Original Article
CRYSTALLIZATION TEST FOR THE DETECTION OF MALIGNANCY OF
THE FEMALE GENITAL TRACT
1 2
Sanobar I. Shaikh , Deepak N. Kawale , Chhaya V.Diwan , AbdulQuadeer , Aruna R. Kharkar
ABSTRACT
Cancer is an ageold disease which has claimed
millions of human lives along with other fatal
diseases . Cancer of the cervix is the most prevalent
form of cancer in developing countries especially the
South -East Asia region.
T h e p r e s e n t s t u d y i s u n d e r t a ke n t o
determine the efficacy of the crystallisation
test for the detection of malignancy in
cancer cases of the female genital tract and
also to determine the efficacy of this test to
detect the precancerous conditions (
Erosion of cervix etc.) of the female genital
tract and to know whether the results of
crystallisation test correlate with the
histopathological diagnosis of cancer and
precancerous cases.
Crystallisation test was carried out in 300
female patients from Gynaecology wards of
Government Medical College and Hospital,
Aurangabad.Out of 300 cases studied ,211
cases were diagnosed cancer patients , 39
cases were in precancerous stage and 50 were
non-cancer cases.The non-cancer cases,
including 10 normal cases were taken as a
control.Out of 211 diagnosed cancer cases,92
were histologicaly proved cancer cases.
Positive results were obtained in 94.7% cases of
malignancy.Positive results were obtained into
84.6% of precancerous cases.
To all the cancer and non-cancer cases of the female
genital tract,Chi-Square test was applied,degree of
freedom was calculated and the significance of the
test i.e. P value was calculated.P value was found to
be far less than 0.001 .This indicates that the
crystallization test is statistically highly significant
test for the detection of malignancy.
Ke y - w o r d s : - C ry s t a l l i z a t i o n , m a l i g n a n c y,
precancerous.
118 International Journal of Basic Medical Science - October 2012, Vol : 3, Issue : 4
3 4 5
INTRODUCTON
The current methods of confirmation of cancerous
neoplasms are histopathological, cytological,
endoscopic techniques and roentgenic studies.
However these studies are valuable only when the
growths are obvious. Pfeiffer (1938) advanced a new
method for cancer detection by specific
crystallization pattern of cupric chloride in
admixture with blood of cancer patient.
The present work has been undertaken to detect
malignant lesions as well as precancerous lesions
eg.(Erosion of cervix etc.) of the female genital tract
and to know whether this test is useful where the
cancerous lesion is inaccessible to biopsy and other
procedures.It is also undertaken to know whether
this test is useful for mass screening programmes
for cancer and whether it can be utilized as scanning
method in cancerous and precancerous conditions.
MATERIAL AND METHODS
When an organic extract is added to a solution of
cupric chloride and the resultant mixture allowed to
crystallize, the crystals formed assume a pattern,
different from that of the crystals which develop
from cupric chloride solution alone. The pattern of
arrangement of crystals varies with the kind and
qualitative characteristic of the organic extract,
and is specific for that extract. The present study
has shown that a definite arrangement of crystals is
produced when blood from normal persons and from
patients with malignancy, is added to cupric chloride
solution.
Crystallizatin test was carried out in 300 females
from Gynaecology ward of Medical College and
Hospital, Aurangabad. Out of 300 cases, 211 were
diagnosed cancer cases, 39 were precancerous
cases and 50 were non-cancer cases.The non-
cancer cases including 10 normal cases were taken
as a control for cancer cases.
 Sanobar I. Shaikh et al, Crystallization Test for the www.ijbms.com

