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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 34  |  Issue : 1  |  Page : 33-37

Efficacy of crystallization test in screening of potentially malignant oral disorders


1 Department of Oral Medicine and Radiology, C.S.M.S.S Dental College and Hospital, Aurangabad, Maharashtra, India
2 Public Government Hospital, Maharashtra, India
3 Department of Public Health Dentistry, S.M.B.T Dental College and Hospital, Sangamner, Maharashtra, India

Date of Submission14-Jan-2021
Date of Decision12-Aug-2021
Date of Acceptance20-Dec-2021
Date of Web Publication25-Mar-2022

Correspondence Address:
Dr. Vijaya Manikrao Ingle
Department of Oral Medicine and Radiology, C.S.M.S.S Dental College and Hospital, Aurangabad, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_12_21

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   Abstract 


Context: Pfeiffer in 1938 observed the crystallization pattern of cupric chloride on admixture with blood of cancer patient. Most of the oral squamous cell carcinoma develops from potentially malignant disorders (PMDs). Aim: The aim of this study is to validate the efficacy of crystallization test in screening of oral PMDs. Setting and Design: The study was carried out in the Department of Oral Medicine and radiology and oral Pathology and Microbiology. Methods and Material: The study included 20 oral PMDs and 20 normal healthy participants. One drop of blood was collected and dilution of 6% hemolyzed blood was done; 0.1 to 0.2 cc of this blood sample is added to 10 cc of 20% cupric chloride solution and further is subjected to crystallization test. Statistical Analysis Used: The Chi-square test was applied and “P” value was found to be significant. This indicates that crystallization test was statistically significant for the detection of oral PMDs. Results: In this study, sensitivity 85% and specificity 90% of crystallization test in screening PMDs was observed. Among 20 PMDs, 17 were positive and 3 were negative and among 20 normal cases, 18 were negative, and 2 were positive for the crystallization test. Positive predictive value and negative predictive value was found to be 89.47% and 85.47% respectively. Conclusion: This test is simple, less invasive, economical and can be used at the community level in mass screening of high-risk individuals more prone to develop potentially malignant oral lesions.

Keywords: Crystallization, oral PMDs, screening


How to cite this article:
Ingle VM, Kale LM, Gawai AS, Jain HR, Mhaske RV. Efficacy of crystallization test in screening of potentially malignant oral disorders. J Indian Acad Oral Med Radiol 2022;34:33-7

How to cite this URL:
Ingle VM, Kale LM, Gawai AS, Jain HR, Mhaske RV. Efficacy of crystallization test in screening of potentially malignant oral disorders. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2022 May 27];34:33-7. Available from: https://www.jiaomr.in/text.asp?2022/34/1/33/340724




   Introduction Top


Oral squamous cell carcinoma accounts for about 90% of all oral cancers and is often not diagnosed until advanced. Proper diagnosis and treatment of PMDs would prevent their further malignant transformation. The survival rates can be improved if detected, diagnosed, and treated at an early stage.[1],[2],[3]

Although there are many methods available for early detection, the need of the hour is, a test should be simple, economical, reliable, less invasive, and used for mass screening of high-risk groups. Previous studies[4],[5] have been conducted to observe the crystallization pattern of cupric chloride on admixture with the blood of cancer patients. This study is an attempt to observe the crystallization pattern of cupric chloride on an admixture of blood of PMD patients and to validate the efficacy of the crystallization test in mass screening of oral PMDs.


   Subjects and Methods Top


The study was carried out in the Department of Oral Medicine and radiology; Oral Pathology and Microbiology. Twenty diagnosed oral PMDs cases which did not receive any therapy before the study and were free of any systemic disease were included. Control group included 20 healthy subjects matched for age and sex having no obvious oral lesions or systemic diseases. Unwilling participants were excluded from the study. All procedures followed as per the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1964 and later versions. Ethical clearance obtained from the institutional review board. Informed consent obtained from study participants after explaining the procedure. The research proposal of Title and synopsis of original research study was discussed in the Ethics committee held on date 4-10-2019 at our institutional review board. The letter/proposal of student is C.S.M.S.S/DCA/OMDR/4/2019 dated 4/10/2019. All the individuals of the study were in the age range of 30 to 75 years. In the potentially malignant group, subjects with leucoplakia followed by oral submucous fibrosis were selected.

