Dilution and Mixing of Allergen Extracts

Basic Concepts , General Guidelines

and Hands-on Training

Lenoir, North Carolina

 Overview

¾ Objectives: Discuss/review, demonstrate

and streamline extract dilution/mixing

¾ Dilution/mixing calculations and procedures

are straightforward and easy-to-master

¾ 2 common mathematical approaches to

dilution and mixing yield identical results

¾ Proper dilution/mixing and effective IT

require awareness of 5 critical factors
 Critical Factors
¾ Quality and consistency of extracts
Compositions, Concentrations, Potencies

¾ Integrity and activity of allergens in extracts and mixtures

Stabilities, Compatibilities, Cross-reactivities

¾ Sterility and aseptic handling of allergen vials

Storage, Workspace, Withdrawal/delivery

¾ Diluent choices, advantages/disadvantages and preferences

Normal saline, Buffered saline, HSA-saline, 10% glycerin

¾ Doing the math

Serial dilutions, Algebraic calculations, 1-step or multi-step
 Extract Stability

¾ Major influences / environmental factors

Time
Temperature
pH
Diluent
Strength / dilution / concentration
Presence of active hydrolytic enzymes
Oxidation / age
 Extract Compatibility

¾ Major influences / interactions

Protein and carbohydrate concentrations

Protease levels and specificities
Allergen susceptibilities
Glycerin/diluent content and pH
Levels of endogenous enzyme inhibitors
 Extract Stability FAQs

¾ Effects of room temperature exposures?

¾ Short exposures to elevated temperatures?

¾ Accidental freezing and thawing?

¾ Diluent differences?

¾ Clinical correlations w/ laboratory test results?

 Extract Compatibility FAQs

¾ Compatibility of allergens with molds/insects?

¾ Compatibilities of cat, mite, ragweed and grasses?

¾ [Protease] from product-product and lot-lot?

¾ Compatibility of molds with insects?

¾ Compatibility of molds with other molds?

 Extract Compatibility Guidelines

¾ NOT OK to mix insects with molds, pollens or mites

NOT OK to mix molds with pollens or mites

¾ OK to mix molds with cat or other molds

¾ OK to combine dust mites, pollens, cat/dog/other eps

¾ Evidence In vivo Skin tests

In vitro ELISA, Immunoblot
Compatibility After mixing and storage with ...
of ...

Insects Molds Mites Rag Grass Trees/ Eps Cat Venom

Weeds
Insects

Molds

Mites
Risky
Ragweed

Grasses

Trees/ Not OK OK
Weeds
Epithelia

Cat

Venoms
 Dating/Re-prep Considerations
¾ Individual extract stabilities are concentration-dependent
Source materials and glycerin concentrations

¾ Extract mixture stabilities are based on cumulative strengths

Correspond to individual allergens at final concentrations

¾ 10-fold reductions in strength reduce stabilities in half

Represent worst-case examples

¾ Stabilities at high dilutions are speculative at best

Below detection/quantitation limits of in vitro potency tests

¾ Practical dating may alter lower doses but IT remains effective

Slightly lower cumulative doses but similar maintenance doses
Treatment Extract Stability Guidelines
Extract Strength/Dilution Dating/storage at 2-8°C
Individuals Rx mixtures Conservative Practical
1:10-1:20 w/v
NA 18-36 months 18-36 months
40K-200K PNU/mL
1:100 w/v
Maintenance conc 6-12 months 12 months
4K-40K PNU/mL
1:1,000 w/v 1:10 of conc 3-6 months 6 months
1:10,000 w/v 1:100 of conc 3-6 weeks 6 months
1:100,000 w/v 1:1,000 of conc 1 week 3 months
1:1,000,000 w/v 1:10,000 of conc 1 week 6 weeks
1:10,000,000 w/v 1:100,000 of conc 1 week 6 weeks
 Types of Dilutions
¾ Serial
Constant dilution factor, one dilution used to make subsequent
Examples: 10-fold series = 0.5 mL extract + 4.5 mL diluent
5-fold series = 1.0 mL extract + 4.0 mL diluent

¾ Endpoint
Variable dilution factors, one step to final targeted dilution
Examples: 20-fold dilution = 0.5 mL extract to 10.0 mL total volume
4-fold dilution = 1.25 mL to 5.0 mL total volume

¾ Combination
Variable dilution factors, multiple steps to final targeted dilution
Example: 500-fold dilution
Step 1 50-fold dilution = 0.2 mL to 10.0 mL
Step 2 10-fold of 50-fold = 1.0 mL (50f) to 10.0 mL
 Extract Strengths/Units
¾ Weight/volume ratio (w/v)
Weight of raw material (g) / volume of extraction/dilution fluid (mL)
Inverse proportions 1:10 w/v = 10x stronger than 1:100 w/v

