Allergen Immunotherapy Extract Preparation Instructional Guide

Allergen Immunotherapy

Extract Preparation

Instructional Guide

Revised October 2019

AUTHORS

Peter Mustillo, MD

Bryan L. Martin, DO, FACAAI

John Oppenheimer, MD, FACAAI

Michael R. Nelson, MD, PhD, FACAAI

Table of Contents

I. Introduction .......................................................................................3

II. Allergen Extracts.................................................................................... 4

III. Allergen Extract Mixing Standards Qualiﬁcations, and Competencies ......................................5

IV. Allergy Immunotherapy Prescriptions..................................................................7

V. Color Coding, Labels and Beyond Use Dates (BUD) ..................................................... 9

VI. Mixing Individual Patient Allergen Extract Treatment Sets................................................10

VII. Stinging Insect Allergen Extract Preparation ...........................................................15

VIII. Allergen Extract Stability and Storage.................................................................16

IX. Summary .........................................................................................17

X. Acknowledgments .................................................................................18

XI. References ........................................................................................18

XII. Appendices

Appendix 1. Effective Dose Range for Allergen Extracts, US Standardized Units ............................20

Appendix 2. Initial and Ongoing Competency Assessment: Allergen Extract Mixing ........................21

Appendix 3. Extract Volumes Needed for 5.0 ml Maintenance Vials.......................................22

Appendix 4. Recommended Documentation for Allergen Immunotherapy Extract Prescription Forms

and Allergen Immunology Extract Prescription Form ........................................23

Appendix 5. USP Section 21 — Compounding Allergenic Extracts ........................................25

Appendix 6. Forms

Competency Assessment — Initial Personnel Training: Allergen Extract Mixing............................30

Competency Assessment — Annual Personnel Training: Allergen Extract Mixing ..........................32

Competency Assessment — Aseptic Technique and Related Practices of Mixing Personnel .................34

Competency Assessment — Hand Hygiene and Garbing Procedures ....................................36

Competency Assessment — Cleaning and Disinfecting Procedures......................................38

Complaints/Adverse Effects .......................................................................40

Gloved Fingertip and Thumb Sampling .............................................................41

Media-ﬁll Testing Checklist ........................................................................42

Primary Engineering Control (PEC) Hood............................................................43

Refrigerator Temperature Log......................................................................44

I. INTRODUCTION

Origins of Allergy Immunotherapy

Allergen immunotherapy was ﬁrst introduced

by Leonard Noon in 1911.

Dr. Noon originally

hypothesized that patients suffering from “hay fever”

were sensitive to a “toxin” contained in grass pollen. He

proposed that patients would beneﬁt by stimulating the

immune system against the toxin by use of inoculations

of pollen extract. These inoculations involve giving

increasing amounts of allergen extracts to reduce

symptoms on re-exposure to those particular allergens.

The procedure has been widely used since its inception

to treat immediate hypersensitivity disorders mediated

by allergen-speciﬁc IgE antibodies. These same basic

principles hold true today, more than 100 years later, for

current allergen immunotherapy.

Efﬁcacy of Allergy Immunotherapy

Multiple studies have demonstrated the effectiveness of

allergen immunotherapy for the following conditions for

both children and adults:

2-25

• Allergic rhinitis

• Allergic conjunctivitis

• Allergic asthma

• Atopic dermatitis

• Insect allergy (Hymenoptera)

Each patient’s immunotherapy prescription is customized

and tailored to their particular allergic triggers, and

the administration schedule (buildup or maintenance)

may vary. The degree of effectiveness may vary for the

individual patient and is in part determined by whether

causative symptoms are predominantly allergic vs non-

allergic — patients may be more likely to have a limited

response if other causes are contributing to impairment.

Other symptomatic causes may include vasomotor

rhinitis, chronic infectious sinusitis, or anatomical

obstruction such as severe adenoidal hypertrophy.

In those whose symptoms are predominantly allergic

in nature, clinical improvement should occur within or

soon after the ﬁrst year of treatment, and this beneﬁt

may improve with continued treatment. The Allergen

Immunotherapy Practice Parameter third update

suggests that, “If clinical improvement is not apparent

after one year of maintenance therapy, possible

reasons for lack of efﬁcacy should be evaluated. If

none are found, discontinuation of immunotherapy

should be considered, and other treatment options

should be pursued.” It has been observed that some

patients may experience a worsening of their asthma,

atopic dermatitis and allergic rhinitis or conjunctivitis

symptoms during treatment, especially during the ﬁrst

few months of therapy.

Duration of Therapy

There is no consensus on when to discontinue

aeroallergen immunotherapy, but beneﬁts are often

maintained for years after stopping therapy in some

individuals, and indeﬁnitely in others. In grass-

pollen allergy, a three-year course of subcutaneous

immunotherapy gave prolonged relief of symptoms.

For many patients with stinging insect allergy, 3-5

years of treatment may be sufﬁcient for sustained

effectiveness. However, patients who have experienced

life-threatening hymenoptera venom reactions should

be considered for life-long treatment.

Adverse Reactions

Allergen immunotherapy is usually well tolerated;

however, adverse reactions do occur, ranging in severity

from mild to severe. Allergen immunotherapy carries a

small but signiﬁcant risk for life-threatening anaphylaxis

and has very rarely been associated with death.

Systemic reactions associated with immunotherapy may

have numerous causes. Risk factors include asthma and

injections administered during periods of symptom

exacerbations. Human error, due to administering

an incorrect dose or the wrong extract to a patient

is another risk factor. Delays in the administration of

epinephrine during a systemic reaction may increase

the risk of fatalities, which highlights the importance of

having trained personnel capable of identifying and

managing reactions.

Each patient should be evaluated prior to the

immunotherapy administration visit to determine

whether any recent health changes such as illness, acute

asthma symptoms, or new medication - which might

require modifying or withholding the immunotherapy

treatment. Clinical judgment is required when altering

the dose or schedule of administration. While allergen

immunotherapy extracts are relatively easy to prepare

and administer, initial and ongoing training will improve

the expertise of health care workers responsible for

mixing and administering immunotherapy and improve

the safety of the patients.

Subcutaneous allergen immunotherapy is not

indicated for patients with food allergies. Although

studies have demonstrated an increased tolerance to

peanut in patients who received subcutaneous peanut

immunotherapy,

27, 28

there was an unacceptably high

rate of systemic reactions (e.g., anaphylaxis) in most of

the patients during treatment.

II. ALLERGEN EXTRACTS

Allergen extracts used for immunotherapy are

manufactured following collection of raw material.

They consist of liquid solutions containing dissolved

allergenic proteins derived from pollens, dust mites,

animal dander and epithelia, molds, cockroach, and/or

venom. Extracts chosen for the treatment vials should be

clinically relevant for patients receiving treatment, based

on perennial and seasonal symptoms, the regional

presence of pollens and molds, and causative allergens

based on exposure.

The manufacturing process typically involves crushing

raw materials and “extracting” allergenic proteins by

addition of solvents capable of isolating the protein

from the solid raw material. This is followed by a

puriﬁcation process, resulting in a liquid solution that is

stable under normal storage condition (approximately

4 degrees C or 39 degrees F), consisting of a mixture

of the diluents or solvents, additives, stabilizer /

preservative, allergenic proteins and other components

of the raw material.

Stock allergen extracts are licensed by the Center for

Biologics Evaluation and Research (CBER) within the

Food and Drug Administration (FDA) in the United

States. Commercially available stock extracts, supplied

by a handful of manufacturers, are used to mix

individual treatment sets and to prepare test panels.

Concentrated stock extracts are available in multiple

forms:

• Aqueous

• Glycerinated

• Lyophilized (freeze dried)

• Acetone precipitated

• Alum precipitated

Glycerinated stock extracts contain 50% glycerin by

deﬁnition. Other liquid-based extracts (i.e., saline,

buffers, liquid diluents) are referred to as aqueous

extracts.

Lyophilized extracts are aqueous extracts that

have been freeze-dried to increase stability during

storage and shipping. When they are reconstituted in

accordance with package insert instructions with an

appropriate diluent just prior to use, they form aqueous

extracts. Hymenoptera venom extracts are typically

available in lyophilized form.

Acetone-precipitated extracts are liquid extracts that

include a processing step of acetone precipitation to

create a high concentration stock solution. The acetone

is used to precipitate proteins from liquid into a solid

form, which is then re-dissolved in a diluent to make the

ﬁnal highly concentrated stock solution.

Alum-precipitated extracts are liquid extracts that

include a processing step involving the addition of

aluminum hydroxide, or alum. Allergenic proteins attach

to the alum and form complexes that serve as depot

when injected into skin, slowing the release of allergens

on injection. The slow-release alum-allergen complexes

may allow for larger doses of extract to be given at less-

frequent intervals and a more rapid buildup to higher

maintenance doses with reduced incidence of systemic

reactions. Local reactions at the site of alum-precipitated

extract injection may be immediate or delayed. Delayed

reactions may start several hours later, with local edema,

erythema (redness), itching and pain.

The cloudy appearance of the extract, which may

contain visible precipitate, is normal and different

than typical aqueous extracts. These extracts require

shaking before use. Furthermore, only certain diluents

can be used to dilute these extracts. The package

insert from stock antigens may be referenced to

identify the appropriate diluents for use with alum-

precipitated extracts. For example, one manufacturer

requires the use of phenol saline diluent for all 10-

fold dilution vials. Ten percent glycerol-saline and

human serum albumin (HSA) diluent usually cannot

be used for alum-precipitated prescriptions because

of interference with the aluminum hydroxide-antigen

absorbed complex. The slow release described above

makes these less effective in skin testing and are thus

used for treatment only.

Diluents are solutions used to keep the allergens in

suspension. They are used to reconstitute lyophilized

extracts, to dilute extracts for diagnostic use, to dilute

vials in treatment sets and to ﬁll maintenance vials to

ﬁnal volume after addition of stock allergen quantities.

Commonly used diluents:

• Glycerin (e.g., 50% glycerin ± phenol)

• Phenol saline (e.g., 0.4% phenol, saline)

• HSA (e.g., 0.03% HSA, 0.4% phenol, saline)

Each diluent has advantages and disadvantages related

to preservation of extract potency and sterility. These

additives are discussed in further detail in this chapter’s

discussion of extract stability.

Standardized Subcutaneous Allergen Extracts

Standardized subcutaneous allergen extracts in the

United States include:

• Cat hair and pelt (BAU/mL potency labeling based

on Fel d 1 content)

• Dust mite (Dermatophagoides pteronyssinus and D.

farinae; potency in AU/ML)

• Short ragweed (potency in AU/mL or wt/vol with

lot-speciﬁc Amb a 1 concentration)

• Grass (Bermuda, Kentucky bluegrass, perennial

rye, orchard, timothy, meadow fescue, red top and

sweet vernal; potency in BAU/mL)

• Hymenoptera venoms (yellow jacket, honey bee,

wasp, yellow hornet, white-faced hornet, and mixed

vespids; potency in µg/mL)

These extracts have been standardized with the intent

of ensuring consistency of allergy content and potency

between manufacturers and lots made from the same

manufacturer. Standardization is based on intradermal

skin test responses in allergic individuals. Speciﬁcally,

reference standards from the FDA CBER are obtained

for standardized allergen extracts by identifying

concentrations that reproducibly produce erythema

with a sum of perpendicular long axes of 50 mm, or

EAL.

These reference standards are then used by

manufacturers to ensure that the allergen content of

each new lot falls within speciﬁed ranges for potency

labeling. Blood tests (immunoassays) have been

developed that correlate allergenic protein content to

skin test reactions and, in some cases, treatment results.

These include measurement of major allergen content

(cat hair Fel d 1 and ragweed Amb a1), total protein/

hyaluronidase/phospholipase content (Hymenoptera

venom) and other assays (pooled sera immunoassay

inhibition activity).

Units of potency applied to standardized extracts vary

and include bioequivalent allergy unit/mL (BAU/mL),

allergy unit/mL (AU/mL), and microgram protein/mL

(µg/mL). Some allergen extract labels also include the

concentration of major allergenic proteins in µg/mL.

Since the standardization is based on allergen content

falling within a speciﬁed range, it still remains possible

for actual allergenic protein content to vary several-fold

for the same potency label. Only a few allergen extracts

have been standardized to date (see Appendix 1 for

effective dosing ranges).

III. ALLERGEN EXTRACT MIXING STANDARDS, QUALIFICATIONS, AND COMPETENCIES

While it would be ideal for allergen mixing personnel

to have a single guideline to refer to, there are

currently two documents that all personnel who mix

allergen extracts must utilize. The most widely adopted

recommendations in the United States for all aspects

of allergen immunotherapy are contained in the 2011

publication, “Allergen immunotherapy: A practice

parameter third update.”