Crystallization test

The procedure involved collecting a drop of blood by pricking the ring finger under aseptic conditions. The blood drop was added to 1 cc of double-distilled water, and a final dilution of 6% hemolyzed blood [Figure 1]c was achieved; 0.1 to 0.2 cc of blood sample was then added to 10 cc of 20% Cupric chloride solution [Figure 1]b made using 20mg cupric chloride powder [Figure 1]a. Immediately, the mixture was poured into the prewarmed petri dish of 10 cm size. The Petri dish was placed in a Bio-Oxygen Demand (BOD) incubator [Figure 1]d (temperature: 28°C–32°C and humidity of 35% to 55%) in an isolated room without any vibratory disturbances for about 18 to 19 h. Later, the crystallization patterns were carefully observed using magnifying lens against daylight. The presence of transverse form was considered as positive crystallization test.
Figure 1: (a) Cucl2 Powder, (b) Cupric chloride solution, (c) 6% hemolyzed blood, (d) BOD incubator

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Sample size estimation

Formula of calculating sample size (Two Independent Samples: Variable measured on NOMINAL scale) Objective: Testing Null Hypothesis P1 = P2 (P stand for percentage in each group).



Whereas,

P1-Probability of positive crystallization test in Group -1 (PMD's) 0.75

P2-Probability of positive crystallization test in Group -2 (Control) 0.3

Z1-Z value associated with set level of alpha (One sided) -1.959964

Z2-Z value associated with set level of beta-0.841621

n1-Minimum sample size per group-19

Assuming all the factors, minimum sample size per group comes around 19 rounded to 20 subjects leading to a total of 40 samples (two study groups) in this study.

Statistical analysis

Statistical analysis was performed using Statistical Package for Social science version 21 for Windows (SPSS Inc, Chicago, IL). Descriptive qualitative data were expressed in percentage/proportion, respectively. Confidence interval was set at 95% and probability of alpha error (level of significance) set at 5%. Power of the study was set at 80%. Pearson Chi square test was used to find significance in relation to screening properties of crystallization test in detecting potentially malignant oral disorders. Sensitivity, Specificity, Positive Predictive Value (PPV) Negative Predictive Value (NPV) and prevalence of conditions were calculated in relation to both groups comparison [Figure 6].
Figure 6: Screening test parameters

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   Results Top


The pattern of crystallization with cupric chloride solution alone included thick crystals with needles arranged randomly in a haphazard manner. Such pattern was called muddle formation by Sabarth and Williams [Figure 2].[6],[7] The pattern of crystallization in the control group was that of an eccentrically placed center of gravity with orderly arranged needles radiating from center toward periphery [Figure 3].
Figure 2: Muddle formation Pattern

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Figure 3: Eccentrically placed center of gravity with orderly arranged needles Pattern

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Among the PMDs, there was presence of “transverse bar” or “TF” which consisted of transverse needles with wing-like formation on either or both sides [Figure 4] and [Figure 5]. The needles emerging from the center failed to pierce the TFs. These TFs did not exhibit any secondary or tertiary branching. Among 20 PMDs, 17 were positive and 3 were negative for crystallization test. Among the 20 normal cases, 18 were negative and 2 were positive.
Figure 4: Transverse needles with wing like formation both sides

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Figure 5: Transverse needles with wing like formation

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[Table 1]. Sensitivity and specificity of crystallization test in screening PMDs were calculated. The positive and negative predictive values for oral potentially malignant group were found to be 89.47 and 85.71, respectively. The Chi-square test was applied and “P” value was found to be < 0.05. This indicates that crystallization test was statistically significant for the detection of oral PMDs [Table 2].
Table 1: Positive/negative crystallization test

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Table 2: Screening test parameters