¾ Protein nitrogen units (PNU)

Acid-precipitable protein, not predictive of allergenic potencies

¾ Bioequivalent Allergy Units (BAU, AU)

Dust mites and grasses IgE binding to multiple protein allergens
Dust mites @ 10,000 AU/mL Most grasses @ 100,000 BAU/mL

¾ Major allergen units/micrograms (Fel d 1/AgE U)

Cat and Short ragweed IgG binding to single dominant proteins
Cat @ 10,000 BAU/mL = 10-20 Fel d 1 U/mL = 40-80 µg Fel d 1/mL
Short ragweed @ 1:20 w/v = 150-300 AgE U/mL (= µg/mL)
 Effective Maintenance IT Doses

¾ Non-standardized allergens w/v or PNU

Pollens 0.5 mL of 1:50-1:250 w/v or 2,000-8,000 PNU/mL
Dose = 1,000-4,000 PNU
Fungi 0.5 mL of 1:50-1:250 w/v or 4,000-20,000 PNU/mL
Dose = 2,000-10,000 PNU

¾ Standardized allergens AU, BAU or AgE U

Dust mites 0.5 mL of 1,000-4,000 AU/mL 500-2,000 AU
Grasses 0.5 mL of 2,000-8,000 BAU/mL 1,000-4,000 BAU
Cat 0.5 mL of 2,000-6,000 BAU/mL 1,000-3,000 BAU
Ragweed 0.5 mL of 12-24 AgE U/mL 6-12 AgE Units
 Dilution/Mixing Approach #1
¾ Conservation of mass approach (CM)
Quantity you want = quantity you have

Formula Vol want x Conc want = Vol have x Conc have

Vw Cw = Vh Ch
Example Want 5 mL at 1:250 w/v, have 1:10 w/v
Vw = 5.0 Vh = ?
Cw = 1/250 = 0.004 Ch = 1/10 = 0.10
(5.0) (0.004) = ? (0.10)
? = (5.0) (0.004) / 0.10 = 0.20

Formulation 0.20 mL of 1:10 w/v extract

5.0 – 0.20 = 4.80 mL of diluent
 Dilution/Mixing Approach #2
¾ Dilution factor/fractional volume approach (DF)

DF = ratio of initial to final concentrations

1:10 w/v to 1:250 w/v = 250 / 10 = 25-fold dilution

(1/10) divided by (1/250) = 0.1 / 0.004 = 25

Same example used with approach #1

Vw = 5.0
DF = 25
Vh = Vw / DF = 5.0 / 25 = 0.20

Formulation 0.20 mL of 1:10 w/v extract

5.0 – 0.20 = 4.80 mL of diluent
 CM Approach 2nd w/v
¾ Quantity you want = quantity you have
Formula Vw Cw = Vh Ch

Example Want 5 mL at 1:50 w/v

Have 1:10 w/v
Vw = 5.0 Vh = ?
Cw = 1/50 = 0.02 Ch = 1/10 = 0.10
(5.0) (0.02) = ? (0.10)
? = (5.0) (0.02) / 0.10 = 1.00

Formulation 1.00 mL of 1:10 w/v extract

5.0 – 1.00 = 4.00 mL of diluent
 DF Approach 2nd w/v
¾ DF = Initial concentration / Final concentration

1:10 w/v to 1:50 w/v = 50 / 10 = 5-fold dilution

(1/10) divided by (1/50) = 0.10 / 0.02 = 5

Vw = 5.0
DF = 5
Vh = Vw / DF = 5.0 / 5 = 1.00

Formulation 1.00 mL of 1:10 w/v extract

5.0 – 1.00 = 4.00 mL of diluent
 CM Approach 1st PNU
¾ Quantity you want = quantity you have
Formula Vw Cw = Vh Ch

Example Want 5 mL at 4,000 PNU/mL

Have 40,000 PNU/mL
Vw = 5.0 Vh = ?
Cw = 4,000 Ch = 40,000
(5.0) (4,000) = ? (40,000)
? = (5.0) (4,000) / 40,000 = 0.50

Formulation 0.50 mL of 40,000 PNU/mL extract

5.0 – 0.50 = 4.50 mL of diluent
 DF Approach 1st PNU
¾ DF = Initial concentration / Final concentration

40,000 PNU/mL to 4,000 PNU/mL

(40,000) divided by (4,000) = 10

Vw = 5.0
DF = 10
Vh = Vw / DF = 5.0 / 10 = 0.50

Formulation 0.50 mL of 40,000 PNU/mL extract

5.0 – 0.50 = 4.50 mL of diluent
 CM Approach 2nd PNU
¾ Quantity you want = quantity you have
Formula Vw Cw = Vh Ch

