920 D3000 new imprint Word template


 

Vol 13, No 1 (2025) 
ISSN 2167-8677 (online) 

DOI 10.5195/d3000/2025.920 

 

	

	

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Iron Overload and Serum and Saliva Ferritin Levels in Individuals with 
Beta Thalassemia Needing Several Blood Transfusions 
 
Anaam Mahdi Hadi1, Layla Sabri Yas2, Shaimaa A Saeed3, Ali G. Al-Dulimi1  
 
1College of Den*stry, Bilad Alrafidain University, Diyala, Iraq 
2College of Den*stry, University of Baghdad, Iraq 
3Specialized Dental Center, Baghdad, Iraq 
 

Abstract 

Iron overload in individuals with beta thalassemia major is mostly caused by blood trans-
fusion treatment. Since the human body lacks a method for excreDng extra iron, iron over-
load is unavoidable in individuals with thalassemia major who require regular blood trans-
fusions. Because exfoliaDve cytology is a rapid, easy, painless, and bloodless treatment, it 
is an aIracDve assessment. The main intracellular iron-storage protein, ferriDn, is released 
in minute amounts into the body's plasma and helps to maintain iron in a soluble, non-
toxic state. In the absence of inflammaDon, the amount of the body's total iron reserves is 
posiDvely correlated with the concentraDon of this plasma (or serum) ferriDn. When com-
pared to blood, saliva offers major biochemical and logisDcal benefits, making it one of the 
most essenDal bodily fluids for diagnosDc purposes. The diseases associated with iron over-
load are aIributed to the significant increase in ferriDn levels in saliva. The objecDves of 
the study were as follows: 1. To measure the levels of iron overload in paDents with beta-
thalassemia major using oral exfoliaDve cytology with the special Perl's Prussian blue stain. 
2. ELISA was used to measure the ferriDn level (iron overload) in the serum and saliva of 
individuals with beta thalassemia major. 3. To compare the amounts of ferriDn in their se-
rum and saliva with the Prussian blue staining posiDvity of each individual. Smears were 
extracted from the buccal mucosa of thirty healthy people in the same age range (6-26 
years) and sixty β-thalassemia major paDents who had received at least ten transfusions. 
Prussian blue stain kit from Perl was used to stain smears. To esDmate ferriDn levels, blood 
and saliva samples were simultaneously obtained from the control and study group. Posi-
Dvity of Prussian blue was evaluated using predetermined grading standards. 48 out of 60 
thalassemic paDents (80%) had Perl's posiDvity, which was posiDvely correlated with ferri-
Dn levels in the saliva and serum. PaDents with β-thalassemia major can have their iron 
overload evaluated using coloring peeled cells from the oral mucosa. 

Open Access 
Cita%on: Hadi AM, et al. (2025) Oral Exfolia%ve Cytology 
as a Means of Es%ma%ng Iron Overload and Comparing 
Serum and Saliva Ferri%n Levels in Individuals with Beta 
Thalassemia Needing Several Blood Transfusions. Den-
%stry 3000. 1:a001 doi:10.5195/d3000.2025.920 
Received: April 28, 2025  
Accepted: May 5, 2025 
Published: June 12, 2025 
Copyright: ©2025 Hadi AM, et al. This is an open access 
ar%cle licensed under a Crea%ve Commons AYribu%on 
Work 4.0 United States License. 
Email: anaammahdi100@gmail.co

Introduc)on 

Beta	thalassemias,	sometimes	referred	to	as	
β	thalassemias,	are	a	group	of	genetic	blood	

disorders.	These	types	of	thalassemia	vary	in	
severity,	 ranging	 from	 severe	 anemia	 to	
cases	 that	 show	 no	 clinical	 symptoms,	 and	
this	 condition	 results	 from	 a	 de<iciency	 or	

absence	of	the	production	of	the	beta	chain	
of	 hemoglobin	 [1].	 Children	 who	 have	 this	
condition	 may	 experience	 problems	 that	
might	 affect	 the	 spleen	 and	 need	 regular	