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   Discussion Top


Biochemical alterations occurring in malignancy can be detected in the blood as it serves as an excellent medium reflecting alteration in the body during malignancy.[6] A specific method for cancer detection was employed by Pfeiffer (1938). According to this method, cupric chloride on admixture with the blood of a cancer patient exhibited specific pattern of crystallization. Pfeiffer's crystallization test is based on the principle that colloidal proteins existing in the dilute form of human blood behave as an impurity when mixed with cupric chloride solution and get transformed into an orderly pattern with radiating needle-like crystals.[8] These patterns were found to be distinctive in health and disease by Pfeiffer. In experimental animals, these patterns were found to be specific and characteristic for each disease.[8] The process of crystallization pattern of blood attracted the research workers across as early as 1648.[9] Glauber studied the crystallization process in mid 17th century and concluded that it is controlled by some “occult” forces. The crystallization test is based purely on physical phenomenon; hence, it was very necessary to have controlled physical conditions, which were provided by the BOD incubator.[9],[10]

These biochemical changes occurring in blood have a molecular basis at the grass root level. There are certain forces at the molecular level maintaining the integrity of molecular structure. In case of malignancy, biochemical alterations bring about change in these physical forces involved in maintaining cohesion of molecules in crystalline pattern. This in turn is responsible for the peculiar crystalline pattern forming tendency with chemical substances. It can, therefore, be expected that these physical forces get altered in any kind of malignancy causing disturbance in molecular integrity.[11],[12] In malignancies, it has been noticed that there is increase in the levels of diamines and polyamines in blood which are intermediate products of protein metabolism. These degraded products of protein metabolism might be responsible for the particular precancer and cancer-specific patterns in the crystallization test.[12] In the present study, crystallization patterns observed were quite similar to the previous studies. Certain variations in patterns mentioned by Sarode et al.[13] like the presence of two or more centers of gravity with wing-like formations were present in study. In this study, side branching arising from central radiating needles were differentiated from TFs by closer examination with magnifying lens similar to the study by Sarode et al.[13] In the Tarigoppula et al.[11] study, sensitivity and specificity of crystallization test for the oral potentially malignant group were 83.33% and 86.84%, respectively. Crystallization test carried out on precancerous conditions of female genital tract had a sensitivity of 84.61% in a study by Shaikh et al.,[5] whereas in this study, sensitivity of 85% and specificity of 90% was observed. Sensitivity of the test carried out on oral cancer patients by Gulati et al.[14] and Kuczkowski et al.[15] were 88 and 71.5, respectively, whereas in genital cancer carried out by Shaikh et al.[5] was 94.7%. In this study in the potentially malignant group, highest number of TFs was observed in leukoplakia followed by submucous fibrosis.[11] Histopathological features such as epithelial dysplasia are most commonly used indicators for malignant transformation. Other parameters are needed for accurate assessment of malignant transformation apart from histopathological assessment.[9],[13],[16]

Typical hallmark of malignancy was shown to be transverse formation by Pfeiffer and subsequently by other workers.[7],[17],[18] A further insight into the formation of crystallization pattern in malignancy was given in detail by Selawry.[19]

In this regard, TFs in oral PMDs can be considered for risk assessment. In this study, 3 patients of oral PMDs group exhibited false negative. Oral PMDs group included leukoplakia and oral submucous fibrosis. On the other hand, 2 of the normal group people exhibited false positive result. Further, the irregular or ill-defined patterns observed for few of the study could be due to the technical sensitivity. As this test is purely based on physical phenomenon and is technically highly sensitive, it should be carried out under strict physical conditions to avoid false positives and negatives as shortcomings. This, however, needs further validation by carrying over larger populations.

Limitations and Future study Prospects

Limitations of this study is that Crystallization test is very technique sensitive. Measurements and temperature should be very strictly controlled for accuracy of test and to avoid possibility of human errors. There can be variations in interpretation from person to person.

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 with more confirmatory tests.

Further studies are needed having large sample to establish the crystallization test to be more reliable as screening tool for oral PMDs. We recommend that the research focuses on crystallization patterns should be conducted in metabolic disorders where alterations in polyamines and diamines are expected.


