Guidance for

Industry

Pre-Storage Leukocyte Reduction

of Whole Blood and

Blood Components Intended for

Transfusion

DRAFT – NOT FOR IMPLEMENTATION

This document is being distributed for comment purposes only.

Comments and suggestions regarding this draft document should be submitted within 90 days

of publication in the Federal Register of the notice announcing the availability of the draft

guidance. Submit comments to Dockets Management Branch (HFA-305), Food and Drug

Administration, 5630 Fishers La., rm. 1061, Rockville, MD 20857. All comments should

be identified with the docket number listed in the notice of availability that publishes in the

Federal Register.

Additional copies of this draft guidance document are available from: the Office of

Communication, Training and Manufacturers Assistance (HFM-40), 1401 Rockville Pike,

Rockville, MD 20852-1448, or by calling 1-800-835-4709 or 301-827-1800, or from the

Internet at http://www.fda.gov/cber/guidelines.htm.

Questions may be directed to Jong-Hoon Lee, M.D. at (301) 827-3543.

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Biologics Evaluation and Research

January, 2001
i.

TABLE OF CONTENTS

I. SCOPE OF RECOMMENDATIONS .............................................................................. 1

II. PURPOSE AND RATIONALE FOR RECOMMENDATIONS.................................... 1

III. BACKGROUND DISCUSSION.................................................................................... 2

A. Changes Compared with the May 1996 Leukocyte Reduction Memorandum............ 2

B. Benefits of Pre-Storage Leukocyte Reduction.............................................................. 2

C. Potential Benefits of Pre-Storage Leukocyte Reduction.............................................. 3

D. Safety Concerns Related to Bedside Filtration............................................................. 4

E. Potential for Leukocyte Reduction to Decrease the Theoretical Risk of nvCJD....... 4

F. Potential for Reducing Other Leukocyte-Associated Infections .................................. 4

IV. MANUFACTURING RECOMMENDATIONS.......................................................... 4

A. Methods and Blood Components ................................................................................... 5

Table 1: Blood Components and Leukocyte Reduction Methods....................................... 6

B. Standard Operating Procedures..................................................................................... 6

1. Leukocyte Reduction Equipment..........................................................................................................................7

2. Leukocyte Reduction Procedures.........................................................................................................................7

3. Process Validation and Quality Monitoring .....................................................................................................8

4. Investigation of Process Failure...........................................................................................................................8

C. Product Labeling.............................................................................................................. 8

Table 2: Component Names for Container Labels............................................................... 9

D. Process Validation........................................................................................................... 9

E. Quality Monitoring........................................................................................................ 10

1. Quality Monitoring Program..............................................................................................................................10

2. Sample Collection and Quality Control Testing.............................................................................................11

3. Expected Results and Actions.............................................................................................................................11

4. Unexpected Results and Actions.........................................................................................................................12

5. Technical Training and Performance Evaluation of Operating Personnel...............................................13

F. Quality Assurance and Manufacturing Records ......................................................... 13

V. REGISTRATION AND LICENSURE........................................................................... 14

Table 3: License Application Contents and Alternatives................................................... 15

A. Apheresis Procedures: Prior Approval Supplements (PAS)........................................ 15

B. Alternatives: Prior Approval Supplement (PAS)........................................................ 16

C. Alternatives: Comparability Protocol (CP) for Multiple Facilities............................ 16

D. Review Divisions within FDA....................................................................................... 16

Table 4: FDA Contact Information..................................................................................... 17

VI. GLOSSARY................................................................................................................... 18

VII. REFERENCES.............................................................................................................. 19
1.

GUIDANCE FOR INDUSTRY

PRE-STORAGE LEUKOCYTE REDUCTION OF WHOLE BLOOD

AND BLOOD COMPONENTS INTENDED FOR TRANSFUSION

This guidance document represents FDA’s current thinking on the manufacture of pre-storage

leukocyte reduced Whole Blood and blood components intended for transfusion. It does not

confer any rights for or on any person and does not operate to bind the FDA or the public. 

An alternative approach may be used if it satisfies the requirement of the applicable statutes

and regulations.

I. SCOPE OF RECOMMENDATIONS

This document describes Food and Drug Administration (FDA) recommendations regarding prestorage leukocyte reduction of Whole Blood and blood components intended for transfusion,

including products made by apheresis. This document provides information to assist licensed facilities

in filing supplements to their biologics licenses to include leukocyte reduced products. It applies to

Whole Blood, and to Red Blood Cells and Platelets, whether made from Whole Blood or by

apheresis. This guidance document supersedes the FDA memorandum issued on May 29, 1996,

entitled “Recommendations and Licensure Requirements for Leukocyte Reduced Blood Products.”

