International Scientific Committee of

Ozone Therapy SOP: ISCO3/MET/00/02

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ISCO3/MET/00/02 Minor Autohemotherapy

Index

Title................................................................................................................................... 2
1.1. Brief background ................................................................................................... 2
1.2. Purpose .................................................................................................................. 2
1.3. Scope ..................................................................................................................... 2
1.4. Acronyms, abbreviations and definitions .............................................................. 2
3. Procedure ...................................................................................................................... 3
3.1 Indications .............................................................................................................. 3
3.2 Contraindications .................................................................................................... 3
3.3 Recommended doses and intervals ......................................................................... 4
3.4 Clinical evaluation .................................................................................................. 4
3.5 Preliminary operations ............................................................................................ 4
3.6 Main procedure ....................................................................................................... 5
3.7 Side effects ............................................................................................................. 5
3.8 Patients Follow-up .................................................................................................. 6
3.9 Effect Mechanism ................................................................................................... 6
4. Contingencies; Corrective Actions ............................................................................ 7
5. References ................................................................................................................... 7
5.1 SOP References ...................................................................................................... 7
5.2 Other References .................................................................................................... 7
6.1 List of recommended medical disposables ......................................................... 8
7. Change History............................................................................................................ 8
8. Document Records ...................................................................................................... 8

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 International Scientific Committee of
Ozone Therapy SOP: ISCO3/MET/00/02
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Title

ISCO3/MET/00/02 Minor Autohemotherapy

1.1. Brief background

Minor AHT without ozone, only injecting whole blood through I.M. route, is a very old,
traditional treatment as unspecified immunomodulatory therapy. Later Dr. Hans Wolff,
suggested to add ozone for activating blood components.

It is one of the methods to apply ozone therapy. It is very safe, has almost no negative side
effects, and permits an ozone dosage range from very low to very high. It is recommended,
either as only treatment or in combination with other forms of ozone therapy, or other
medication, for a rather large number of diseases or conditions.

1.2. Purpose

The purpose of this SOP is to describe the procedure for a Minor Autohemotherapy (MiAHT)
session with ozone.

1.3. Scope
This procedure specifies the blood collecting technique, doses, volume of gas and frequency of
application of ozone.

1.4. Acronyms, abbreviations and definitions

G6PD Glucose 6 phosphate dehydrogenase

Gamma Non appropriate but frequently word to designate microgram
MiAHT Minor Autohemotherapy
SOP Standard Operation Procedure
Total doses Total amount, in micrograms, of ozone given per session, calculated as volume
in mL multiplied by concentration in µg/mL

2. Responsibility

Physician Patients’ Clinical records registration

Assessment of the indication, contraindications
Request the informed consent (ISCO3/QAU/00/21) and the privacy consent
Applications and monitoring
Recording all data on medical records
Prescription of investigations to assess the effectiveness of the treatment (e.g.
biochemical or immunological assays)
Reporting any late complications
Patient follow-up

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Nurses Accommodate the patients

Preparation of the clinical procedure
Supervision of patients, and vital signs control (temperature and pressure)
Detects and alerts the doctor to anomalies due to possible reactions
Notification of possible complications

A MiAHT session should be done by a physician, adequately trained in ozone therapy. Also a
trustworthy assistant, nurse, or paramedical professional, may do the procedure, provided this
person is adequately trained for this work. It is the physician’s responsibility to see that all steps
of the procedure are done in the correct manner, in order to always avoid errors, accidents, and
to prevent incidents.

3. Procedure
3.1 Indications
Mainly benefits in immune system; either boost or suppression like Acne vulgaris (common
acne), allergies, adjuvant in cancer therapy, unspecific immune activation, infections,
rheumatoid arthritis, Herpes infections, Herpes zoster, Post herpetic neuralgia etc. Basically
indications of using Antibiotic, Antiviral, Antifungal agents or Corticosteroids in conventional
medicine can be indication reasons for MiAHT as well.

3.2 Contraindications
Absolute contraindication: Favism: Glucose-6-phosphate dehydrogenase deficiency (favism,
acute haemolytic anaemia).*
Relative contraindications / special situations:
 Uncompensated diabetes
 Acute myocardial infarction
 Pregnancy in the first 3 months
 Uncompensated toxic hyperthyroidism - Basedow Graves status
 Thrombocytopenia less than 50.000 and serious coagulation disorders
 Severe Cardiovascular instability
 Acute alcohol intoxication
 Acute infarction of myocardium
 Massive and acute hemorrhage
 During convulsive states
 Hemochromatosis
 Patients receiving treatment with copper or iron.