This joint effort by experts

from the College and AAAAI and focuses on evidence-

based recommendations to optimize immunotherapy

efﬁcacy and safety. All health care providers involved

in immunotherapy preparation and administration

should be oriented to the contents of this practice

parameter, which contains practical clinical information

and sample forms. In this document, TABLE VII. Allergen

Immunotherapy extract preparation guidelines may be

found on page S36.

The second publication, USP Chapter <797>, focuses

on maintaining sterility in the compounding of

allergen extracts, including requirements for training

and testing of personnel. USP 797 sets standards for

preparing sterile preparations, including allergen extract

preparation. The requirements for compounding of

allergenic extracts are laid out in USP 797 Section 21.

Required qualiﬁcations of mixing personnel may be

found in Section 21 on pages 30 — 31, and facilities

requirements found on page 31. These standards,

though extensive and more rigorous than earlier USP

797 requirements, remain less rigorous than standards

required for typical sterile drug compounding in

pharmacies.

Practitioner Qualiﬁcations

There are many positions within the allergy immunology

ofﬁce that have an impact on the safety of allergen

immunotherapy. These include:

1. Supervising physician: responsible for overseeing

and ensuring the competency of staff preparing and

administering allergen immunotherapy.

2. Designated supervisor: person with training

and expertise in allergen immunotherapy who is

responsible for ensuring that personnel who will

be preparing allergen immunotherapy are trained,

evaluated and supervised.

3. Compounding personnel: those who mix allergenic

extracts. Before beginning to independently prepare

allergenic extracts, all compounding personnel must

complete training and demonstrate knowledge of

principles and skills for sterile compounding.

4. Shot personnel: those who administer allergy

immunotherapy injections. State laws may differ with

regard to approved personnel. Knowledge of how to

appropriately administer immunotherapy extracts is

critical for patient safety, making personnel training

a requirement. Recognizing the signs and symptoms

of anaphylaxis are also critical for any facility involved

in the delivery of allergen immunotherapy. Efforts

at standardization, including color coding vials,

standardizing labeling and documentation are

intended to decrease the risk of dosing errors for any

facility administering immunotherapy.

Training, Competency Assessment and Documentation

Training of personnel involved in the mixing of

allergen extracts and/or administration of allergen

immunotherapy is a critical requirement for safety

and efﬁcacy. Content of the training should include

core cognitive knowledge as well as demonstration of

procedure performance competency.

There are a variety of ways to receive training in allergen

immunotherapy preparation and administration.

Formats may vary from lectures to hands-on training to

meet the needs of each learner and include:

• On-the-job training from a qualiﬁed co-worker or

supervisor.

• Workshops and seminars offered at the College’s

Annual Meeting.

• Educational offerings via the College Learning

Connection, including the Allergen Extract

Preparation Quiz.

• Manuals from allergen extract manufacturers.

• Allergen Immunotherapy: A Practice Parameter

Third Update and other journal articles.

• Allergen Extract Mixing Toolkit.

Suggested qualiﬁcations of extract preparation

personnel based on the Practice Parameter on Allergen

Immunotherapy and applicable USP Chapter <797>

standards include ability to:

• Demonstrate understanding of appropriate hand

hygiene, garbing, surface disinfection, aseptic

technique, achieving and/or maintaining sterility,

calculating/measuring/mixing, use of equipment

and documentation.

• Pass a written test on aseptic technique and extract

preparation.

• Annually pass a media-ﬁll or equivalent test

verifying use of aseptic technique.

• Annually pass a gloved ﬁngertip-thumb sampling

test verifying hand sterility after passing three initial

tests.

• Be reinstructed and reevaluated if failing the written

test, media-ﬁll test or gloved ﬁngertip-thumb

sampling test.

To be compliant with USP Chapter 797 Section 21

requirements, allergist ofﬁces must keep records

of training, assessment results, evaluations and

qualiﬁcations for all compounding personnel, including

any corrective actions following assessments and

evaluations. Appendix 2 contains a sample document

for assessing and documenting competency of

personnel in the preparation of allergen immunotherapy

treatment sets. It is adapted from competency elements

for allergy technicians / nursing personnel at the U.S.

Army Centralized Allergen Extract Laboratory. These

competency elements are based on recommendations

of the Joint Commission on Accreditation of Hospital

Organizations, or JCAHO, requirements. As with all

sample forms, this serves as an example — each mixing

center should develop site-speciﬁc standard operating

procedures and competency standards, with forms that

meet the needs of their speciﬁc practice.

IV. ALLERGEN IMMUNOTHERAPY PRESCRIPTIONS

Allergen immunotherapy prescriptions specify the

precise contents of individual treatment sets for

patients receiving immunotherapy. They may be written

or electronic but should contain several essential

elements. Standardization of content will promote

proper preparation, minimize risk for errors in allergy

shot administration and facilitate patient transfers of

care. Employing the prescribing principles that follow

will result in a product that is safe and effective, and can

ensure maintenance of expected potency through the

expiration date. Each prescription / immunotherapy

record should contain:

• Two patient identiﬁers (i.e., Name and Date of Birth

or Medical Record Number)

• Patient contact information

• Name of prescriber

• Date of prescription

• Name, concentration and volume for each allergen

• Name and volume of diluents

• Schedule for administration (including adjustments

for interruptions and reactions)

All prescriptions should be reviewed for accuracy prior

to preparation.

Optimal mixing of allergens to create an individual

patient treatment set should be based on:

• Use of relevant allergens for each patient

• Dosing of allergen extracts within minimum

effective dose ranges (Appendix 1)

• Avoiding combining extracts that may adversely

affect overall potency

° Separate high protease extracts (mold, cockroach)

from pollens

° Avoid mixing venom extracts with aeroallergen

extracts.

Selection and adjustment of doses

using knowledge of cross-reactivity

Mixing high-protease extracts with most other

aeroallergens will result in a loss of potency that can

affect immunotherapy efﬁcacy. Aeroallergens with

known high cross-reactivity allow prescribers to treat

with fewer allergens while providing coverage for

a large number of related allergens. For example,

treatment with one or two northern pasture grass

allergen extracts should be sufﬁcient to provide beneﬁt

for the more than 10 cross-reactive northern grass

species.

Figure 1 is an example of a completed allergen

immunotherapy prescription. In this example, the

desired maintenance dose for cat was 2000 BAU for a

0.5-mL injected dose from a 5-mL maintenance vial (a

blank form is available in the Appendix).

2 mL of standardized cat extract (10,000 BAU/

mL) is needed to achieve a ﬁnal maintenance vial

concentration of 4000 BAU/mL and an injection dose of

2000 BAU. To do this use the step-by-step calculations

below.

Step-by-step calculations are as follows:

• Maintenance vial concentration of cat = injection

dose/injection volume = 2000 BAU/0.5 mL = 4000

BAU/mL

• V1 x C1 = V2 x C2 (maintenance vial volume x

maintenance vial concentration = stock volume x

stock concentration)

• 5mL x 4000 BAU/mL = V2 x 10,000 BAU/mL [V2 =

stock volume = (5 x 4K)/10K = 2 mL]

• Repeat for each antigen in vial

• Total antigen volume = cat 2 mL + D. farinae 0.5 mL

+ D. pteronyssinus 0.5 mL + timothy 0.4 mL + short

ragweed 0.2 mL

• Diluent volume = (maintenance vial volume) – (sum

antigen volumes) = 5 mL – 3.6 mL = 1.4 mL

• Final maintenance vial contents:

Antigen Concentration Dose

Cat 4000 BAU/ml 2000 BAU

Ragweed 17.5 microgram/mL 8.75 microgram/

mL Amb A 1

D. farina 1000 AU/mL 500 AU

D. pteronyssinus 1000 AU/mL 500 AU

FIGURE 1. EXAMPLE — ALLERGEN IMMUNOTHERAPY EXTRACT PRESCRIPTION (blank form available in Appendix)

Patient Name: Mary Wheeze Prescribing Physician: Dr. Allergist

Patient Number: Address:

Birth Date: Telephone:

Telephone: Fax:

Antigen

Number

Extract Name

Allergen or Diluent

(Common name of

Genus/species)**

Concentration and Type

Manufacturer’s Extract

(AU, BAU, W/V, PNU)/

(50%, G, Aq, Ly, AP)

Volume of

Manufacturer’s

Extract to Add

Extract

Manufacturer

Lot

Number

Expiration

Date

Short Ragweed 1:10 w/v G

(350 mcg Amb a1

0.25 ml Greer 11111 12/25/2021

D. farina 10,000 AU/ml G 0.5 ml Allermed 22222 12/26/2021

D. Pteronyssinus 10,000 AU/ml G 0.5 ml Antigen labs 33333 2/7/2022

Cat 10,000 BAU/ml G 2 ml Hollister-Stier 44444 12/5/2021

Timothy Grass 100,000 BAU/ml G 0.4 ml ALK 55555 12/3/2021

Diluent

HAS 1.35 ml ALK 66666 12/5/2021

Total

Volume

**Components of mixes listed on a separate sheet.

By Use Date ____________________________________________________________________________________________________________

Storage Requirements ___________________________________________________________________________________________________

Results of Quality Control (e.g. visual inspections, second veriﬁcation of questions) _____________________________________________

Allergen Extract Name:

R, Dm, C, G

Bottle Name Abbreviations

Tree: T Mold: M

Grass: G Cat: C

Weed: W Dog: D

Ragweed: R Cockroach: CR

Mixture: Mx Dust Mite: Dm

Speciﬁc Instructions:

Prescribing Physician’s Signature Date

Volume to add = Maintenance Concentration x Total Volume

Conc. of Manufacturer’s Extract

Maintenance concentration and subsequent dilutions

reported as volume/volume (v/v) dilutions with maintenance

concentrations=1:1 v/v

BAU=Bioequivalent Allergy Unit, AU=Allergy Unit

PNY=Protein Nitrogen Unit

W/V=Weight per Volume Ratio

G=50% Glycerinated

Aq=Aqueous, Ly=Lyophilized

AP=Alum precipitated, AcP=Acetone precipitated

Maintenance Concentrate Prescription Form

Prepared by: Great Nurse Date Prepared: 11/01/2019 Time: 10 a.m.

Dates of subsequent dilutions from maintenance concentration with expirations dates:

Vial __________ From Vial __________ on ____/____/____ Expiration date: ____/____/____

*Adapted from Allergen immunotherapy: A practice parameter second update. J Allergy Clin Immunol 2007;120: S77.

V. COLOR CODING, LABELS AND BEYOND USE DATES

The Allergen Immunotherapy Practice Parameters

and Joint Commission National Patient Safety goals

emphasize the need for clear and consistent labeling.

Standardization of allergen immunotherapy label

contents and vial coding helps improve communication

between care providers and patients, and likely prevent

errors in extract administration.

Each patient’s treatment vial label should contain at

minimum:

• Patient name, plus a second identiﬁer (e.g., date of

birth)

• Concentration in vol/vol

• Color code and vial number, per the Practice

Parameters

• Expiration or beyond use date (BUD)

• Storage conditions

Immunotherapy treatment vial concentrations are

now labeled in vol/vol, with 1:1 vol/vol representing

the maintenance concentrate. Alternatively, the vial

concentration can be labeled in actual units (e.g., 1000

BAU, 100 BAU), but this system may be complicated

if allergens with different potency units are used (e.g.,

wt/vol, BAU, AU or PNU) and these differences make

it difﬁcult to interpret the vial label. All vials in the

treatment set should be numbered and/or color coded

in the following manner as outlined in the most recent

practice parameters (Figure 2, page S44):

RED Maintenance 1:1 vol/vol #1

concentrate

YELLOW 10-fold dilution 1:10 vol/vol #2

BLUE 100-fold dilution 1:100 vol/vol #3

GREEN 1000-fold dilution 1:1000 vol/vol #4

SILVER 10,000-fold dilution 1:10,000 vol/vol #5

The highest concentration should be labeled #1 and

the next 10-fold dilution (i.e., yellow vial) would be

labeled #2, and so forth. This system was designed to

eliminate variation from patient to patient that occurs

when labeling vials of higher concentrations with larger

numbers. This practice resulted in patients often having

a different number on their maintenance vial that was

based on the total number of dilutions prepared.

Vial expiration dates should follow the manufacturer’s

recommendations. The expiration date for a treatment

vial should be no later than the earliest expiration

date recommended for any stock allergen or diluent

contained in each prescription vial. Less-concentrated

extracts are more sensitive to temperature and might

not maintain potency until the listed expiration date;

1:10 to 1:200 dilutions of stock extracts are generally

stable for at least 12 months. This usually includes at

least the patient’s red maintenance treatment vial and

the 1:10 vol/vol, or yellow, vial. Expiration dates for

venom extracts may be shorter; perhaps this is due

to the use of diluents with low levels of glycerin. The

venom extract package inserts provide guidelines for

expiration dates for the different dilutions.

Expiration dating periods for allergen extract products

are regulated by the FDA. Even under ideal refrigerated

conditions, some loss of potency occurs over time.

