Contact-Thermography and Live Blood Analysis and Clot Retraction Test Techniques–response to...

Contact-Thermography and Live Blood Analysis and Clot Retraction Test Techniques – response to queries Prepared by: Luis Vitetta1 Marilyn Johnson 2 Fernando Cortizo 3 Avni Sali4

Graduate School of Integrative Medicine, Swinburne University 1 Director of Research 2 Research Assistant 3 Senior Lecturer and Research Supervisor 4 Professor and Head of School

Contact-Thermography and Live Blood Analysis Techniques – response to queries Vitetta L, Johnson M, Cortizo F, Sali A. Graduate School of Integrative Medicine

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Contact-Thermography and Live Blood Analysis Techniques – response to queries

Graduate School of Integrative Medicine Swinburne University 9 Frederick Street Hawthorn Vic 3122 Telephone: + 61 3 9214 5296 Facsimile: + 61 3 9214 8009 Email: [email protected] Website: www.swin.edu.au/gsim

© 2003


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Overall Summary:

We have been asked to comment in the form of an official scientific report on

three separate screening techniques that are part of a current proceeding by the

ACCC against Australian Biologics – Testing Services Pty Ltd.

The screening techniques in question include:

1. contact thermography

2. live blood analysis

3. clot retraction test

For this review of the scientific evidence we have utilised the rating system that is

recommended by the Quality of Care and Health Outcomes Committee and has been

adapted from the system developed by the US Preventive Services Task Force,

which has also been adopted by the National Health and Medical Research Council

of Australia. The rating system consists of the following:

• Level I Evidence is obtained from a systematic review of all relevant

randomised controlled trials – meta analyses.

• Level II Evidence is obtained from at least one properly designed

randomised controlled clinical trial.

• Level III Evidence is obtained from well designed controlled trials without

randomisation or from well designed cohort or case control analytic design

studies, preferably from more than one centre or research group or from

multiple time series with or without an intervention.

• Level IV Evidence represents the opinions of respected authorities based

on clinical experience, descriptive studies or reports of expert committees.

As far as contact thermography is concerned there is ample level II, III and IV

evidence to show that this is a most important modality that can be considered to

be most useful in determining peripheral blood flow in numerous disease states.


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We refer you to our previously prepared document.1 Further, the evidence to date

demonstrates that contact thermography has applications which have been

successfully applied in the diagnosis and treatment of various disease states that

include breast cancer, cardiovascular disease, diabetes, and general blood flow

problems. Contact thermography has general adjuvant diagnostic properties that

need to be fully exploited by the medical profession. Moreover, contact

thermography has been advocated as a rapid, non-invasive diagnostic modality in

evaluating patients at risk for breast cancer.91 In that study which was conducted

approximately twenty years ago it was reported that thermograms should be

reported as either being normal or abnormal, criteria that would allow the modality

to show a high degree of reliability.

We did not identify any research evidence at levels I to III associated with the

Live Blood Analysis Test on searching the peer reviewed medical literature.

However, this should not constitute a lack of validity or scientific principle

associated with this technique. Our report comprises level IV evidence and is part

of an expert opinion in the area of the Live Blood Analysis Test. Alterations in

erythrocyte (red blood cells) and leukocyte (white blood cells) morphology can be

easily detected and the results have a high level of reproducibility when expert

technicians are used to employ the technique.

Along similar lines of scientific reasoning, recently, the erythrocyte agglutination

test has been successfully employed to demonstrate combined leukocyte and

erythrocyte aggregation in the peripheral venous blood.2-6 Further, also recently it

was concluded that the association between increased erythrocyte

adhesiveness/aggregation, higher concentrations of acute phase proteins, and

increased atherosclerotic risk factors points to a possible clinical applicability of

the erythrocyte adhesiveness/aggregation test to reveal the presence of both low-


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grade subclinical smoldering inflammation and morbid biology in individuals with risk

factors for atherosclerosis.7

The Live Blood Analysis Test requires further scientific validation however, from

our own research on the technique we have shown that there is a high degree of

correlation between screening results obtained by The Live Blood Analysis Test and

Standard Diagnostic Medical (haematological) Tests in the area of erythrocyte and

leukocyte morphology [Refer Appendix I). Further, the detection of specific

precipitates in peripheral blood identified with the Live Blood Analysis Test has

shown strong correlations with gastrointestinal dysfunction and immune function

[Graduate School of Integrative Medicine, Swinburne University Database].

In our experience The Live Blood Analysis Test that employs the dark field

illumination microscopy technique is a most useful and accurate method as a first

line screening for digestive system and immune function dysfunction. Hence the

employment of dark field illumination microscopy provides the observer the ability

to distinguish all the major subtypes of white blood cells. This leads to a complete

differential screening – analysis of the leukocytes and thus is gained a significant

insight into the immunologic function of the patient. The Live Blood Analysis Test

that employs dark field illumination microscopy method allows the screening of

blood cells in a single session and clinical setting.