* The prevalence of Glucose 6 phosphate dehydrogenase (G6PD) deficiency varies among ethnic groups with overall
lower frequency in the Americas (3.4%), Europe (3.9%), and the Pacific (2.9%) as compared to sub-Saharan Africa
(7.5%), the Middle East (6.0%), and Asia (4.7%).1 Test of G6PD is recommended prior to O3 therapy in order to
avoid complications (Nkhoma et al. 2009).

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MiAHT approach must be carefully balanced in patients under anticoagulant or antiaggregating

treatment. If MiAHT if finally performed, special and prolonged compression has to be applied
at the injection site.

3.3 Recommended doses intervals

MiAHT may be given at most variable intervals, from daily to weekly or monthly. Also as
cycles to be given once or more times per year.

The volume of blood to use varies between 2 mL and 10 mL.

Frequency of treatment: The number of treatment sessions and the ozone dosage administered
will depend on the general condition of the patient, age and main disease.

Ozone concentrations for MiAHT

The ISCO3 recommended dose for MiAHT are range from 10 μg/NmL to 40 μg/NmL (see table
below), concentrations above 60 µg/mL should be avoided because of the increased risk of
hemolysis, reduction of 2,3 DPG and anti-oxidant and a consequent inability to activate
immune-competent cells. This is the dose ranges used by German, Russian and Madrid
Declaration (Viebahn-Hänsler et al. 2012; Maslennikov et al., 2008; ISCO3/QAU/01/03). 2).
theoretically, the vigorous mix of blood and gas leads to hemolysis. So, hemolysis for high O 3
concentration (and small blood volume) is not relevant here and very small concentrations could
be not useful (Bocci, 2005).

High Medium Low

O3
Minor Auto C. (µg/mL) 40-30 15-20 5-10
Hemotherapy V. (mL) 5
Dose (µg) 200-150 75-100 25-50

Note: German Guideline: Vol. 10 mL blood + 10 mL O3 at a concentration of 10-20 µg/mL

(Viebahn-Hänsler et al. 2012); Russian Guideline Vol. 5-10 mL blood + 10-15 mL O3 at a
concentration of 10-40 µg/mL (Maslennikov et al., 2008); Madrid Declaration as shown in the
table (ISCO3/QAU/01/03); Bocci Vol. 5 mL blood + 5 mL O3 at a concentration 40-80 µg/mL
(Bocci, 2005).

3.4 Clinical evaluation

A clinical and/or laboratory evaluation is necessary to establish a precise diagnosis and to

permit comparisons between the patient’s status before, during and after ozone therapy.

3.5 Preliminary operations

The patient must be fully informed in advance about the method itself, about all the steps of the
procedure, about the desired effect(s) and also about the possible unwanted side effects. Also a

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written Term of Informed Consent should be read, understood and signed by the patient or the
person responsible for the patient.

Device: must fit the standard requirement ISCO3/DEV/00/01

Syringes: Plastic-based devices, intended to contain blood, must meet the ISO 15747 standard:
2005 (This is the European Union regulation). All containers and devices used in O3x must be
ozone-resistant and must not release phthalates

3.6 Main procedure

Its principle is similar to major auto hemotherapy. Medical ozone gas and patient’s own whole
blood are mixed in a sterile, pyrogen-free disposable syringe and then reinjected to the patients.
Comparing to Major Auto Hemotherapy, Minor Auto Hemotherapy has some differences.
1- Volume: In general 2 - 10 mL (mostly 5 mL) blood
2- Injection Site: Intramuscular mostly (or Subcutaneous)
3- Anti Coagulant: It is not necessary to use in Minor Auto Hemotherapy
4- Ozone Volume: It can be equal or a little more than the blood volume.
5- Ozone Dose: It must be set by the physician depending on the case but in general dose
should be in therapeutic dose ranges.

In minor autohemotherapy (MiAHT), under aseptic conditions, patient’s whole blood is mixed
with ozone in desired amount and at necessary concentration. Mixing procedure can be done
two ways:

1- Drawing blood into a syringe and taking ozone into another syringe (dose and amount of
ozone vary depending on the treatment). Then inserting ozone into the blood containing
syringe or the opposite – blood into the ozone syringe.
2- Ozone can be taken into a syringe and then blood directly can be drawn from patients venous
and then ozone – blood are mixed at the same syringe.

