Date:     May 29, 1996

From:     Director, Center for Biologics Evaluation and Research

Subject:  Recommendations and Licensure Requirements for
          Leukocyte-Reduced Blood Products

To:       All Registered Blood Establishments


I.   INTRODUCTION

During the past 10 years, leukocyte-reduced blood and blood
products have played an increasing role in the management of a
variety of transfusion-related adverse reactions and diseases. 
In the United States, approximately 20% of the blood products
administered are supplied or infused as leukocyte-reduced
products.1

Recent advances in leukocyte reduction technology have
contributed to the ease and effectiveness of removing leukocytes
from blood and blood products including Whole Blood, Red Blood
Cells (RBCs), Platelets, and Platelets, Pheresis.  These
developments have also generated the need for guidance as
questions have arisen related to terminology, quality control,
product standards, efficacy claims by manufacturers, optimal
conditions for leukocyte reduction, and the clinical need or
benefit of using leukocyte-reduced products.

This memorandum provides general recommendations and explains
licensure requirements in order to assist blood establishments in
preparing leukocyte-reduced blood and blood products by methods
consistent with current industry standards and practices and to
facilitate licensure of these products.  The document also serves
as a basis for future discussions with blood establishments,
manufacturers, researchers, and other interested parties.

With developments in leukocyte reduction technology ongoing,
practices and standards are subject to change based on additional
research data.  Accordingly, the recommendations in this
memorandum are evolving and are intended as guidance only.  This
memorandum does not bind the agency or create any rights,
privileges, or benefits for, or in, any person.  However, it does
represent the agency's current thinking with regard to preparing
leukocyte reduced blood products in blood establishments.


II.  BACKGROUND

Recently, filtration and improved cytapheresis technologies
without secondary processing have been increasingly used for the
preparation of leukocyte-reduced blood products.
Based on the current standard of practice in transfusion
medicine, many physicians use leukocyte-reduced blood products
for a variety of clinical indications.  Blood products subjected
only to leukocyte reduction techniques have been implicated in
cases of transfusion-associated graft-versus-host disease
(TA-GVHD), and therefore, leukocyte reduction does not always
prevent TA-GVHD.2  Gamma irradiation of blood products containing
viable lymphocytes continues to be the most appropriate approach
to the prevention of TA-GVHD.3

III. RECOMMENDATIONS FOR MANUFACTURING

All leukocyte-reduced blood products should be manufactured and
processed following applicable current Good Manufacturing
Practices (cGMPs) codified in Title 21, Code of Federal
Regulations (21 CFR), Parts 600 through 680 and Parts 210 and
211.  Leukocyte filters used for manufacturing are usually
product-specific and are not interchangeable.  In addition, blood
products that are leukocyte-reduced by filtration as part of
manufacture (prestorage leukocyte reduction) should continue to
be transfused with an administration set containing a standard
blood filter.
 
Leukocyte-reduced products may be prepared in blood
establishments after blood collection and prior to storage. 
Although the optimal time and temperature for leukocyte reduction
has not been established, several researchers suggest that
prestorage leukocyte removal may have several advantages over
filtration immediately prior to transfusion.  Benefits of
prestorage leukocyte reduction are reported to be fewer toxic and
immunogenic cell products, possible elimination of the
immunomodulating effects of blood transfusion on tumor growth
enhancement, lower levels of pro-inflammatory cytokines and
partial removal of microbial contaminants.4-14  Furthermore, when
leukocyte reduction is performed in blood establishments rather
than at the bedside, product quality control may be standardized,
and inventories of leukocyte-reduced blood products may be
maintained. 

A.   Prestorage Preparation of Leukocyte-Reduced Blood Products

     1.  Closed Systems

     The closed system can be achieved by apparatus that
     includes, but is not limited to: 
     (a) manufacturing with an in-line filter included in the
     blood bag collection set; (b) attaching a leukocyte
     reduction filter to a blood component container, collection
     set or a cytapheresis collection set with a Food and Drug
     Administration (FDA)-cleared Sterile Connecting Device
     (STCD);15  (c) using a functionally closed system such as a
     platelet collection system that is totally closed at the
     point of manufacture except for preconnected solutions
     and/or preconnected FDA approved bacteriostatic filters on
     spike and port connections; or  (d) preparing a product such
     as Platelets, Pheresis with cytapheresis equipment that does
     not require the addition of a filter system in order to
     produce a leukocyte reduced product.  If the closed system
     is maintained, the expiration date of the final leukocyte
     reduced product remains the same as that of the original
     product.
    