Further, for the benefit of patients The Live Blood Analysis Test requires the

employment of scientific technicians with a detailed knowledge of pathological

haematology and who communicate in the first instance with qualified medical

practitioners for the proper management of their patients. As our knowledge is

enhanced from further research and development in this area of medical research,

the general practice clinician who represents the first line contact with the


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community is hence provided with a powerful, sensitive, and comprehensive

screening health method that in the first instance can be used as a screening tool

for promoting lifestyle changes in their patients with the ultimate goal being one of

disease prevention and health promotion.

Responses to Queries:

1. thermography does not provide assessment of the degree of illness

(slight to serious) (ACCC interpretation “thermography does not

provide an assessment of the degree of all illness)

We have reviewed this screening technique in a previous report that

has been delivered to Australian Biologics.

In Summary: from our interpretation of the evidence of this

technique we believe contact thermography to be a most useful

adjuvant screening procedure for a number of different medical

problems.

At no time has there been an interpretation that this screening

technique would provide an assessment of the degree of all illness.

Moreover there is no current medical test/procedure that was or is

currently available, that provides an assessment or measure of the

degree of ALL illness.

Excerpt from our review:1 In terms of breast pathology we conclude in

our report that from the evidence that is currently available that contact

thermography may constitute a low cost adjuvant screening intervention that

could be a yearly evaluation as part of a routine physical assessment for

women who find mammography a painful and difficult procedure. Therefore


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as soon as a suspicious (positive) breast thermal examination is performed,

the appropriate follow-up diagnostic and clinical testing can be ordered. This

would include mammography and other imaging tests, clinical laboratory

procedures, nutritional and lifestyle evaluation and training in breast self

examination. Thermography is a simple, non-invasive, highly accurate,

inexpensive form of screening imaging technique as well as being a ‘breast

friendly’ procedure.

Moreover, as thermography is capable of measuring changes in temperature

that can reflect blood supply and inflammation which are general

physiological changes, it is then possible to envisage its adaptation to screen

other physiological changes that are associated with blood flow. An

additional search of the current medical literature showed that contact

thermography has been applied to other medical conditions. Namely,

cardiovascular disease, comorbidities associated with diabetes and as an

adjuvant method to better define cancer diagnoses.

We have identified numerous studies from the medical literature that hence

emphasize the importance of this technique in medical practice.1-175

In addition to breast screening studies we have identified other peer

reviewed studies that have employed contact thermography in other areas

of medicine and these include:

cardiovascular disease and circulation/blood flow

• Coloured contact thermography has been employed as an additional

method for the diagnosis of diseases of the abdominal aorta and its

major branches.171


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• Thermography has been used to study myocardial temperature during

infusion of cold cardioplegic solutions. Slow cooling was recorded

distal to coronary stenosis or occlusions, thereby indicating

insufficient protection of the myocarium in these areas169, 171-173

• In a recent study of thermograms in army personnel regional

hothermia was investigated with thermograms.160 Further, in another

recent study of thermograms in army personnel by the same group of

researchers investigated thermograms during and after basic military

training evaluating the temperature changes in thermograms whilst

differentiating between those with normal foot parameters, those

with musculoskeletal stress and others with foot stress. The

thermograph showed that normal foot cooling differed between the

three groups. In conclusion thermography did not reveal an exact

diagnosis however it was of greatest benefit monitoring severity and

healing of the foot.160

• Liquid Crystal contact Thermography has been shown to be very

reliable for as a method of investigating skin blood flow and for a

screening method in the diagnosis of deep venous thrombosis.59,164

• The use of thermography has been used to evaluate the peripheral

circulatory function in the diagnosis of diabetic complications.163

Thermography has been employed to observe wound temperature and

hence monitor healing in surgical patients. 162, 163

Contact thermography in conjunction with Doppler sonograph resulted

in the highest diagnostic accuracy when investigating retrograde

blood flow in the internal spermatic vein.152


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Diabetic neuropathy

• Contact thermography has been successfully employed to investigate

and identify diabetic patients who are at increased risk for

neuropathic foot ulceration.16

2. thermography can only assess some aspects of peripheral blood flow

and it does provide an assessment of the circulation, disturbances of

cerebral and/or peripheral blood circulation and vascular occlusion.

(ACCC interpretation – “thermography can only assess some aspects of

peripheral blood flow and it does not provide an assessment of the

circulation of the blood as a whole”

There is substantial scientific evidence that thermography can assess

various aspects of peripheral blood flow.1, 8-165 That is, that

microcirculatory disturbances can be clinically assessed by thermography.

Further, the patho-physiological analysis of the peripheral circulation using

thermography has been investigated as an example of functional body

imaging. Moreover, since vascular damage is the main complication of

diseases such as diabetes, in studies with lower limb amputations for

diabetic foot have shown that the amputation level of the limb was

determined by skin thermography. Thermography was useful in avoiding

deep and systemic circulatory problems which are commonly associated with

diabetic limb amputations. Hence, any interpretation that thermography is

only useful for some aspects of peripheral blood flow and that it does not

provide an assessment of the circulation of the blood as a whole is without

merit as there are numerous peer reviewed studies indicating its successful

employment.


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3. thermography does not provide assessment of the strain a person is

able to bear before he be subject to an operation or vaccination

Thermography provides a useful assessment of peripheral blood flow

abnormalities. Therefore when combined with medical expertise these

results may have a useful adjuvant and indirect application in the assessment

of the surgical patient.