   Conclusion Top


Thus, our study concludes crystallization test to be an effective, simple, less invasive, and less time-consuming screening method in detection of oral PMDs. This test can be used at community level in mass screening of high-risk individuals who are more prone to develop potentially malignant oral lesions. These high-risk individuals should be subjected to more confirmatory tests to evaluate their malignancy potential. However, further studies are required on larger samples to validate the effectiveness of crystallization test in screening of oral PMDs.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Key message

Crystallization test as screening tool for oral PMDs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Mercadante V, Paderni C, Campisi G. Novel non-invasive adjunctive techniques for early oral cancer diagnosis and oral lesions examination. Curr Pharm Des 2012;18:5442-51.  Back to cited text no. 1
    
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Mortazavi H, Baharvand M, Mehdipour M. Oral potentially malignant disorders: An overview of more than 20 entities. J Dent Res Dent Clin Dent Prospects 2014;8:6-14.  Back to cited text no. 3
    
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Warnakulasuriya S, Johnson NW, Van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 2007;36:575-80.  Back to cited text no. 4
    
5.
Shaikh SI, Kawale DN, Diwan CV, Quadeer A, Kharkar AR. Crystallization test for detection of malignancy of female genital tract. Int J Basic Med Sci 2012;3:118-24.  Back to cited text no. 5
    
6.
Burkhardt A. Advanced methods in the evaluation of premalignant lesions and carcinomas of the oral mucosa. J Oral Pathol1985;14:751-78.  Back to cited text no. 6
    
7.
Sabarth E, Williams HN. Sensitive Crystallization Processes: A Demonstration of Formative Forces in Blood. 2nd ed. Spring Valley, New York: Anthroposophic Press; 1975.  Back to cited text no. 7
    
8.
Gruner OC. Experience with the Pfeffer crystallization method for diagnosis of cancer. Can Med Assoc J 1940;43:99-106.  Back to cited text no. 8
    
9.
Pegg AE, Williams-Ashman HG. On the role of S-adenosyl-Lmethionine in the biosynthesis of spermidine by rat prostate. J Biol Chem 1969;244:682-93.  Back to cited text no. 9
    
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Bali SL, Marathe RR. Crystallization test for early detection of malignancy. Int Arch BIOMed Clin Res 2017;3:46-9.  Back to cited text no. 10
    
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Tarigoppula RKVN, Ahmed Mujib BR, Naik R. Efficacy of crystallization test in screening of oral precancer and cancer. Indian J Dent Res 2018;29:556-61.  Back to cited text no. 11
[PUBMED]  [Full text]  
12.
Quadeer A. New Approach for Detection of Malignancy by Crystallization TEST.PhD thesis for (medicine)degree in Anatomy Nagpur University; 1988.  Back to cited text no. 12
    
13.
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. 13
[PUBMED]  [Full text]  
14.
Gulati SP, Sachdeva OP, Sachdeva A, Adlakha RP, Kakkar V. Crystallization test for the detection of head and neck cancer. ORL J OtorhinolaryngolRelat Spec 1994;56:283-6.  Back to cited text no. 14
    
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Kuczkowski J, Zaorski P, Betlejewski A. Crystallization test in patients with head and neck neoplasms. Otolaryngol Pol 1995;49:121-4.  Back to cited text no. 15
    
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Reibel J. Prognosis of oral premalignant lesions: Significance of clinical, histopathological, and molecular biological characteristics. Crit Rev Oral Biol Med 2003;14:4762.  Back to cited text no. 16
    
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Quadeer, A. A simple blood test as a diagnostic tool for detection of susceptibility to malignancy. Jr Anat Soc Ind 1985;34:33.  Back to cited text no. 17
    
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Prehn, RT. Effect of carcinogens on normal cells. In: Brain L, Norris FH, editors. The remote effects of cancer on the Nervous System. I. New York, London: Grune and Stratton; 1964. p. 168.  Back to cited text no. 18
    
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Selawry A. Die Kupferchlorid Crystallization, 1957.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2]



 

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