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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 3  |  Page : 440-444

An insight into crystallization test: A neoteric approach for screening premalignant and malignant lesions


Department of Oral and Maxillofacial Pathology, Maulana Azad Institute of Dental Sciences, New Delhi, India

Date of Submission16-Mar-2017
Date of Decision20-Jan-2018
Date of Acceptance25-Feb-2018
Date of Web Publication04-Sep-2018

Correspondence Address:
Aadithya B Urs
Department of Oral and Maxillofacial Pathology, Maulana Azad Institute of Dental Sciences, BSZ Marg, New Delhi - 110 002
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_275_17

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 > Abstract 


Introduction: Crystallization test is based on the principle that, when a salt crystallizes out of an aqueous solution, the crystal growth is influenced by the presence of other substances in the solution, such as blood or plant extracts. If a mixture of copper chloride solution with a small amount of whole blood is allowed to crystallize under controlled experimental conditions, an aggregate of crystals forms. Crystallization method can be used as a diagnostic aid to provide information about the systemic conditions and general health of the patient.
Aim: This study aims to study the patterns of crystallization and to further determine the efficacy of crystallization test as a screening modality in premalignant lesions and oral squamous cell carcinoma (OSCC).
Materials and Methods: Fifty patients of OSCC, 50 patients of premalignant lesions, and 50 healthy individuals were selected. One drop of blood was collected from the study groups to perform crystallization using cupric chloride.
Statistical Analysis Used: Statistical analysis was performed using Chi-square test, Student's t-test (two-tailed), and analysis of variance.
Results: The different patterns of crystals formed were studied and statistically analyzed.
Conclusion:Based on the study, it was concluded that Crystallization test can be used as an effective screening modality for detection of premalignant lesions and OSCC.

Keywords: Crystallization test, crystals patterns, cupric chloride, oral squamous cell carcinoma


How to cite this article:
Rawat G, Kureel K, Urs AB. An insight into crystallization test: A neoteric approach for screening premalignant and malignant lesions. J Can Res Ther 2020;16:440-4

How to cite this URL:
Rawat G, Kureel K, Urs AB. An insight into crystallization test: A neoteric approach for screening premalignant and malignant lesions. J Can Res Ther [serial online] 2020 [cited 2020 Aug 7];16:440-4. Available from: http://www.cancerjournal.net/text.asp?2020/16/3/440/229634




 > Introduction Top


Oral squamous cell carcinoma (OSCC) is the most common malignant disorder of the oral cavity, accounting for over 90% of all malignant neoplasms in this region.[1],[2],[3] OSCC includes a group of neoplasms affecting any region of the oral cavity, pharyngeal region, and salivary glands.[1] It accounts for 2%–4% of all cancers worldwide and is ranked as the sixth leading cause of cancer mortality globally.[2],[3],[4] About 30%–80% of the oral malignancies arise from premalignant disorders. The most common premalignant lesions affecting the oral cavity include leukoplakia and oral submucous fibrosis which have malignant transformation rates of 0.13%–17.5% and 2.3%–7.6%, respectively.[5],[6]

Early diagnosis and prompt treatment of oral malignancies is the most effective means of reducing rates of mortality and morbidity from this disease. Various methods such as histopathological, cytological, and endoscopic techniques are used for diagnosis of malignancies. However, it is of utmost importance to have a diagnostic method which is simple, economical, reliable, less time-consuming, and less invasive, especially in developing countries like ours where there is high prevalence of premalignant lesions and OSCC.[7]

In 1938, Pfeiffer proposed a method for cancer detection by analyzing specific crystallization pattern of cupric chloride in admixture with blood of cancer patient. This method can be applied to whole blood. The basis of the test was that when a salt crystallizes out of an aqueous solution, the other substance in the solution like blood, influence the growth of the crystals.

If a mixture of copper chloride is allowed to crystallize with some amount of whole blood under controlled experimental conditions on a glass plate, aggregate of crystals are formed, specific for the blood used. Pfeiffer had defined a characteristic pattern for cancer as built upon a straight line (short or long) to the center of which impinge a few lines at angles varying from 5° to 30°.[8] In few other studies, the presence of “transverse bar formations” crystallization pattern was considered to be a distinctive finding in malignancy.[9]

The aim of the present study was to study the patterns of crystallization and to further determine the efficacy of crystallization test as a screening modality in premalignant lesions and OSCC.