The potency and stability of these products are not

guaranteed beyond their labeled expiration date.

Nonstandard extract products are assigned expiration

dates in accordance with FDA regulations (21 CFR,

Section 610.53) with regard to whether products are

glycerinated or non-glycerinated. A total of six years

from the time of extraction is allotted to 50% glycerin

bulk extracts. This six-year period is divided into a

maximum of three years for manufacturer storage

and three years for shipped vial expiration dating.

Non-glycerinated products are allowed only a total of

three years or half the expiration the time allotted for

glycerinated extracts —18 months for manufacturer

storage and 18 months for shipped expiration dating.

Following is an example of expiration dates for

diagnostic and treatment sets prepared by the

U.S. Army Centralized Allergen Extract Laboratory,

which are based on stock concentrate manufacturer

recommendations from its suppliers.

Diagnostic Products Expiration Date*

Prick Test Materials 1 Year

ID Test Materials 6 Months

Immunotherapy Treatment Sets

1:10 W/V-1:5000 W/V 1 Year

1:50,000 W/V and weaker 3-6 Months**

1000 PNU/ml — 20,000 PNU/ml 1 Year

<1000 PNU/ml 3-6 Months**

500 AU/ml and Stronger 1 Year

< 500 AU/ml 3-6 months**

1000 BAU/ml and Stronger 1 Year

< 1000 BAU/ml 3-6 months**

*Use earliest of stock extract label expiration date or date

below

**The stability of lower extract concentrations (e.g.,

1:1000 and 1:10,000 vol/vol) has not been extensively

studied. Loss of potency in these lower concentrations

may be due to absorption of the allergenic proteins to

the glass wall. Human serum albumin may have a more

protective effect against this cause of loss of potency

than other diluents such as normal saline.

Reconstituted Venom Freeze Dried Preparations

100mcg/ml 6 or 12 months*

1-10mcg/ml 1 month

0.1mcg/ml 14 days

<0.1mcg/ml 24 hours

*Varies with company. Guidelines for dilution expiration

dating are in the extract package insert

VI. MIXING INDIVIDUAL PATIENT ALLERGEN EXTRACT TREATMENT SETS

General Principles

Every clinic should develop a speciﬁc standard

operating procedure (SOP) document or manual to

ensure standardization and safe practices of allergen

extract mixing. Responsible providers developing the

procedures should consult stock extract manufacturer

recommendations and the most recent Allergen

Immunotherapy Practice Parameter Update to

incorporate the most up-to-date recommendations. USP

797 requirements must also be reviewed and followed.

The mixing of antigens in a syringe is not recommended

because of the potential for cross-contamination of

extracts.

Initial Preparation

1) Designate a dedicated allergenic extracts

compounding area (AECA) with a visible perimeter

that establishes the boundaries of the AECA, and

meets the following conditions (as required by USP

797):

• Access restricted to authorized personnel.

• No other activity permitted during compounding.

• All surfaces are cleanable and kept clean.

• Carpet is not allowed.

• Surfaces must be impervious to cleaning and

sanitizing agents; must be smooth, non-shedding,

and free of cracks or crevices.

• Overhangs should be avoided or must be easily

cleaned.

• Work areas must be well lit, temperature and

humidity controlled for comfort of compounding

personnel.

• Work surface must be cleaned and disinfected

before and after each compounding session;

disinfected between each new set, as well as at the

time of any spill or contamination.

• During compounding, at that time.

2) Before beginning compounding, ensure your

personnel performs hand hygiene and garbing

procedures (that should include the minimum

requirements noted below) and according to your

clinic’s SOPs.

• Low-lint garment with sleeves that ﬁt snugly around

the wrists and that is enclosed at the neck (e.g.,

gowns or coveralls);

• Low-lint, disposable covers for head that cover the

hair and ears and, if applicable, disposable cover

for facial hair;

• Face mask; and

• Sterile powder-free gloves.

3) Ensure compounding personnel rub sterile 70% IPA

onto all surfaces of the gloves and allow them to dry

thoroughly throughout the compounding process.

4) Clearly identify expiration dating standards for your

clinic.

5) Become familiar with stock allergen ordering and

storage procedures.

6) Orient personnel to stock allergen extracts,

refrigerator storage designated mixing location,

mixing equipment, prescriptions, documentation and

packaging.

7) Ensure appropriate personnel are trained on SOP and

safety measures and have the required competencies

to independently mix allergen extracts.

Pre-mixing Preparation

1) Verify that a supervising physician is present in the

same building as the mixing location(s).

2) Cleanse and maintain an aseptic work environment

using 70% isopropanol as recommended in USP 797

Allergen Extract Preparation

3) Prepare vial labels in accordance with prescription

and verify accuracy of:

• Name and second identiﬁer

• Concentration

• By use date (BUD) is consistent with clinic

procedures and source antigens.

4) Verify color-coded vials with designated

concentration

Labels may be applied after mixing. For example, the

label for the empty maintenance concentrate (red) vial

(or all vials) can be left off until all contents are injected

into the vial to improve visibility during checks for

impurities, ﬁnal volume, color comparison of dilution

series, and better aseptic technique.

Example Allergen Extract Mixing Step-By-Step

Procedures

This sample set of procedures provides general guide-

lines that can be used as a starting point to develop

procedures that best ﬁt a speciﬁc clinic/facility needs.

Mixing the Maintenance (Red) Vial (see Appendix 3)

1) Select new empty sterile vials (usually 5, 8 or 10 mL)

for each vial in patient’s treatment set, using color

codes to line up strongest (maintenance/red) to most

dilute.

2) Select stock extract for each antigen contained in the

prescription, and stock diluents.

• Check stock antigens for turbidity/particulate

matter. If present, consult package insert or

manufacturer guidelines including possible

recommendations for resuspension or ﬁltering.

• For prolonged mixing sessions, return unused

stock extracts to refrigerator or cooling tray (2º-8ºC)

between prescriptions or during extended breaks.

3) Place a new syringe by each stock antigen vial and

the diluent.

• A separate syringe is used for each antigen and

diluent.

• Label each syringe (i.e. abbreviation for antigen or

diluents)

• Avoid storing stock extracts in a syringe for

extended periods due to risk of potency loss and

misidentiﬁcation.

4) Document lot number and manufacturer for each

antigen

5) Ensure that label expiration date does not exceed

earliest stock vial extract or diluent expiration date.

6) Perform Hand Hygiene per USP 797 standards.

• Remove visible debris from underneath ﬁngernails

under warm running water using a disposable nail

cleaner

• Wash hands and forearms up to the elbows with

soap and water for at least 30 seconds

• Dry hands and forearms to the elbows completely

with low-lint disposable towels or wipers.

7) Personnel garbing requirements

• Don hair and facial hair covers, gowns and face

masks

• Use alcohol- based surgical hand scrub prior to

gloving.

• Don powder-free sterile gloves compatible with

70% isopropyl alcohol.

8) Disinfect gloves with isopropyl alcohol and allow

them to dry before mixing (and intermittently for

lengthy mixing).

9) Wipe vials and/or ampules with 70% isopropyl

alcohol and allow to dry.

10) Maintain aseptic technique by minimizing contact

with secretions, skin, glove ﬁngertips and other

potential sources of contamination during the

mixing process.

11) Draw the correct amount of each antigen and the

diluent into the syringe, and place each syringe by

the respective stock antigen vial.

12) Verify drawn doses are correct volume and antigen.

(Quality checkpoint opportunity: have a co-worker

verify, if available. This is a best practice, but this is

not required by USP 797.)

13) Sequentially inject contents of antigens into the

maintenance concentrate (red) vial.

• Empty syringes should be discarded immediately

into an appropriate sharps disposal container

• If the sterile maintenance vial is not a vacuum vial,

an equal volume of air may need to be withdrawn

prior to injecting stock extract volumes

14) After mixing is complete, conduct ﬁnal quality

assurance check (best practice would include mixer

and trained co-worker), including:

• Solution color check.

• Label check.

• Vial color-code check.

• Liquid turbidity, precipitate and consistency check.

• Vial physical integrity (leaks, cracks and so on)

check.

15) If applicable, package treatment set for transport or

shipping.

16) Document preparation details according to clinic-

speciﬁc procedures on prescription or preparation

form and in mixing log as follows:

• Name of preparer and date prepared.

• Stock allergen extract manufacturer, lot number

and beyond-use or expiration date.

• Mixing log, to be maintained in the unlikely event

of a stock allergen recall or for extract or adverse-

event troubleshooting.

Notes Regarding Alum-Precipitated Extracts

• Diluent: Alum-precipitated extracts generally

require phenol saline diluent for all 10-fold dilution

vials. Ten percent glycerol-saline or HSA diluent

cannot be used for alum- precipitated prescriptions

as it interferes with the aluminum hydroxide-

antigen absorbed complex.

• For alum-precipitated extract treatment vials,

consider applying a small “shake well” label, as

the alum-precipitated antigens are very viscous

in nature. Precipitated alum- antigen complex will

settle to the bottom of the vial.

• Unlike aqueous and glycerinated extracts that

generally do not lose potency with ﬁltering, large

antigen-alum complexes may be lost during the

ﬁltering process, with the result being a result

in loss of potency. Therefore, do not ﬁlter alum-

precipitated extracts.

Preparing Serial 10-Fold Dilutions

• Serial 10-fold dilutions are prepared to complete a

patient’s initial allergen immunotherapy treatment

vial set. Dilutions are made by serial dilution

(taking from a parent vial and placing into a new

vial preﬁlled with diluent to create a 10-fold dilution

1/10 the amount of allergen contained in the

parent vial). This newly diluted vial then becomes

the “new” parent vial, and dilutions are repeated

until the desired number of 10-fold dilutions is

achieved.

• The volume used to make serial dilutions from

parent vials depends on both the desired dilution

(10-fold in this case) and the ﬁnal volume.

Typical treatment set vials are 2, 5, 8 or 10 mL.

Treatment set vials are available with original or

snap-on colored caps to create sets according to

the recommended color scheme. Vials also are

available empty or preﬁlled with diluents suitable

for intradermal or subcutaneous administration.

Preﬁlled volumes correspond to the amount of

diluent needed to make a 10-fold dilution.

• To determine how much should be taken from the

parent vial for a 10-fold dilution for ﬁnal volume

X, divide X by ten (i.e., for a 10 ml vial: 10ml/10

= 1 ml). Then calculate the amount of diluent

needed by subtracting this X/10 volume from the

ﬁnal volume X (i.e. 10 ml – 1 ml = 9 ml). The ﬁnal

concentration of the diluted vial is 1/10th that of the

parent vial.

Sample preparation of a 5-mL SERIAL 10-Fold Dilution

Verify that the labeling and order (color coded, label

concentration) for vials are correct.

1) Ensure the maintenance vial is mixed by inverting or

rolling.

2) Using a fresh syringe and aseptic technique, remove

0.5 mL from the mixed 5-mL maintenance concentrate

red or 1:1 vol/vol vial.

3) Using aseptic technique, inject this 0.5 mL form

the maintenance vial into the 4.5-mL preﬁlled (10%

glycerol-saline or HSA) yellow or 1:10 vol/vol vial.

This vial will consist of 5mL of a 10-fold dilution of the

maintenance concentration vial.

4) Ensure the newly made 10-fold diluted (yellow) vial is

mixed by inverting or rolling.

5) Subsequent 10-fold dilutions are done in the same

manner for the remainder of the vials in the treatment

set (0.5 mL into 4.5 mL of the 10-fold, labeled 10%

glycerol-saline preﬁlled vial):

1. 0.5 mL from yellow 1:10 vol/vol into a 4.5 mL

diluent-ﬁlled blue 1:100 vol/vol vial

2. 0.5 mL from blue 1:100 vol/vol into 4.5 mL diluent-

ﬁlled green 1:1000 vol/vol vial

3. 0.5 mL from green 1:1000 vol/vol into 4.5 mL

diluent-ﬁlled silver 1:10,000 vol/vol vial

4. And so on for additional more dilute (silver) vials

5. Whereas using a fresh syringe for each dilution

transfer is often preferred, use of the same syringe

for serial dilution transfers is an alternative if a

“mix/rinse” step is included. A mix/rinse step

consists of pulling up a syringe volume (1mL for

a 1-mL syringe) from the vial just injected and re-

injected into the same vial without removing the

syringe. This is often repeated (i.e., for a total of

three times) prior to pulling up the ﬁnal volume for

the transfer to the next dilute vial. (Reminder: This

is for making dilutions only. Do not reuse syringes

or mix/rinse between different stock solutions

when mixing the initial maintenance vial.)

Table 1. Calculations for making Extract Dilutions

Table II. Calculations for making Extract Dilutions* (from Practice Parameters page S10)

All dilutions can be calculated by using the following formula:

V1 X C1 = V2 X C2,

where

V1 = Final volume you want to prepare

C1 = Concentration (wt/vol or PNU) of extract you want to prepare

V2 = Volume of extract you will need for dilution

C2 = Concentration of extract you will use.