II. PURPOSE AND RATIONALE FOR RECOMMENDATIONS

These recommendations update current Good Manufacturing Practices (cGMP) applicable to prestorage leukocyte reduced Whole Blood and blood components for transfusion. Additionally, the

Agency supports the use of leukocyte reduced blood and blood components and seeks to streamline

the licensing procedure for leukocyte reduced blood products to assist blood establishments in

making pre-storage leukocyte reduced blood products more widely available.

Although adverse transfusion reactions have been associated with leukocyte reduction by filtration

[1,2], advances in blood cell separation technology enable the safe and substantial reduction of

residual leukocytes. Such continuing improvements in technology have made it possible to provide

purer Red Blood Cells and Platelets. The Agency believes that increased availability of pre-storage

leukocyte reduced products will contribute to safety. The potential additional benefits of using

leukocyte reduced blood products suggest that consideration should be given to making leukocyte

reduced blood products more widely available.

FDA therefore is issuing recommendations for pre-storage leukocyte reduction of Whole Blood and

blood components for transfusion, including recommendations for quality monitoring of the leukocyte

reduction process. FDA believes that these manufacturing and quality assurance recommendations

are consistent with current industry practices and standards.
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III. BACKGROUND DISCUSSION

A. Changes Compared with the May 1996 Leukocyte Reduction Memorandum

In a May 29, 1996 memorandum, FDA issued recommendations on leukocyte

reduction, a manufacturing step performed under controlled and monitored laboratory

conditions. Leukocyte reduced components were to contain <5.0 x 106 residual white

blood cells per blood component, with at least 85 percent retention of the original

therapeutic cells. The memorandum was consistent with the outcome of a public

workshop held in 1995 on that subject. This guidance is intended to update the

manufacturing and quality assurance recommendations for pre-storage leukocyte

reduction to further improve the purity, consistency, and safety of leukocyte reduced

blood components. This guidance differs from the earlier May 1996 FDA

memorandum in that it recommends the following: 

(1) to assure product safety and efficacy, the leukocyte reduced product

should contain <1.0 x106 residual white blood cells;

(2) to use statistical methods for quality control testing in monitoring the

leukocyte reduction process, to assure with 95% confidence that at

least 95% of the products meet intended product specifications;

(3) to increase the frequency of quality monitoring;

(4) to reduce product loss by identifying repeated causes of filter failures;

(5) to directly test every leukocyte reduced component intended to be used

in lieu of cytomegalovirus (CMV) negative units for residual leukocytes;

(6) to provide multiple licensing options to decrease reporting burden; and

(7) to consider routine donor screening for sickle cell trait.

This guidance document presents FDA’s current thinking on pre-storage leukocyte

reduction of Whole Blood and blood components intended for transfusion. It was

shaped in part by the discussions held at a FDA workshop entitled “Implementation of

Universal Leukocyte Reduction” (December 10, 1999, Bethesda, Maryland), and the

66th meeting of the Blood Products Advisory Committee (BPAC; June16, 2000;

Silver Spring, Maryland).

B. Benefits of Pre-Storage Leukocyte Reduction

In current medical practice, leukocyte reduced Whole Blood and blood components

have been shown to be beneficial [3-7]: 

(1) to reduce immunization to leukocyte antigens that may complicate care

of patients who undergo transplantation or chronic transfusion therapy

(e.g. patients with aplastic anemia or hematologic malignancies);

(2) to reduce transmission of CMV to patients at increased risk of CMV

disease (e.g. chemotherapy recipients for whom severe neutropenia is
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expected, recipients of hematopoietic progenitor cell replacement

therapy, CMV seronegative recipients of CMV seronegative solid

organ grafts, and low birth weight premature infants); and

(3) to reduce recurrent febrile, non-hemolytic transfusion reaction

(FNHTR)(e.g. patients with history of two or more febrile reactions to

transfusion).

Based on the identified benefits, FDA believes that leukocyte reduced products should

be provided to patients with these defined indications, and that pre-storage leukocyte

reduced products should be provided in preference to bedside filtered blood products

based on evidence of their superior quality and safety.

Limited data suggest that leukocyte reduction may have additional, more general

benefits by avoidance of immunomodulatory effects, and as a safeguard against

transmission of known and as yet undiscovered leukocyte associated pathogens. The

known and potential benefits of leukocyte reduction were discussed by FDA’s Blood

Products Advisory Committee (BPAC) at its public meeting in September 1998. At

that meeting, a majority of the voting committee members found that the benefit-to-risk

ratio associated with leukocyte reduction is sufficient to justify the universal leukocyte

reduction of transfusion blood components, irrespective of the theoretical

considerations for transfusion-transmitted Creutzfeldt-Jakob disease (CJD). 

Consistent with the advice of the BPAC, FDA is providing guidance to streamline the

procedures for licensure of leukocyte reduced products in order to assist the industry

in making these products more widely available. FDA supports the increased use of

leukocyte reduced blood.