 > Materials and Methods Top


This study was conducted in the Department of Oral Pathology and Microbiology, Maulana Azad Institute of Dental Sciences, New Delhi. The study was approved by the institutional ethical committee board of Maulana Azad Institute of Dental Sciences, New Delhi.

Patient selection

Clinically, patients with leukoplakia and OSCC in the age range from 18 years to 80 years were included in the study. The patients were selected after obtaining written informed consent from each. A total of 150 patients with OSCC (Group A; n = 50), leukoplakia (Group B; n = 50) along with healthy age- and sex-matched individuals (control, Group C; n = 50) were included in the study.

All the included OSCC cases were free of any other systemic disorder. Patients not willing to participate, already undergoing treatment, recurrent cases of OSCC, patients suffering from conditions/situations contraindicated for biopsy were not included in the study. Histopathological grading was carried out using Broders grading system. Patients diagnosed with verrucous carcinoma after histopathological examination were excluded from the study.

Procedure

Preparation of cupric chloride solution

Twenty gram of fine powder of cupric chloride (CuCl2.2H2O) was dissolved in 100 ml of distilled water to form a 20% solution of cupric chloride. The solution was freshly prepared for every case.

Collection of blood sample

Blood samples were collected by pricking the ring finger from all the study groups. One drop of blood was added in 1 ml of distilled water, and it was left for 30 min to hemolyze at room temperature to give a final dilution of 6% hemolyzed blood.

Crystallization test

Ten milliliter of 20% cupric chloride solution was taken in a test tube. A volume of 0.1 ml of hemolyzed blood was added to this test tube at room temperature. The mixture was poured into a flat bottom prewarmed petri-dish of 10 cm diameter, which was then placed into BOD incubator at 28°C–32°C. While handling the petri-dishes care was taken to hold them from the side and not to touch the inner surface. The crystallization was allowed to take place for 18–19 h without any vibration disturbances. The crystallization patterns were carefully studied by holding them against daylight. The crystal patterns formed were analyzed, and interpretations were made.

Statistical analysis

All values were expressed as mean ± standard deviation Statistical analysis was performed by using Chi-square test, Student's t-test (two-tailed), and analysis of variance. P <0.05 was considered as statistically significant.


 > Results Top


Interpretation of crystal patterns

The crystallization pattern of cupric chloride solution alone showed thick crystals with needles arranged at arbitrary angles. This pattern is called as “Muddle formation”/“Messy Pattern.”

In our study, the crystals showing orderly arrangement of radiating crystals emanating from a primary eccentric center of gravity toward the periphery were categorized as orderly arrangement pattern. This pattern was found in 38 cases (76%) control group out of total 50 cases studied [Figure 1]a.
Figure 1: (a) Orderly arrangement of cupric chloride crystals in control. (b) Star form pattern of cupric chloride crystals in control

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The crystals having a radial arrangement of the crystal needles with many eccentric centers of gravity toward the periphery were categorized as Star-form pattern. This pattern was observed in 24% controls, 4% OSCC cases, and 8% leukoplakia cases [Figure 1]b.

About 96% of the OSCC cases showed Hollow formation with transverse bars pattern, i.e., the crystals showed hollow formations along with sharply set off transverse bars which consists of transverse needles but no secondary or tertiary branching [Figure 2].
Figure 2: Depicts hollow formations with transverse bar pattern of crystals in oral squamous cell carcinoma patient

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Ninety-two percent of leukoplakia cases showed Leaf like pattern which consisted of a primary branch with numerous secondary branches arising from it and absence of transverse bars [Figure 3].
Figure 3: Depicts leaf like pattern of cupric chloride crystals in leukoplakia patient

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In the OSCC group, hollow formations with transverse bars were observed in 48 cases (96%) and 2 cases (4%) showed star form pattern (no transverse bars). The presence of transverse bars in OSCC cases was considered as positive crystallization test. Thus, 48 cases of OSCC showed positive crystallization, i.e., 96% reliability and 4% false negative (OSCC cases without transverse bar formations in crystallization test) [Table 1].
Table 1: Positive crystallization test in oral squamous cell carcinoma group on the basis of transverse bar