Example Want 5 mL at 10,000 PNU/mL

Have 40,000 PNU/mL
Vw = 5.0 Vh = ?
Cw = 10,000 Ch = 40,000
(5.0) (10,000) = ? (40,000)
? = (5.0) (10,000) / 40,000 = 1.25

Formulation 1.25 mL of 40,000 PNU/mL extract

5.0 – 1.25 = 3.75 mL of diluent
 DF Approach 2nd PNU
¾ DF = Initial concentration / Final concentration

40,000 PNU/mL to 10,000 PNU/mL

(40,000) divided by (10,000) = 4.0

Vw = 5.0
DF = 4.0
Vh = Vw / DF = 5.0 / 4.0 = 1.25

Formulation 1.25 mL of 40,000 PNU/mL extract

5.0 – 1.25 = 3.75 mL of diluent
 Prepare IT Vials by Serial Dilution
¾ Vial colors/labels and concentrations
Red Vial 1 Maintenance concentrate
Yellow Vial 2 1/10 of maintenance concentrate
Blue Vial 3 1/100 of maintenance concentrate
Green Vial 4 1/1,000 of maint. concentrate
Silver Vial 5 1/10,000 of maint. concentrate

¾ Maintenance concentrates
< 5 components ideal, > 10 components not recommended
Final component conc = initial conc / DF
Components act like diluent for other components
Calculations for each component as in previous examples
 Prepare IT Vials by Serial Dilution
¾ Maintenance concentrate (Vial 1), 5.0 mL total volume
Ext A 1:250 w/v 1:10 = 1/25 = 0.20 mL
Ext B 1:50 w/v 1:10 = 1/5 = 1.00 mL
Ext C 4,000 PNU/mL 40K = 1/10 = 0.50 mL
Ext D 10,000 PNU/mL 40K = 1/4 = 1.25 mL
Diluent 5.0 – (0.2 + 1.0 + 0.5 + 1.25) = 2.05 mL

¾ Subsequent vials in treatment set, 5.0 mL total volumes

Vial 2 0.50 mL Vial 1 + 4.50 mL diluent
Vial 3 0.50 mL Vial 2 + 4.50 mL diluent
Vial 4 0.50 mL Vial 3 + 4.50 mL diluent
Vial 5 0.50 mL Vial 4 + 4.50 mL diluent
 CM Approach 1st BAU
¾ Quantity you want = quantity you have
Formula Vw Cw = Vh Ch

Example Want 5 mL at 4,000 BAU/mL Cat

Have 10,000 BAU/mL Cat
Vw = 5.0 Vh = ?
Cw = 4,000 Ch = 10,000
(5.0) (4,000) = ? (10,000)
? = (5.0) (4,000) / 10,000 = 2.00

Formulation 2.00 mL of 10,000 BAU/mL Cat

5.0 – 2.00 = 3.00 mL of diluent
 DF Approach 1st BAU
¾ DF = Initial concentration / Final concentration

10,000 BAU/mL Cat to 4,000 BAU/mL

(10,000) divided by (4,000) = 2.5

Vw = 5.0
DF = 2.5
Vh = Vw / DF = 5.0 / 2.5 = 2.00

Formulation 2.00 mL of 10,000 BAU/mL Cat

5.0 – 2.00 = 3.00 mL of diluent
 CM Approach 2nd BAU
¾ Quantity you want = quantity you have
Formula Vw Cw = Vh Ch

Example Want 5 mL at 2,000 BAU/mL Grass

Have 100,000 BAU/mL Grass
Vw = 5.0 Vh = ?
Cw = 2,000 Ch = 100,000
(5.0) (2,000) = ? (100,000)
? = (5.0) (2,000) / 100,000 = 0.10

Formulation 0.10 mL of 100,000 BAU/mL Grass

5.0 – 0.10 = 4.90 mL of diluent
 DF Approach 2nd BAU
¾ DF = Initial concentration / Final concentration

100,000 BAU/mL Grass to 2,000 BAU/mL

(100,000) divided by (2,000) = 50

Vw = 5.0
DF = 50
Vh = Vw / DF = 5.0 / 50 = 0.10

Formulation 0.10 mL of 100,000 BAU/mL Grass

5.0 – 0.10 = 4.90 mL of diluent
 CM Approach AU
¾ Quantity you want = quantity you have
Formula Vw Cw = Vh Ch

Example Want 5 mL at 1,500 AU/mL Mite

Have 10,000 AU/mL Mite
Vw = 5.0 Vh = ?
Cw = 1,500 Ch = 10,000
(5.0) (1,500) = ? (10,000)
? = (5.0) (1,500) / 10,000 = 0.75

Formulation 0.75 mL of 10,000 AU/mL Mite

5.0 – 0.75 = 4.25 mL of diluent
 DF Approach AU
¾ DF = Initial concentration / Final concentration