4. thermography does not provide an assessment of the success of

therapies ACCC interpretation – “thermography does not provide an

assessment of the success of all therapies”

As an example of thermography providing success of a therapy can be seen

in a recent study121 that assessed temporary stellate ganglion block

thermographically and its effectiveness during cardio-surgical procedures.

The assumption behind this method was that the increase in the

temperature of the upper extremity on the side of the blockade was due to

the broadening of the arterial bed. It was shown that thermography was a

useful method for the assessment of stellate blockade effectiveness.

Effective blockade resulted in increased blood flow in the radial artery.

Further, low temperature is an important factor in protecting the

myocardium during an operation on the heart. This can be difficult to

accomplish if the cold cardioplegic solution is hindered by occlusions or

stenosis of the coronary arteries. Thermography has been used to study

myocardial temperature during infusion of cold cardioplegic solution. Slow

cooling was recorded distal to coronary stenosis or occlusions, thereby

indicating insufficient protection of the myocarium in these areas.162-165


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5. thermography is not suitable for diagnostic purposes in the cardiac

field

Thermography has been employed extensively in the cardiac field. Namely,

thermography has been used to study myocardial temperature during

infusion of cold cardioplegic solutions. Slow cooling was recorded distal to

coronary stenosis or occlusions, thereby indicating insufficient protection of

the myocarium in these areas.162-165 Further, in order to understand, treat,

and prevent acute coronary syndromes it has been reported that there is a

need to improve the ability to identify the rupture-prone, vulnerable

atherosclerotic coronary plaque. In a recent review it was noted that

thermography used in clinical trials provided useful results.176 Moreover, it

has also been recently reported that an atherosclerotic plaque is considered

vulnerable when it is at higher risk of inducing acute cardiac events. The

early detection and follow-up of the vulnerable plaque are crucial to prevent

these events from happening. Arterial wall thermography, which traces the

heat signature of the activated macrophages, is a new and promising method

in this direction.177

6. thermography is not suitable for analytical purposes in the cardiac

field

Thermography is a very suitable analytical technique in the cardiac field.

The identification of vulnerable plaque is one of the primary goals in

cardiology during the last years. Several techniques have been developed

for the anatomic and functional assessment of atherosclerotic plaques.

Thermography is a new method for the evaluation of the inflammatory

process locally within the atherosclerotic plaque. Several animal and clinical

studies demonstrated the value of thermography not only for the detection

of inflamed atherosclerotic plaques, but its use in new fields like in the

evaluation of inflammation in the coronary vascular bed and the


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cardiovascular system. A recent article has reviewed the developments and

the clinical implications of thermography.178

7. thermography does not represent true preventative medicine (ACCC

interpretation of this “thermography does not prevent people from

becoming ill”)

To claim outright that contact thermography has no preventative medicine

benefits is both unsubstantiated and erroneous given the current peer

reviewed literature that is available. To claim that it is a technique that

prevents people from becoming ill is further erroneously misrepresented.

Contact thermography is a very useful adjuvant technique that can assist

the clinician to provide guidance to the patient with breast cancer or a

suspicious breast lump.1 Further, it has been shown that contact

thermography has other useful applications in medicine as has been pointed

previously in points 4 and 5.

8. thermography does not provide an indication of unrecognised disease,

hidden cause and dangerous sequelae (complications).

From the available evidence it has already been shown that contact

thermography has been successfully used to monitor newly developing breast

disease.1 These results support contact thermography as being useful in

detecting previously unrecognised disease. Therefore contact thermography

does provide some evidence for previously undiagnosed disease with serious

sequalae (eg. Breast cancer).

9. thermography does not provide a valuable aid in monitoring the

restoration of health.


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This statement is inaccurate. Refer further to points 1, 2, 3, 4, and 8

previously.

10. CRT does not indicate the degree of oxidation in the body

11. CRT cannot determine whether a patient’s treatment regime is

effective within a short period of time by way of a simple finger prick

test (ACCC interpretation of this – “CRT is not capable of accurately

measuring the effectiveness of a treatment regime”)

12. CRT does not indicate

(i) the degree of free radicals

(ii) the function of the heart

(iii) the function of the ovary

(iv) the function of the prostate

(v) the function of any other human organ

(vi) physical stress

(vii) psychological stress

(viii) allergies

(ix) arthritis

(x) vitamin levels

(xi) mineral levels

(xii) heavy metal level

With respect to items 10, 11 and 12 we note that the clot retraction test

(CRT) or Bolen’s Test has been extensively investigated in Europe.179-195 That

the CRT cannot accurately measure the effectiveness of a treatment regime

is inaccurate. Our research indicates otherwise.


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Reactive Oxygen Toxic Species are by-products of virtually every

dysfunction or challenge to cells. Most of these reactive oxygen species are

normally produced as a consequence of metabolic function however abnormal

species are known to be produced. Their causes may be either exogenous or

endogenous. The term free radical has been generally used to encompass

these species. Reactive Oxygen Toxic Species are molecular substances

that have modifications of the oxygen molecule, are highly reactive to

biochemical structures and include all free radicals. The most important

Reactive Oxygen Toxic Species which is not a free radical is hydrogen

peroxide.