  Iron Overload and Serum and Saliva Ferri1n Levels in Individuals with Beta Thalassemia Needing Several Blood Transfusions 

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blood	transfusions	for	the	rest	of	their	lives.	
Certain	children	may	bene<it	from	bone	mar-
row	 transplants	 [2].	 Two	 factors	 that	 con-
tribute	to	or	exacerbate	iron	overload	in	pa-
tients	 include	 frequent	 blood	 transfusions.	
Organ	damage	must	be	avoided	with	the	use	
of	 iron	 chelation	 therapy.	 Treatments	 for	
thalassemia	 major	 are	 becoming	 more	 ad-
vanced,	allowing	patients	to	live	longer	with	
appropriate	care	[3,4].	Maintaining	a	hemo-
globin	level	above	9–10.5	g/dl	is	the	recom-
mended	 course	 of	 therapy	 for	 thalassemia	
major.	 There	 are	 several	 problems	 associ-
ated	 with	 long-term	 transfusions	 [5].	 The	
body	stores	iron	mostly	in	ferritin	[6].	A	pos-
itive	 acute	 phase	 response	 protein,	 ferritin	
no	longer	re<lects	the	amount	of	the	iron	re-
serve	 when	 concentrations	 rise	 during	 in-
<lammation	[7].	With	considerable	biochem-
ical	and	logistical	bene<its	over	blood,	saliva	
is	one	of	the	most	crucial	bodily	<luids	for	di-
agnostic	purposes.	According	to	Bigler	et	al.	
(2009)	 [8],	 saliva	 collection	 is	 a	 common	
procedure	 that	 is	 safe,	 noninvasive,	 and	
somewhat	easy	for	patients	to	undergo	with-
out	experiencing	any	pain.	

Ferritin	levels	in	saliva	are	known	[9,10],	and	
in	iron	overload	conditions,	salivary	ferritin	
levels	rise	noticeably.	However,	the	ratio	of	
salivary	 to	 serum	 ferritin	 does	 not	 change,	
indicating	 that	 salivary	 and	 serum	 ferritin	
grow	 proportionately	 while	 maintaining	 a	
stable	value	[11,12].	

When	 a	 patient	with	 persistent	 anemia	 re-
quires	frequent	blood	transfusions,	transfu-
sional	iron	excess	occurs.	While	the	body	ex-
cretes	only	around	1	mg	of	iron	per	day	[13],	
transfusions	 include	 over	 250	 mg	 of	 iron	
[14].	According	to	Greer	et	al.	(2013)	[3],	af-
<licted	 patients’	 liver,	 heart,	 endocrine	
glands,	and	lungs	all	have	deposits	of	hemo-
siderin.	Çaliskan	et	al.	 (2011)	[15]	and	Raj-
put	et	al.	(2010)	[16]	have	reported	the	dis-
covery	of	iron	deposits	in	the	gingivae.		

The	following	tests	are	used	to	monitor	the	
patient's	iron	overload	status	and	the	effec-
tiveness	of	iron	chelation	therapy:	serum	fer-
ritin	level,	serum	iron	level,	total	iron-bind-
ing	 capacity,	 non-transferrin-bound	 iron	
level	in	serum,	liver	biopsy	to	assess	paren-
chymal	and	reticuloendothelial	stores,	in	ad-
dition	to	computed	tomography	of	the	liver	
or	magnetic	resonance	imaging,	and	cardiac	

imaging	 using	magnetic	 resonance	 imaging	
(T2	*	or	Ferriscan	technique)	[17].	

The	process	of	exfoliative	cytology	is	rapid,	
easy,	 painless,	 bloodless,	 and	 noninvasive	
[18].	It	is	predicated	on	the	microscopic	ex-
amination	of	epithelial	cells	following	the	<ix-
ation	and	staining	process.	There	are	two	ap-
proaches	 used:	 the	 direct	 technique,	which	
involves	rubbing	mucosal	surface	cells,	and	
the	 indirect	way,	which	 involves	 aspirating	
individuals	with	self-exfoliated	cells	[19].	Us-
ing	 an	 affordable	method	 called	 exfoliative	
cytology	[20],	this	study	aims	to	quantify	the	
iron	excess	in	such	individuals.	