Out of 211 diagnosed cancer cases, 92 were showed a single eccentrically placed centre of
histologically proved cancer cases.Crystallization gravity. (Figure 3.)
test was carried out in all the cases to know 3) The crystal pattern of the mixture of cupric
whether it was positive or negative for cancer, chloride with blood from a cancer patient and a
whether the localization of crystals in the genital precancerous patient showed sharply set off
one, was proper or not and whether the results of transverse bar formation.(Figure 4 & 5)
crystallization test correlate with
The whole crystallization pattern represents the
thehistopathological diagnosis of cancer and
complete human body with definite localization of the
precancerous cases.
various parts of the body,as shown in Figure-
The blood samples were collected from non-cancer 1.Accordingly, the crystallization field is divided into
patients, histologically proved cancer patients and four quadrants by drawing vertical and horizontal
suspected cancer patients (precancerous cases),by axes intersecting at centre of gravity which is
pricking the ring finger with usual aseptic localized eccentrically in the field towards one
precautions.Eight drops of blood from a cancer side.This is known as the localization map,which
patient were allowed to fall on a special filter paper outlines the various parts of the body.
(Whatman No.1) on an area approximately 1 cm in
In figure-1 , only the four quadrants and the genital
diameter and were left to dry.The parts of the filter
zone or zone of sexual organs and peripheral or
paper with dried drops of blood were cut out, and
border zone are shown.
were dissolved in 8 ml of distilled water in a test
tube.Four drops of this diluted blood solution were
added to 10 ml of 20% filtered cupric FIGURE-1
chloridesolution in a calibrated test tube and mixed
gently. The mixture was poured gently in a flat
bottom petridish (10 cm diameter )known as “Assay
Petridish”.
Two more petridishes were prepared as control.In
one petridish 10 ml of filtered cupric chloride
solution was poured.In the other petridish, mixture
of 4 drops of blood solution from normal healthy
person with 10 ml of filtered cupric chloride solution
was poured.Thepetridishes were carefully arranged
inside the crystallization chamber i.e. incubator at
A-A : Vertical axis M-M : Horizontal axis.
320C for a period of 19 hours, for complete
The intersection of the A-A and M-M axes = centre of
crystallization under controlled humidity.The test gravity.Number 1,2,3,4 show four quadrants of the
was also carried out in 250cancer and precancerous crystallization field.
cases and 50 non-cancer cases. The hatched portion = peripheral or border zone.
OBSERVATIONS S = Genital zone or zone of sexual organs.

Following crystal patterns were observed in the

petridishes under study as seen by the dissecting
microscope:
1) The crystal pattern with pure cupric chloride
solution showed anumber of crystals with many
centres of gravity. (Figure 2.)
2) The crystal pattern of the mixture of cupric
chloride and blood from a normal healthy person

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Crystallization Test STATISTICAL ANALYSIS

Sr. No. Of
Name of the cancer
no. cases Positive Negative To all the cancer and non- cancer cases of the female
Pattern Pattern
genital tract, Chi-square test was applied, degree of
01 Carcinoma of cervix 131 126 5
freedom was calculated and the significance of the
02 Malignant ovarian tumour 57 53 4
test i.e. P value was calculated. The P value was
03 Carcinoma of endometrium 15 13 2 found to be far less than 0.001. This indicates that
04 Carcinoma of vulva and vagina 4 4 0 the crystallization test is statistically highly
05 Rhabdomyosarcoma of uterus 1 1 0 significant test for the detection of malignancy.
06 Dysgerminoma of ovary 1 1 0
Histopathological
No. Positive Negative Diagnosis
07 Choriocarcinoma 2 2 0 Proved cancer
of pattern for pattern for
cases
cases cancer cancer Positive Negative
Total 211 200 11
Carcinoma of cervix 42 42 0 41 1
TABLE-1 : DIGNOSED CANCER CASES FROM FEMALE GENITAL TRACT
Malignant ovarian
40 40 0 40 0
Percentage of positivity = 94.7%Sensitivity of test = 94.7 % tumour
Carcinoma of
5 5 0 5 0
endometrium
Carcinoma of vulva
2 2 0 2 0
Crystallization Test and vagina
Sr. No. of Dysgerminoma of
Precancerous cases 1 1 0 1 0
no. cases Positive Negative ovary
Pattern Pattern
Choriocarcinoma 1 1 0 1 0

01 Erosion of cervix 38 32 6 Rhabdomyosarcoma

1 1 0 1 0
of uterus
Hypertrophic Lichenification
02 1 1 0 Total 92 92 0 91 1
pruritis of vulva