Both of the methods above, have the same effect. But inserting ozone into blood in syringe
results serious bubbling which means either blood loss or injecting air – oxygen into the muscle.
Also there will be two syringes used. First to take ozone into the syringe and then drawing
blood to the same syringe, mixing gently, drawing the residual gas out and injecting is the best
recommended way. When medical ozone is mixed with whole blood, ozone reacts with blood in
seconds. The residual gas in the syringe is left over oxygen and it is meaningless to inject this
volume to the patient.

After mixing ozone and blood gently for about 1 min, it can be injected via I.M. route. Some
physician can prefer S.C. route. There is no clinical data comparing I.M. or S.C. route but in
general I.M. route is preferred. Because in S.C. method, later some color changes can occur on
the skin.

3.7 Side effects

No systematic side effect is reported. Only light local side effects can be observed. Several
processes, such as serum reabsorption, fibrinolysis, via lymphatic vessels and a mild sterile

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inflammatory reaction, are likely to take place as occasionally suggested by a slight swelling at
the injection site reported by some patients during the next few days.

3.8 Patients Follow-up

Patients may be followed and re-evaluated from time to time, from the clinical / subjective point
of view and/or with laboratory and/or image examinations.

3.9 Effect Mechanism

The hypothetical action mechanism of minor autohemotherapy in infectious diseases will

involves that ozone can fragment most virions present in blood, without regard to preserving
cellular elements. This treated blood, injected intramuscularly, carries fragments of viral
envelope and nucleic acids which find their way into the general circulation and to the immune
network. The latter, if still relatively operational, begins to manufacture appropriate antibodies
which in turn, serve to counter the evolution of the infection. The interesting feature of this
technique is that antibodies thus manufactured are individualized to the particular patient
receiving the treatment, since they are derived from their own viral stock, In view of the high
mutability of retroviruses, each patient carries a unique viral strain. Minor autohemotherapy can
thus be conceptualized as a method of auto vaccination providing a high degree of antibody
specificity (V. Bocci 1999).

The minor autohemotherapy specifically stimulates the body’s second line of defense that is
important in autoimmune diseases and long-time chronic illness. After a period of time when
the body needs to fight a long-term illness, the first line of defense becomes weakened. Ozone
therapy, especially the minor autohemotherapy has the ability to reactivate the second line of
defense, especially the natural killer cells (V. Bocci 1999).

Chemotactic compounds released at the site may stimulate the local infiltration of monocytes
and neutrophils which take up hemolyzed erythrocytes and denatured proteins. Activated
monocytes and lymphocytes may release interferons and interleukins either in loco or along the
lymphatic system, upregulating the physiological cytokine response (Bocci, 1981; 1988). Thus
it would be quite interesting to evaluate some immunological parameters and some other heat
shock proteins (Tamura et al., 1997) that may enhance immune reactivity and explain the
beneficial effects (V. Bocci 1999).

Ozonated, hence primed, leukocytes may either infiltrate the tissue or/and may return via
lymphatics into the blood pool or into other lymphoid microenvironments. Most of the
erythrocytes will be slowly broken down locally and will provide substrates for rebuilding the
extracellular matrix but, most importantly, heme will induce the synthesis of stress proteins,
particularly heme oxygenase I (HO-I or HSP-32). This is a most protective enzyme that, by
enhancing the release of CO and bilirubin, facilitates the local circulation and neutralizes
oxidant compounds. Finally, the twice ozonated platelets will be activated and release locally a
wealth of growth factors (platelet-derived growth factor-PDGF, basic-fibroblast growth factor,
b-FGF, transforming growth factor b1, TGFb1), which will greatly help tissue reconstruction
(Gracer and Bocci, 2005).

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Ozone Therapy SOP: ISCO3/MET/00/02
Tel/Fax (+34) 913515175. Cell Phone (+34) 669685429
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4. Contingencies; Corrective Actions

Some patients react very sensitively to the pain caused by the insertion of a needle. They may
feel dizzy, may sweat profusely, and even faint. It is very convenient to have an electric table
which allows a Trendelenburg position. Emergency bottles of saline, injectable glucose,
injectable vitamin C should be at hand. In extreme cases, an oxygen mask might be required.