Materials and Methods           

								In	the	Hereditary	Blood	Disease	Center,	
Gynecology,	 Obstetrics,	 and	 Pediatrics	 De-
partment	of	 Ibin	Albaladi	Hospital	 in	Bagh-
dad,	 a	 cross-sectional	 research	was	 carried	
out.	The	research	on	prominent	cases	of	β-
thalassemia	who	received	ten	or	more	blood	
transfusions	was	carried	out	from	January	to	
April	2024.	The	Baghdad	University,	College	
of	Dentistry's	institutional	ethics	committee	
gave	 its	 approval.	 The	hours	 of	 the	 sample	
sessions	 were	 restricted	 to	 9:00	 a.m.	 to	
11:00	a.m.	Data	from	patient	<iles	were	used	
to	determine	the	diagnosis	and	total	number	
of	transfusions.		

The	Sample	

Ninety	 volunteers	 of	 both	 genders,	 ranging	
in	age	from	6	to	26	years,	were	included	in	
this	 study	 and	 were	 categorized	 into	 two	
main	 groups:		
60	beta	thalassemia	major	patients	were	di-
vided	into	the	following	study	groups:	a.	Fif-
teen	 patients	 (15)	 who	 had	 received	 re-
peated	 transfusions	 but	 were	 not	 getting	
iron	 chelation	 therapy	 (treatment-free).		
b.	Forty-<ive	patients	(45)	receiving	iron	che-
lation	therapy	were	divided	into	subgroups	
based	 on	how	 long	 their	 treatments	would	
last	 (5–10),	 (11–15),	 and	 (16–20)	 years.		
2.	30	clinically	and	haematologically	healthy	
people	made	up	the	control	group.		

Inclusion	Criteria	

1.	 Patients	 between	 the	 ages	 of	 26	 and	 26	
who	regularly	get	blood	transfusions	with	a	
transfusion	 count	 of	 at	 least	 10.		
2.	The	patient's	inclusion	was	con<irmed	by	

hemoglobin	 electrophoresis.		
3.	 Control	 group:	 Individuals	 chosen	 based	
on	a	clinical	examination,	history,	and	blood	
tests	that	were	within	normal	limits.		

Exclusion	Criteria	

Individual	who	has	not	yet	received	a	blood	
transfusion	 and	 who	 has	 a	 history	 of	 any	
other	 serious	 illnesses.	 Hepatitis,	 megalo-
blastic	anemia,	iron	de<iciency	anemia,	can-
cer,	and	veri<ied	acute	and	chronic	 liver	 in-
jury	should	all	be	absent	in	control	persons.	

Sample	Collection						

At	the	same	time,	swabs,	saliva	samples,	and	
blood	samples	were	collected.	Patients	were	
instructed	 to	 rinse	 their	 mouths	 with	 dis-
tilled	water	to	remove	any	residues.,	both	in	
the	 research	 group	 and	 the	 control	 group.	
Patients'	 buccal	mucosa	was	 cleansed	with	
gauze,	and	cells	were	then	extracted	using	an	
interdental	brush	that	was	used	repeatedly	
in	 a	 straight	 line	 and	with	 very	 little	 pres-
sure.	According	to	Thomas	et	al.	(2009)	[21]	
and	Farhan	 and	Yas	 (2018)	 [22],	 scrapings	
were	spread	out	across	a	vast	area	on	a	clean,	
dry,	frosted	glass	slide	to	prevent	cell	clump-
ing.	Following	the	<ixation	of	the	slides	 in	a	
coplin	 jar	 with	 70%	 ethanol	 alcohol,	 the	
slides	were	sent	to	a	lab	for	iron	staining	us-
ing	an	abcam	150674	kit.		