     2.  Open Systems

     The open system involves attaching a filter to one of the
     ports on the blood bag collection set using aseptic
     technique.  After entering the container in this manner, the
     red blood cells have a 24-hour expiration date when stored
     at 1-60 C.  Platelets that are pooled and then filtered have
     an expiration date of 4 hours after entering the containers
     to pool the product.16

     3.  Platelets, Pheresis

     The expiration date of the leukocyte-reduced platelet
     product will depend upon whether the platelet product was
     collected in an open system or a functionally closed system
     as described above.  External needles or satellite bags may
     only be attached to these systems using an FDA-cleared STCD. 
     Platelets, Pheresis collected in an open system expire 24
     hours from the termination of the procedure.  Platelets,
     Pheresis collected in an approved 5 day functionally closed
     system expire on midnight of the fifth day.17

B.  Standard Operating Procedures (SOPs)

The SOPs for manufacturing leukocyte-reduced blood products
should include the following information:

1.   Name and manufacturer of the equipment used for leukocyte
     reduction.  Prestorage filters or direct cytapheresis
     equipment should be FDA-cleared for the specific product, 
     manufactured and intended for use in prestorage leukocyte
     reduction.  Filters intended for bedside filtration should
     not be used for pre-storage filtration.

2.   Leukocyte reduction procedures consistent with
     manufacturers' instructions including quality control
     procedures.  Methods of investigation of quality control
     failures.

3.   Time period when leukocyte reduction is performed, i.e.,
     during the initial 8-hour room temperature hold or after
     refrigeration consistent with manufacturers' instructions. 

4.   A description of all procedures related to the use of STCDs
     when these devices are used in leukocyte reduction.

5.   Procedures for handling units that do not meet the criteria
     for labeling the product as leukocyte-reduced, following the
     leukocyte reduction procedure.


IV.  LABELING

Previous labeling for leukocyte-reduced products included the
terms, "Leukocytes Removed," "Leukocyte Poor," and "Leukocyte
Depleted" which are no longer considered appropriate by FDA.  The
following labeling reflects the current terminology approved by
the FDA for leukocyte-reduced products.

A.   Whole Blood
 
Whole blood products prepared by a method known to leave a
residual leukocyte count of less than 5X106 per container and
retain a minimum of 85% of the original product should be
labeled, " Whole Blood, Leukocytes Reduced," including
appropriate modifiers, e.g., CPD Whole Blood, Leukocytes Reduced,
etc.  The product code formerly used for "Whole Blood, Leukocytes
Removed" should be used for "Whole Blood, Leukocytes Reduced."

B.   Red Blood Cells

Red blood cell products prepared by a method known to leave a
residual leukocyte count of less than  5X106 per container and
retain a minimum of 85% of the original product should be
labeled, "Red Blood Cells, Leukocytes Reduced", including
appropriate modifiers, e.g., CPD Red Blood Cells, Leukocytes,
Reduced, etc.  The product codes formerly used for "Red Blood
Cells, Leukocytes Removed" should be used for "Red Blood Cells,
Leukocytes Reduced."

C.   Platelets 

Platelet products prepared from whole blood by a method known to
leave a residual leukocyte count of less than 8.3 X 105 per
individual container and retain a minimum of 85% of the unpooled
product should be labeled "Platelets, Leukocytes Reduced."  
Leukocyte reduced pooled platelet concentrates are not considered
products licensable by the FDA.

D.   Platelets, Pheresis

Platelets, Pheresis products prepared by cytapheresis without
secondary processing (such as filtration) having a residual
leukocyte count of less than 5 X 106 per container should be
labeled, "Platelets, Pheresis, Leukocytes Reduced."  Platelet
Pheresis products, leukocyte reduced by filtration with a
residual leukocyte count of less than 5X106 per container and
retaining a minimum of 85% of the original product should be
labeled, "Platelets, Pheresis, Leukocytes Reduced."

Products further modified from the original products listed
above, e.g., Irradiated, Divided, etc. should put the additional
words after "Leukocytes Reduced" on the label to indicate the
modification.


E.   Circular of Information

The Circular of Information constitutes labeling for blood
products and contains indications, contraindications, side
effects, hazards, dosage, and administration of leukocyte-reduced
products.18  This information is regularly updated by the blood
industry and is subject to approval by the FDA.   Currently,
blood establishments should add an addendum to the Circular of
Information to meet the standards defined in this memorandum. 
Licensed establishments should submit this information with their
product license application (PLA).

V.   QUALITY CONTROL PROCEDURES

Process validation is a requirement of the Current Good
Manufacturing Practices Regulations for Finished Pharmaceuticals,
21 CFR Parts 210 and 211, mentioned above, and of the Good
Manufacturing Practice Regulations for Medical Devices, 21 CFR
820, and therefore, is applicable to the manufacture of
pharmaceuticals and medical devices.19  Manufacturers of
leukocyte reduced products should perform extensive quality
control (QC) procedures to assure that leukocyte reduction
equipment meets specifications and labeling claims.
  