C. Potential Benefits of Pre-Storage Leukocyte Reduction

Leukocyte reduction has been reported to reduce FNHTR, human leukocyte antigen

(HLA) alloimmunization, and CMV transmission [3-7]. In addition, a growing list of

potential, but not established, benefits of leukocyte reduction includes reduction of: 

(1) transfusion-associated immunomodulation [11];

(2) bacterial overgrowth [3, 12];

(3) viral reactivation [3, 12];

(4) reperfusion injury following cardiopulmonary bypass [12];

(5) red blood cell and platelet storage lesions [13]; and

(6) the theoretical risk of transfusion-transmitted CJD and new variant CJD

(nvCJD) [14]. 

At present, leukocyte reduction is not considered appropriate for the prevention of

transfusion-associated graft-versus-host disease (TA-GVHD) owing to the availability

of irradiation as the definitive and preferred method against this serious adverse

transfusion outcome [15]. In addition to eliminating the potential for precipitous
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hypotension, pre-storage leukocyte reduction allows the leukocyte reduction process

to be monitored under controlled conditions that assure product purity, consistency,

and safety. 

D. Safety Concerns Related to Bedside Filtration

Bedside filtration remains available as a leukocyte reduction method to physicians

prescribing transfusion therapy. As a post-storage procedure, however, bedside

filtration has been associated with precipitous hypotension in the transfusion recipient,

an infrequent yet serious adverse effect not clearly associated with pre-storage

leukocyte reduction [8]. Patients on medications that inhibit the angiotensin converting

enzyme (ACE inhibitors) appear to be particularly susceptible. Also, it is recognized

that bedside filtration may fail to adequately remove leukocytes due to uncontrolled

filtration times and temperature. Therefore, pre-storage leukocyte reduction is

generally preferable to bedside filtration [8-10].

E. Potential for Leukocyte Reduction to Decrease the Theoretical Risk of

nvCJD

Limited scientific data have suggested the possibility that leukocyte reduction may be a

useful precautionary measure against the theoretical risk of transfusion-transmitted

nvCJD [14]. Over the last two years, some international blood authorities have

implemented or are seriously considering the implementation of the regulatory

requirement to leukocyte reduce all Red Blood Cells and Platelets intended for

transfusion (universal leukocyte reduction, ULR), as well as plasma for further

manufacturing in some cases. Countries implementing these requirements include the

United Kingdom, Ireland, France, Germany, Austria, Portugal, and Canada.

However, FDA’s Transmissible Spongiform Encephalopathies Advisory Committee

(TSEAC) advised on June 2, 2000, that current scientific data are inadequate to affirm

the effectiveness of leukocyte reduction in significantly reducing the infectivity of CJD

and nvCJD in blood components.

F. Potential for Reducing Other Leukocyte-Associated Infections

The potential to reduce infection by pathogens that are primarily white blood cell

associated may extend beyond CMV [6,7] to include other known and unknown

agents potentially transmitted by white blood cells [14].

IV. MANUFACTURING RECOMMENDATIONS

All pre-storage leukocyte reduced blood components must be manufactured in accordance

with cGMP [21 CFR Parts 210-211, 600-680]. Leukocyte reduction filters are typically

component-specific; a filter intended for one component type should not be used with other
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component types [16]. When leukocyte reduction is performed according to cGMP and

without breaching closure, the expiration date of the component remains unchanged. If a

filter is attached to a blood container using aseptic technique but without an FDA-cleared

sterile tubing connection device (STCD), the container is considered an open system. 

Leukocyte reduced Whole Blood and Red Blood Cells prepared in an open system and

stored at 1 - 6 oC have an expiration of 24 hours. Platelet units that are entered, pooled and

filtered have an expiration of 4 hours [21 CFR 606.122]. Currently, Pooled Platelets,

regardless of their method of preparation and processing have an expiration of 4 hours. All

blood components including those leukocyte reduced prior to storage should be

administered through a standard blood filter designed to remove clots and/or

microaggregates that form during blood storage [16,17].

A. Methods and Blood Components

Whole Blood and blood components intended for transfusion may be leukocyte

reduced prior to storage using any of the following closed system methods: 

(1) filtration through an in-line filter integral to the blood collection set;

(2) filtration through a filter system attached to a blood component

container using an FDA-cleared STCD [18];

(3) direct leukocyte reduction, simultaneous with Platelets, Pheresis

collection, using automated cytapheresis without filtration.

Leukocyte reduced blood components are listed in Table 1, along with corresponding

product specifications (maximum residual leukocyte content, minimum amount of

therapeutic product) and applicable leukocyte reduction methods. Investigations

should be performed on leukocyte reduced blood products that fail to retain at least

85% of the original therapeutic component.
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Table 1: Blood Components and Leukocyte Reduction Methods

(LR = Leukocytes Reduced; STCD = Sterile Tubing Connecting Device)

Blood Components Recommended

Maximum Residual

Leukocyte Content

Recommended

Minimum Therapeutic

Product Post Filtration

Applicable Methods

Whole Blood, LR 1.0 x 106160 ml[16] red blood cells STCD-filtration;

In-line filtration

Red Blood Cells, LR 1.0 x 106160 ml[16] red blood cells STCD-filtration;

In-line filtration

Preparation from Whole

Blood, L.R.