TOTAL 39 33 6
Table 4 shows that the results of crystallization
TABLE-2 : PRECANCEROUS CASES FROM FEMALE GENITAL TRACT test correlate very well with thehistopathological
Percentage of positivity = 84.61% Sensitivity of test = 84.61% diagnosis. This indicates that the crystallization
test has detected all the proved cancer cases
except one which is a false positive result.
Crystallization Test
Sr. No. of
Diagnosis Positive Histopathological
no. cases Positive Negative Negative Diagnosis
Precancerous No. of pattern
Pattern Pattern pattern
cases cases for
for cancer Positive Negative
cancer
01 Normal healthy individuals 10 0 10
Erosion of
38 32 6 5 33
cervix
02 Fibroid of uterus 15 4 11
Hypertrophic
Dysfunctional uterine lichenification 1 1 0 1 0
03 2 0 2 pruritus of vulva
bleeding
Total 39 33 6 6 33
04 Benign ovarian tumour 3 0 3
TABLE-5 : CORRELATION OF POSITIVE CRYSTALLIZATION TEST FOR CANCER,
Cervical polyp with severe WITH THE HISTOPATHOLOGICAL DIGNOSIS OF PRECANCEROUS CASES
05 8 0 8
anaemia
Procidentia or Total
06
prolapse of uterus
11 1 10 Table-5 Shows that the results of crystallization
test correlate very less with the
07 Ovarian cyst 1 0 1
histopathologicaldiagnosis.This does not indicate
Total 50 5 45 that the cryastallization test has failed to detect
the precancerous cases but it probably indicates
TABLE-3 : NON-CANCER CASES FROM FEMALE GENITAL TRACT
that these cases may develop malignancy at any
Percentage of negativity = 90 %Specificity of test = 90 %
time in future.Such precancerous cases could not
be followed up further because in these cases,
operation of total hysterectomy was done.

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Total no. of cancer

Proper Improper Percentage of proper
and
localization localization localization
precancerous cases

250 241 9 96.4%

TABLE-6 : PERCENTAGE OF PROPER LOCALIZATION OF CRYSTALS IN

THE GENITAL ZONE , BY THE CRYSTALLIZATION TEST IN CANCER
AND PRECANCEROUS CASES

Table - 6 shows that the percentage of proper

localization in the cancer and precancerous cases of
the female genital tract is 96.4 % i.e. all the
transverse crystals are localized to the genital zone.

Total no of proved Proper Improper Percentage of proper

cancer cases localization localization localization

92 91 1 98.91%
TABLE-7 : PERCENTAGE OF PROPER LOCALIZATION OF CRYSTALS
IN THE GENITAL ZONE IN PROVED CANCER CASES,
BY THE CRYSTALLIZATION TEST

Table – 7 shows that the percentage of proper

localization in the proved cancer cases of the female FIG.1:Crystallization pattern of cupric chloride solution alone.
genital tract is 98.91 % i.e. all the transverse
crystals are localizedto the genital zone.

Total no. of Proper Improper Percentage of

non- cancer cases localization localization proper localization

50 48 2 96 %

TABLE-8 : PERCENTAGE OF PROPER LOCALIZATION OF CRYSTALS

IN THE GENITAL ZONE IN NON-CANCER CASES,
BY THE CRYSTALLIZATION TEST.

TABLE – 8 shows that the percentage of proper

localization of crystals in non-cancer cases is 96 %
i.e. all the crystals are localized to the genital zone.
From table 6,7 and 8 it is seen that, out of 250
cancer and precancerous cases of the female genital
FIG.2: Crystallization pattern of a mixture of cupric chloride with blood
tract, 241 cases showed proper localization and from a normal healthy person.
only 9 cases showed improper localization. Out of 92
proved cancer cases, 91 cases showed proper
localization and only 1 case showed improper
localization. Out of 50 non-cancer cases, 48 cases
showed proper localization and 2 cases showed
improper localization. The failure may be attributed
to lack of ideal experimental conditions or some fault
in technique.