In addition, manufacturers should continue to generate data to
support the claims that their production process remains stable
and consistent over time and any variation does not exceed
product specifications. 

It is essential that the validation program is documented and
that the documentation is properly maintained at the
manufacturing site.19

Current Good Manufacturing Practices (cGMPs) for blood and blood
product manufacturers require that end users develop and follow
written QC procedures to verify that the technology has been
applied correctly and that finished blood products meet
established criteria.  QC procedures should include periodic
monitoring of residual leukocyte counts using validated methods,
described below in section C, that have the appropriate
sensitivity to consistently detect the limits of expected numbers
of residual leukocytes and to accurately measure the quantity of
the original product remaining after leukocyte reduction.   

A.  Samples

A representative sample should be collected from leukocyte
reduced products by using an FDA- cleared STCD to attach
additional tubing or a suitable storage container, or from
freshly stripped and filled tubing segments.  Device
manufacturer's directions or validated sample collection
procedures should be followed.  Samples should be handled,
prepared and processed without delay according to the
requirements of the counting method to ensure that a true and
representative count is obtained. 

B.   Sampling Plan

     Each blood establishment should develop an appropriate
     sampling plan for testing leukocyte-reduced products.
     Establishments may sample and count the total required
     number of donor units per month from units collected within
     the previous 30 days.  The sampling plan should include
     testing a minimum of  1% of the establishment's monthly
     production of each leukocyte-reduced blood product (Whole
     Blood, Red Blood Cells, Platelets, or Platelets, Pheresis),
     or for establishments producing under 400 units per month, 4
     units per month of each leukocyte-reduced blood product.20 
     All units tested (100%) should meet the standard as
     described in the section on labeling for use as a
     leukocyte-reduced blood product.  Units which fail to meet
     the minimum criteria may be used for transfusion provided
     that a closed system is maintained and the products are not
     labeled as leukocytes reduced.  Products should not be 
     refiltered routinely as this may cause additional loss of 
     the original product intended for transfusion.  The sampling 
     plan should provide a level of confidence that assures a 
     high probability of detecting errors in application of 
     leukocyte reduction technology.  The units selected for 
     counting should be selected at random to adequately 
     represent the larger population of units from which they 
     were selected.21 

C.   Counting Methods

     Leukocyte reduced blood products should contain less than 5
     X 106 residual leukocytes, except for platelet concentrates
     which should contain less than 8.3X105 residual leukocytes. 
     Special methods of cell counting are required to detect such
     low numbers of leukocytes.  Automated hematology cell
     counting devices may not accurately determine the residual
     leukocytes in leukocyte reduced products.  The counting
     methods that should be used for evaluating low numbers of
     residual leukocytes are as follows:

     1.  Manual methods:

          Methods using a Nageotte counting chamber and Turk's or
          Plaxan's staining solutions alone or in conjunction
          with Zap-oglobin , or centrifugation are comparable in
          sensitivity and reliability for counting less than 5
          leukocytes/uL.22-28

     2.   Automated method:

          Flow cytometry, using propidium iodide alone or
          combined with thiazole orange, compares favorably in
          sensitivity to the Nageotte chamber method and provides
          the advantages of automation and the ability to
          determine phenotypes of residual leukocytes which may
          be of interest to clinicians.25,29  

          Other validated methods may also be acceptable provided
          that they achieve the desired level of sensitivity for
          counting the residual leukocytes. 


D.   Expected Results

Leukocyte reduced products meet the standard for leukocyte
reduction when less than 5 X 106 leukocytes per container (unit)
remain after preparation and a minimum of 85% of Whole Blood or
Red Blood Cells is retained.  Individual Platelet concentrates
meet the standard when less than 8.3 X 105 leukocytes per
container remain after preparation and 85% of the product is
retained.  Platelets, Pheresis products, leukocyte reduced by
filtration meet the standard when less than 5 X 106 leukocytes
per container remain after preparation and 85% of the product is
retained.  Platelet Pheresis products prepared by cytapheresis
without secondary processing (such as filtration) meet the
standard when less than 5 X 106  leukocytes per container remain
after preparation.  The counting method validation should include
consideration of the time between blood sampling and counting. 
The units tested should meet the established criteria defined in
the sampling plan so that product specifications meet the
requirements for safety and effectiveness in accordance with the
product label as a leukocyte reduced product.  Standard operating
procedures (SOPs) should include procedures for handling units
that fail QC testing and for initiating an investigation of the
failures so that corrective action can be instituted. The results
of the investigation and corrective action should be documented. 
Units that fail QC testing but retain a minimum of 85% of the
original product may be used as non-leukocyte reduced products
provided a closed system is maintained and they are not labeled
as leukocyte reduced.  They should not be routinely reprocessed
to remove leukocytes.  If QC testing and evaluation of the
leukocyte reduction process indicates that the process is no
longer stable and/or not capable of meeting the criteria for
leukocyte reduced products, blood establishments should examine
the preparation and processing of leukocyte reduced products and
proficiency in the use of the counting method.  If necessary,
technical advice should be sought from the cytapheresis equipment
or filter manufacturer.