Platelets, Pheresis, LR 1.0 x 1063.0 x 1011 platelets Automated

Plateletpheresis;

STCD-filtration;

In-line filtration

Automated

plateletpheresis

Platelets, LR 1.6 x 1055.5 x 1010 platelets STCD-filtration;

In-line filtration

1

 A unit of Platelets derived from Whole Blood is infrequently leukocyte reduced. For an adult transfusion

recipient, multiple units of Platelets (4-6) are pooled into a single platelet dose. The total dose (4-6 units) should

contain <1.0 x 106 residual leukocytes. In order to preserve product shelf life, multiple units of Platelets are

typically pooled after storage and prior to filtration, rather than filtering individually prior to pooling. 

Although plasma components, when manufactured under cGMP, may have a residual

leukocyte content of less than 1.0 x 106 per unit, data to support the recognition of

such products has not been submitted for CBER review and approval. Currently

accepted and validated cell counting procedures may need to be modified for plasma

products. Plasma components (including Fresh Frozen Plasma and Cryoprecipitated

Antihemophilic Factor) may be labeled “Leukocytes Reduced” provided that

manufacturing process validation and quality monitoring as described in Sections D

and E includes these components.

B. Standard Operating Procedures

Written standard operating procedures (SOP) for leukocyte reduction must be

maintained [21 CFR 606.100(b)]. The SOPs should identify: 

(1) the blood products to be leukocyte reduced;

(2) blood product specifications;
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(3) leukocyte reduction methods;

(4) leukocyte reduction equipment and its manufacturer;

(5) the labeling and disposition of products failing to meet product

specifications;

(6) the methods for identifying donor specific repeated process failures.

In addition, the following aspects of the leukocyte reduction processes should be

described in sufficient detail to ensure manufacturing consistency.

1. Leukocyte Reduction Equipment

FDA regulations require that equipment used in the collection or processing of

blood and blood components “shall perform in the manner for which it was

designed.” [21 CFR 606.60(a)]. An establishment must maintain written SOPs

for all steps in the collection and processing of blood and blood components [21

CFR 606.100(b)]. Accordingly, an establishment must incorporate the device

manufacturer’s instructions for use into its SOPs. Regulations further require

establishments to maintain records “concurrently with the performance of each

significant step in the collection [and] processing of blood and blood

components.” [21 CFR 606.160(a)(1)].

The SOP should indicate that the leukocyte reduction equipment (blood

collection sets, filters, or apheresis machines) is either cleared or approved for its

intended use by CBER. Filters intended for bedside filtration should not be used

for pre-storage leukocyte reduction. The SOP should accurately incorporate the

equipment manufacturer’s instructions for use.

2. Leukocyte Reduction Procedures

FDA recommends that the SOP describe at least the following:

(1) the time interval, according to the filter manufacturers' directions

for use, but no more than 72 hours, within which leukocyte

reduction is to be performed following blood collection;

(2) the temperatures at which leukocyte reduction is to be performed;

(3) the use of an FDA-cleared STCD according to FDA-cleared

instructions for use [18]; and

(4) the time interval, within which filtration should be completed after

beginning filtration [19,20].
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3. Process Validation and Quality Monitoring

The SOP should include procedures for process validation and quality

monitoring. Each leukocyte reduction process should be validated initially and

monitored periodically to detect process failures that may compromise blood

product quality (See Sections D and E).

4. Investigation of Process Failure

The SOP should describe how leukocyte reduction process failure is to be

investigated and resolved. Process failure may be apparent during manufacturing

or may be detected through quality control testing. The SOP should address the

disposition of blood units manufactured during a potential or confirmed unstable

process (See Sections D and E).

A blood component that requires a filtration time longer than the time interval specified

in the manufacturer’s directions for use may be released for transfusion use as

leukocyte reduced only if direct quality control testing of that unit confirms acceptable

product specifications. Products that fail to meet the processing specifications in the

manufacturer’s directions for use or that fail to meet product specifications should be

thoroughly investigated. For example, products from an individual donor may

repeatedly have extended filtration times. Additional investigation including

manufacturing record review should be conducted to determine if the unit could have

been detected earlier in the manufacturing process.

Leukocyte reduction by filtration often fails for Whole Blood or Red Blood Cells

collected from donors with sickle cell trait [21-28]. Routine donor screening for sickle

trait or use of a validated alternative method should be considered for all donors. 