To	determine	whether	the	blue	iron	granules	
are	present	in	the	cytoplasm,	the	sample	was	
<irst	examined	using	a	10x	magni<ication	lens	
and	 then	 with	 a	 40x	 magni<ication	 lens.	
For	grading	purposes,	ten	high	power	<ields	
(objective	 lens	 x40)	 that	met	 the	 following	
requirements	 were	 selected:		
1.	 Cells	 that	 are	 widely	 apart	 and	 barely	
overlap.		
2.	In	each	high-power	<ield	(x40),	a	minimum	
of	20	epithelial	 cells	 and	a	maximum	of	30	
cells.		
3.	 Whether	 blue	 intracytoplasmic	 granules	
are	 present	 or	 absent	 [20].		
A	 camera	 that	was	 <ixed	 to	 the	microscope	
was	 used	 to	 take	 the	 pictures.	 Achromatic	
objective	of	40X	was	used	to	take	all	the	cell	
photos.	The	computer	was	used	to	save	the	
captured	images,	and	Image	J	software	ver-
sion	1.52	a	was	used	for	analysis.	The	aver-
age	 results	 were	 calculated	 from	 ten	 high-



  Iron Overload and Serum and Saliva Ferri1n Levels in Individuals with Beta Thalassemia Needing Several Blood Transfusions 

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strength	areas	(40X),	and	then	the	appropri-
ate	score	was	determined	based	on	that.	

• Grade	0	-	No	particles			

• 	Grade	I	-	Less	than	5	particles	in	the	high	
<ield	of	vision			

• Grade	II	-	5-10	particles	in	the	high	<ield	of	
vision			

• Grade	III	-	10-20	particles	in	the	high	<ield	of	
vision			

• Grade	IV	-	Visible	clumps	in	less	than	3	high	
<ields	of	vision			

• Grade	V	-	Coarse	particles	or	visible	clumps	
in	3	or	more	high	<ields	of	vision	[20].			

Bone	marrow	used	as	positive	control	tissue.	

The	study	and	control	groups	had	their	ve-
nous	blood	drawn	at	the	same	time	as	exfoli-
ative	 cytology.	The	 sample	was	 centrifuged	
for	 20	 minutes	 at	 4000	 rpm	 to	 produce	 a	
clear	 supernatant.	 The	 hospital	 next	 used	
VIDAS®	Ferritin,	an	automated	quantitative	
test	 that	 uses	 the	 Enzyme	 Linked	 Fluores-
cent	Assay	technology	with	a	ferritin	kit	(bio-
Mérieux)	to	determine	the	level	of	ferritin	in	
human	serum	or	plasma.		

										Saliva	was	taken	from	each	participant	
while	they	were	at	rest	and	not	aroused.	One	
hour	before	to	the	sample	collection,	the	par-
ticipants	were	told	not	 to	eat	or	drink	any-
thing	and	to	rinse	their	mouths	with	water	to	
get	rid	of	any	debris.	Under	supervision,	the	
participants	 were	 told	 to	 sit	 comfortably	
with	their	heads	slightly	leaned	forward,	al-
low	 saliva	 to	 collect	 on	 the	 <loor	 of	 their	
mouths,	and	then	expel	it	through	expectora-
tion.	During	the	<ive-minute	saliva	collection	
time,	 the	 samples	were	 coded,	 and	 the	 pa-
tient's	 identity	 was	 recorded.	 The	 samples	
were	processed	and	stored	in	a	sterile	plastic	
specimen	container	[23].	

Saliva	 samples	were	 collected,	 then	 centri-
fuged	for	20	minutes	at	4000	rpm,	with	the	
clear	 supernatant	 removed	 and	 put	 in	 Ep-
pendorf	tubes.	The	samples	were	then	deep	
frozen	at	-20°C	until	the	required	number	of	
samples	were	obtained.	The	E-EL-H0168	Hu-
man	FE	(Ferritin)	ELISA	kit/Elabscience	was	

then	used	to	analyze	saliva	samples	in	the	la-
boratory.		