Oxidation of biomolecules as a result of Reactive Oxygen Toxic Species can

play a role in susceptibility to a number of diseases. Reactive Oxygen Toxic

Species may result in a metabolic challenge to every cell in the body. It is

the interactions of these toxins with body cells which produce the systemic

effects of metabolic dysfunctions and challenges. Moreover, in affecting

the body’s basic cellular structures and biochemical pathways they react

with blood constituents to form various metabolic by-products which can be

detected. These morphological changes in the blood as well as the

constituents of metabolic by-products and the specific Reactive Oxygen

Toxic Species vary as a function of the metabolic dysfunction or challenge,

strength of the immune system and the level of Reactive Oxygen Toxic

Species being generated.

Oxidative stress is caused by the presence of free radical or radical-

generating agents in concentrations that overwhelm natural radical-blocking

or -scavenging mechanisms. Sources of oxidative stress include exogenous

factors, such as cigarette smoke, and endogenous factors, such as the


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oxidative burst from activated macrophages. Antioxidant mechanisms

include antioxidant enzymes and plasma antioxidants, many of which are

determined by dietary antioxidant intake. Oxidative stress, in turn, can

cause oxidative damage to DNA, proteins, and lipids, and many clinical

conditions are associated with increased indices of oxidant stress; this

suggests that overwhelming the antioxidant defence system initiates and

propagates processes involved in the pathogenesis of many diseases.196-198

Free radicals are in fact potent deleterious agents causing cell death or

other forms of irreversible damage, eg, free radicals appear to modify <

~10,000 DNA base pairs every day.197

From a research perspective the Graduate School of Integrative Medicine,

Swinburne University is focusing on fully investigating this technique with

the Live Blood Analysis Test to further set guidelines for its implementation.

We have currently data available for approximately 2500 patients of which

160 patients have had these tests correlated with standard blood tests

In summary we have observed through the CRT test, physiological and

biochemical changes that were consistent with medical investigations and

reports provided by physicians that were consistent with the degree of

free radicals associated with a normal patient profile, the function of

the heart, the function of the ovary, the function of the prostate, the

function of any other human organ, physical stress, psychological

stress, allergies, arthritis, vitamin C levels, mineral levels, and heavy

metal levels. (Refer Appendix I - Plates 1 to 11 for examples of these).

Further, we have identified and correlated the following results obtained

from CRTs.


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1. Reactive Oxygen Toxic Species – these are white masses

seen in drops and if relevant organs are affected these are

noted and a detailed report provided to medical practitioner.

2. Heinz bodies – small black dots seen in Reactive Oxygen

Toxic Species masses – possibly due to lysing red blood cells.

3. Adrenal stress – small translucent masses spread throughout

the drop of blood.

4. Reactive Oxygen Toxic Species Linkage – indicative of

inflammation process.

5. Allergic reactions – indicated by spreading of small masses in

the centre of the drop of blood.

6. Advanced cancer profiles.

Currently this technique is being employed to follow up patients diagnosed by

Medical Practitioners with conditions such as chronic fatigue, hormonal

dysfunction, psychological distress, physical stress, cancer and a

micronutrient inbalance. We have employed qualified technicians with

expertise in haematology to conduct and report on the Live Blood Analysis

Test and the CRT.

13. LBA does not provide valuable information about

(i) the human digestive system

(ii) the human immune system

14. LBA does not show

(i) the oxygen carrying capacity of red cells

(ii) the efficiency of protein metabolism

(iii) the efficiency of fat metabolism


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(iv) liver function

(v) gall bladder function

(vi) the degree of bowel toxicity

With respect to items 13 and 14, The Live Blood Analysis Test encompasses

innovative scientific research in areas of science and medicine not previously

part of conventional medicine. We have not identified any peer reviewed

research articles at the level I to III evidence that were associated with

the Live Blood Analysis Test. However, this should not constitute a lack of

validity or scientific principle associated with this technique. Our own

experience of several years investigating the Live Blood Analysis Test

constitutes the best evidence that is deemed level IV evidence and this will

serve as a template for further discussions herein.

Orthodox medicine has always attempted to treat the affected part of the

body only, in the belief that by doing so illness would be eliminated. Medical

statistics are based solely on conventional medical treatments giving rise to

a significant decrease in acute infectious diseases whilst chronic diseases

such as cancer, cardiovascular disease and viral infections have increased

significantly over the past 50 years.203 This trend has fostered the

development of new immunology and endocrinology based research ideas that

have given rise to the disciplines of psychoneuroimmunology and

psychoneuroendocrinology. As a result, immune system functions, hormonal

function and their relationship to the nervous system have been related to

the effect of the body as a ‘whole’. Hence, adverse environmental sequelae

(eg. heavy metal toxicity), high energy nutrition with poor food choices,

psychological, life and traumatic stresses have been shown to have negative

impacts on overall health. Within this framework and scientific paradigm,

The Live Blood Analysis Test can primarily aid the physician screen, then


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diagnose and then monitor progress of their patients’ chronic conditions as

they recommend and follow up lifestyle changes that are made to their

patients.

