Mixing

Dilution and Mixing of Allergen Extracts Basic Concepts , General Guidelines and Hands-on Training

Lenoir, North Carolina

Overview ¾

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Objectives: Discuss/review, demonstrate and streamline extract dilution/mixing Dilution/mixing calculations and procedures are straightforward and easy-to-master 2 common mathematical approaches to dilution and mixing yield identical results Proper dilution/mixing and effective IT require awareness of 5 critical factors

Critical Factors ¾

Quality and consistency of extracts Compositions, Concentrations, Potencies

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Integrity and activity of allergens in extracts and mixtures Stabilities, Compatibilities, Cross-reactivities

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Sterility and aseptic handling of allergen vials Storage, Workspace, Withdrawal/delivery

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Diluent choices, advantages/disadvantages and preferences Normal saline, Buffered saline, HSA-saline, 10% glycerin

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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?

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Short exposures to elevated temperatures?

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Accidental freezing and thawing?

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Diluent differences?

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Clinical correlations w/ laboratory test results?

Extract Compatibility FAQs ¾

Compatibility of allergens with molds/insects?

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Compatibilities of cat, mite, ragweed and grasses?

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

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Compatibility of molds with insects?

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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

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OK to mix molds with cat or other molds

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

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Evidence

In vivo

Skin tests

In vitro

ELISA, Immunoblot

Compatibility of ...

After mixing and storage with ... Insects

Molds

Mites

Rag

Grass

Trees/ Weeds

Insects Molds Mites Ragweed

Risky

Grasses Trees/ Weeds Epithelia Cat Venoms

Not OK

OK

Eps

Cat

Venom

Dating/Re-prep Considerations ¾

Individual extract stabilities are concentration-dependent Source materials and glycerin concentrations

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Extract mixture stabilities are based on cumulative strengths Correspond to individual allergens at final concentrations

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10-fold reductions in strength reduce stabilities in half Represent worst-case examples

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Stabilities at high dilutions are speculative at best Below detection/quantitation limits of in vitro potency tests

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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 40K-200K PNU/mL

NA

18-36 months

18-36 months

1:100 w/v 4K-40K PNU/mL

Maintenance conc

6-12 months

12 months

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

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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

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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

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Protein nitrogen units (PNU) Acid-precipitable protein, not predictive of allergenic potencies

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Bioequivalent Allergy Units (BAU, AU) Dust mites and grasses Dust mites @ 10,000 AU/mL

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IgE binding to multiple protein allergens Most grasses @ 100,000 BAU/mL

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

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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

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 Vh = ? Vw = 5.0 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 ¾

2nd w/v

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 Vh = ? Vw = 5.0 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 ¾

2nd w/v

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 ¾

1st PNU

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 Vh = ? Vw = 5.0 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 ¾

1st PNU

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 ¾

2nd PNU

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 Vh = ? Vw = 5.0 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 ¾

2nd PNU

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 Yellow Blue Green Silver

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Vial 1 Vial 2 Vial 3 Vial 4 Vial 5

Maintenance concentrate 1/10 of maintenance concentrate 1/100 of maintenance concentrate 1/1,000 of maint. concentrate 1/10,000 of maint. concentrate

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 Ext B Ext C Ext D Diluent

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1:250 w/v 1:10 = 1/25 = 0.20 mL 1:50 w/v 1:10 = 1/5 = 1.00 mL 4,000 PNU/mL 40K = 1/10 = 0.50 mL 10,000 PNU/mL 40K = 1/4 = 1.25 mL 5.0 – (0.2 + 1.0 + 0.5 + 1.25) = 2.05 mL

Subsequent vials in treatment set, 5.0 mL total volumes Vial 2 Vial 3 Vial 4 Vial 5

0.50 mL Vial 1 0.50 mL Vial 2 0.50 mL Vial 3 0.50 mL Vial 4

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4.50 mL diluent 4.50 mL diluent 4.50 mL diluent 4.50 mL diluent