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In the leukoplakia group, Leaf-like pattern was observed in 46 cases (92%) while 4 cases (8%) exhibited star form pattern. Transverse bar formations were absent in all the 50 cases of leukoplakia. The presence of leaf like pattern in leukoplakia cases was considered as positive crystallization test. Thus, 46 cases of leukoplakia showed positive crystallization i.e., 92% reliability and 8% false negative [Table 2].
Table 2: Positive crystallization test in leukoplakia group on the basis of leaf like pattern

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Comparison of histopathology and crystallization patterns

Histologically proven cases of OSCC showed that number of transverse bar formations varied with the different histologic grades of OSCC. Grade I tumors (well differentiated – 78% cases) had lesser transverse bar formations compared to Grade II (moderately differentiated – 12% cases) and Grade III (poorly differentiated – 10% cases) tumors [Table 3].
Table 3: Oral squamous cell carcinoma group: Comparison of histopathological grade and crystallization patterns

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Histological assessment of cases of leukoplakia group showed hyperorthokeratosis in 50% (25) cases, hyperparakeratosis in 16% (8), and hyperkeratosis in 30% (15) while chronic inflammation was observed in 4% (2) cases. Forty-six cases of leukoplakia group showed leaf like pattern whereas four cases showed star form pattern in which two were hyperorthokeratosis and two were chronic inflammation histopathologically [Table 4].
Table 4: Leukoplakia group: Comparison of histopathological finding and crystallization patterns

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Sensitivity of crystallization test for OSCC and leukoplakia was found to be 96% and 92%, respectively. Specificity was 100% for both study groups.


 > Discussion Top


Kopaczewski (1933) studied on crystallization test and concluded that the different patterns of crystallization produced by organic and inorganic salts by addition of different colloidal solution were due to different rates and amplitude of molecular movements involved in the evaporation process.[8] Pfeiffer (1938) first developed the crystallization test using cupric chloride solution. It was based on the importance of physical and molecular forces in maintaining the integrity of the molecules and the chemicals. The typical hallmark of crystallization pattern in malignancy was shown to be “transverse form” formation.[9] Gruner concluded that the different crystallization patterns in health and disease are due to the pivotal role of colloidal proteins in dilute solution of blood. The specific pattern in cancer reflects the specific nature of the abnormal proteins with changes in the position of amino and sulfhydryl groups.[8] Gulati et al. and Kuczkowski et al. carried out this test in head and neck malignancies and concluded that it is a simple, reliable, economical, less time-consuming, and less invasive diagnostic procedure, which can be used for mass screening program.[7]

The results obtained in our study were similar to that as described by Pfeiffer in context of cancer. We also found that predominantly hollow formation with transverse bar pattern was seen in OSCC. On analyzing the crystallization patterns according to the histologic grade of OSCC, it was found that Grade III tumors (poorly differentiated – 12% cases) showed numerous transverse bar formations compared to Grade II (moderately differentiated – 12% cases) and Grade I (well–differentiated – 78% cases) tumors, with latter having least transverse bars. Therefore, in the present study, it was observed that the number of transverse bars increased as the tumor grade advanced from Grade I to Grade II to Grade III tumors. However, two histologically proven cases of OSCC showed star form pattern with the absence of transverse bar formations. These contrasting findings may be due to failure in maintaining the required physical conditions in these cases as basis of crystallization test is entirely a physical phenomenon.

On analysis of crystal patterns obtained by addition of blood to cupric chloride solution in samples of leukoplakia patients, it showed an altogether different picture in 92% cases which was termed as leaf like pattern. Since, this type of test has not been studied in oral premalignancy earlier, hence, the term leaf like was used to describe this distinct pattern, as comprehended from the appearance of the crystals on petri-dish. Histologically, two cases of leukoplakia were diagnosed as chronic inflammatory lesions. These two chronic inflammatory lesions and two hyperorthokeratosis failed to show leaf like pattern which we have found unique and diagnostic for leukoplakia. These unlikely results may be attributed to the crystallization test being purely dependent on physical interactions. Due to lack of any data in the available past literature, comparison with results of other studies was not possible.