In case of other side effects follow the instructions of ISCO3/CLI/00/01 "Fist Aids in ozone
therapy (Inhalator exposition and accidental over dose)" and report the side effect using
ISCO3/REC/00/03 "The ISCO3 Safety Information and Adverse Event Reporting Program
Form".

5. References

5.1 SOP References

ISCO3/QAU/00/21. Informed Consent Form in Ozone Therapy.

ISCO3/DEV/00/01 Guidelines and Recommendations for Medical Professionals Planning to
Acquire a Medical Ozone Generator.
ISCO3/CLI/00/01. Fist Aids in ozone therapy (Inhalatory exposition and accidental over dose)
ISCO3/REC/00/03 The ISCO3 Safety Information and Adverse Event Reporting Program
Form.
ISCO3/QAU/01/03. Madrid Declaration on Ozone Therapy 2015-2020 Eng. Schwartz-Tapia
A, Martínez-Sánchez G, Sabah F, Alvarado-Guémez F, Bazzano-Mastrelli N, Bikina O,
Borroto-Rodrígez V, Cakir R, Clavo B, González-Sánchez E, Grechkanev G, Najm Dawood
A H, Izzo A, Konrad H, Masini M, Peretiagyn S, Pereyra, V R, Ruiz Reyes D, Shallenberger
F, Vongay V, Xirezhati A, Quintero-Marino, R. Madrid Declaration on Ozone Therapy.
2th ed. Madrid: ISCO3; ISBN 978-84-606-8312-4; 2015. 50 p.

5.2 Other References

1. Bocci, V., 1981. Production and role of interferon in physiological conditions, Biol. Rev. 56:49-
85
2. Bocci, V., 1988, Roles of interferon produced in physiological conditions. A speculative review,
Immunology 64:1-9.
3. Bocci V, Valacchi G, Rossi R, et al. Studies on the biological effects of ozone: 9. Effects of
ozone on human platelets. Platelets. 1999;10(2-3):110-6.
4. Bocci V, 2005, Ozone A New Medical Drug, 42 – 44.
5. Gracer , V. Bocci. Can the combination of localized ‘‘proliferative therapy’’ with ‘‘minor
ozonated autohemotherapy’’ restore the natural healing process? Medical Hypotheses (2005)
65, 752–759
6. Maslennikov, O.V., Kontorshikova, C.N. & Gribkova, I.A. Ozone therapy in Practice. Health
Manual, Ministry Health Service of The Russian Federation The State Medical Academy Of
Nizhny Novgorod, Russia. http://www.absoluteozone.com/assets/ozone_therapy_in_practice.pdf,
(2008).
7. Nkhoma ET, Poole C, Vannappagari V, Hall SA, Beutler E. The global prevalence of glucose-6-
phosphate dehydrogenase deficiency: a systematic review and meta-analysis. Blood Cells Mol
Dis. 2009 May-Jun;42(3):267-78.

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 International Scientific Committee of
Ozone Therapy SOP: ISCO3/MET/00/02
Tel/Fax (+34) 913515175. Cell Phone (+34) 669685429
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Avenida Juan Andrés 60. Local 1 – Bajo Izquierdo 28035, Date: 31/03/2016
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8. Tamura, Y., Peng P., Liu K., Daou M., and Srivastava P. K., 1997, Immunotherapy of tumors
withautologous tumor-derived heat shock protein preparations, Science 278:117-120.
9. Viebahn-Hänsler R, Fernández OSL, Fahmy Z. Ozone in Medicine: The Low- Dose Ozone
Concept. Guidelines and Treatment Strategies. Ozone Science & Engineering. 2012;34(6):408-
24.
10. Warkentin TE, Greinacher A. Heparin-induced thrombocytopenia and cardiac surgery. Ann
Thorac Surg. 2003 Dec;76(6):2121-31.

6. Documentation and Attachments

6.1 List of recommended medical disposables

Siliconated Luer lock syringe of 20 mL

Gloves and disinfectant solution
Sterile antimicrobial filter

7. Change History

Effective Previous
SOP no. Significant Changes
Date SOP no.
ISCO3/MET/00/02 19/01/2016 Draft. First version
31/03/2016 Final version First version

8. Document Records

Name Title Signature Date

Author Ruhi Cakir, M.D. Member ISCO3 31/03/2016

E.mail. [email protected]

Authoriser / ISCO3 Board and members All members

Approved 2015-2020

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