Statistical	Analysis	

Data	analysis	was	conducted	using	 the	Sta-
tistical	Package	for	the	Social	Sciences	(SPSS)	
version	22.0.	The	chi-square	test	was	applied	
to	compare	the	control	group	and	the	study	
group,	 and	 Analysis	 of	 Variance	 (ANOVA),	
Spearman's	 correlation	 coef<icient,	 and	 the	
F-test	were	calculated	to	relate	the	positivity	
of	the	oral	swab	to	serum	and	saliva	ferritin	
levels.	

	

Figure	 1.	 Perl’s	 Prussian	 blue	 staining	 of	
squamous	 epithelial	 cells	 from	 buccal	 mu-
cosa	 showing	 blue	 intracytoplasmic	 iron	
granules,	 counter	 stain	used	 is	nuclear	 fast	
red	stain.	

Results 

Among	the	60	patients	in	the	research	group,	
48	patients	(80%)	showed	a	positive	result	
for	 the	blue	Prussian	dye	 test	 in	 the	exfoli-
ated	oral	mucosal	cells.	Meanwhile,	all	thirty	
participants	 in	 the	 control	 group	 (100%)	
showed	 a	 negative	 reaction	 to	 the	 blue	



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Prussian	dye.	The	results	of	the	Prussian	dye	
test	 were	 compared	 between	 the	 research	
and	control	groups	using	the	chi-square	test,	
and	as	shown	in	Table	1,	there	was	a	statisti-
cally	signi<icant	difference	between	the	two	
groups	at	a	p-value	of	>0.001.		

Serum	Ferritin	(ng/ml)	Marker	

In	 reference	 to	 "Serum	 Ferritin"	 ng/ml	
across	 the	 various	 groups,	 the	 non-treat-
ment	 group	 has	 the	 highest	 mean	 value	
(8194.3±3549.7)	 ng/ml	with	 a	 large	 varia-
tion	 when	 compared	 to	 the	 other	 treated	
study	 groups,	 which	 have	 mean	 levels	 of	
(5274.4±2940.9)	 ng/ml,	 (5062.7±1926.6)	
ng/ml,	 and	 (4567.3±2102.9)	 ng/ml	 for	 the	
5–10,	 11–15,	 and	 16–20	 years	 accordingly	
(Table	2).		

Saliva	Ferritin		

In	 relation	 to	 "Saliva	 Ferritin"	 ng/ml,	 no	
treatment	group	recorded	the	highest	mean	
value	 (25.347±4.273)	 ng/ml.	 Then,	 treated	
groups	 5-10	 years,	 11-15	 years,	 and	 16-20	
years	 period	 followed,	 with	 mean	 values	
(11.319±3.395)	 ng/ml,	 (5.021±2.025)	
ng/ml,	 and	 (2.061±1.167)	 ng/ml,	 respec-
tively,	 and	 lastly,	 the	 control	 group	
(0.146±0.058)	ng/ml.	This	is	shown	in	Table	
3.	

In@luence	between	Saliva	and	Serum	Ferritin				

A	correlation	between	 the	examined	mark-
ers	was	found	(R=0.94254;	P<0.001).	

Comparison	of	Pearl's	Stain	Grades	in	Exfolia-
tive	 Cytology	 and	 Serum	and	 Saliva	 Ferritin	
Levels	in	the	Study	Groups	

The	current	study's	results,	when	compared	
using	ANOVA,	 the	F	 test,	and	the	spearman	
correlation	coef<icient,	revealed	a	non-signif-
icant	 association	 between	 "Saliva	 ferritin"	
and	the	grades	of	buccal	smears,	but	a	signif-
icant	 correlation	 (P<0.05)	 between	 "Serum	
ferritin"	and	the	exfoliative	cytology	grades.		