Example: Solve for V2; (V1 X C1)/C2 = V2.

To determine the concentration of an item in a mixture:

1. determine which formula you need to use;

2. choose the numbers/fractions that will be inserted into the formula for V1, C1, V2, and C2;

3. change all wt/vol fractions to a decimal number and insert into the formula (see below); and

4. multiply ﬁrst and then divide to get the answer.

To express concentration as a percentage:

1:10 wt/vol 1/10 = 0.1 X 100 = 10% solution

1:20 wt/vol 1/20 = 0.05 X 100 = 5% solution

1:40 wt/vol 1/40 = 0.025 X 100 = 2.5% solution

Example:

V1 = 5 mL Final volume you want to prepare

C1 = 1:200 Concentration you want to prepare

V2 = Unknown Volume of extract you will need for dilution

C2 = 1:10 Concentration of extract you will use

Add values into formula:

V1 X C1 = V2 X C2 5 X (1/200) = V2 X (1/10)

5 X (0.005) = V2 X (0.1)

V2 = (V1 X C1)/C2 V2 = 0.025/0.1 = 0.25

To determine amount of diluent needed:

V1 — V2 5 – 0.25 = 4.75 mL

Adapted from the Greer Allergy Compendium. Lenoir (NC): Greer Laboratories: 2005. p. 71. Permission provided by Robert Esch, PhD.

Allergen Extract Treatment Set Preparation Hints

1) Do not mix prescriptions for more than one patient at

a time.

2) Train multiple qualiﬁed personnel in allergen extract

preparation in case of absences and for participation

in quality checks.

3) Avoid putting hand lotion on before the

compounding of allergen extract and skin test

antigens. Lotion tends to harbor bacteria.

4) Regularly review operating procedures for opportunities

to make the process safer and more efﬁcient.

5) Establish a regular inventory check.

• Identify stock allergen extracts, diluents and mixing

supplies in need of reordering

• Check for expiring stock allergen extracts, diluents

and mixing supplies

6) Return antigen stock trays to the refrigerator when

away from the compounding area for an extended

period of time.

7) Minimize distractions during extract preparation.

8) Stock refrigerators are not to be used for food or

drink storage.

9) Particulates and precipitates suspended in an extract

solution are not uncommon.

• These particulates and precipitates often do not

cause any signiﬁcant loss in potency. Consult

manufacturer recommendations in package insert

or bulletins for additional information.

• Attempted re-suspension by agitation (shaking

or rolling) may be indicated in accordance with

the package insert and your clinic operating

procedures.

10) Diluted allergen immunotherapy vials (yellow,

blue, green, and silver) should not made by pulling

directly from a manufacturer’s concentrated stock

vial extract.

• The primary reason for this is the potential for error

that is increased progressively with each dilution. For

dilute vials, a very small amount of allergen would

need to be pulled from the stock extract vial, and it is

virtually impossible to achieve the precision needed

for the most dilute vials.

• Thus, a dilution vial prepared by this method

may contain inaccurate amounts of extract and

potentially increase the risk of adverse events

during vial transitions within the buildup phase.

Additional Quality Assurance Checks

Additional quality assurance checks before allergen

extract shipping and use ideally are conﬁrmed by a co-

worker. Vials should be inspected for:

• Vial serial color dilution that matches label

concentration and vial color coding

• Vial integrity (no cracks, leaks, and so on)

• Vial content (particulate matter, ﬁll volume, and so

on)

• Label accuracy – “ﬁve rights” as described below

Verify that the label contains the 5 rights

• Right name,

• Right content (allergens),

• Right concentration, right alphanumeric number

in the right order with most concentrated = 1 (if

numbers used),

• Right expiration date (dilute vials may have earlier

expiration dates than more concentrated vials).

• Right color, a solution color check for each vial

should be conducted.

The solution in the maintenance concentrate vial should

be the darkest in color, and vials should be lighter in

color with each 10-fold dilution. The weakest strength

vial should contain the lightest-colored solution. When

using color-coded vials, a vial color code check should

be performed. Vials in the treatment set should be

arranged in order (red/maintenance, yellow, blue,

green and silver). For each color-coded vial, label

concentration, in vol/vol, or number should match what

is recommended in the Practice Parameters for the color

code (see Tables XI and XII from Practice Parameters

All vials should also undergo a content check. Vials

should be ﬁlled to the expected volume. Solutions

within each vial should be inspected for the presence of

particulate or solid materials and cloudiness. If found,

vials may be contaminated or contain precipitated

raw allergen extract contents. Contamination may

be bacterial or other microbial source, but may

also be a result of introduced solid materials like

the rare occurrence of vial stopper fragments from

manufacturing or repeated puncturing. Any abnormal

ﬁnding during any of these checks should be followed

by an investigation for the cause and, in most instances,

starting over and remixing that patient’s vial set.

Table 2. Preparing Allergen Extract Treatment Sets (2 antigens, 0.5 ml stock of each)

Dilution label/

color

Label Conc

vol/vol

Extract

added

Diluent

added

Ragweed

w/v

Grass

BAU/ml

stock extract 1:10 100,000

1 (red/maint) 1:1 0.5 mL of each stock 4.0 mL 1:100 10,000

2 (yellow) 1:10 0.5 mL red (1) vial 4.5 mL 1:1,000 1,000

3 (blue) 1:100 0.5 mL yellow (2) vial 4.5 mL 1:10.000 100

4 (green) 1:1,000 0.5 mL blue (3) vial 4.5 mL 1:100,000 10

5 (silver) 1:10,000 0.5 mL green (4) vial 4.5 mL 1:1,000,000 1

*Treatment set for maintenance concentrate with 0.5 ml stock conc. grass (100,000 BAU/ml) & ragweed (1:10 w/v) in 5ml

VII. STINGING INSECT ALLERGEN EXTRACT PREPARATION

Extracts are available for ﬁve winged Hymenoptera

species at a concentration of 100 µg/mL: honey bee,

wasp, yellow jacket, yellow hornet and white-faced

hornet. The last three (yellow jacket, yellow hornet

and white-faced hornet) are closely related members

of the Vespidae family and have also been combined

in a single “mixed vespid” extract at a reconstituted

concentration of 300 µg/mL. Lyophilized or freeze-

dried stinging insect venom extracts are available

commercially for diagnostic testing and patient

treatment. These extracts are composed of venom

isolated directly from dissected venom sacs.

Previously manufactured extracts using whole insect

body as opposed to concentrated venom proved

not to be as effective as extracts made from venom.

Accordingly, handling of these extracts is limited to

reconstitution and dilution. The same principles and

requirements for labeling apply with the exception of

number/color coding and use of vol/vol concentration.

The concentration of these extracts and all dilutions is

expressed in µg/mL. Reconstitution and dilution of all

insect venom extracts is most commonly performed with

HSA (HSA/phenol) diluent.

Extracts are also available for imported ﬁre ant

Hymenoptera species. Two ﬁre ant species, Solenopsis

richteri and S. invicta, are commercially available as

individual extracts for testing or treatment, or as a

ﬁre ant mix containing both species. Fire ant extracts

are made from whole ﬁre ant bodies. Fire ant venom

extracts are being investigated for clinical use but

require a signiﬁcant amount of time and resources for

mass production. Fire ant stock concentrate extracts

typically are available as non-standardized glycerinated

extracts in wt/vol concentrations (i.e., 1:20 wt/vol). The

Practice Parameters for Insect Allergy contains survey

data on common ﬁre ant maintenance doses ranging

from 0.5 mL of 1:100 wt/vol to 0.5 mL of 1:10 wt/vol

maintenance concentrate, with most using 0.5 mL of a

1:100 wt/vol maintenance concentrate.

Insect venom (and ﬁre ant) extracts generally should

not be mixed with other venom or aeroallergen extracts

for either testing or treatment because of the lack of

sufﬁcient stability, safety and efﬁcacy studies to support

mixing. The only FDA-approved mixture is commercially

available mixed vespid extract containing 100 µg/mL of

each of the three common vespids.

VIII. ALLERGEN EXTRACT STABILITY AND STORAGE

The stability and potency of allergen extracts can be

compromised by elevated temperatures, contamination

and protease degradation of key allergenic proteins

responsible for the efﬁcacy of immunotherapy.

31-34,37

Several measures are taken by stock extract manufacturers

and healthcare personnel to minimize the risk of loss of

potency of extracts during normal storage and use.

Dilution of extracts alone can affect the long-term

potency of extracts. For example, diluted extracts have

lower concentrations of important preservatives and

stabilizers. Furthermore, lower concentrations of proteins

decrease three-dimensional protein structure stabilization

achieved through protein-interactions present at higher

protein concentrations. Finally, dilutions may also magnify

the effect of allergenic protein loss as a result of binding

to sites on glass vials that is essentially insigniﬁcant at

higher protein concentrations.

There are several “routine” operating procedures that

when performed consistently should promote extract

stability and reduce errors associated with the use of

outdated materials:

• Routinely check expiration dates on all products.

• Ensure that the stock inventory in refrigerators is

routinely rotated such that expiring products are

placed in the front and used ﬁrst.

• Verify that the expiration dates on labels for

treatment and diagnostic sets are no later than the

stock extract used with the earliest expiration date.

• Discard or separate products that have expired.

• Ensure that personal allergen extract storage trays

are stored at recommended temperatures.

• Ensure that extracts are kept cool during extended

periods of mixing.

Manufacturer processing steps include additives that

stabilize the allergenic proteins and preservatives

that prevent contamination of the stock extract and

individual patient treatment sets derived from them.

Preservatives are added to allergen extract solutions

to prevent microbial growth in the event that bacteria

or fungi are introduced into the solution during the

preparation process or when needles are inserted

into vials for administration of immunotherapy. All

allergen extracts must contain preservatives that are

bacteriostatic. Bacteriostatic agents prevent the growth

of microbial contaminants like bacteria, but do not

necessarily kill microorganisms.

Sterilization and pasteurization processes that kill

microorganisms are less commonly used.

Phenol is a common bacteriostatic preservative added

to allergen extracts and is used at a ﬁnal concentration

of approximately 0.4%. One possible ill effect of using

phenol is that it may denature (unfold or breakdown)

allergenic proteins even if in a 50% glycerin.

31,33

HSA

may protect against phenol’s adverse effects on

allergenic proteins.

Other recognized preservatives

such as thimerosal and methylparaben are not generally

used in allergen extract preparation.

Stabilizers are added to diluents to maintain the

structure of allergens in solution and prevent sticking or

adherence to the glass vials in which they are contained.

Common stabilizers include glycerin and HSA. Fifty

percent glycerin is often considered the best stabilizer

alternative and is also considered a preservative,

whereas HSA is not a preservative. Glycerin potently

stabilizes proteins in solution and inhibits proteases

found in some allergen extracts, and is bacteriostatic at

concentrations ≥20%.

Preservative and stabilizing properties decrease as the

concentration of glycerin is decreased. Glycerin, when

injected subcutaneously, can cause local irritation and

result is a burning sensation.

Allergen extracts are stored in refrigerators at a

temperature of 4ºC or in accordance with manufacturer

recommendations. A temperature range of 2˚- 8ºC is

considered acceptable by most experts. Given the

expense and temperature sensitivity of stock allergen

extract concentrates and mixed patient treatment

sets, it is also reasonable to conduct some form of

temperature monitoring to ensure that extracts are not

exposed to temperature extremes. For example, a log

of daily temperatures (See Refrigerator Temperature

Log Form) can be maintained or an automated

continuous temperature monitoring device can be

installed. Facilities might also consider installing

temperature alarms.

Many allergen extracts are heat sensitive. The loss in

potency when allergen extracts are exposed to high

temperatures (i.e., over 78ºF or 26ºC) may be due to the

heat-labile (-sensitive) proteins that unfold or degrade

at these temperatures. Loss of potency can also occur at

lower temperatures, including room temperature (i.e.,

68ºF – 72ºF and 22ºC). This is possibly due to proteases

in the extract that are activated at these temperatures

and degrade relevant allergen proteins in the extract.

Allergen extracts exposed to room temperature over

time may thus lose potency, such as extracts frequently

left out of the refrigerator for long periods during

testing or treatment. For example, skin testing trays

with extracts that are taken out of the refrigerator in

the morning every day and not replaced until the clinic

closes in the evening may suffer from reductions in

potency unless the trays are cooled while out of the

refrigerator. Short intervals for testing or treatment rarely

result in clinically signiﬁcant losses of potency. Fifty

percent glycerin may help protect against the effects

of prolonged exposure to room temperature, possibly

due to its effect on proteolytic enzymes. Less is known

about the effects of freezing (<0ºC) on allergen extract

potency, but at least one study found a moderate loss of

potency when an extract was stored frozen and thawed

for use.