The Live Blood Test that adopts the technique of dark field microscopy can

successfully detect various alterations of erythrocyte or leukocyte

morphology. Further, it can also be used to identify compon(ts(te-5.ek)2.8(ocyte 0.073 -2.0011 TD0.00171 Tc0.someir )45(a( t)d cyc6(mi-6.)-6.5e2.8(o.6(26]TJ10.6(26]T189.96 572.7.4399 88.22039Tm-0.0007.43TD09(ne )]TJ10.6779 0 202.4Tj67.867.42 9022035 TD0.00145 Tc0.0(m)9(mlrtizolowinso4(nh)6.6( dry)-6.5(( l)5.rs.6(ct2(p)1.6(ot)1.2(ion6(cts(suc6(ct2(p)1.6inso4(mntif7(mn4 t)u-7.274.8n)-1.4(t-6.uc6(ct(( l)5eir8i-6uuc6(ct2.8(6.6( a p8n)-1athe0.3ie.8n)-1t whiy)-6.5h ca)-4.0 70.07te )]TJ0 -2.0973 TD0.0018 Tc-0.04.3(ya( l8(thge)-6.4126(is (-)0.nr8i)-6ben4 t)d )5.6(o )54((f)--6.)1(c)-ow)1(c)-en4 t)d )56( u )56(p( lt)d )5.6wf)--6i4126)-6..2( h)1(s)5.6(s.4126(isen4 t)r).6754.3(3( .6(f)2(nr8i)-v).6780.nr8i)-6..2( i4126)-onr8i)-ar8i1((f)--6 s (-)0ar8i1(en4 t)04.3(ya.4126(io.)1(c)-og).67-6i47(3(3( ar8i1((f)--6 .4126(ien4 t)s6..2( s)1(c3g)4he0. )15.55(n )]TJ-0.073 -2Tm-0.0007 TD0 Tc( )Tj0 -2.093 TD0.00131 Tc0.D4.34Gradu8)r)9u8)5( (da)-1(c)-en4 26.)-68(e8n)-12(p14(s)mnso4()-1(c)-cr)9u8)os).675.6)9u)4.3( c9(p(72.2(a4 t)d p14(s)o).2745.6( w)-68(en4 26t2(o)-5(p)1.6a)-1(c)-lmnso4(5(ons)5.8(9 )5.6-can al4e Sch14(s)oo).274e8n)-12(pcan h) t)6rad3)7.1-6bl4e Sen4 26.nr72.2 us).675.ed(3( en4 26x)mi-6t2(o)-en4 26ns).675.if)--6v).671(ech14(s)y a)mi4(nrmog)e8nan


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and activity is also well observed. Blood platelets, fibrin crystals (appearing

as thin slightly refractile needles or spicules), and chylous material are all

observed and clearly discerned.

At this point I would like to extend this discussion to include our research

experience of The Live Blood Analysis Test.

What can The Live Blood Analysis Test do?

1. micronutrient deficiencies

The relative sizes of erythrocytes can reveal important factors in

cytogenesis that can affect all body cells. Vitamins such as folic acid and B12

are essential for the synthesis of nucleic acids. A deficiency in both of

these vitamins can lead to asynchronous development of all body cells as the

development of the nucleus will be delayed while the maturation of the

cytoplasm continues relatively unaffected. This pathogenic process may

progress as new generations of abnormally large cells are formed. The

relatively uniform size of red cells provides an unambiguous and ready

contrast from the generation of cells affected by such a state of deficiency

presenting with larger sized cells. These large red cells are termed

macrocytes and display diameters exceeding normal values and in the range

of 8.5 – 9.0 µm.200, 205, 207 This erythrocyte abnormality is the principle

characteristic of frank folate and or vitamin B12 deficiency in pernicious

anemia where the macrocytes take on an oval conformation.187 The relatively

short life of red blood cells provides for this characteristic before many

other tissues are significantly affected. If the deficiency state is not

corrected an overt disease termed megaloblastic anemia will ensue. Similarly

neutrophilic white blood cells and platelets will present with an abnormally

large morphology that can further assist the screening of the haematologic


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condition. This can be simply discerned under dark field illumination

microscopy. In a study reported in the Journal of Clinical Pathology199 it was

reported that significant nuclear hypersegmentation of the neutrophil

provides a reliable and sensitive parameter by which to screen for folic acid

deficiencies. The normal neutrophil may have as many as four nuclear

segments. These nuclear segments are sharply contrasted from the highly

refractive granules of the neutrophil cytoplasm. Hence hypersegmentation

is defined by the presence of five or more nuclear segments within the

neutrophil. Thus the discernment of a significant increase in number of

nuclear segments in the hypersegmented neutrophil provides a screening

parameter of folic acid deficiency second only to a folic acid serum assay or

red blood cell folate assay in sensitivity.