VI.  TRAINING/CONTINUING EDUCATION

Staff preparing leukocyte reduced products should be well-trained
to perform their duties and retrained, as needed, on a continuing
basis.  Training should include the following:

1.   General information about product handling and consequences
     to recipients if the products do not meet the
     specifications.
2.   Preparation methods.
3.   Collection, storage, preparation and stability of quality
     control samples.         
4.   Labeling procedures.
5.   Quality control testing procedures.
6.   Equipment preventive maintenance.
7.   Routine in-service education.

VII. RECORDS

Records must be maintained according to 21 CFR 606.160 and
reviewed to assure that an accurate history of all work
performed, training, preventive maintenance, quality assurance,
and continuing education are maintained.  Additionally, records
should include a list of filters used; their identification or
lot numbers; dates received; dates of expiration; filter
manufacturers' package inserts and validation data for the
leukocyte counting method used.

Incident logs and preventive maintenance records should be kept
for all cytapheresis equipment.

VIII.     LICENSURE REQUIREMENTS

Pursuant to 21 CFR, Part 607, any establishments routinely
manufacturing leukocyte reduced blood products must register (if
not already registered) with the FDA within 5 days of initiating
this activity and annually thereafter using Form FDA 2830.  In
addition, any establishments shipping these products in
interstate commerce must be licensed in accordance with Section
351 of the Public Health Service Act.  FDA registration is not
required for products that will be leukocyte reduced at the
bedside.

Licensed blood establishments should request and wait for
approval of a supplement to their applicable product licenses
prior to implementation.   The following forms and information
should be submitted to the FDA at the address provided in Part
IX:

1.   Whole Blood, Form FDA 3098; or Red Blood Cells, Form FDA
     3098a; for Platelets, Form FDA 3098c; and, if applicable,
     for Platelets, Pheresis, Form FDA 3098e.

2.   SOPs for the preparation of products including the
     information described above in Part IIIB.

3.   Cell counting method and where it is performed. If not
     performed at the blood establishment, a laboratory certified
     by the Health Care Financing Administration (HCFA) may be
     used. A written agreement should be on file permitting
     authorized FDA inspectors to inspect the testing laboratory
     (Form FDA 3098c, section 6c).

4.   Two months of QC data for each product demonstrating the
     adequacy of the method used for preparing leukocyte-reduced
     products by providing the residual leukocyte counts and
     hematocrit or platelet counts of the product remaining after
     leukocyte reduction.  Documentation of monthly QC should be
     available for review at the time of FDA inspections.
  
5.   Product labels consistent with these recommendations and
     Draft Revision of FDA Guideline for the Uniform Labeling of
     Blood and Blood Products, 1989.  The product codes listed
     for Whole blood and Red Blood Cells, Leukocytes Removed by
     Filtration may be used for Whole Blood and Red Blood Cells,
     Leukocytes Reduced.
  
6.   Evidence of supervisory review of production and QC records.

     Until Forms FDA 3098, 3098a, 3098c, and 3098e are updated,
     the licensure information requested and quality control
     recommendations stated in this memorandum supersede those
     described on the forms for leukocyte reduced products. 


Licensed establishments with approved supplements to manufacture
Red Blood Cells, Leukocytes Removed by Filtration using
FDA-cleared
filters, only need to submit labeling to reflect the labeling
changes discussed in this memorandum.  Licensed establishments
changing to a different FDA-cleared filter, including those
requiring the use of an STCD and using the same anticoagulant,
do not have to notify the FDA.

IX.  ADDITIONAL INFORMATION

Questions concerning whole blood and red blood cells that are
leukocyte reduced may be submitted to the following address:

               Director, Division of Blood Applications (HFM-370)
               Food and Drug Administration
               Center for Biologics Evaluation and Research
               c/o Document Control Center (HFM-99)
               1401 Rockville Pike, Suite 200N
               Rockville, MD 20852-1448
               Voice telephone number: (301) 827-3543,
               Fax number: (301) 827-2857

Questions concerning platelets and platelet pheresis products
that are leukocyte reduced may be addressed to the Director,
Division of Hematology, (HFM-330) at the above address.  Voice
telephone number (301) 496-4396, FAX number (301) 402-2780.

                                                  


                                   ____________________________
                                   Kathryn C. Zoon, Ph.D.





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