An increase in the number of multiple freeze-

thaw cycles increases the observed loss in potency

of extracts. Thus, extracts that are accidentally frozen

should be replaced with new extract prior to use.

Some extracts contain proteolytic enzymes or proteases

that can degrade proteins needed for allergen extract

effectiveness. Tree, grass and weed pollens and some

pet danders are particularly susceptible to these

proteases. For this reason, the most recent Practice

Parameters recommend the separation of extracts

with high proteolytic enzyme activities, such as mold

and cockroach, from other extracts, such as pollens.

Dust mite extracts do not appear to cause signiﬁcant

degradation of pollen or animal dander extracts and

thus can be mixed together.

Investigations have shown that extracts stored in vials

only partially ﬁlled with solution are less stable. In

other words, 1 mL of extract in a 10-mL vial will lose

potency more rapidly than 10 mL of extract in a 10-

mL vial. This volume effect is more pronounced with

higher dilutions. For this reason, it is reasonable to

consider reordering and preparing treatment and

diagnostic materials as the extract volume in current

vials diminishes.

IX. SUMMARY

The preparation of allergen immunotherapy extracts is

a technical skill that requires training and a high level

of attention to detail. Errors may cause life-threatening

allergic reactions in patients receiving immunotherapy.

Using a team approach to develop clinic/facility-

speciﬁc policies and procedures and verify ongoing

competency will ultimately improve the quality and

precision of allergen immunotherapy preparation.

Ongoing review of these procedures will lead to

increased knowledge of and adherence by individuals

preparing allergen extracts. These steps will ensure

the end product is appropriately prepared according

to the most recent standards and manufacturer

recommendations. Thorough knowledge and training will

promote the safety of the patients entrusted to our care

and of those performing allergen extract preparation.

There are several major themes that new personnel

assigned to prepare allergen extracts should become

familiar with. These include, but are not limited to:

• Contamination is prevented by adequate training

and the use of using aseptic technique

• Accurate labels and color coding are highly

recommended to prevent errors

• Use of quality assurance checks throughout the

mixing process is highly recommended

• Initial treatment sets consist of a maintenance vial

and a series of 10-fold dilutions

• Stinging insect and aeroallergen extracts should

not be mixed

All personnel involved in allergen extract preparation

should be familiar with the contents of the most recent

Immunotherapy Practice Parameter and 2019 Updated

USP 797 Standards for allergen extract mixing. A

companion examination has been developed based on

this training document to assist in satisfying competency

assessment and documentation requirements. It is

currently available via the College Learning Connection.

XI. REFERENCES

1. Noon, L. (1911). Prophylactic inoculation against hay

fever. Lancet, 1, 1572-73.

2. Ross, R. N., Nelson, H.S., Finegold, I. (2000). Effec-

tiveness of speciﬁc immunotherapy in the treatment

of asthma: A meta-analysis of prospective, random-

ized, double-blind, placebo- controlled studies. Clin

Ther, 22, 329-41.

3. Ross, R. N., Nelson, H.S., Finegold, (2000). Effective-

ness of speciﬁc immunotherapy in the treatment of

allergic rhinitis: An analysis of randomized, prospec-

tive, single- or double-blind, placebo-controlled

studies. Clin Ther, 22, 342-350.

4. Ross, R. N., H.S., Finegold, I. (2000). Effectiveness of

speciﬁc immunotherapy in the treatment of hyme-

noptera venom hypersensitivity: A meta-analysis.

Clin Ther, 22, 351-8.

5. Calderon, M. A., Alves, B., Jacobson, M., Hurwitz,

B., Sheikh, A., Durham, S. (2007). Allergen injection

immunotherapy for seasonal allergic rhinitis. The Co-

chrane Database of Systematic Reviews, Jan 24(1).

CD001936.

6. Abramson, M. J., Puy, R. M., Weiner, J. M. (2003).

Allergen immunotherapy for asthma. The Cochrane

Database of Systematic Reviews. (4). $%001186.

7. Lockey, R., Bukantz, S., Bousquet, J. (Eds.) (2004).

Allergens and allergen immunotherapy. (3rd Ed).

8. Ewan, P. W., Alexander, M. M., Snape, C., Ind, P. W.,

Agrell, B., Drebord, S. (1988). Effective hyposensi-

tization in allergic rhinitis using a potent partially

puriﬁed extract of house dust mite. Clin Allergy, 18,

501-8.

9. Olsen, O. T., Larsen, K. R., Jacobsan, L., Svendsen, U.

G. (1997). A 1-year placebo-controlled, double-blind

house dust-mite immunotherapy study in asthmatic

adults. Allergy, 52, 853-859.

10. Haugaard, L., Dahl, R., Jacobsen, L. (1993). A con-

trolled dose-response study of immunotherapy with

standardized, partially puriﬁed extract of house dust

mite: Clinical efﬁcacy and side effects. J Allergy Clin

Immunol, 91, 709-22.

11. Ohman, J. L., Jr., Sundin, B. (1987). Standardized

allergenic extracts derived from mammals. Clin Rev

Allergy, 5, 37-47.

12. Lombardero, M., Carreira, J., Duffort, O. (1988).

Monoclonal antibody based radioimmunoassay for

the quantitation of the main cat allergen (Fel d I or

Cat-1). J Immunol Methods, 108, 71-76.

13. American Academy of Allergy, Asthma and Immu-

nology (AAAAI) (1977). The use of standardized

allergen extracts. J Allergy Clin Immunol, 99, 583-6.

X. ACKNOWLEDGMENTS

ORIGINAL DOCUMENT CREATED IN 2007

Authored by:

Michael R. Nelson, MD, PhD, FAAAAI, FACAAI

Edited by:

Linda Cox, MD, FAAAAI

Tao T. Le, MD, MHS, FACAAI, FAAAAI

With Acknowledgments to:

Ms. Susan Kosisky, Ms. Anita Bienlein, Dr. Ceclia Mikita,

Mr. Eric Riddock, TSgt Adam Hughlett, TSgt Debra Horne

of the US Army Centralized Allergen Extract Laboratory at

Walter Reed Army Medical Center. Dr. Bryan Martin and

Dr. Renata Engler of the Walter Reed Vaccine Healthcare

Centers Network. Dr. Gary Gross and Dr. Don Aaronson,

MD of Advocacy Council of ACAAI; Dr. Linda Cox and

colleagues of the AAAAI Immunotherapy and Allergy

Diagnostics Committee and ACAAI Immunotherapy and

Diagnostics Committee and the members of ACAAI/

AAAAI Task Force on Practice Parameters.

DOCUMENT REVISED IN 2017

ACAAI Immunotherapy & Diagnostics Committee

John J. Oppenheimer, MD — Chair

Mike Tankersley, MD

Trever Burnett, MD

David B. Engler, MD

Warren V. Filley, MD

Desiree E. Larenas-Linnemann, MD

Alnoor A. Malick, MD

Anil Nanda, MD

Michael R. Nelson, MD, PhD

Ruby Pawankar MD, PhD, FRCP

Jorge A. Quel, MD

Noel Rodriguez, MD

Millard Tierce, IV, DO

Kevin M. White, MD

Ira Finegold, MD

Cheryl Bernstein, RN

14. Van Metre, T. E., Jr., Marsh, D. G., Adkinson, N. F.,

Jr., et al. (1989). Immunotherapy decreases skin

sensitivity to cat extract. J Allergy Clin Immunol, 83,

888-899.

15. Van Metre, T. T., Jr., Marsh, D. G., Adkinson, N. G., Jr.,

et al. (1988). Immunotherapy for cat asthma.

J Allergy Clin Immunol, 82, 1055-1068.

16. Ewbank, P. A., Murray, J., Sanders, K., Curran-Eberett,

D., Dreskin, S., Nelson, H. S. (2003). A double-blind,

placebo-controlled immunotherapy dose-response

study with standardized cat extract. J Allergy Clin

Immunol, 111, 155-161.

17. Dolz, I., Martinez-Cocera, C., Bartolome, J. M., Cimar-

ra, M. (1996). A double-blind, placebo-controlled

study of immunotherapy with grass-pollen extract

Alutard SQ During a 3-year period with initial rush

immunotherapy. Allergy, 51, 489-500.

18. Walker, S. M., Pajno, G. B., Lima, M. T., Wilson, D. R.,

Durham, S. R. (2001). Grass pollen immunotherapy

for seasonal rhinitis and asthma: A randomized,

controlled trial. J Allergy Clin Immunol, 107, 87-93.

19. Frew, A. J., Powell, R. J., Corrigan C. J., Durham, S. R.

(2006). Efﬁcacy and safety of speciﬁc immunother-

apy with SQ allergen extract in treatment-resistant

seasonal allergic rhinoconjunctivitis. J Allergy Clin

Immunol, 117, 319-325.

20. Leynadier, F., Banoun, L., Dollois, B., et al. (2001). Im-

munotherapy with a calcium phosphate-adsorbed

ﬁve-grass pollen extract in Seasonal rhinoconjuncti-

vitis: A double-blind, placebo-controlled study. Clin

Exp Allergy, 31, 988-996.

21. Lockey, R., Slater, J., Esch, E. Preparation of standard-

ization of allergen extracts. In F. Adkinson, J. Yungin-

ger, W. Busse, B. Bochner, S. Holgate, & E. Simmons

(Eds.0. (2003). Middelton’s allergy principles and

practice (6th ed.). St. Louis: Mosby.

22. Creticos, P., Adkinson, N. F., Jr., Kagey-Sabotka, A.,

et al. (1985). Nasal challenge with ragweed pollen in

hay fever patients: Effect of immunotherapy. J Clin

Invest, 76, 2247-2253.

23. Creticos, P. S., Marsh, D. G., Proud, D., et al. (1989).

Responses to ragweed-pollen nasal challenge be-

fore and after immunotherapy.

J Allergy Clin Immunol, 84, 197-205.

24. Lent, A. M., Harbekc, R., Strand, M., et al. (2006). Im-

munologic response to administration of standard-

ized dog allergen extract at differing doses. J Allergy

Clin Immunol, 118, 1249-1256.

25. Durham, S. R., Walker, S. M., Varga, E. M., et al.

(1999). Long-term clinical efﬁcacy of grass-pollen

immunotherapy. N Engl J Med, 341, 468- 475.

26. Cox, L., Nelson, H., Lockey, R., et al. (2011). Allergen

immunotherapy: A practice parameter third update.

J Allergy Clin Immunol, 127 (1 Suppl,) S1-S55. Erra-

tum. (2011). J Allergy Clin Immunol, 127, 840.

27. Oppenheimer, J. J., Nelson, H. S., Bock, S. A., Chris-

tensen, F., Leung, D. Y. (1992). Treatment of peanut

allergy with rush immunotherapy.

J Allergy Clin Immunol, 90, 256-262.

28. Nelson, H. S., Lahr, J., Rule, R., Bock, A., Leung, D.

(1997). Treatment of anaphylactic sensitivity to pea-

nuts by immunotherapy with injections of aqueous

peanut extract. J Allergy Clin Immunol, 99, 744-751.

29. Turkeltaub, P. Allergenic extracts. II. In vivo standard-

ization. In E. Middleton, JR., E. F. Ellis, N. F. Adkinson,

Jr., & J. W. Yunginger (Eds.). (1988). Allergy: Princi-

ples and practice (3rd ed.). St. Louis: Mosby.

30. USP Sterile Compounding Committee (2008, 2019).

USP <797> Guidebook to Pharmaceutical Com-

pounding — Sterile Preparations. U.S. Pharmacopeia

31. Vijay, H. M., Young, N. M., Bernstein, I. L. (1987).

Studies on Alternaria tenuis extracts under a variety

of storage conditions. Int Arch Allergy Appl Immu-

nol, 83, 325-328.

32. Grier, T. J., LeFevre, D. M., Duncan, E. A., Esch, R. E.

(2007). Stability of standardized grass, dust mite, cat,

and short ragweed allergens after mixing with mold

or cockroach extracts. Ann Allergy Asthma Immunol,

99, 151-160.

33. Niemeijer, N. R., Kauffman, H. F., van Hove, W., Du-

bois, A. E., de Monchy, J. G. (1996). Effect of dilution,

temperature, and preservatives on the long-term

stability of standardized inhalant allergen extracts.

Ann Allergy Asthma Immunol, 76, 535-540.

34. Weber, R. W. (2008). Guidelines for using pollen

cross-reactivity in formulating allergen immunother-

apy. J Allergy Clin Immunol, 122, 219-221.

35. Hunt, K. J., Valentine, M. D., Sobotka, A. K., et al.

(1978, July 27). A controlled trial of immunotherapy

in insect hypersensitivity. N Engl J Med, 299(4), 157-

161.

36. Golden, B. K., Mofﬁtt, J., Nicklas, R. A., et al. (2011).

Stinging insect hypersensitivity: A practice parame-

ter update 2011. J Allergy Clin Immunol, 127, 852-

854.