Further, the dark field illumination microscopy method used in The Live

Blood Cell Analysis technique also provides a ready made discernment of

abnormally small red blood cells by a physical contrast with normal sized

cells. Microcytes are red cells that are usually less than 6µm in diameter

and are liberated as such into the peripheral blood or may be the result of

cellular fragmentation.200, 205 Microcytes are the classical hallmark of iron

deficiency anemia.205 Microcytosis results from the incorporation of

insufficient levels of haemoglobin into maturing erythrocytes and can also

result from aluminium or lead excess body burden200, 202 where these two

potentially toxic metals have been reported to interrupt porphyrin synthesis.

2. Unbalanced and excessive oxidation of the internal environment of

the body is manifest by features that affect red blood cells. The lipid

component of the cellular membranes is readily degraded by excessive

oxidation. The oxidised lipids which comprise the majority of the structural


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property of the cellular membranes lose much of their structural integrity

and strength. The red cell may then assume a change in conformational

shape that is most probably due to the inability to counter osmotic pressure

changes caused by the cytoplasm as a response to the oxidative degradation

of the lipids within the cell membranes. Conditions of excessive oxidation

may overwhelm the red cell mechanism of antioxidation that protects the

cell and its haemoglobin. This denaturation commonly results in the

formation of precipitate aggregates of the protein globin. These bodies of

aggregated globin are easily discerned within the red cell and are termed

Heinz bodies. These haematological features can be distinguished from

many ‘non-live’ and stained specimens. Further, making use of the dark field

illumination technique of Live Blood Analysis of a blood sample it is possible

to observe the aggregation of red blood cells.

Moreover one also is able to observe the aggregation of thrombocytes

(platelets) and thus enable the discernment of an important indicator of

potential thrombosis. Thrombocyte aggregation and ‘stickiness’ is one of the

hallmarks of possible impending cardiovascular disease that may lead to

stroke and heart attack. Dark field illumination microscopy of a live blood

sample that is unstained or altered by anticoagulants may demonstrate the

potentially thrombotic activation of thrombocytes with the observation of

aggregates. With proper technique and knowledge in the area of

haematological pathology this analysis enables a powerful indicator of risk

for an ischemic crisis not reliably available from other routine means of

study. If significant aggregation of thrombocytes is indicated effective

preventive measures by a medical physician can be employed (eg. prescribing

of aspirin). This form of analysis has important sequelae in potentially

screening at risk populations for such indicators of risk.


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3. Digestive System and Liver Problems

Dietary fats and oils are absorbed by an active formation of micelles in the

small intestine. At this stage these micelles are termed chylomicrons with

an approximate size of 1µm. The chylous material enters the systemic

circulation from the lymphatic system at the left subclavian vein and can be

demonstrated in the peripheral blood after approximately 15 to 20 minutes

of ingestion. In individuals with malabsorption the appearance of the lipid

micelles in the blood may be delayed or diminished in proportion to the lipid

content amount of a meal. The chylomicrons are normally transformed into

smaller micelles with high efficiency by the healthy liver and are then

termed chylous material. In instances of significant liver dysfunction the

efficiency of this process may be impaired and chylomicrons may be

detected in the peripheral blood in greater proportions than in trace

amounts. Usually these liver modified micelles will range in size from

between 0.1µm to 0.4µm and can be readily visualised and identified by dark

field microscopy. Under dark field illumination microscopy they will appear

as small highly refractile particles. Moreover, the relative size of the

chylous particle indicates its constituent lipids. Hence, the smaller particles

will typically be of the higher density lipoproteins while the larger micelle

particles will be composed largely of the lower density lipoproteins.

Conventional medical research has shown that increased proportions of low

density lipoproteins have been associated with increased risk for

atherosclerosis and cardiovascular disease. Conversely, high levels of high

density lipoproteins have been associated with a reduction in risk.


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The chylous material is normally removed from the circulation by cellular

absorption after approximately four hours. In individuals with a lipid

intolerance may demonstrate significant amounts of chylous material in the

peripheral blood after six or more hours of fasting. The relative ease of

discerning the blood chylous material under dark field microscopy

illumination enables a screening analysis of lipid absorption and tolerance.

Such results are then referred back to medical practitioners for follow up

and or further quantitative analytical testing.

4. Immune function

Conditions of inflammation or a significant immune response typically

increase the concentration of the blood protein by the introduction of acute

phase globulins such as histamine or immunoglobulins. Increasing the blood

concentration of these proteins may produce a cohesive linkage of individual

red cells into ordered chains termed rouleaux (vide infra Appendix I –

Patient A Plate A) or into tightly aggregated masses in approximate

proportion to blood concentrations of these globulins. The cohesive cross –

linkage of the red cells can significantly impede the microcirculatory

perfusion of the tissues’ capillaries. A profound degree of rouleaux is a

common feature in the clinical condition termed shock that may result from

extensive physical trauma.

Rouleaux may be evaluated from preparations of peripheral blood by

observation with dark field microscopy of stacked chains of red cells by the

percentage of involvement and the degree of complexity. This type of

screening may be of clinical importance in the evaluation of soft tissue

trauma or to reveal unknown indications of inflammation that may be present

in the instance of a malignancy. The quantitative degree of rouleaux is


24

indicated by the sedimentation rate procedure of common haematological

practice. Hence while the sedimentation rate is the quantitative procedure

of choice for analysis, it is a less direct method of analysis. Dark field

illumination microscopy provides a means of discerning a haematological

condition that is a first line screening procedure.