Such testing should reduce the number of products lost to filter failures. To address

process failures, the SOP should include testing the blood unit for sickle cell trait when

process failure is apparent, either at filtration or at quality control testing. Typically,

process failure will be apparent during filtration and further investigation may detect the

donor with sickle cell trait. Rarely, sickle cell trait units may be detected at quality

control testing. Additional investigation including manufacturing record review should

be conducted to determine if the unit could have been detected earlier in the

manufacturing process. In either instance, leukocyte reduction procedures need not

be revised, but the blood unit should not be released for transfusion unless directly

tested and found acceptable.

C. Product Labeling

The Circular of Information for the Use of Human Blood and Blood Components

(The Circular) describes in detail the indications, contraindications, side effects,

hazards, dosage, and administration of leukocyte reduced products [16]. The
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Circular is periodically revised by the blood industry subject to FDA review and

acceptance. The container label must follow a format accepted by FDA [21 CFR

606.121]. Table 2 lists the proper names of the major leukocyte reduced blood

products, followed by the corresponding International Society of Blood Transfusion

(ISBT) Code 128 name [29]. Until ISBT Code 128 terminology is adopted, product

labels should include the name of the appropriate anticoagulant storage solution as

modifiers (e.g., CPDA-1 Whole Blood, Leukocytes Reduced). Product modifications

(e.g., Irradiated, Frozen) should be indicated after the phrase “Leukocytes Reduced.”

 The phrases “Leukocytes Removed,” “Leukocyte Poor,” “Leukocytes Depleted,”

and other similar terms should not be used in product labeling.

Table 2: Component Names for Container Labels

Proper Name ISBT 128 Name

Whole Blood Leukocytes Reduced WHOLE BLOOD LEUKOCYTE REDUCED

Red Blood Cells Leukocytes Reduced RED BLOOD CELLS LEUKOCYTES REDUCED

APHERESIS RED BLOOD CELLS LEUKOCYTES

REDUCED

Platelets, Pheresis Leukocytes Reduced APHERESIS PLATELETS LEUKOCYTES

REDUCED

Platelets Leukocytes Reduced PLATELETS LEUKOCYTES REDUCED

D. Process Validation

Process validation requirements for finished pharmaceuticals [21 CFR 210, 211]

apply to the manufacture of leukocyte reduced blood and blood components, and the

use of leukocyte reduction equipment, respectively. "The Guideline on General

Principles of Process Validation" provides general direction in this area [30].

Under 21 CFR 210/211, manufacturers must prepare a written validation protocol

which specifies the procedures and tests to be conducted and the data to be collected

that will provide a high degree of assurance that each leukocyte reduction process will

consistently produce a product meeting its predetermined specifications and quality
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characteristics. Elements incorporated into a leukocyte reduction validation process,

should include but not be limited to:

(1) blood collection times, platelet content, plasma content;

(2) intrinsic donor characteristics (e.g. sickle cell trait) and initial blood

collection;

(3) equipment quality and proper handling and use;

(4) operating procedure (e.g., storage time between blood collection

and filtration; filtration time, pressure, and temperature; equipment

priming).

Each leukocyte reduction process should be tested initially in sufficient detail to

establish process stability and acceptable process performance. Following successful

validation, each leukocyte reduction process should be tested at least weekly to detect

a potential unstable manufacturing process that may compromise blood product

quality.

E. Quality Monitoring

1. Quality Monitoring Program

A facility should establish and follow a statistical monitoring plan that assures, at

95% confidence level, that more than 95% of the units intended to be labeled as

leukocyte reduced meet product specifications [31,32]. One example of a plan

that satisfies these goals and is acceptable to FDA without prior FDA review is

presented below.

Statistical plan

Initially, a facility tests 60 consecutive units for each of the following major

process variations: 

(1) filtration of Whole Blood;

(2) filtration of Red Blood Cells;

(3) filtration of Platelets;

(4) filtration of Platelets, Pheresis

(5) direct collection of leukocyte reduced Platelets, Pheresis

Testing 60 consecutive units for each process will assure compliance if all of the

products meet the product specifications. Additional testing will be important if a

failure is identified.

Using more than one variation of filter, SOP (e.g. different temperature) or

apheresis instrument increases the total number of process variations which would

require initial validation and weekly evaluation; quality control testing should be
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performed separately for each of the 4 major leukocyte reduction process

variations. A blood center should determine its monthly production rate for each

type of product and leukocyte reduction process. The minimum number of

products tested for each type of product and process should be 20 per month.

This will enable a specific number of products to be tested every week. Testing

of 5 units per week for each leukocyte reduction process should allow early

detection of an unstable process thus averting potentially costly product retrievals.

 If all testing is acceptable, the facility assures at 95% confidence, on a floating

three month basis, that more than 95% of the blood units manufactured meet

product specifications. The testing of 60 units (with all units acceptable) should be

repeated whenever an unacceptable unit is identified or a leukocyte reduction

process is significantly changed.