37. Soldatova, L. N., Paupore, E. J., Burk, S. H., Pastor,

R. W., Slater, J. E. (2000). The stability of house dust

mite allergens in glycerinated extracts. J Allergy Clin

Immunol, 105, 482-488.

38. Grier, TJ. How’s my dosing? A one-step, math-free

guide for comparing your clinic’s maintenance

immunotherapy doses to current practice parameter

recommendations. Ann Allergy Asthma Immunol

108 (2012) 201-205

XII. APPENDICES

Appendix 1. Effective Dose Range for Allergen Extracts, US Standardized Units

Table IX. Probable effective dose range for standardized and non-standardized US-licensed allergen extracts

Allergenic extract

Labeled potency

or concentration

Probable effective

dose range

Range of estimated major allergen

content in US-licensed extracts

Dust mites: D farinae and

D pteronyssinus

3,000, 5,000, 10,000, and

30,000 AU/mL

500-2,000 AU 10,000 AU/mL

20-160 µg/Ml Der p 1, Der f 1*

2-180 µg/mL Der p 2, Der f 2*

78-206 µg./mL Der p 1, Der f 1†

13-147 µg/mL Der p 2, Der f 2†

Cat hair 5,000 and 10,000

BAU/mL

1,000-4,000 BAU 10,000 AU/mL

20-50 µg/mL Fel d 1*‡

30-100 µg/mL cat albumin§

Cat pelt 5,000-10,000 BAU/mL 1,000-4,000 BAU 10,000 BAU/mL

20-50 µg/mL Fel d 1 *‡

400-2,000 µg/mL cat albumin§

Grass, standardized 100,000 BAU/mL 1,000-4,000 BAU 100,000 BAU/mL

425-1,100 µg/mL Phl p 5*

506-2,346 µg/mL group 1||

Bermuda 10,000 BAU/mL 300-1,500 BAU 10,000 BAU/mL

141-422 Cyn d 1 µg/mL*

Short ragweed 1:10, 1:20 wt/vol,

100,000 AU/mL

6-12 µg of Amb a 1 or 1,000-

4,000 AU

1:10 wt/vol

300 µg/mL Amb a 1‡

Concentration of Amb a 1 is

on the label of wt/vol extracts

Nonstandardized

AP Dog

1:100 wt/vol 15 µg of Can f 1 80-400 µg/mL Can f 1†

10-20 µg/mL dog albumin¶

Nonstandardized extract,

dog

1:10 and 1:20 wt/vol 15 µg of Can f 1 0.5 to 10 µg/mL Can f 1†

<12-1,500 µg/mL dog albumin¶

Nonstandardized

extracts: pollen

1:10 to 1:40 wt/vol or

10,000-40,000 PNU/mL

0.5 mL of 1:100 or 1:200 wt/vol NA

Nonstandardized

extracts: mold/fungi,

cockroach

1:10 to 1:40 wt/vol or

10,000-40,000 PNU/mL

Highest tolerated dose NA

Hymenoptera venom 100 µg/mL single venom

300 µg/mL in mixed

vespid extract

50-200 µg of each venom 100-300 µg/mL of venom protein

Imported ﬁre ant 1:10 to 1:20 wt/vol

whole-body extract

0.5 mL of a 1:100 wt/vol to 0.5

mL of a 1:10 wt/vol extract

NA, Information not available.

ALK-Abelló ELISA.

†Indoor Biotechnology ELISA.

‡FDA radial immunodiffusion assay.

§Greer Radial Immunodiffusion assay.

||Greer ELISA.

¶Hollister-Stier ELISA using Innovative Research, Inc, reagents.

Appendix 2. Initial and Ongoing Competency Assessment: Allergen Extract Mixing

Extract Preparation Task Date Validated

by:

Comments, Notes, Additional Testing

Personnel Training documented

Passed initial and annual written test on aseptic

technique & extract preparation

Passed initial and annual Media-ﬁll test verifying

aseptic technique

Successfully completed initial gloved ﬁngertip

and thumb sampling 3 separate times and then

at least annually thereafter

Understands & demonstrates appropriate hand

hygiene

Understands and appropriately dons required

garb

Reviews prescriptions for accuracy

Checks expiration dating of antigens and diluents

Cleans mixing surface appropriately

Checks stocks & mixed extracts for turbidity/

particulate matter

Swabs vial stoppers with 70% IPA

Draws up appropriate amounts of extract

Disposes of syringes in an appropriate manner

Documents lot # and preparation details per

clinic SOP

Creates appropriate labels

Stores extracts at appropriate temperatures

Packages materials and supplies in a neat and

efﬁcient manner.

I understand that of all the topics listed, I will be allowed to perform only those for my skill level/scope of practice and only after I

have demonstrated competency.

Employee Signature _____________________________________________ Date _________________________________

Appendix 3. Extract Volumes Needed for 5.0 ml Maintenance Vials with 0.5 ml injection volume (From Grier TJ. “How’s

my dosing? A one-step, math free guide for comparing your clinic’s maintenance immunotherapy doses to current

practice parameter recommendations” Ann Allergy Asthma Immunol 108 (2012) 201-205.

Extract/concentrate strength Volume of concentrate needed per vial (mL)

Category Concentrate Min Mid Max

Pollens, fungi, insects 1:10 w/v 0.25 0.375 0.50

1:20 w/v 0.50 0.75 1.00

1:40 w/v 1.00 1.50 2.00

Short ragweed

200 AgE U/mL 0.30 0.45 0.60

Cat 10,000 BAU/mL 1.00 2.50 4.00

Dog AP 1:100 w/v NA NA 1.00

Dog epithelia 1:10 w/v 0.25 0.375 0.50

1:20 w/v 0.50 0.75 1.00

Dust mites

30,000 AU/mL 0.17 0.42 0.67

10,000 AU/mL 0.50 1.25 2.00

Pasture grasses

100,000 BAU/mL 0.10 0.25 0.40

10,000 BAU/mL 1.00 2.50 4.00

Bermuda grass 10,000 BAU/mL 0.30 0.90 1.50

Abbreviations: w/v, weight-to-volume ratio; AgE. Antigen E or Amb a 1; U Unit; AU, Allergy Unit; BAU. Bioequivalent Allergy Unit;

NA. Not applicable.

Minimum recommended dose was not speciﬁed for this extract category.

ªAlso applies to ragweed mix (short + giant) products at 1:20 w/v and approx. 100AgE U/mL.

Also applies to dust mite mix and pasture grass mix (eg, KORT,7 grass) products at the same AU/mL or BAU/mL strengths.

Appendix 4. Recommended Documentation for Allergen Immunotherapy Extract Prescription Forms

The purpose of the allergen immunotherapy

prescription form is to deﬁne the contents of the

allergen immunotherapy extract in enough detail that it

could be precisely duplicated. The following information

should be on an immunotherapy prescription form:

Patient information:

• Patient name, patient number (if applicable), birth

date, telephone number, and picture (if available)

should be included.

Preparation information:

• Name of person and signature preparing the

allergen immunotherapy extract should be

included.

• Date of preparation should be recorded.

• Bottle name should be included (eg, trees and

grass). If abbreviations are used, a legend should

be included to describe the meaning of the

abbreviations.

Allergen immunotherapy extract content information:

The following information for each allergen should be

included on the form in a separate column:

• Content of the allergen immunotherapy extract,

including common name or genus and species of

individual antigens and detail of all mixes, should

be included..

• Concentration of available manufacturer’s extract.

• Volume of manufacturer’s extract add added to

achieve the projected effective concentration.

This can be calculated by dividing the projected

effective concentration by the concentration of

available manufacturer’s extract times the total

volume.

• The type of diluent (if used).

• Extract manufacturer.

• Lot number should be included.

• Expiration date should be recorded and not

exceed the expiration date of any of the individual

components.

Appendix 4. Allergen Immunology Extract Prescription Form*

Patient Name: Prescribing Physician:

Patient Number: Address:

Birth Date: Telephone:

Telephone: Fax:

Antigen

Number

Extract Name

Allergen or Diluent

(Common name of

Genus/species)**

Concentration and Type

Manufacturer’s Extract

(AU, BAU, W/V, PNU)/

(50%, G, Aq, Ly, AP)

Volume of

Manufacturer’s

Extract to Add

Extract

Manufacturer

Lot

Number

Expiration

Date

Diluent

Total

Volume

**Components of mixes listed on a separate sheet.

By Use Date ____________________________________________________________________________________________________________

Storage Requirements ___________________________________________________________________________________________________

Results of Quality Control (e.g. visual inspections, second veriﬁcation of questions) _____________________________________________

Allergen Extract Name:

Bottle Name Abbreviations

Tree: T Mold: M

Grass: G Cat: C

Weed: W Dog: D

Ragweed: R Cockroach: CR

Mixture: Mx Dust Mite: Dm

Speciﬁc Instructions:

Prescribing Physician’s Signature Date

Volume to add = Maintenance Concentration x Total Volume

Conc. of Manufacturer’s Extract

Maintenance concentration and subsequent dilutions

reported as volume/volume (v/v) dilutions with maintenance

concentrations=1:1 v/v

BAU=Bioequivalent Allergy Unit, AU=Allergy Unit

PNY=Protein Nitrogen Unit

W/V=Weight per Volume Ratio

G=50% Glycerinated

Aq=Aqueous, Ly=Lyophilized

AP=Alum precipitated, AcP=Acetone precipitated

Maintenance Concentrate Prescription Form

Prepared by: ____________________________ Date Prepared: ___/___/___ Time: __________

Dates of subsequent dilutions from maintenance concentration with expirations dates:

Vial __________ From Vial __________ on ____/____/____ Expiration date: ____/____/____

USP Section 21 – Compounding Allergenic Extracts

*Final 2019 standards for allergen extract compounding under USP Chapter 797.

Allergenic extract prescription sets must follow

standards at least as stringent as those in this section:

Personnel Qualiﬁcations

• A designated person with training and expertise

in allergen immunotherapy is responsible for

ensuring that personnel who will be preparing

allergen immunotherapy are trained, evaluated and

supervised.

• Before beginning to independently prepare

allergenic extracts, all compounding personnel

must complete training and be able to

demonstrate knowledge of principles and skills for

sterile compounding.

• Annual personnel training and competency must

be documented. Personnel must demonstrate

proﬁciency in these procedures by passing written

or electronic testing before they can be allowed to

compound allergenic extract prescription sets.

• Before being allowed to independently compound,

all compounders must successfully complete

gloved ﬁngertip and thumb sampling on both

hands (see USP Chapter <797>; Box 2-1 and Table

1), no fewer than 3 separate times. Each ﬁngertip

and thumb evaluation must occur after performing

separate and complete hand hygiene and garbing

procedure. After the initial competency evaluation,

compounding personnel must successfully

complete gloved ﬁngertip and thumb sampling at

least every 12 months thereafter.

• Compounding personnel must have their sterile

technique and related practices evaluated every 12

months as demonstrated by successful completion

of a media-ﬁll test (see USP Chapter <797>; Box

2-2).

• Personnel who fail competency evaluations must

successfully pass reevaluations in the deﬁcient

area(s) before they can resume compounding of

allergenic extract prescription sets. The designated

person(s) must identify the cause of failure and

determine appropriate retraining requirements.

• Personnel who have not compounded an

allergenic extract prescription set in more than 6

months must be evaluated in all core competencies

before resuming compounding duties.

Personnel Hygiene and Garbing

• Before beginning compounding of allergen

immunotherapy prescription sets, personnel must

perform hand hygiene (see USP Chapter <797>;

Box 3-1 and garbing procedure according to

facility SOPs.

• The Minimum garb requirements include:

° Low-lint garment with sleeves that ﬁt snugly

around the wrists and that is enclosed at the neck

(e.g., gowns or coveralls).

° Low-lint, disposable covers for head that cover

the hair and ears and, if applicable, disposable

cover for facial hair.

° Face mask.

° Sterile powder-free gloves.

• Compounding personnel must rub sterile 70%

IPA onto all surfaces of the gloves and allow them

to dry thoroughly throughout the compounding

process.

Facilities

• The compounding process must occur in an ISO

Class 5 PEC or in a dedicated allergenic extract

compounding area (AECA). The PEC or AECA used

to compound prescription sets must be located

away from unsealed windows, doors that connect

to the outdoors, and trafﬁc ﬂow, all of which may

adversely affect the air quality. Neither a PEC nor

an AECA may be located where environmental

control challenges (e.g., restrooms, warehouses,

or food preparation areas) could negatively affect

the air quality. The PEC or the work surfaces in the

AECA must be located at least 1 meter away from a

sink. The impact of activities that will be conducted

around or adjacent to the PEC or AECA must be

considered carefully when designing such an area.

° If used, the PEC must be certiﬁed every 6 months

(see 5. Certiﬁcation and Recertiﬁcation).

° If used, a visible perimeter must establish the

boundaries of the AECA.