Leukocytes or white blood cells are nucleated cells normally present in the

circulating peripheral blood. The principle function of these cells is

immunologic. This principle function normally involves body defence against

principally infectious agents that comprise bacteria and viruses or any other

foreign agent. White blood cells may be classified according to their

morphological appearances or their immunological functions. At the most

basic level of classification these cells can be classified as either

granulocytes or agranulocytes. Hence, granulocytes represent a distinct

classification of white blood cells that are involved with immunological

surveillance that is carried out by a phagocytosis mechanism that is

augmented by the antigen – immunoglobulin and complement systems. These

cells contain high concentrations of granules containing proteases and

peroxides that are used to destroy phagocytised target antigens (vide infra

Appendix I – Patient D Plate A).

These granules are readily distinguishable by dark field microscopy by the

high cytoplasmic refractivity. Three classes of granulocytes are defined

from their relative staining affinities. The neutrophil may be characterised

by the regular, fine, and white granules densely occupying the cytoplasm of a

blood cell that is typically polynuclear. Neutrophils are normally the most

abundant subtype of granulocytes comprising ~40 – 65% of the total white

blood cell count. Neutrophils are mobilised within a few hours of exposure


25

to an immune challenge, principally to an elevated white blood cell count early

during an immune response and challenge by an antigen(s). The neutrophil

provides an important function by their potent phagocytosis, albeit

unselective without the specific directing influence of the more specific

immunoglobulins. The nuclei of the neutrophils are poorly refractive by dark

field illumination microscopy and sharply contrasted from the white

cytoplasmic granules. This high contrast provides a simple method of

counting the nuclear segments and enables an index of the relative maturity

of individual neutrophils.

The neutrophil is capable of multidirectional motility (vide infra Appendix I –

Patient E - Plates A to E) that enables an active surveillance and a migration

from the systemic circulation into interstitial spaces and the mucosa. A

diminished capacity for this active immune surveillance can be indicated by

the lack of apparent motility of the neutrophils after several minutes in the

Live Blood Analysis Test specimen.

We have examined extensively the motility of neutrophils and have observed

that neutrophil motility that is not recovered within a few minutes following

preparation of the blood sample for Live Blood Analysis constitutes a sample

from a patient that may have a diminished capacity for this active immune

surveillance response. Further, we have observed a diminished capacity for

neutrophil motility in patients diagnosed by a physician to have depression

(unpublished data and communication).

The eosinophil is a classification of the granulocyte based upon an affinity

for the acidic stain eosin. Eosinophils are the 4th most abundant subtype of

white blood cell, normally constituting 0 – 4% of the total white blood cell


26

differential count. These cells have been demonstrated to have a response

to foreign protein substances such as allergic antigens, protozoan or fungal

infections and certain chemical exposures. Certain malignancies will

demonstrate an increase in the ratio of eosinophils. The physical features

of an eosinophil’s cytoplasmic granules enable selective identification of this

white blood cell subtype. Hence, the eosinophil’s cytoplasmic granules are

very refractive and appear more brilliant pink - yellow tinged in colour and

are approximately twice the size of the cytoplasmic granules of the

neutrophils. Active eosinophils may be indicated by a hypodense character

that may be viewed under dark field illumination microscopy by a more

diffuse distribution of the bright granules in the eosinophil cytoplasm.

Basophils are normally those white blood cells with the lowest presence in

the blood. This subgroup of leukocytes comprises less then 1% of the total

white cell differential count in the peripheral blood. These cells are distinct

but similar to the tissue mast cells. They contain large granules that are

reported to contain histamine that may be released as the cells degranulate

typically as part of immediate hypersentitive reaction. Under dark field

illumination microscopy the basophil can be clearly distinguished from other

granulocytes by the comparatively large size of their cytoplasmic granules,

typically appearing hollow and yellow in colour.

We have correlated an increase in the number of eosinophils and basophils

observed with the Live Blood Analysis Test in patients previously diagnosed

by a physician to be allergic to an environmental stimulus.

Dark field illumination microscopy also allows the distinction of lymphocytes

to be observed. Lymphocytes are a classification of the agranulocytes that


27

normally represent the second most abundant subtype of white blood cell

observed. Lymphocytes normally range from ~18 – 45% of the total

differential white blood cell count. The lymphocytes may be differentiated

by dark field microscopy by their single nucleus and notable thick nuclear

membrane and diffusely distributed off – white cytoplasmic granules.

Moreover, natural killer cells can be distinguished from other lymphocytes

by a distinctive cytoplasmic marker, that is a small brightly refractive

cytoplasmic granule/particle structure (vide infra Appendix I – Dark Field

Microscopy Blood Cell Images).

The monocytes are a further classification of the agranulocytes that

normally represent the third most abundant form of white blood cells. The

monocytes normally range from 4 – 8% of the total differential white blood

cell count. The principal activity of the monocyte is reported to be a

selective phagocytosis of micro-organisms or cellular debris than that of the

neutrophil. Blood monocytes are the principle source of leukocyte interferon

and lysozyme. It has also been reported that these cells are transitory cells

progressing to full development as a macrophage and hence incorporation

into the reticuloendothelial system. The monocyte can be distinguished by

dark field microscopy from the single nucleus presenting a roughened tree

bark like texture with a thick and well defined nuclear membrane and a

diffuse distribution of cytoplasmic granules seen as blue-grey in colour.