CM Approach ¾

1st BAU

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 Vh = ? Vw = 5.0 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 ¾

1st BAU

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 ¾

2nd BAU

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 Vh = ? Vw = 5.0 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 ¾

2nd BAU

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 ¾

AU

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 Vh = ? Vw = 5.0 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 ¾

AU

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 Mite Grass Diluent

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4,000 BAU/mL 10K = 1/2.5 = 2.00 mL 1,500 AU/mL 10K = 1/6.67 = 0.75 mL 2,000 BAU/mL 100K = 1/50 = 0.10 mL 5.0 - (2.0 + 0.75 + 0.1) = 2.15 mL

Subsequent vials in treatment set, 5.0 mL total volumes Vial 2 Vial 3 Vial 4 Vial 5

0.50 mL Vial 1 0.50 mL Vial 2 0.50 mL Vial 3 0.50 mL Vial 4

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4.50 mL diluent 4.50 mL diluent 4.50 mL diluent 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?

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10 mL total, 1:100 w/v or 5,000 PNU/mL per component Alternaria Red maple Johnson Nettle Aspergillus Juniper

1:10 w/v, 120,000 PNU/mL stock vial 1:10 w/v, 60,000 PNU/mL stock vial 1:20 w/v, 70,000 PNU/mL stock vial 1:20 w/v, 30,000 PNU/mL stock vial 1:10 w/v, 90,000 PNU/mL stock vial 1:10 w/v, 12,000 PNU/mL stock vial

Exercise Results ¾

20 mL total volume Dog Bermuda Ragweed Cat

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1/5 = 4.00 mL 1/3 = 6.67 mL 1/10 = 2.00 mL 1/3 = 6.67 mL

Final: Final: Final: Final:

1:50 w/v 3.3K BAU/mL 48 AgE U/mL 3.3K BAU/mL

1/10 = 1.00 mL 1/6 = 1.67 mL 1/25 = 0.40 mL 1/5 = 2.00 mL

Final: Final: Final: Final:

1:100 w/v 1.7K BAU/mL 19 AgE U/mL 2K BAU/mL

1/20 = 0.25 mL 1/10 = 0.50 mL 1/50 = 0.10 mL 1/8 = 0.63 mL

Final: Final: Final: Final:

1:200 w/v 1K BAU/mL 9.5 AgE U/mL 1.3K BAU/mL

10 mL total volume Dog Bermuda Ragweed Cat

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1:10 w/v 10K BAU/mL 475 AgE U/mL 10K BAU/mL

1:10 w/v 10K BAU/mL 475 AgE U/mL 10K BAU/mL

5 mL total volume Dog Bermuda Ragweed Cat

1:10 w/v 10K BAU/mL 475 AgE U/mL 10K BAU/mL

Exercise Results ¾

10 mL total, 1:100 w/v per component Alternaria Red maple Johnson Nettle Aspergillus Juniper

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1:10 w/v 1:10 w/v 1:20 w/v 1:20 w/v 1:10 w/v 1:10 w/v

1:100 = 1/10 dilution = 1.00 mL 1:100 = 1/10 dilution = 1.00 mL 1:100 = 1/5 dilution = 2.00 mL 1:100 = 1/5 dilution = 2.00 mL 1:100 = 1/10 dilution = 1.00 mL 1:100 = 1/10 dilution = 1.00 mL

10 mL total, 5,000 PNU/mL per component Alternaria Red maple Johnson Nettle Aspergillus Juniper

120,000 PNU/mL 60,000 PNU/mL 70,000 PNU/mL 30,000 PNU/mL 90,000 PNU/mL 12,000 PNU/mL

5,000 = 1:24 dilution = 0.42 mL 5,000 = 1:12 dilution = 0.83 mL 5,000 = 1:14 dilution = 0.71 mL 5,000 = 1/6 dilution = 1.67 mL 5,000 = 1/18 dilution = 0.55 mL 5,000 = 1/2.4 dilution = 4.17 mL