Better differential diagnosis of iron deficiency anemia from beta-thalassemia trait/Demir eksikligi anemisinin beta-talasemi tasiyiciligindan ayirici tanisinin daha iyi yapilmasi.
Abstract: Objective: Iron deficiency anemia (IDA) and beta-thalassemia trait ([beta]-TT) are the most common forms of microcytic anemia. This study was conducted to compare the validity of various discrimination indices in differentiating [beta]-TT from IDA by calculating their sensitivity, specificity and Youden's index.

Methods: Totally 323 subjects (173 children and 150 adults) with microcytic anemia were involved in this study. We calculated 10 discrimination indices in all patients with IDA and [beta]-TT. We divided the patients into two different groups as younger or older than 10 years.

Results: None of the indices showed sensitivity and specificity of 100% in the patients older than 10 years, and in the patients younger than 10 years, only Shine & Lal index showed sensitivity close to 90% and specificity of 100%. The most accurate discriminative index for patients younger than 10 years was Shine & Lal and for those older than 10 years it was RDW index. According to Youden's index, Shine & Lal and RBC count showed the greatest diagnostic value in patients younger than 10 years and RDW and RBC count indices in those older than 10 years.

Conclusion: None of the indices was completely sensitive and specific in differentiation between [beta]-TT and IDA. Mean and median mean cell Hb density (MCHD) were very close to normal values in both IDA and [beta]-TT patients, but in the case of mean density of Hb/liter (MDHL), we found that the mean and median were significantly higher than normal values in [beta]-TT and lower than normal values in IDA patients. In our study, Youden's index of RBC and Shine & Lal were the highest and most reliable indices in differentiating [beta]-TT from IDA in the patients younger than 10 years. For patients older than 10 years, the most reliable discrimination indices were RBC and RDW. (Turk J Hematol 2009; 26: 138-45)

Key words: [beta]-Thalassemia trait, iron deficiency anemia, red blood cell, indices, discriminative indices

Amac: Demir eksikligi anemisi (DEA) ve beta-talasemi tasiyiciligi, mikrositik aneminin en sik nedenleridir. Bu calisma, [beta]-talasemi tasiyiciliginin ([beta]-TT) DEA'dan ayriminda kullanilan ayirt edici indekslerin duyarliliklarini, ozgulluklerini ve Youden indekslerini hesaplayarak, ayirici tanidaki onemlerini karsilastirmak amaciyla yurutulmustur.

Materyal ve Metod: Mikrositik anemili toplam 323 kisi (173 cocuk ve 150 eriskin) bu calismaya dahil edilmistir. DEA ve [beta]-TT'li tum hastalarda 10 ayirt edici indeks degerlendirilmistir. Hastalar 10 yasindan kucuk ve 10 yasindan buyuk olmak uzere iki farkli gruba ayrilmistir.

Bulgular: Indekslerden hicbiri 11 yasindan buyuk hastalarda %100 duyarlilik ve ozgulluk gostermemis, sadece Shine ve Lal indeksleri 10 yasindan kucuk hastalarda %90'a yakin bir duyarlilik ve %100 ozgulluk gostermistir. En hassas ayirt edici indeks olarak 10 yasindan kucuk hastalar icin Shine ve Lal indeksleri, 11 yasindan buyuk hastalar icin ise RDWI indeksi bulunmustur. Shine ve Lal ve kirmizi kan hucresi sayisi icin Youden indeksinin ve RDWI ve kirmizi kan hucresi sayisi icin Youden indeksinin, sirasiyla 10 yasindan kucuk ve 10 yasindan buyuk hastalar icin en yuksek tanisal degere sahip oldugu gorulmustur.