Hence the employment of dark field illumination microscopy provides the

observer the ability to distinguish all the major subtypes of white blood

cells. This leads to a complete differential screening – analysis of the

leukocytes and thus is gained a significant insight into the immunologic

function of the patient. The Live Blood Analysis Test that employs dark


28

field illumination microscopy method allows the screening of blood cells in a

single session and clinical setting. As our knowledge is enhanced from

further research and development in this area of medical research, the

general practice clinician who represents the first line contact with the

community is hence provided with a powerful, sensitive, and comprehensive

screening health method that in the first instance can be used as a

screening tool for promoting lifestyle changes in their patients with the

ultimate goal being one of disease prevention and health promotion.


29

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Appendix I Live Blood Analysis Images from the Graduate School of Integrative Medicine, Swinburne Universtity. Sample images of live blood samples before and after interventions by General Practitioners practicing nutritional medicine are herewith presented. Part of a Database of 2500 patients with correlations so far conducted on 160 patients. Patient A / Plate A – before intervention (dark field microscopy)

Patient A / Plate B – after intervention ~ 6 months (dark field microscopy)


46

Patient B / Plate A – before intervention (direct light microscopy)

Patient B / Plate B – after intervention (direct light microscopy)


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Patient C / Plate A – Patient seeks Medical advice from a General Practitioner because of chronic diarrhoea. GP suspects and then diagnosis gastrointestinal dysfunction associated with an irritable bowel. Live Blood Analysis Test confirms the presence of large bowel toxicity crystals (yellow arrows). Further, the patient also exhibits poor nutritional absorption represented by erythrocytes appearing as ‘Ghost’ like cells (green arrows). Compare with ‘healthy’ erythrocytes in a patient with no bowel toxicity [Plate D]. Note granulocyte (red arrow).

Patient D / Plate A – no bowel toxicity


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Patient E / Plates A – E: Time photography plates of neutrophil motility (red arrow - site of specific point of reference). A. (0 secs – time reference)

B. (+ 15 secs)

C. (+ 20 secs)

D. (+ 22 secs)

E. (+ 25 secs)


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Dark Field Microscopy Images:

A. Neutrophil (magnification factor X 600)

B. Eosinophil (magnification factor X 600)

C. NK cell (Natural Killer) lymphocyte [red arrows denote

distinctive refractive cytoplasmic granule/particle markers] (magnification factor X 600)

D. Non NK cell lymphocytes [note absence of refractive

granule/particle] – Blood monocytes (red arrow) (magnification factor X 600)


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Clot Retraction Test (Bolen’s test) Plate 1 – A Normal Patient Profile


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Clot Retraction Test (Bolen’s test) Plate 2 - Patient with “line fractures or spokes” (arrows) observed on clot retraction test – patient at risk for osteoporosis with hypercalcemia confirmed by a standard blood test showing an elevated blood calcium level.


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Plate 3 – CRT of blood sample of a patient with small to medium Reactive Oxygen Toxic Species masses (arrows) – in this patient indicative of adrenal stress.


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Plate 4 – CRT of blood sample of a patient with Reactive Oxygen Toxic Species linkage (arrows) – when the masses tend to become linked is often an indication of inflammation – for this patient with an inflammation - query arthritis and report to medical practitioner.


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Plate 5 – CRT of blood sample of a patient with Reactive Oxygen Toxic Species (arrows) correlated with hormonal dysfunction - report to medical practitioner for further follow up.


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Plate 6 – CRT of blood sample of a patient with an allergy profile.


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Plate 7 – CRT of blood sample of a patient with an inflammatory profile.


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Plate 8 – CRT of blood sample of a patient with a primary bile duct cancer. Refer Arrows – areas of extensive free radical activity.


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Plate 9 – CRT of blood sample of a patient with suspected heart disease. Refer Arrows – areas of extensive free radical activity.


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Plate 10 – CRT of blood samples of patients with different vitamin C consumptions. Refer Arrows – red blood cell with and without dispersion.

a) – normal\elevated vitamin C consumption

b) – low vitamin C consumption


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Appendix II

Current Data from the Graduate School of Integrative Medicine (N = 160 patients)

Live Blood Analysis Test comparisons with Standard Haematological* Tests

and General Practitioner Diagnoses

Condition

LBA Test

(Observed)

Standard Haematological

Tests r

Microcytosis √ 0.90* Macrocytosis √ 0.90* Immune Function

- multi-lobed granulocytes - neutrophil motility - eosinophilia (allergies)

√ √ √

0.85* 0.70* 0.80

Digestive System - platelet adhesion - liver function - erythrocyte aggregation - rouleaux formation - severe erythrocyte aggregation - essential fatty acids deficiency

√ √ √ √ √ √ √

0.80* 0.80 0.85 0.85* 0.65 0.70

Fungal infections √ 0.70

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