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FIG.3:Crystallization pattern of a mixture of cupric chloride with blood
from a cancer patient i. e. Malignant ovarian tumour. Arrow shows
transverse crystal in the genital zone of the localization map.
FIG.4: Crystallization pattern of a mixture of cupric chloride with blood from
a patient suffering from Dysfunctional uterine bleeding with cervical erosion.
Arrows show transverse crystals in the genital zone of the localization map.
DISCUSSION
The principle underlying Pfeiffer's crystallization test
is that the colloidal proteins present in an extremely
dilute solution of human blood act as an
impuritywhen mixed with cupric chloride solution and
are amazingly transformed into well co-
ordinatedpattern as long radiating needle like
crystals1.After a series of experiments he noted
that the crystallization pattern of cupric chloride
solution differs substantially when the blood of a
diseased individual is added to it.Thus he observed
star formation in inflammation , a hollow glass in
benign condition and transverse formation in
cancer.The classical experiments of Gruner (1940)
122 International Journal of Basic Medical Science - October 2012, Vol : 3, Issue : 4
Sanobar I. Shaikh et al, Crystallization Test for the
and others have subsequently led to establish a
simple test for early detection of cancer , because
the positive results were demonstrated in case of
susceptible strains of animals even though they
were clinically free from any cancerous growth1.
The patterns which crystals assume after the
addition of blood to a solution of cupric chloride under
specified conditions have been shown by E.Pfeiffer
(1938) to be distinctive between health and disease.
In experimental animals, the pattern produced by
adding blood to cupric chloride is rendered abnormal
and is distinctive and characteristic for each
disease.( Gruner,1940.)
Kopaczewski (1933) has attributed the
crystallization pattern to different rates and
different amplitudes of molecular movement during
the process of evaporation and trikinetic forces
operating through a variety ofmaterials.The
reaction is mainly physical and no new chemical
substance is formed.
The illustrative experiments of Sabarth and
Williams(1975), highlight the fact that the dominant
role of pattern formation force of some
constituents of blood solution, is responsible for the
typical crystallization pattern when blood from
normal healthy individuals is added to cupric chloride
solution2.
The results of different workers, Pfeiffer and Miley
(1939); Morris D.L. and Morris C.T.(1939); Gruner
(1940) ; Selawry (1940) ;Teichmann (1965);
Koopmans (1969);Sabarth and Williams(1975)
under different laboratory conditions, have the
common feature of similar pattern formation in
normal healthy individuals. The same pattern in
normal healthy individuals isalso observed in the
present study.
The crystallization pattern is modified or completely
changed when the salts crystallize from solution
containing impurities as reported by many workers.
[Du Nouy (1926,1928;Michaud (1936)3,4]
The addition of blood to cupric chloride solution
essentially means addition of an impurity.It is very
difficult to pinpoint the actual substance acting as
an impurity in case of blood as even one drop of blood
contains number of vitally important substances like
 Sanobar I. Shaikh et al, Crystallization Test for the
various ions, hormones,enzymes,proteins and
cellular components3,4.Trumpps and Rascher (1936)
pin pointed proteinous colloid as the root cause of
modified crystallization pattern in biological systems
in human beings5.
Koepf and Selawry (1962) reported the extreme
sensitivity of cupric chloride to proteins. They were
of the view that the proteins in colloidal status
probably play an important role in the pattern
formation mechanism6.In cancer, specific pattern of
cupric chloride crystals was observed (Pfeiffer
1935,1939;Gruner 1940 ; Selawry
1957,1968;Koef and Selawry 1962; Koopmans
1969) . Similar pattern was also observed in the
present study.
Bachrach and Plesser (1985) observed elevated
levels of naturally occuring polyamines and diamines
in the body fluids incluidingblood, in cancer7.This
information further stresses the positive role of
presence of elevated protein levels for detection of
cancer .Thus it was confirmed that the protein or
the degradation product of protein , is responsible
for particular cancer specific pattern, the pattern in
inflammation , in allergic or benign conditions and in
normal conditions.6
Gruner(1940) pointed out that the crysatallization
test aims at detecting the precursor metabolic
changes.Russell (1978) reported high
concentrations of polyamines and diamines in cancer
cells which are intermediate products of protein
metabolism.The excretion of these amines in urine is
also found to be elevated in cancer patients7. Russell
(1971) and Bachrach and Plesser (1985)
suggested that these amines could serve as
markers of malignancy and probably these are
responsible for the positive crystallization test8,9.
Pfeiffer (1938) has demonstrated positive results in
75% of cases,while positive results have been
demonstrated in 91.1% cases by Gruner
(1940).Quadeer (1980) has demonstrated positive
results in 94.15 % cases.In the present work, out of
