| Abstract|| |
Aim: To determine the efficacy of crystallization test for screening oral squamous cell carcinoma (OSCC) with clinico-pathologic correlation.
Materials and Methods: Fifty patients of OSCC and 30 healthy individuals were selected. One drop of blood was collected into 1 cc of doubled distilled water at room temperature to give a final dilution of 6% hemolyzed blood. 0.1 to 0.2 cc of blood sample was then subjected to crystallization test using 10% cupric chloride solution.
Results: In healthy control group, the typical pattern was an eccentrically placed center of gravity with needles arranged in radiating fashion, while in OSCC group, there was 'transverse form' (TF) formation. The sensitivity and specificity of this test was found to be 96% and 96.66%, respectively. The positive and negative predictive values were found to be 97.96% and 98.55%, respectively. The mean TF frequency was found to be increasing from grade I (3.20 ± 1.5%) to grade II (653 ± 2.23%) and difference was statistically significant (P = 0.0001). However, there was no correlation between mean TF frequency and TNM stages of OSCC.
Conclusion: Crystallization test is a simple, reliable, economical, less time-consuming, and less invasive screening procedure, which can be used for early detection of OSCC.
Keywords: Crystallization test, oral cancer, oral squamous cell carcinoma, screening test
|How to cite this article:|
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
Oral cancer is the tenth most common cancer in the world, accounting for 3-5% of all malignancies.  About 90% of oral cancers are squamous cell carcinoma arising from the oral epithelium. Most of them (67% to 77%) are not diagnosed until they have attained at least the T2 stage (>2 cm). The failure to achieve early diagnosis and greater frequency of advanced stages contribute to the continued poor prognosis of this lesion.  It is generally believed that cancer mortality can be reduced if they are detected, diagnosed, and treated at early stages.  Nowadays, various methods are available for early detection of oral cancer, but the need of the hour is that the test ought to be simple, economical, reliable, less time-consuming, and less invasive.
|How to cite this URL:|
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 [serial online] 2013 [cited 2019 May 19];24:464-7. Available from: http://www.ijdr.in/text.asp?2013/24/4/464/118398
The phenomena of crystallization had always attracted the workers over centuries. Kopaczewski (1933) had worked on this subject 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.  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' (TF) formation.  Gruner (1940) 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 sulphydryl groups.  Gulati SP 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.
The present study was carried out to determine the efficacy of the crystallization test for screening of oral squamous cell carcinoma (OSCC) and to find out the correlation, if any, between number of TF and clinical and histopathological grades of OSCC.
| Materials and Methods|| |
This study was carried out in the Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Aurangabad. Fifty-five patients who were subsequently diagnosed histopathologically as OSCC and who had not received any therapy prior to study were included. All OSCC cases were subjected to screening for systemic diseases by an experienced physician. Suspected cases were subjected to further appropriate imaging and biochemical investigations to rule out any systemic disease. All the included OSCC cases were free of any other systemic disorder. Histopathological grading was carried out by using Broaders grading system. AJCC system for lip and oral cavity cancer was used for TNM staging. Thirty age- and sex-matched healthy subjects having no obvious oral lesions and systemic diseases were selected as control group.
In the present study, all the subjects were in the age range of 30 years to 75 years, with mean age of 55.14 years. The peak age incidence of OSCC group was noted in 7 th decade of life. The OSCC group consisted of 31 (62%) males and 19 (38%) females. The anatomical sites of OSCC were found to be overlapping with alveolar ridge being the most common site (16 cases) followed by the buccal mucosa and tongue (10 cases each). Gingiva and palate were involved in 6 cases each, whereas floor of mouth and buccal vestibule were involved in 5 and 4 cases, respectively. The retromolar area was involved in 3 cases.
Blood sample was collected by pricking the ring finger with usual aseptic precautions. One drop of blood was collected into 1 cc of doubled distilled water at room temperature to give a final dilution of 6% hemolyzed blood. 0.1 to 0.2 cc of blood sample was then added to the 10 cc of 20% cupric chloride solution at room temperature. The mixture was then poured immediately in the pre-warmed flat-bottom petri-dish of 10 cm diameter. The petri-dish was then placed in BOD incubator (Temperature: 28°C to 32°C and humidity: 35% to 55%) in an isolated room. Crystallization was allowed to take place without any vibration disturbances for 18 to 19 hours. The crystallization patterns were carefully studied through hand lens by holding them against daylight. The presence of TF was considered as positive crystallization test. The number of TFs was also counted so as to correlate them with histopathological grades and clinical stages.
All values were expressed as mean ± SD. Statistical analysis was performed by using Chi square test, Student's t-test (two tailed), and analysis of variance (ANOVA). Significance was set at P < 0.05.
| Results and Observation|| |
The clinical staging was done according to the TNM classification system, according to which 5 (10%) cases were in stage II, 12 (24%) cases were in III, and 33 (66%) cases were in stage IV. The histopathological grading was done according to Anneroth et al. (1987), which showed 22 (44%) cases in grade I and 28 (56%) in grade II.