› Access to the AECA during compounding must

be restricted to authorized personnel.

› During compounding activities, no other activity

is permitted in the AECA.

Appendix 5. USP Section 21 — Compounding Allergenic Extracts

› The surfaces of walls, ﬂoors, ﬁxtures, shelving,

counters, and cabinets in the AECA must be

cleanable.

› Carpet is not allowed in the AECA.

› Surfaces should be resistant to damage by

cleaning and sanitizing agents.

› The surfaces in the AECA upon which the

allergenic extract prescription sets are prepared

must be smooth, impervious, free from cracks

and crevices, and non-shedding to allow for

easy cleaning and disinfecting.

› Dust-collecting overhangs such as utility pipes,

ledges, and windowsills should be minimized. If

overhangs or ledges are present, they must be

easily cleanable.

› The AECA must be designed and controlled

to provide a well-lit working environment, with

temperature and humidity controls for the

comfort of compounding personnel wearing the

required garb.

Cleaning and Disinfecting

• In a PEC, all interior surfaces of the PEC must be

cleaned and disinfected daily and when surface

contamination is known or suspected. Apply sterile

70% IPA to the horizontal work surface between

each prescription set.

• In an AECA, all work surfaces in the AECA where

direct compounding is occurring must be

cleaned and disinfected daily and when surface

contamination is known or suspected. Apply sterile

70% IPA to the horizontal work surface between

each prescription set.

° If present, walls, doors, and door frames within

the perimeter of the AECA must be cleaned

and disinfected monthly and when surface

contamination is known or suspected.

° Ceilings within the perimeter of the AECA must

be cleaned and disinfected when visibly soiled

and when surface contamination is known or

suspected.

• Vial stoppers on packages of conventionally

manufactured sterile ingredients must be wiped

with sterile 70% IPA to ensure that the critical sites

are wet and allowed to dry before they are used to

compound allergenic extracts prescription sets.

Establishing BUDs

• The by-use-date (BUD) for the prescription set must

be no later than the earliest expiration date of any

allergenic extract or any diluent that is part of the

prescription set, and the BUD must not exceed 1

year from the date the prescription set is mixed or

diluted.

Labeling

• The label of each vial of an allergenic extract

prescription set must display the following

prominently and understandably:

° Patient name.

° Type and fractional dilution of each vial, with a

corresponding vial number.

° BUD.

° Storage conditions.

Shipping and Transport

• If shipping or transporting allergenic extract

prescriptions sets, compounding personnel must

select modes of transport that are expected to

deliver properly packed prescription sets in an

undamaged, sterile, and stable condition.

• When shipping or transporting allergen extract

prescriptions sets that require special handling,

personnel must include speciﬁc handling

instructions on the exterior of the container.

Documentation

• All facilities where allergenic extract prescription

sets are prepared must have and maintain written

or electronic documentation to include, but not

limited to the following:

° SOPs describing all aspects of the compounding

process.

° Personnel training records, competency

assessments, and qualiﬁcation records including

corrective actions for any failures.

° Certiﬁcation reports of the PEC, if used, including

corrective actions for any failures.

° Temperature logs for the refrigerator(s).

° Compounding records for individual allergenic

extract prescription sets (see USP Chapter

<797>; Box 21-1).

° Information related to complaints and adverse

events.

° Investigations and corrective actions.

*Adapted from USP General Chapter <797> Pharmaceutical

Compounding-Sterile Preparations; Section 21. Compounding

Allergenic Extracts

Box 2-1. Gloved Fingertip and Thumb Sampling Procedures

• Use one sampling device per hand (e.g., plates, paddles, or slides) containing general microbial growth agar [e.g.,

trypticase soy agar (TSA)] supplemented with neutralizing additives (e.g., lecithin and polysorbate 80) as the agar

supports both bacterial and fungal growth.

• Label each sampling device with a personnel identiﬁer, whether it was from the right or left hand, and the date

and time of sampling.

• Do not apply sterile 70% isopropyl alcohol (IPA) to gloves immediately before touching the sampling device

because this could cause a false-negative result.

• Using a separate sampling device for each hand, collect samples from all gloved ﬁngers and thumbs from both

hands by rolling ﬁnger pads and thumb pad over the agar surface.

• Incubate the sampling device at a temperature of 30°- 35°for no less than 48 hours and then at 20°- 25° for no

less than 5 additional days. Store media devices during incubation to prevent condensate from dropping onto the

agar and affecting the accuracy of the cfu reading (e.g., invert plates).

• Record the number of cfu per hand (left hand, right hand).

• Determine whether the cfu action level is exceeded by counting the total number of cfu from both hands.

Return to Allergy Standards

Table 1. Action Levels for Gloved Fingertip and Thumb Sampling

Gloved Fingertip and Thumb Sampling Action Levels (total number of cfu from both hands)

Initial sampling after garbing >0

Subsequent sampling after media-ﬁll testing >3

Action levels are based on the total cfu count from both hands. Return to Allergy Standards

Box 2-2. Media-Fill Testing Procedures

• If all of the starting components are sterile to begin with, manipulate them in a manner that simulates sterile-to-

sterile compounding activities, and transfer the sterile soybean – Casein digest medium in non-bacteriostatic water

to make a 3% nonsterile solution. Manipulate it in a manner that simulates nonsterile-to-sterile compounding

activities. Prepare at least 1 container as the positive control to demonstrate growth promotion, which is indicated

by visible turbidity upon incubation.

• Once the compounding simulation is completed and the ﬁnal containers are ﬁlled with the test media, incubate

them in an incubator for 7 days at 20°- 25° followed by 7 days at 30°- 35° to detect a broad spectrum of

microorganisms.

• Failure is indicated by visible turbidity or other visual manifestations of growth in the media in one or more

container-closure unit(s) on or before 14 days.

Return to Allergy Standards

Box 3-1. Hand Washing Procedures

• Remove visible debris from underneath ﬁngernails under warm running water using a disposable nail cleaner.

• Wash hands and forearms up to the elbows with soap and water for at least 30 seconds.

• Dry hands and forearms to the elbows completely with low-lint disposable towels or wipers.

Return to Allergy Standards

Box 3-2. Hand Sanitizing Procedures

• Apply an alcohol-based hand rub to dry skin following the manufacturer’s instructions for the volume of product

to use.

• Apply product to one hand and rub hands together, covering all surfaces of hands and ﬁngers, until hands are dry.

• Allow hands to dry thoroughly before conning sterile gloves.

Return to Allergy Standards

Box 21-1. Compounding Records for individual Allergenic Extract Prescription Sets

Compounding Records must include at least the following information:

• Name, concentration, volume, vendor or manufacturer, lot number, and expiration date for each component.

• Date and time of preparation of the allergenic extract.

• Assigned internal identiﬁcation number.

• A method to identify the individuals involved in the compounding process and verifying the ﬁnal CSP.

• Total quantity compounded.

• Assigned BUD and storage requirements.

• Results of QC procedures (e.g., visual inspection, second veriﬁcation of quantities).

Return to Allergy Standards

Appendix 6. Forms

Competency Assessment — Initial Personnel Training: Allergen Extract Mixing..............................30

Competency Assessment — Annual Personnel Training: Allergen Extract Mixing ............................32

Competency Assessment — Aseptic Technique and Related Practices of Mixing Personnel ...................34

Competency Assessment — Hand Hygiene and Garbing Procedures ......................................36

Competency Assessment — Cleaning and Disinfecting Procedures .......................................38

Complaints/Adverse Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Gloved Fingertip and Thumb Sampling ...............................................................41

Media-ﬁll Testing Checklist ..........................................................................42

Primary Engineering Control (PEC) Hood .............................................................43

Refrigerator Temperature Log .......................................................................44

Competency Assessment –

Initial

Personnel Training: Allergen Extract Mixing

Name: Job Title:

Extract Preparation Task Date Validated by Comments, Notes, Additional Testing

1. Passed initial written test on aseptic technique and extract preparation. See Form A1.

2. Passed initial Media-ﬁll test verifying aseptic technique. Attach lab report.

3. Successfully completed initial gloved ﬁngertip and thumb sampling three separate

times. Attach lab report. TOTAL R cfu + L cfu = 0.

Test 1: R cfu + L cfu =

Test 2: R cfu + L cfu =

Test 3: R cfu + L cfu =

4. Understands & demonstrates appropriate hand hygiene. See Form A2.

5. Understands and appropriately dons required garb. See Form A2.

6. Reviews prescriptions for accuracy.

7. Checks expiration dates of antigens and diluents.

8. Cleans mixing surface appropriately. See Form A3.

9. Checks stocks & mixed extracts for turbidity/particulate matter.

10. Swabs vial stoppers with 70% IPA.

11. Draws up appropriate amounts of extract.

12. Disposes of syringes in an appropriate manner.

13. Documents lot # and preparation details per clinic SOP.

14. Creates appropriate labels.

15. Stores extracts at appropriate temperatures.

16. Packages materials and supplies in a neat and efﬁcient manner.

I understand that of all the topics listed, I will be allowed to perform only those for my skill level/scope of practice and only after I have demonstrated

competency. Furthermore, I must successfully pass reevaluations in *deﬁcient area(s) before I can resume compounding allergenic extract.

_______________________________________

Employee signature

_______________________________________

Date

Self-Assessment: Evaluation/Validation Methodologies:

1 = Experienced T = Tests

2 = Needs practice/assistance D = Demonstration

3 = Never done V = Verbal

NA = Not applicable I = Interactive Class

*Corrective actions on separate page.

Competency Assessment – Corrective Action

Name: Job Title:

Extract Preparation Task Date Validated by Actions Taken

1. Passed written test on aseptic technique and extract preparation.

2. Passed Media-ﬁll test verifying aseptic technique.

3. Successfully completed gloved ﬁngertip and thumb sampling three separate times.

4. Understands & demonstrates appropriate hand hygiene.

5. Understands and appropriately dons required garb.*

6. Reviews prescriptions for accuracy.

7. Checks expiration dates of antigens and diluents.

8. Cleans mixing surface appropriately.

9. Checks stocks & mixed extracts for turbidity/particulate matter.

10. Swabs vial stoppers with 70% IPA.

11. Draws up appropriate amounts of extract.

12. Disposes of syringes in an appropriate manner.

13. Documents lot # and preparation details per clinic SOP.

14. Creates appropriate labels.

15. Stores extracts at appropriate temperatures.

16. Packages materials and supplies in a neat and efﬁcient manner.

Competency Assessment –

Annual

Personnel Training: Allergen Extract Mixing

Name: Job Title:

Extract Preparation Task Date Validated by Comments, Notes, Additional Testing

1. Passed annual written test on aseptic technique and extract preparation. See Form A1.

2. Passed annual Media-ﬁll test verifying aseptic technique. Attach lab report.

3. Successfully completed annual gloved ﬁngertip and thumb sampling three

separate times. Attach lab report. TOTAL R cfu + L cfu = 0.

Test 1: R cfu + L cfu =

4. Understands & demonstrates appropriate hand hygiene. See Form A2.

5. Understands and appropriately dons required garb. See Form A2.

6. Reviews prescriptions for accuracy.

7. Checks expiration dates of antigens and diluents.

8. Cleans mixing surface appropriately. See Form A3.

9. Checks stocks & mixed extracts for turbidity/particulate matter.

10. Swabs vial stoppers with 70% IPA.

11. Draws up appropriate amounts of extract.

12. Disposes of syringes in an appropriate manner.

13. Documents lot # and preparation details per clinic SOP.

14. Creates appropriate labels.

15. Stores extracts at appropriate temperatures.

16. Packages materials and supplies in a neat and efﬁcient manner.

I understand that of all the topics listed, I will be allowed to perform only those for my skill level/scope of practice and only after I have demonstrated

competency. Furthermore, I must successfully pass reevaluations in *deﬁcient area(s) before I can resume compounding allergenic extract.

_______________________________________

Employee signature

_______________________________________

Date

Self-Assessment: Evaluation/Validation Methodologies:

1 = Experienced T = Tests

2 = Needs practice/assistance D = Demonstration

3 = Never done V = Verbal

NA = Not applicable I = Interactive Class

*Corrective actions on separate page.

Competency Assessment – Corrective Action

Name: Job Title:

Task Date Validated by Actions Taken

1. Passed annual written test on aseptic technique and extract preparation. See Form A1.

2. Passed annual Media-ﬁll test verifying aseptic technique. Attach lab report.

3. Successfully completed annual gloved ﬁngertip and thumb sampling three

separate times. Attach lab report. TOTAL R cfu + L cfu = 0.