Pfeiffer concluded after studying numerous crystallization patterns that the crystal patterns formed after addition of blood to cupric chloride solution was distinctive between health and disease. These patterns are affected by all kinds of impurities and especially if the substances are in the colloidal state, it is possible to identify their presence not only by chemical means but also by crystallization.[8]

Copper is present in varied coenzymes of human body and chloride ions play an important role in osmoregulation. It is known that cupric chloride reacts more sensitively in admixture when compared to other salts such as magnesium sulfate. It has been noted that there is excess excretion of polyamines and diamine in urine of cancer patients. Hence, it is possible that there is crucial role of proteins in the pattern formation. Hence, the colloidal proteins present in extremely diluted blood can act as impurities when added to cupric chloride solution.[7],[10]

Pfeiffer further revealed that the pattern produced by the addition of blood to the cupric chloride is rendered abnormal if the subject is in a pathological state. He conducted experiments in diagnosis of cancer in man after he found encouraging results in diagnosis of tuberculosis in cattle. The pattern characteristic for cancer has been defined by Pfeiffer. The higher the numbers of groupings occur in a plate, and more explicit they appear, higher was the malignancy grade. It is the act of crystallization which serves as the detector of molecular forces coming into operation only at that time because of the constitution of the added material.[8]

In malignancy, there are high concentration of polyamines and diamines in the blood. These amines are the intermediate products of protein metabolism. It was observed that the colloidal proteins in dilute solution of blood play a pivotal role in formation of different crystallization in health and disease. Thus, it can be reasoned that the proteins or degraded products of proteins, i.e., polyamines and diamines may be responsible for particular cancer-specific pattern in crystallization test.[10]

In the present study, the reliability in control group was found to be 96.66%. Similar results in control group were reported by Pfeiffer (91%), Gruner (96%), Quadeer A (100%), Shaikh S (90%) and Gulati et al. (92%).[7],[8],[9],[11],[12] The reliability in OSCC group was found to be 96%. There are very few studies in malignancies wherein, the study on head and neck malignancies by Gulati et al. and Kuczkowski et al. reported quite similar results (83.3% and 96%, respectively).

Pfeiffer (91%), Gruner (90.1%), Quadeer A (94.14%), and Shaikh S (94.78%)[12] (have reported analogous results in various other malignancies affecting the body.[8],[9]


 > Conclusion Top


Crystallization test can be considered as a reliable and less invasive screening aid for premalignant and malignant lesions affecting the oral cavity and can be used to identify high-risk group patients during mass screening programs done at the community level.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Markopoulos AK. Current aspects on oral squamous cell carcinoma. Open Dent J 2012;6:126-30.  Back to cited text no. 1
    
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Carvalho AL, Singh B, Spiro RH, Kowalski LP, Shah JP. Cancer of the oral cavity: A comparison between institutions in a developing and a developed nation. Head Neck 2004;26:31-8.  Back to cited text no. 3
    
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Sharma P, Saxena S, Aggarwal P. Trends in the epidemiology of oral squamous cell carcinoma in Western UP: An institutional study. Indian J Dent Res 2010;21:316-9.  Back to cited text no. 4
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Yardimci G, Kutlubay Z, Engin B, Tuzun Y. Precancerous lesions of oral mucosa. World J Clin Cases 2014;2:866-72.  Back to cited text no. 5
    
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Katarkar A, Mukherjee S, Khan MH, Ray JG, Chaudhuri K. Comparative evaluation of genotoxicity by micronucleus assay in the buccal mucosa over comet assay in peripheral blood in oral precancer and cancer patients. Mutagenesis 2014;29:325-34.  Back to cited text no. 6
    
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Gulati SP, Sachdeva OP, Sachdeva A, Adlakha RP, Kakkar V. Crystallization test for the detection of head and neck cancer. ORL J Otorhinolaryngol Relat Spec 1994;56:283-6.  Back to cited text no. 7
    
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Gruner OC. Experience with the Pfeiffer crystallization method for the diagnosis of cancer. Can Med Assoc J 1940;43:99-106.  Back to cited text no. 8
    
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Sarode SC, Sarode GS, Barpande S, Tupkari JV. Efficacy of crystallization test for screening of oral squamous cell carcinoma with clinico-pathological correlation. Indian J Dent Res 2013;24:464-7.  Back to cited text no. 9
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Savory J, Shipe JR. Serum and urine polyamines in cancer. Ann Clin Lab Sci 1975;5:110-4.  Back to cited text no. 10
    
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Quadeer A. New approach for detection of malignancy by crystallization test. Ph.D. thesis for (medicine) degree in Anatomy, Nagpur University, 1988.   Back to cited text no. 11
    
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Shaikh SI. Crystallization test for detection of malignancy in the female genital tract. Dissertation for MS (Anatomy). Dr. Babasaheb Marathwada University, Aurangabad, May 1992.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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