211 diagnosed cancer cases , 200 gave positive
results (94.78%) and out of 39 precancerous
cases,33 gave positive results (84.61%) .Out of 50
non-cancer cases, only 5 cases showed positive
pattern for cancer.
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It has been observed in the present study that the
percentage of positivity of cancer and precancerous
cases in 93.2 % which is termed as sensitivity of the
test. The percentage of negativity of transverse
forms in non-cancer cases is 90% which is termed
as specificity of the test.
In the present study,39 precancerous cases were
studied for cancer pattern.Out of 39 precancerous
cases ,33 cases showed the typical cancer
pattern,while 6 cases failed to give it and the
percentage of positivity was found to be 84.61%.
Selawry and his co-workers (1957),studied 52
precancerous cases .Out of 52 precancerous cases
,43 cases showed the typical cancer pattern while 9
cases failed to give it and the percentage of
positivity was found to be 82.69 %.
Localization of transverse crystals in specific zones
of the localization map were also observed in the
present study.
Quadeer (1980) studied the localization of crystals
in specific zones of the field in cancer cases of
various systems and the percentage of proper
localization was found to be 90.76%. In the present
study , 250 cancer and precancerous cases of the
female genital tract were observed for the proper
localization of crystals in the genital zone of the
localization map, and the percentage of proper
localization was found to be 96.4%. The percentage
of proper localization of crystals in specific zone in
non- cancer cases was found to be 96%.
The positive cases of cancer by the crystallization
test were correlated withthe histopathological
diagnosis of proved cancer cases.Out of 92 proved
cancer cases, 91 cases showed positive
histopathological diagnosis and only 1 case showed
negative result. This shows that the results of
crystallization test correlate very well with the
histopathologicaldiagnosis.This indicates that the
crystallization test has detected all the proved
cancer cases except 1 which is a false positive
result.It was also observed that the results of
crystallization test correlate very less with the
histopathological diagnosis of precancerous
cases.This does not indicate that the crystallization
test has failed to detect the precancerous cases
123
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but it probably indicates that these cases may
develop malignancy at any time in future.Such
precancerous cases could not be followed up further
,because in these cases ,operation of total
hysterectomy was done.
CONCLUSIONS
At the end of the experiments of crystallization
test, the following conclusions were drawn :-
a) Crystallization test can be used for detecting all
cancer cases of the female genital tract.
b) Cryatallization test is helpful for early detection
of cancer i.e.detection of precancerous cases
and for detection of cancer in apparently normal
individuals with no clinical manifestation of
cancer.
c) The great value of this test is in detecting the
presence of malignancy of the female genital
tract , where the lesion is inaccessible to biopsy
and other procedures.
d) Crystallization test helps in providing clue for
prognosis.
e) Crystallization test is also helpful in detecting
individuals who have genetic predisposition for
cancer, long before the appearance of
cancerous changes.
BIBLIOGRAPHY
1. Gruner, O.C. Experience with the Pfeiffer
Crystallization Method for the diagnosis of
Cancer.J.Cand , Med. Assn. 1940; 43:99-106.
1. Sanobar I. Shaikh
Associate Professor, Department of Anatomy
Govt. Medical College, Aurangabad.
2. Deepak N. Kawale
Senior Resident, Department of Anatomy,
Govt. Medical College, Aurangabad.
3. Chhaya V.Diwan
Professor and Head, Department of Anatomy
Govt. Medical College, Aurangabad.
124 International Journal of Basic Medical Science - October 2012, Vol : 3, Issue : 4
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2. Sabarth E. and Williams H.N. Sensitive
Crystallization Processes – as Demonstration
of Formative Forces in the Blood. 2nd ED.
Anthroposophic Press, Spring Valley ,New
York,1975.
3. Du Nouy P.L.(1926,1928) Quoted by Morris
D.L. and Morris C.T.(1939).
4. Michaud F. (1936) Quoted by Morris D.L. and
Morris C.T.(1939).
5. Morris D.L. and Morris C.T.(1939) The
Modification of Cupric Chloride Crystallization
Patterns by Traces of Proteins. J. of Biological
Chemistry ,1939;43:5.
6. Rascher S. and Trumpp J. Experiments with
Crystallographic Carcinoma
Diagnosis.Mu.Med.Wo'schr.1939;86,14:545.
7. Koepf H. Selawry A. Application of the
Diagnostic Crystallization Method for the
Investigation of Quality of Food and
Fodders.Bio-Dynamics, 1962 & 1963;64& 65.
8. Bachrach V. and Plesser Y.M. A Sensitive, Rapid
,Chemiluminescence Based Method for
Determination of Diamines and
P o l y a m i n e s . A n a l y t i c a l B i o c h e m i s t r y,
1986;152:423-431.
9. Russell D.H.(1971,1978)Quoted by BachrachV.
And Plesser Y.M. (1986).
10. Quadeer A. Crystallization test for Detection of
Malignancy.J.Anat.Soc.Ind.1980;29:2
4. AbdulQuadeer
Associate Professor,
Department of Anatomy
Govt. Medical College,Nagpur.
5. Aruna R. Kharkar
Associate Professor,
Department of Anatomy
MGM Medical College, Aurangabad.