4. Understands & demonstrates appropriate hand hygiene. See Form A2.

5. Understands and appropriately dons required garb. See Form A2.

6. Reviews prescriptions for accuracy.

7. Checks expiration dates of antigens and diluents.

8. Cleans mixing surface appropriately. See Form A3.

9. Checks stocks & mixed extracts for turbidity/particulate matter.

10. Swabs vial stoppers with 70% IPA.

11. Draws up appropriate amounts of extract.

12. Disposes of syringes in an appropriate manner.

13. Documents lot # and preparation details per clinic SOP.

14. Creates appropriate labels.

15. Stores extracts at appropriate temperatures.

16. Packages materials and supplies in a neat and efﬁcient manner.

Form A1: Competency Assessment – Aseptic Technique and Related Practices of Mixing Personnel

Name: Job Title:

Facility: Location:

All mixing personnel must perform media-ﬁll testing to assess their sterile technique and related practices initially and every 12 months thereafter.

TASK (X = acceptable practice; N/A = not applicable; N/O = not observed) X N/A N/O

1. Completed hand hygiene and garbing competency assessment.

2. Performs proper hand hygiene, garbing and gloving procedures according to SOPs.

3. Disinfects work surfaces with sterile 70% IPA.

4. Disinfects components/vials with sterile 70% IPA.

5. Only introduces essential materials in a proper arrangement in work area.

6. Ensures syringes and needles are only opened in work area.

7. Does not expose critical sites to contact contamination.

8. Disinfects stoppers, injection ports and ampul necks by wiping with sterile 70% IPA and allows time to dry.

9. Afﬁxes needles to syringes without contact contamination.

10. Punctures vial stoppers and spikes infusion ports without contact contamination.

11. Correctly prepares labels.

12. Disinfects sterile gloves routinely by wiping with sterile 70% IPA during prolonged mixing.

13. Disposes of sharps and waste according to institutional policy.

The person assessed is immediately informed of all unacceptable activities and informed of corrective actions.

_______________________________________ ____________________________________ _________________________

Signature of person assessed Printed name Date

_______________________________________ ____________________________________ _________________________

Signature of Qualified Evaluator Printed name Date

Form A1: Competency Assessment – Corrective Action

Name: Job Title:

Task Date Validated by Actions Taken

1. Completed hand hygiene and garbing competency assessment.

2. Performs proper hand hygiene, garbing and gloving procedures according to SOPs.

3. Disinfects work surfaces with sterile 70% IPA.

4. Disinfects components/vials with sterile 70% IPA.

5. Only introduces essential materials in a proper arrangement in work area.

6. Ensures syringes and needles are only opened in work area.

7. Does not expose critical sites to contact contamination.

8. Disinfects stoppers, injection ports and ampul necks by wiping with sterile 70% IPA

and allows time to dry.

9. Afﬁxes needles to syringes without contact contamination.

10. Punctures vial stoppers and spikes infusion ports without contact contamination.

11. Correctly prepares labels.

12. Disinfects sterile gloves routinely by wiping with sterile 70% IPA during

prolonged mixing.

13. Disposes of sharps and waste according to institutional policy.

college.acaai.org/extract

Form A2: Competency Assessment – Hand Hygiene and Garbing Practices

Name: Job Title:

Facility: Location:

Individuals entering a compounding area must take appropriate steps to minimize microbial contamination of the environment and the SCPs, including hand hygiene, garbing and consideration of

needed materials brought into the compounding area. Before entering a compounding area, individuals must remove any items that are not easily cleanable or that are not necessary for compounding.

TASK (X = acceptable practice; N/A = not applicable; N/O = not observed) X N/A N/O

Remove debris from underneath ﬁngernails under running water using a disposable nail cleaner.

Wash hands and forearms up to the elbows with soap and water for at least 30 seconds.

Dry hands and forearms to the elbows completely with low-lint disposable towels or wipers.

The order of hand washing and garbing depends on the placement of the sink. The order of garbing must be determined by the facility and documented in the facility’s SOP. Hands must be

sanitized with alcohol-based hand rub before donning sterile gloves. Sterile gloves must be donned in a classiﬁed room or SCA.

Apply an alcohol-based hand rub to dry skin following the manufacturer’s instructions for the volume of product to use.

Apply product to one hand and rub hands together, covering all surfaces of hands and ﬁngers, until hands are dry.

Allow hands to dry thoroughly before donning sterile gloves.

Dons appropriate size gloves; inspects to ensure a tight ﬁt and that there are no defects, holes or tears – apply sterile 70% IPA to gloves – allow to dry

before compounding.

While compounding, routinely disinfects gloves with sterile 70% IPS after touching items or surfaces that may contaminate gloves .

Any person entering a compounding area must be properly garbed in accordance with the facility’s SOPs. Garb must be replaced immediately if it becomes visibly soiled or if its integrity is compromised.

Low-lint garment with sleeves that ﬁt snugly around the wrists and that is enclosed at the neck (e.g., gowns or coveralls).

Low-lint, disposable covers for head that cover the hair and ears, and if applicable, disposable cover for facial hair.

Face mask.

Sterile powder-free gloves.

Compounding personnel must rub sterile 70% IPA onto all surfaces of the gloves and allow them to dry thoroughly throughout the compounding process.

Removes PPE on the clean side of the AECA.

Removes gloves and performs hand hygiene.

Removes and discards gown, mask, head cover and beard cover (if used).

The person assessed is immediately informed of all unacceptable activities and informed of corrective actions.

_______________________________________ ____________________________________ _________________________

Signature of person assessed Printed name Date

_______________________________________ ____________________________________ _________________________

Signature of Qualified Evaluator Printed name Date

Form A2: Competency Assessment – Corrective Action

Name: Job Title:

Task Date Validated by Actions Taken

Remove debris from underneath ﬁngernails under running water using a disposable

nail cleaner.

Wash hands and forearms up to the elbows with soap and water for at least 30

seconds.

Dry hands and forearms to the elbows completely with low-lint disposable towels or

wipers.

Apply an alcohol-based hand rub to dry skin following the manufacturer’s

instructions for the volume of product to use.

Apply product to one hand and rub hands together, covering all surfaces of hands

and ﬁngers, until hands are dry.

Allow hands to dry thoroughly before donning sterile gloves.

Dons appropriate size gloves; inspects to ensure a tight ﬁt and that there are

no defects, holes or tears – apply sterile 70% IPA to gloves – allow to dry before

compounding.

While compounding, routinely disinfects gloves with sterile 70% IPS after touching

items or surfaces that may contaminate gloves .

Low-lint garment with sleeves that ﬁt snugly around the wrists and that is enclosed at

the neck (e.g., gowns or coveralls).

Low-lint, disposable covers for head that cover the hair and ears, and if applicable,

disposable cover for facial hair.

Face mask.

Sterile powder-free gloves.

Removes PPE on the clean side of the AECA.

Removes gloves and performs hand hygiene.

Removes and discards gown, mask, head cover and beard cover (if used).

Form A3: Competency Assessment – Cleaning and Disinfecting Procedures

Name: Job Title:

Facility: Location:

DAILY TASKS (X = acceptable practice; N/A = not applicable; N/O = not observed) X N/A N/O

Follows garbing procedures when performing any cleaning activities.

In a PEC, all interior surfaces of the PEC must be cleaned and disinfected daily and when surface contamination is known or suspected. Apply sterile

70% IPA to the horizontal work surface between each prescription set.

In an AECA, all work surfaces in the AECA where direct mixing is occurring must be cleaned and disinfected daily and when surface contamination is

known or suspected. Apply sterile 70% IPA to the horizontal work surface between each prescription set.

Mops ﬂoors, starting at wall opposite the entrance; moves carts as needed to clean entire ﬂoor surface. Use of a microﬁber cleaning system is an

acceptable alternative to mops.

MONTHLY TASKS

Follows garbing procedures when performing any cleaning activities

Performs monthly cleaning on a designated day.

If present, walls, doors and door frames within the perimeter of the AECA must be cleaned and disinfected monthly and when surface contamination is

known or suspected

Ceilings within the perimeter of the AECA must be cleaned and disinfected when visibly soiled and when surface contamination is known or suspected.

Documents all cleaning activities as to who performed such activities with date and time noted.

The person assessed is immediately informed of all unacceptable activities and informed of corrective actions.

_______________________________________ ____________________________________ _________________________

Signature of person assessed Printed name Date

_______________________________________ ____________________________________ _________________________

Signature of Qualified Evaluator Printed name Date

Form A3: Competency Assessment – Corrective Action

Name: Job Title:

Task Date Validated by Actions Taken

Follows garbing procedures when performing any cleaning activities.

In a PEC, all interior surfaces of the PEC must be cleaned and disinfected daily and

when surface contamination is known or suspected. Apply sterile 70% IPA to the

horizontal work surface between each prescription set.

In an AECA, all work surfaces in the AECA where direct mixing is occurring must

be cleaned and disinfected daily and when surface contamination is known or

suspected. Apply sterile 70% IPA to the horizontal work surface between each

prescription set.

Mops ﬂoors, starting at wall opposite the entrance; moves carts as needed to clean

entire ﬂoor surface. Use of a microﬁber cleaning system is an acceptable alternative

to mops.

Follows garbing procedures when performing any cleaning activities

Performs monthly cleaning on a designated day.

If present, walls, doors and door frames within the perimeter of the AECA must

be cleaned and disinfected monthly and when surface contamination is known or

suspected

Ceilings within the perimeter of the AECA must be cleaned and disinfected when

visibly soiled and when surface contamination is known or suspected.

Documents all cleaning activities as to who performed such activities with date and

time noted.

Complaints/Adverse Events

Complainant/Adverse Event: Date:

Complaint received: Orally Written (attach)

Describe complaint/adverse event and any additional information:

Describe action taken and results:

Describe action taken to avoid same issue in the future:

_______________________________________ ____________________________________ _________________________

Signature Printed name Date

Gloved Fingertip and Thumb Sampling

Name: Job Title:

Date: Time: Check Notes

Gather and prepare sampling plates – one plate per hand.

Label each sampling plate with a personnel identiﬁer (i.e., initials); Right or Left hand.

DO NOT APPLY STERILE 70% ISOPROPYL ALCOHOL TO GLOVES IMMEDIATELY BEFORE

TOUCHING THE SAMPLING DEVICE, WHICH COULD CAUSE A FALSE-NEGATIVE RESULT.

Wipe the work surface with sterile 70% isopropyl alcohol; allow to dry.

Don garbing, including sterile gloves.

Using a separate sampling plate per hand, roll all ﬁnger pads & thumb pad over agar surface.

Cover and invert plates to prevent condensation from forming on agar; incubate at 30-

35°C for no less than 48 hours and then at 20-25°C for no less than ﬁve additional days.

Thumb Index Middle Ring Pinky

Record the number of cfu per Right hand.

Record the number of cfu per Left hand.

Attach report. TOTAL cfu

Action Levels for Gloved Fingertip and Thumb Sampling

Gloved Fingertip & Thumb Sampling. Action Levels (total cfu count from both hands)

Initial sampling after garbing. 0

Subsequent sampling after media-ﬁll testing (every 6 months). ≤3

Successful completion of initial gloved fingertip and thumb sampling is defined as zero colony-forming units (cfu). Successful completion of subsequent gloved fingertip and

thumb sampling after media-fill testing is defined as ≤3 cfu (total from both hands).

Action levels are based on the total cfu count from both hands.

_______________________________________ ____________________________________ _________________________

Signature Printed name Date

Media-ﬁll Testing Checklist

Name: Media:

Date: Time: Manufacturer: Exp. Date: Lot #:

TASK Check Notes

Don garbing, including sterile gloves.

Wipe the work surface with sterile 70% isopropyl alcohol; allow to dry.

For sterile components (i.e., commercially sold media-ﬁll kits) do not dilute the media

further, unless directed by manufacturer. Obtain certiﬁcate of analysis (COA). Using a

sterile syringe, transfer media to vial to simulate sterile-to-sterile compounding activities.

If non-sterile components are used, dissolve nonsterile soybean-casein digest powder in

non-bacteriostatic water to make a 3% nonsterile solution. (prepare one vial as a control to

demonstrate growth promotion) Using a sterile syringe, transfer media to vial and simulate

nonsterile to sterile compounding activities.

Once the compounding simulation is completed, incubate vials.

First incubate at 20-25°C for 7 days. Next incubate at 30-35°C for 7 additional days.

Starting Temp.: Date: Starting Temp.: Date:

Failure is indicated by visible turbidity or other visual growth in the media in one or more vials on or before 14 days.

_______________________________________ ____________________________________ _________________________

Observer Signature Printed name Date

_______________________________________ ____________________________________ _________________________

Person who Reads/Documents Results Printed name Date

Primary Engineering Control (PEC) Hood

Name of Practice:

Location of PEC:

Date

Initial Certiﬁcation Report Attached

6-Month recertiﬁcation Report Attached

_______________________________________ ____________________________________ _________________________

Signature Printed name Date

Refrigerator Temperature Log

Refrigerator Name/Location:

Acceptable Temperature Range: 2-8°C

Date Time Temperature Initials

_______________________________________ ____________________________________ _________________________

Signature Printed name Date