Out	of	60	patients,	a	maximum	of	22	(36.6%)	
had	 a	 mean	 serum	 ferritin	 level	 of	
5102.9±2357.5	 ng/ml,	 indicating	 Grade	 IV	
positive.	 Thirteen	 patients	 (21.6%)	 had	 a	
mean	serum	ferritin	level	of	5953.7±3254.1)	
ng/ml,	indicating	Grade	III	positivity.	Subse-
quently,	 12	 patients	 (20%)	 had	 a	 mean	

serum	ferritin	level	of	7791.0±3179.3	ng/ml,	
falling	into	grade	(0).	Eight	patients	(13.3%),	
on	the	other	hand,	had	a	mean	serum	ferritin	
level	of	4334.9±2005.3	ng/ml,	belonging	to	
grade	II.	Subsequently,	3	patients	(5%)	had	a	
mean	blood	 ferritin	 level	of	8307.2±3428.9	
ng/ml	 (grade	 V).	 Table	 4	 illustrates	 that	 a	
mere	2.3%	of	 individuals	exhibited	grade	I,	
with	 a	 mean	 blood	 ferritin	 level	 of	
1862.7%±4688.3%	ng/ml.	

Discussion 

Cells	were	collected	from	the	oral	mucosa	of	
48	out	of	60	patients	suffering	 from	severe	
thalassemia	 (80%)	 in	 this	 investigation	
showed	 positive	 results	 for	 Perls'	 Prussian	
blue	response.	 It	was	also	noted	 that	Perls'	
Prussian	 blue	 positive	was	 absent	 from	 all	
the	control	participants.	Like	the	<indings	of	
Gururaj	 and	 Sundharam	 et	 al.	 (2003)	 [24],	
who	reported	100%	Perls'	staining	positivity	
in	the	10	patients	they	studied,	their	results	
also	 demonstrated	 Prussian	 blue	 staining	
positivity	in	Perls.		

According	to	Nandprasad	et	al.	(2009)	[25],	
65	out	of	100	patients	had	65%	Perls'	posi-
tivity;	 Bhat	 et	 al.	 (2013)	 [26]	 observed	
71.7%	 positivity	 (43	 out	 of	 60	 patients);	
Chittamsetty	 et	 al.	 (2013)	 [27]	 reported	
72.5%	(29	out	of	40	β-thalassemia	major	pa-
tients);	and	Gupta	et	al.	(2014)	[28]	observed	
61.6%	Perls'	positivity	(37	out	of	60	cases).	
Findings	 from	Leekha	et	al.	 (2016)	[29]	re-
vealed	that	35	(87.5%)	of	40	patients	had	fa-
vorable	results.	Furthermore,	98%	of	Perls'	
positive	 (49	 out	 of	 50	 instances)	 was	 rec-
orded	by	Gajaria	et	al.	(2017)	[20].	

The	observed	positive	<indings	variance	can	
be	explained	by	 the	variations	 in	 the	mean	
blood	ferritin	levels	of	the	patients	involved	
in	these	investigations,	as	well	as	the	overall	
sample	size	differences	among	studies	[20].	
Additionally,	 a	 comparison	 of	 the	 study	
group's	 Perls'	 Prussian	 blue	 staining	 with	
that	of	the	control	group	demonstrated	that	
the	study	group's	positivity	was	not	coinci-
dental.	As	a	result,	there	is	a	strong	correla-
tion	 between	 the	 positive	 iron	 staining	 in	
oral	 smears	 of	 patients	with	 beta-thalasse-
mia	major	who	receive	frequent	blood	trans-
fusions	and	suffer	from	hemosiderosis.	This	
indicates	that	iron	accumulates	in	the	squa-
mous	 epithelial	 cells	 of	 the	 oral	mucosa	 in	

cases	of	hemosiderosis.,	and	that	iron	over-
load	may	be	measured	by	scraping	the	cells.	
The	 controls	 did	 not	 have	 iron	 excess	 and	
were	in	good	health.	