The crystallization pattern of cupric chloride solution alone showed thick textured crystals with needles arranged at arbitrary angles. The needles either showed side branching in fan-shaped manner or lengthwise linear growth. Secondary and tertiary branches were also observed. Sabarth and Williams (1975)  called such pattern as "Muddle formation" [Figure 1].
|Figure 1: Crystallization patterns of cupric chloride solution alone showing muddle formation|
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The crystallization pattern in control group showed a single eccentrically situated center of gravity with orderly arrangement of radiating crystals emanating from the center towards the periphery [Figure 2].
|Figure 2: Crystallization patterns of cupric chloride solution in control group showing center of gravity (black arrow) and radiating crystals|
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The crystallization pattern in OSCC group showed a sharply set off TF, which consists of transverse needles with wing-like formation on either or both sides. Secondary and tertiary branching formation was remarkably absent in TF. The needles of the central radiation were failed to pierce through the TF [Figure 3] and [Figure 4].
|Figure 3: Crystallization patterns of cupric chloride solution in Grade I OSCC showing few TF formation (white arrow)|
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|Figure 4: Crystallization patterns of cupric chloride solution in Grade II OSCC showing numerous TF formation (white arrow)|
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Out of 50 OSCC cases, 48 cases showed positive crystallization test i.e., 96% reliability and 4% false negative (OSCC cases without TF formation in crystallization test). Out of 30 control subjects, 29 showed negative crystallization test i.e., 96.66% reliability. The positive and negative predictive values were found to be 97.96% and 93.55%, respectively. The chi-square test was applied, and P value was found to be 0.0001 [Table 1]. This indicates that crystallization test was statistically highly significant for detection of OSCC.
The number of TF was counted and was expressed as TF frequency for correlation with histopathological grades and clinical stages of OSCC. The mean TF frequency was found to be increasing from grade I (3.20 ± 15%) to grade II (6.53 ± 2.23%), and difference was statistically significant (P = 0.0001) [Figure 3] and [Figure 4]. Similarly, the mean TF frequency in clinical stage II was 5.8 ± 4.658%, whereas that in stage III and IV were 5.75 ± 2.417% and 4.90 ± 2.315%, respectively. This difference was statistically insignificant.
| Discussion|| |
In malignancy, number of cell products, especially the components of the cell surface and enzymes involved in the metabolism of nucleic acids, are shed or freed within the blood circulation. Thus, blood acts as a unique medium, which reflects various biochemical changes occurring in the body due to malignancy.  Hence, blood can be used as a less invasive diagnostic tool.
The biochemical change occurring in the blood in malignancy has molecular basis at grass root level. It is well-known that molecular forces govern the integrity of molecular structure. In malignancy, these biochemical changes bring about the change in the molecular forces. Similar type of molecular forces also acts to maintain the cohesion of molecules in the crystalline form, which are responsible for the peculiar pattern forming tendency in any particular crystalline substance. It can, therefore, be anticipated that any malignancy in the body system can be detected through the agency of physical forces, which maintain the integrity of molecular structure and that of chemical substances. , It has been observed that in malignancy, there are high concentration of polyamines and diamines in the blood. These amines are the intermediate product of protein metabolism.  It was found 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 concluded that the proteins or degraded products of proteins i.e. polyamines and diamines may be responsible for particular cancer-specific pattern in crystallization test i.e., TF.
In the present study, crystallization patterns obtained was quite similar to those reported by earlier workers. However, the variations such as presence of two or more centers of gravity with wing-like formations with or without blank spaces between them were also observed. Such variations were also noted by Sabarth and Williams (1975).  Also, in the present study, side branches were observed arising from the central radiating needles that sometime resembled TF. On careful examination with the help of magnifying lens, they were clearly seen to be arising from the central radiating crystals, as side branchings.
In the present study, the reliability in control group was found to be 96.66%. Similar results in control group were reported by Pfeiffer E  (91%), Gruner OC  (96%), Quadeer A  (100%), Shaikh S (90%),  and Gulati SP et al.  (92%). The reliability in OSCC group was found to be 96%. The study on head and neck malignancies by Gulati SP  and Kuczkowski J et al. reported quite similar results (83.3% and 96%, respectively). The results of other different malignancies of the body were also quite similar [Pfeiffer E  (91%), Gruner OC  (90.1), Quadeer A  (94.14%), and Shaikh S  (94.78%)].
The mean TFs were correlated well with the histopathological grade 1 and grade II, and the difference was found to be statistically significant. But, there was no such correlation found within the clinical stages. Since the available literature does not present any data regarding such correlation, the comparison with the previous studies was not possible. However, maximum number of TFs were reported in the advanced stages of malignancy. ,
In the present study, 2 cases of OSCC showed false negative result and 1 control case showed false positivity. As the crystallization test is based purely on physical phenomena and is technically very sensitive, these shortcomings in the result may be due to failure to maintain the required physical conditions in these cases.
Thus, it is concluded from the present study that crystallization test is a simple, reliable, economical, less time-consuming, and less invasive screening test, which can be used for early detection of OSCC. This test can be applied at community level, especially for high-risk group patients, which will guide us to identify the potential candidates. These potential candidates can be further evaluated for oral cancer with more confirmatory tests. However, further studies are needed on larger sample size to establish crystallization test as a more reliable screening tool. We also recommend that the research focused on crystallization patterns in metabolic disorders where alterations in polyamines and diamines are expected.
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Sachin C Sarode
Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Maheshnagar, Pimpri, Pune
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]