2. Sample Collection and Quality Control Testing

Blood samples should be collected, processed, and tested within 24 hours of

leukocyte reduction. Sampling procedures should include thorough mixing of

blood and the use of freshly filled tubing segments. Sample testing should be

performed using validated procedures at a registered blood center or at a

laboratory certified by the Health Care Financing Administration (HCFA) for

testing leukocyte reduced blood products. The following methods may be used

in enumerating low numbers of residual leukocytes and in calculating product

recovery.

Manual methods using a Nageotte counting chamber and Turk's staining solution

(alone or in conjunction with a recommended red blood cell lysing reagent and/or

centrifugation) may be adequately sensitive and reliable for counting leukocytes at

less than 0.5-1.0 cells/uL [33-36, 40]. Automated methods using FDA-cleared

instrumentation compares favorably in sensitivity, precision, reliability, and

efficiency to the manual Nageotte chamber method but may lose sensitivity if

sample dilutions are necessary [37-40]. Product content may be calculated

simply by multiplying product volume in mL (net weight of blood unit in grams

divided by the specific gravity correction factor) by cell concentration (hematocrit

or platelet count per mL)[31].

3. Expected Results and Actions

Results from weekly process testing should provide data that demonstrate with

95% confidence level that more than 95% of blood units contain < 1.0 x 106

residual leukocytes per unit (or < 1.6 x 105 residual leukocytes for single units of

Platelets derived from Whole Blood). (This standard is met on an ongoing threemonth basis as long as zero failures are encountered on quality control testing of

at least 5 units per week for each product and process variation.) Leukocyte
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reduced units also should meet the standards for minimum product content

specified in Table 1. Additionally, filtration should result in at least 85%

therapeutic product recovery. (Failure to recover at least 85% of the therapeutic

product does not render a given product unsuitable, however, this failure should

trigger an investigation of the filtration process.)

4. Unexpected Results and Actions

The frequency and the extent of process control testing failure may be helpful in

investigating process stability. Operating near the minimum process standard

(i.e., 95% confidence that more than 95% of units will meet product

specifications) may require frequent process investigations. Upon identification of

a unit that does not meet intended leukocyte reduced product specifications, the

identified blood unit could be released for transfusion without a change in the

expiration date, provided:

(1) the container closure has not been breached;

(2) the label accurately reflects the content of therapeutic cells and

residual leukocytes (e.g., “Low Volume”, “other than standard

content”, and/or NOT labeled “Leukocytes Reduced”); and

(3) the container is labeled, “Do Not Leukocyte Reduce.”

The effect of repeated filtration, including bedside filtration, on the safety and

efficacy of blood components has not been established.

When an unacceptable unit is detected, the minimum process standard (more than

95% of units acceptable, at the 95% confidence level) is no longer assured. 

Investigation should be conducted and corrective actions should be taken if

needed [40]. Technical advice may be sought from the manufacturer of the

leukocyte reduction equipment (filter or apheresis machine). The investigation

with or without corrective actions should be documented. The leukocyte

reduction process should be revalidated using 60 consecutive units after the

correction of a process deficiency [31]. If a process deficiency is found, all units

affected by the deficiency should be identified. For the units that remain under

facility control, direct quality control testing should be performed to the extent

possible for each unit, and the container label should be corrected as needed. If

direct testing cannot be performed due to age of the product, (e.g., accurate

white blood cell counts can no longer be obtained) the container label should be

corrected so that the product is no longer labeled as "Leukocytes Reduced." For

the units that have been released, consignees should be notified within 24 hours.

Users of the leukocyte reduction equipment should report equipment failures to

the equipment manufacturer who must review, investigate, and follow up the

complaint [21 CFR 211.198 and 820.198]. If a leukocyte reduction process has
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been confirmed to be deficient, manufacturers of the leukocyte reduced blood

products should inform the consignees of the previously distributed, potentially

unsuitable blood units [21 CFR 7.49] within 24 hours. Results of the process

investigation and follow up consultation on product safety and efficacy should be

provided as soon as possible. Licensed manufacturers of leukocyte reduced

blood products must notify FDA of biologic product deviations in the

manufacture of such products [21 CFR 600.14 and 606.171]. All unlicensed

manufacturers must report manufacturing biologic product deviations to FDA [21

CFR 606.171].

5. Technical Training and Performance Evaluation of Operating Personnel

Control over operational variables must be maintained through initial and

continued staff training in conjunction with routine staff participation in leukocyte

reduction and periodic staff performance assessment [21 CFR 606.20 (b)]. 

Training should include the following:

(1) equipment use and maintenance;

(2) clinical consequences of blood units that do not meet product

specifications;

(3) collection, preparation, storage, and stability of quality control

samples;

(4) SOP for leukocyte reduction, product labeling, and quality

monitoring.

F. Quality Assurance and Manufacturing Records

A quality assurance unit should provide oversight of product manufacturing. All

manufacturing records should be reviewed and approved by the quality assurance unit.