	The	lack	of	positive	instances	in	the	research	
group	 may	 have	 resulted	 from	 improper	
staining	 methods	 or	 early	 shedding	 of	 the	
impacted	 squamous	 epithelial	 cells,	 even	
though	the	patients	had	elevated	serum	fer-
ritin	 levels.	 The	 creation	 of	 iron	 storage	
pools	is	one	of	the	processes	that	determines	
the	 excess	 quantities	 of	 iron	 that	 collect	 in	
different	 tissues.	Perls'	Prussian	blue	stain-
ing	positivity	is	always	impacted	by	the	vari-
ation	in	ferritin	amounts	generated	in	exfoli-
ated	buccal	mucosal	cells	[20].	According	to	
Delaby	et	al.	(2005)	[30],	iron	initially	builds	
up	 in	 reticuloendothelial	macrophages	 and	
then	spreads	to	parenchymal	cells	when	he-
mosiderosis	happens.		

As	 compared	 to	 control	 participants,	 the	
study	 groups'	 mean	 blood	 ferritin	 levels	
were	considerably	higher,	with	the	no	treat-
ment	group	having	the	highest	mean	value.	
These	 <indings	are	 consistent	with	Canatan	
and	Akdeniz	 (2012)	 [12]	 and	Hassan	 et	 al.	
(2013)	[31].	

In	 agreement	 with	 Canatan	 and	 Akdeniz	
(2012)	 [12],	 the	 study	 groups'	mean	 saliva	
ferritin	levels	were	signi<icantly	higher	than	
those	of	the	control	subjects.	The	controlled	
group	also	showed	highly	signi<icant	differ-
ences,	 with	 a	 P-value	 of	 less	 than	 0.001,	
when	compared	to	the	study	groups.	Accord-
ing	 to	 Jaganthan	et	al.	 (2012)	 [10],	salivary	
ferritin	 levels	 signi<icantly	 increase	 in	 iron	
overload	illnesses.		

Because	saliva	contains	enzymes	that	are	de-
pendent	on	iron,	the	biological	system	main-
tains	 larger	 amounts	 of	 salivary	 ferritin,	
which	helps	to	preserve	iron	through	saliva	
[32].	There	is	not	enough	information	in	the	
literature	to	pinpoint	the	precise	pathophys-
iology	of	salivary	ferritin	elevation.	Although	
ferritin	 [12]	 and	 salivary	 components	 [33]	
may	be	similar	in	both	serum	and	saliva,	the	
synthesis	and	secretion	of	parotid	gland	aci-
nar	cells	may	affect	salivary	component	lev-
els,	 making	 them	 inconsistent	 with	 serum	
[34].	



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5	

The	current	investigation	found	a	signi<icant	
association	 (p>0.05)	between	 the	mean	se-
rum	 ferritin	 levels	 in	 the	 treated	 and	 non-
treated	 groups	 and	 the	 grades	 of	 buccal	
smear	 positive.	 This	 <inding	 suggested	 that	
the	 mean	 serum	 ferritin	 levels	 and	 buccal	
smear	 grades	 were	 associated,	 and	 the	 re-
sults	were	found	to	be	comparable	to	those	
of	Gajaria	et	al.	(2017)	[20].	The	grade	of	buc-
cal	 smear	 positive	 was	 shown	 to	 have	 a	
strong	 correlation	with	 the	 patients'	 blood	
ferritin	levels;	as	a	result,	patients	receiving	
regular	transfusions	can	utilize	this	straight-
forward,	 non-invasive	 diagnostic	 to	 track	
iron	excess.		

Many	 factors,	 such	 as	 the	number	of	 blood	
transfusions	the	patient	has	received,	the	age	
at	 which	 iron	 chelation	 therapy	 began,	
whether	the	treatment	is	taken	regularly,	so-
cial	 and	 economic	 status,	 nutritional	 de<i-
ciencies,	and	other	comorbidities,	affect	the	
increase	of	iron	in	this	group	of	patients	[20].		
Regarding	the	"Saliva	ferritin"	marker,	there	
is	no	discernible	relationship	with	the	grades	
of	buccal	smears.	We	are	not	aware	of	any	re-
search	that	relates	salivary	ferritin	levels	to	
Perls'	 Prussian	blue	 staining	 grades	of	 oral	
exfoliative	cytology.	

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identieication	 of	 salivary	 transferrin	 as	 a	 bi-
omarker	for	early	detection	of	oral	cancer.	An-
alytica	chimica	act.	2010,	681.1-2:	41-48.	