10,000 AU/mL Mite to 1,500 AU/mL

(10,000) divided by (1,500) = 6.67

Vw = 5.0
DF = 6.67
Vh = Vw / DF = 5.0 / 6.67 = 0.75

Formulation 0.75 mL of 10,000 AU/mL Mite

5.0 – 0.75 = 4.25 mL of diluent
 Serial Dilution Example #2
¾ Maintenance concentrate (Vial 1), 5.0 mL total volume
Cat 4,000 BAU/mL 10K = 1/2.5 = 2.00 mL
Mite 1,500 AU/mL 10K = 1/6.67 = 0.75 mL
Grass 2,000 BAU/mL 100K = 1/50 = 0.10 mL
Diluent 5.0 - (2.0 + 0.75 + 0.1) = 2.15 mL

¾ Subsequent vials in treatment set, 5.0 mL total volumes

Vial 2 0.50 mL Vial 1 + 4.50 mL diluent
Vial 3 0.50 mL Vial 2 + 4.50 mL diluent
Vial 4 0.50 mL Vial 3 + 4.50 mL diluent
Vial 5 0.50 mL Vial 4 + 4.50 mL diluent
 More Vial 1 Exercises
¾ 20, 10 or 5 mL total volumes
Dog 1/5, 1/10 or 1/20 dil from 1:10 w/v stock vial
Bermuda 1/3, 1/6 or 1/10 dil from 10K BAU/mL stock
Ragweed 1/10, 1/25 or 1/50 dil from 475 AgE U/mL stock
Cat 1/3, 1/5 or 1/8 dil from 10K BAU/mL stock

Formulations and final concentrations of each component?

¾ 10 mL total, 1:100 w/v or 5,000 PNU/mL per component

Alternaria 1:10 w/v, 120,000 PNU/mL stock vial
Red maple 1:10 w/v, 60,000 PNU/mL stock vial
Johnson 1:20 w/v, 70,000 PNU/mL stock vial
Nettle 1:20 w/v, 30,000 PNU/mL stock vial
Aspergillus 1:10 w/v, 90,000 PNU/mL stock vial
Juniper 1:10 w/v, 12,000 PNU/mL stock vial
 Exercise Results
¾ 20 mL total volume
Dog 1:10 w/v 1/5 = 4.00 mL Final: 1:50 w/v
Bermuda 10K BAU/mL 1/3 = 6.67 mL Final: 3.3K BAU/mL
Ragweed 475 AgE U/mL 1/10 = 2.00 mL Final: 48 AgE U/mL
Cat 10K BAU/mL 1/3 = 6.67 mL Final: 3.3K BAU/mL

¾ 10 mL total volume
Dog 1:10 w/v 1/10 = 1.00 mL Final: 1:100 w/v
Bermuda 10K BAU/mL 1/6 = 1.67 mL Final: 1.7K BAU/mL
Ragweed 475 AgE U/mL 1/25 = 0.40 mL Final: 19 AgE U/mL
Cat 10K BAU/mL 1/5 = 2.00 mL Final: 2K BAU/mL

¾ 5 mL total volume
Dog 1:10 w/v 1/20 = 0.25 mL Final: 1:200 w/v
Bermuda 10K BAU/mL 1/10 = 0.50 mL Final: 1K BAU/mL
Ragweed 475 AgE U/mL 1/50 = 0.10 mL Final: 9.5 AgE U/mL
Cat 10K BAU/mL 1/8 = 0.63 mL Final: 1.3K BAU/mL
 Exercise Results
¾ 10 mL total, 1:100 w/v per component
Alternaria 1:10 w/v 1:100 = 1/10 dilution = 1.00 mL
Red maple 1:10 w/v 1:100 = 1/10 dilution = 1.00 mL
Johnson 1:20 w/v 1:100 = 1/5 dilution = 2.00 mL
Nettle 1:20 w/v 1:100 = 1/5 dilution = 2.00 mL
Aspergillus 1:10 w/v 1:100 = 1/10 dilution = 1.00 mL
Juniper 1:10 w/v 1:100 = 1/10 dilution = 1.00 mL

¾ 10 mL total, 5,000 PNU/mL per component

Alternaria 120,000 PNU/mL 5,000 = 1:24 dilution = 0.42 mL
Red maple 60,000 PNU/mL 5,000 = 1:12 dilution = 0.83 mL
Johnson 70,000 PNU/mL 5,000 = 1:14 dilution = 0.71 mL
Nettle 30,000 PNU/mL 5,000 = 1/6 dilution = 1.67 mL
Aspergillus 90,000 PNU/mL 5,000 = 1/18 dilution = 0.55 mL
Juniper 12,000 PNU/mL 5,000 = 1/2.4 dilution = 4.17 mL