 Manufacturing records must be maintained and reviewed as part of quality assurance

[21 CFR 606.160 and 21 CFR 211.192]. Process validation, quality monitoring,

routine production, special investigation, corrective action, and repeat process

validation should be performed according to previously established center SOP. 

Records of the performance of these procedures should be sufficiently detailed to

facilitate process control. At minimum, the following identifying information should be

included: 

(1) responsible personnel;

(2) relevant dates and times;

(3) leukocyte reduction equipment (filter, apheresis machine, or apheresis

soft goods), together with lot numbers, receipt dates, product inserts, and

equipment validation data;

(4) validation data for each leukocyte count method used;

(5) validation data for each leukocyte reduction process.
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V. REGISTRATION AND LICENSURE

Pursuant to 21 CFR Part 607, an establishment that routinely manufactures leukocyte reduced

blood products must register with the FDA within 5 days of initiating this activity and annually

thereafter using Form FDA 2830. An establishment that distributes these products in interstate

commerce also must be licensed for each leukocyte reduced blood product, in accordance with

Section 351 of the Public Health Service Act.

A licensed facility that changes its Whole Blood manufacturing procedures to include leukocyte

reduction in accordance with all recommendations contained in Section IV may supplement its

biologics license to include leukocyte reduction by submitting the following as changes being

effected in 30 days (CBE-30) [21 CFR 601.12 (c)(5)]:

(1) Form FDA 356h, indicating the submission as CBE-30;

(2) product labeling for each product;

(3) quality control data from the initial validation of each leukocyte reduction

process;

(4) evidence of quality assurance oversight, including review and approval of

manufacturing records by a quality assurance unit;

(5) a statement that the facility is following this guidance.

Licensing of leukocyte reduction is facility-specific; an applicant should submit a separate

request for each facility. As a CBE-30 license application supplement, interstate product

distribution may begin 30 days after FDA’s receipt of the supplement filing unless notification is

received from FDA that a prior approval supplement is required.

A licensed facility that seeks to manufacture leukocyte reduced blood using automated

apheresis procedures (21 CFR 640.21-23) or using alternatives to the recommendations

contained in Section IV should submit an application as a prior approval supplement (PAS) [21

CFR 601.12 (b)]. The PAS should describe each alternative method in sufficient detail for a

meaningful FDA review. 

An applicant seeking licensure for multiple facilities may submit as a PAS a comparability

protocol (CP) [21 CFR 601.12 (e)] which describes elements common to all facilities. When

FDA approves the CP, the applicant may then submit facility-specific data as CBE-30 in

accordance with the approved CP. Each of these licensing options is further described in Table

3:

(1) a PAS which describes apheresis methods or alternatives to this guidance;

(2) a PAS for a CP which describes identical processing at multiple facilities;

(3) a CBE-30 supplement when leukocyte reduction is performed by filtration

after manual collection in accordance with all recommendations contained in

Section IV.
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Table 3: License Application Contents and Alternatives

(PAS = prior approval supplement; CBE-30 = changes being effected in 30 days)

Submission Elements

PAS

Apheresis or

Alternative to

Guidance

Comparability Protocol CBE-30

Manual

Collection

and Follows

Guidance

Protocol

(PAS)

Data

(CBE-30)

Form FDA 356h as cover

(additional cover letter optional) X X X X

Listing, leukocyte reduced products

Product specifications X X

Standard operating procedures

(leukocyte reduction) X X

Form FDA 2567

Product labels including Circular X X X X

Description of process validation

Quality monitoring plan X X

Quality control data

(initial process validation) X X X

Evidence of quality assurance

oversight, manufacturing and records X X X

Protocol and rationale

(for multiple facilities) X

A. Apheresis Procedures: Prior Approval Supplements (PAS)

If apheresis procedures are used, the procedures for collection and processing shall be

as described in a biologics license application or a supplement to a biologic license

application (21 CFR 640.21-23). The license application should be organized in a

manner similar to Section IV to facilitate rapid evaluation. Applications should be

submitted as a PAS. Interstate product distribution may not begin until the PAS

submission has been reviewed and approved by FDA.
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B. Alternatives: Prior Approval Supplement (PAS)

The recommendations in Section IV are intended to serve as factors FDA considers

acceptable in evaluating a license application for leukoreduction of Whole Blood and

blood components. Alternatives may be acceptable if adequately supported and

deemed to provide public health protection equivalent to or more stringent than FDA

recommendations. The license application should be organized in a manner similar to

Section IV to facilitate rapid evaluation. Alternative proposals should be submitted as

a PAS. Interstate product distribution may not begin until the PAS submission has

been reviewed and approved by FDA.

C. Alternatives: Comparability Protocol (CP) for Multiple Facilities

For multiple facilities under the direction and control of the same applicant, a CP may

be submitted to consolidate reporting. The CP itself should be submitted as a PAS. 