Table	1.	Comparison	of	the	reaction	of	Prussian	blue	of	pearls	between	the	study	group	and	the	control	group	using	the	contingency	coef<icient.	

Groups	 Exfoliative	cytology	staining	No&%	 Total	No.&%	 C.S.	(*)	P-value	

Negative	 Positive	

Control	 30	 100%	 0	 0.0%		 30	 100%	 C.C.	=	0.625	

P=0.000	Patients	 12	 20%	 48	 80	%	 60	 100%	

Total	 42	 46.6%	 48	 53.3%	 90	 100%	

(*)	SigniIicant	at	P<0.001;	Testing	based	on	chi-square.	

Table	2.	Summary	statistics	of	serum	ferritin	(ng/ml)	readings	distributed	in	different	groups. 

Groups	 No.	 Mean	 Standard	De-
viation	

Standard	Er-
ror	

95%	C.I.	for	Mean	 Minimum	 Maximum	

Lower	
Boundary	

Upper	Boundary	

Control	 30	 71.653	 47.2	 8.6	 54.0	 89.3	 17.6	 180.8	

No	treatment	 15	 8194.3	 3549.7	 916.5	 6228.6	 10160.1	 2492.0	 12000.0	

5-10	years	 15	 5274.4	 2940.9	 759.3	 3645.8	 6903.1	 1531.5	 11929.4	

11-15	years	 15	 5062.7	 1926.6	 497.5	 3995.8	 6129.6	 1969.1	 8059.8	

16-20	years	 15	 4567.3	 2102.9	 543.0	 3402.8	 5731.8	 1515.1	 7688.6	

	

Table	3.	Summary	statistics	of	saliva	ferritin	(ng/ml)	readings	distributed	in	different	groups.	

Groups No.	 Mean	 Standard	Deviation	 Standard	Error	 95%	C.I.	for	Mean	 Minimum	 Maximum	

Lower	Boundary	 Upper	Boundary	

Control	 30	 0.146	 0.058	 0.011	 0.125	 0.168	 0.100	 0.300	

No	treatment	 15	 25.347	 4.273	 1.103	 22.980	 27.713	 15.050	 30.12	

(5		_	10)	yrs.	 15	 11.319	 3.395	 0.877	 9.439	 13.199	 6.530	 17.05	

(11		_	15)	yrs.	 15	 5.021	 2.025	 0.523	 3.899	 6.142	 1.310	 9.500	

(16	_	20)	yrs.	 15	 2.061	 1.167	 0.301	 1.415	 2.708	 1.100	 5.200	

Table	4.	Comparison	of	exfoliative	cytology	grades	with	average	serum	and	salivary	ferritin	levels	in	study	groups.	

Marker	 Grade	 No.	of	Patients	 Mean	 Standard	

Deviation	

Standard	
Error	

Minimum	 Maximum	 P-value	

Serum	Ferritin	ng/ml	 0	 12	 7791	 3179	 917	 2501	 12000	 F=3.333	

P=0.011		I	 2	 1862	 468	 331	 1532	 2194	



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II	 8	 4334	 2005	 709	 1984	 7374	

III	 13	 5953	 3254	 902	 2501	 12000	

IV	 22	 5102	 2357	 502	 1515	 11725	

V	 3	 8307	 3428	 1979	 5224	 12000	

Saliva	Ferritin	ng/ml	 0	 12	 10.7	 10.4	 3.0	 1.2	 30.1	 F=1.625	

P=0.169		I	 2	 9.5	 8.3	 5.9	 3.6	 15.4	

II	 8	 7.9	 4.1	 1.5	 1.1	 15.3	

III	 13	 10.7	 11.1	 3.1	 1.1	 29.7	

IV	 22	 10.5	 9.2	 2.0	 1.1	 28.9	

V	 3	 25.2	 2.0	 1.2	 22.9	 26.5	

Testing	based	on	ANOVA,	F	test	and	spearman	correlation	coefIicient.