold insoluble proteins, therefore, it is important to closely monitor thaw. We implemented this by checking the FFP at approximately 1 hour 30 minutes and adding additional time as needed. When checking the products keep manipulation of the product to a minimum, we don’t want to re-suspend factors prior to centrifugation. Heat is also an important factor that can negatively affecting the recovery.
Process Improvement: Centrifuge
Next step we investigated was centrifugation. Factors such as time, speed (RPM), and brake settings alter the force, applied to the precipitates. We also investigated the fact since we have a chilled centrifuge, we should also chill the cup liners to prevent further unnecessary heat transfer. Here in the component laboratory we have access to two different centrifuges for manufacturing so it is also important to have the settings match each other.
As a result of testing we increased RPM, time and adjusted brake settings for both centrifuges, allowing products to spin faster and longer to get the most precipitates collected to the bottom of the product bag.
Process Improvement: Separation
The next change was looking at separation of cryo precipitates and byproducts. Previous manufacture separation of cryo was by inversion and draining. We noticed any free precipitates that did not get compacted into the pellet would drain into the supernatant. To counterbalance this, we created a shelf for the spun FFP to lie on and gravity drain horizontally. This helps prevent the free precipitates from draining and instead stay in the precipitates bag.
We further emphasized keeping the product cold by laying the precipitate bag on top of coolants on a raised shelf while it’s draining.
We increased single cryo weight to 25-30g with an optimum of 28g. The helps ensure we are only adding 10-15mL of supernatant to re-suspend the precipitates prior to pooling. Increased weight also aided in removing all the precipitates from the product bag.
Process Improvement: Pooling
The next process we re-evaluated was pooling. Pooling ensures 5 single cryo are sterilely connected without being exposed to air or contaminates.
This video shows the process of connecting a single cryo to a pooling set. This instrument works by placing tubing from two separate products into a clamp. Then a wafer is heated, this slices the tubing while maintaining sterility, and welds them together without any contamination. This process is repeated until all five single cryo are connected to the pooling harness set. Changes to the pooling process included the use of a scraper, no rinsing, and reducing heat transfer.
This second video shows the process, which implemented the use of a scraper, to mix the cryo precipitates and plasma. Using a scraper, each single unit is scraped and mixed until the precipitates are re-suspended as shown. Then we visually inspect each bag. This is done to ensure there are no precipitates stuck to the product bag. If precipitates are found, further mixing with the scraper is required. The single cryo are then drained by rolling into the pooling bag. This process is repeated for all five single cryo units.
The implemented process also advises no rinsing of the products unless there are precipitates that need to be further mixed and put into solution. This further reduces manipulation of the product between bags and prevents loss of product in tubing lines and product bags.
The previous manufacturing process required manual manipulation with our hands. Re-suspending the precipitates was done by massaging the single cryo with our hands. Therefore, the new process of using a scraper for mixing reduced hand heat transfer. The precipitated factors are greatly affected by temperature, as exposure to excessive heat will significantly decrease the efficacy of the final product.
As you can see in the video, we now roll the bags empty to make sure all the products are removed from the individual bags into the pooling harness set bag. Excess air is expressed from the final bag to prevent bag brakeage when it’s refrozen, and the pooling bag is heat sealed off, thus creating a 5 unit cryo pool.
This new manufacturing process was validated to an increased average fibrinogen of >= 1,450mg per cryo pool and was implemented November 5, 2018.
Part of the ongoing process improvement, we are continuing to monitor our results since implementation. Pre-intervention of our Factor 8 average results were 823 International Units per pool. Post-intervention of Factor 8 average results are 782 International Units per pool. We are not as concerned about this decrease because it is still well above regulatory requirements. Cryoprecipitate is not typically used for the treatment of Factor 8 deficiencies because factor 8 concentrates are readily available for use. However, these values will continue to be monitored.
Pre-implementation fibrinogen average results were 1,389 mg per pool. Post-implementation fibrinogen average is now 1574mg per pool.
Process Improvement Conclusion
In conclusion, the process improvement will help standardize the cryoprecipitate dosage and benefit the patient, as the needs of the patient come first.
The process change lead to decreasing adult standard dose of two pools to one pool per transfusion. Furthermore, it reduced donor exposure from 10 donors to 5 donors. Exposure to multiple donors creates an increased incidence of transfusion reactions and potentially a higher risk of disease transfer. The process change might also increase inventory, which is especially helpful when cryo usage is higher than normal. It could also be potentially cost saving for patients, when they would only pay for 1 cryo pool, and shorter transfusion time.
With continual monitoring and potential improvements the dosage change was recently effective.
To summarize what we presented:
- Cryoprecipitate is cold insoluble proteins that precipitated from FFP.
- Frozen cryo is stored at negative 18°C or colder and a shelf life of one year.
- Thawed cryo is stored at 20-24°C. A closed system cryo pool has a shelf life of 6 hours.
- Indications for use are for patients needing fibrinogen replacement and occasionally for inherited disorders.
- Standard dose is now 5 units of single cryo or 1 cryo pool.
We hope you enjoyed the presentation. Here are our references, for your prospective process improvement ideas and educational reading! We hope you found this presentation informative.
- McCullough, J: Transfusion medicine. Third edition. West Sussex, UK: John Wiley & Sons, Ltd. 2012.
- Menitove J., Spero J, Richards W, et al: Pre-pooled cryoprecipitate for treatment of hemophilia A. Transfusion 1983 May-Jun;23(3):265-7.
- Shaz B, Hiller C: Transfusion medicine and hemostasis: clinical and laboratory aspects. Second edition. San Diego, CA, Elsevier Science, 2013.
- Slichter S, Counts B, Henderson R, Harker L: Preparation of cryoprecipitated factor VIII concentrates. Transfusion 16:616-626, 1976.
- Standards for Blood Banks and Transfusion Services. Thirty-first edition. Bethesda, MD: American Association of Blood Banks,
- Taswell H, Saeed S, Committee on Quality Control: Principles and practices of quality control in the blood bank. American Association of Blood Banks, 1980.
- Technical Manual. Nineteenth edition. American Association of Blood Banks, 2017.
- United States, Office of Federal Register: Code of federal regulations, title 21, parts 600 to 799 food and drug. Washington: Office of the Federal Register, National Archives and Records Administration. 2017.