The submission should include the following: 

(1) indicate which information is common to all facilities;

(2) provide the rationale for expecting the components

manufactured at different facilities to be comparable;

(3) provide a plan (protocol) for performing quality control

testing; and

(4) the criteria for evaluating the test results, to confirm that

manufacturing at a particular facility is acceptable and

comparable.

When approved, the CP allows facility-specific information to be described as CBE30. As with CBE-30 reporting of manufacturing in accordance with FDA

recommendations, interstate distribution of products manufactured at a facility covered

under the CP may begin 30 days after FDA’s receipt of the CBE-30 filing from that

facility prior to approval, unless FDA indicates that a PAS should be filed or denies

approval of the CP.

D. Review Divisions within FDA

Two divisions within the FDA review biologics license applications for leukocyte

reduction in the manufacture of Whole Blood and blood components. Questions about

licensing and specific review questions about leukocyte reduction of Whole Blood or

Red Blood Cells should be directed to the Division of Blood Applications (DBA);

specific review questions regarding leukocyte reduction of Platelets or Platelets,

Pheresis should be directed to the Division of Hematology (DH). Questions may be

directed also to the Office of Communications, Training, and Manufacturers

Assistance (OCTMA) as an initial general point of contact. All registration forms
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(Form FDA 2830) and licensure applications/supplements should be submitted to the

Director, Center for Biologics Evaluation and Research (CBER). Table 4 presents

FDA contact information regarding leukocyte reduction.

Table 4: FDA Contact Information

Submissions:

• Registrations

• License Applications

Director, Center for Biologics Evaluation and Research, HFM-370,

Food and Drug Administration,

c/o Document Control Center, HFM-99,

1401 Rockville Pike, Suite 200N,

Rockville, MD 20852-1448.

General questions

Director, OCTMA, HFM-40,

Food and Drug Administration,

c/o Document Control Center, HFM-99,

1401 Rockville Pike, Suite 200N,

Rockville, MD 20852-1448,

Voice (301) 827-2000; Fax (301) 827-3843.

Review Questions:

• Licensing

• Red Blood Cells

• Whole Blood

• Platelets

• Platelets, Pheresis

Director, Division of Blood Applications, HFM-370,

Food and Drug Administration,

c/o Document Control Center, HFM-99,

1401 Rockville Pike, Suite 200N,

Rockville, MD 20852-1448,

Voice (301) 827-3543; Fax (301) 827-3534.
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VI. GLOSSARY

applicant: Any person or entity who has submitted an application to manufacture a product

subject to licensure under Section 351 of the Public Health Service Act [42 USC 262].

container closure: The seal of a final product container which assures product sterility.

current good manufacturing practice (cGMP): Methods used in, and the facilities or

controls used for, the manufacture, processing, packing or holding of a drug including, but

not limited to, blood components, to assure that such product meets the requirements of the

Federal Food, Drug and Cosmetic Act as to their safety and has the identity and strength,

and meets the quality and purity characteristics, which it purports or is represented to

possess [21 CFR 210.1]

leukocyte reduction equipment: Equipment used during blood manufacturing to reduce

the leukocyte content in accordance with established product specifications. The equipment

is typically 510(k)-cleared by CBER (pre-storage leukocyte reduction filters and apheresis

machines) but may also be approved as a new drug (blood collection sets with integral in-line

filters) owing to the anticoagulant/storage solution component of the equipment.

process validation: Establishing confidence and documenting evidence that a specific

process consistently functions within established limits towards manufacturing of products

that meet predefined specifications.

quality assurance (QA) program: An organization’s comprehensive system for

manufacturing safe, effective, and quality products according to regulatory standards. This

program includes preventing, detecting, and correcting deficiencies that may compromise

product quality.

quality assurance (QA) unit: One or more individuals designated by, and reporting

directly to, management with defined authority and responsibility to assure that all quality

assurance policies are carried out in the organization.

quality monitoring; quality control (QC) testing: As a component of a quality assurance

program, the direct monitoring of products or activities to confirm that product manufacturing

is in accordance with intended product specifications.

sterile tubing connection device (STCD): A device used to sterilely join blood tubing or

segments without breaching container closure.
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Transfusion 2000; 40 (Suppl) 55S.

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35. Rubella P, Dzik W. Multicenter evaluation of methods for counting residual

white cells in leukocyte-depleted red blood cells. Vox Sanguinis 1994; 66:25-32.
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36. Narvios AB, Alvarez FE, Glassman A, Lichtiger B. Assessing the efficiency

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37. Sheckler V, Loken M. Routine quantitation of white cells as low as 0.001 per

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38. Dzik WH, Ragosta A, Cusak WF. Flow-cytometric method for counting very

low numbers of leukocytes in platelet products. Vox Sanguinis 1990; 59:153-159.

39. Dzik S, Moroff G, Dumont L, et al. A multicenter study evaluating three

methods for counting residual WBCs in WBC-reduced blood components: Nageotte

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40. Dzik WH. Leukocyte counting during process control of leukoreduced blood

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