Sonuc ve Tartisma: Indekslerden hicbiri [beta]-TT ve DEA ayriminda tam olarak duyarli ve ozgul degildi. Hem DEA hem de [beta]-TT hastalari icin ortalama ve ortanca MCHD degerleri normale cok yakindi, fakat MHDL durumunda ortalama ve ortancalarin, [beta]-TT icin normal degerlerden anlamli olarak yuksek ve EDA hastalarinin degerlerlerinden dusuk oldugunu bulduk. Calismamizda, Shine ve Lal ve kirmizi kan hucresi sayisinin Youden indeksi 10 yasindan kucuk hastalarda [beta]-TT ve DEA ayriminda en yuksek ve en guvenilir ayirt edici indeks, 11 yasindan buyuk hastalar icin ise kirmizi kan hucresi ve RDWI en guvenilir indeksler olarak bulunmustur. (Turk J Hematol 2009; 26: 138-45)

Anahtar kelimeler: [beta]-talasemi tasiyiciligi, demir eksikligi anemisi, kirmizi kan hucresi, Indeksler, ayirici indeksler
Article Type: Report
Subject: Thalassemia (Diagnosis)
Thalassemia (Research)
Diagnosis, Differential (Methods)
Diagnosis, Differential (Usage)
Iron deficiency anemia (Diagnosis)
Authors: Rahim, Fakher
Keikhaei, Bijan
Pub Date: 09/01/2009
Publication: Name: Turkish Journal of Hematology Publisher: Aves Yayincilik Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 Aves Yayincilik ISSN: 1300-7777
Issue: Date: Sept, 2009
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Turkey Geographic Code: 7TURK Turkey
Accession Number: 207866380
Full Text: Introduction

The most commonly encountered disorders with mild microcytic anemia are iron deficiency anemia (IDA) and thalassemia trait (TT) [1,2]. The gene frequency of [beta]-thalassemia, however, is high and varies considerably from area to area, having its highest rate of more than 10% around the Caspian Sea and Persian Gulf. [alpha]-thalassemia is very rare in Iran, and there is no clear report of the prevalence for the country as a whole. Results of genotyping for the two most common [alpha]-thalassemia single gene deletions (-[[alpha].sup.3.7] and -[alpha].sup.4.2]) showed that the -[[alpha].sup.3.7] deletion was a common cause of microcytic and hypochromic anemia in Iran [3]. The prevalence of the disorder in other areas is between 4-8%. In Isfahan, a city built around the river Zayandeh-Rood in the central part of Iran, the frequency rises to about 8%. In the Fars Province, in southern Iran, the gene frequency is also high and reaches 8-10% [4]. To date, many discrimination indices have been reported using red blood cell (RBC) indices obtained by automated blood count. Many authors have calculated the sensitivity and specificity of these indices in the distinction between IDA and TT [2]. They proposed that the diagnosis could be established without having to resort to the more time-consuming methods such as transferrin saturation (TS), ferritin and hemoglobin [A.sub.2] (Hb[A.sub.2]) levels. However, none of these indices showed a sensitivity and specificity of 100% in prediction of IDA and TT. Some showed considerable sensitivity for IDA or TT, but not specificity [5]. Youden's index provides an appropriate measure of validity of a particular technique or question by taking into account both sensitivity and specificity [6,7]. In this study, we compared the validity of various indices in differentiating [beta]-TT from IDA by calculating their sensitivity, specificity and Youden's index. In some published articles, the validity of all of the defined discrimination indices were compared in the same patient groups, and it was shown that the RBC count is one of the two most accurate indices available [8-11].

Materials and Methods

Totally 323 subjects (173 children and 150 adults) with microcytic anemia were involved in this study. We divided the patients into two categories according to age as 1 to 10 years and 11 to 57 years. The division was done using a method based on difference in red cell indices such as mean corpuscular volume (MCV) published by Hermiston and Mentzer [12]. Venous blood was drawn with the usual precautions after the patient had been lying quietly for at least 20 minutes. The hematological analyses were carried out with a Coulter Counter (M530, Coulter Electronics Ltd., Luton, UK) particle counter. Hb concentration, RBC count, and total leukocyte count were recorded, and liquid chromatography technique was used for determination of Hb[A.sub.2] [13]. Serum iron (SI), serum iron binding capacity (SIBC), serum ferritin, and Hb[A.sub.2] values were determined in all. Diagnosis of IDA was based on a Hb level of <11.4 and <12.9 g/dl and MCV of <81 and <84.8 fl in those younger or older than 10 years, respectively, and a ferritin value of <10 ng/ml and TS of <12% in both age groups. According to these criteria, 114 children and 56 adults of the 323 patients were diagnosed as IDA. The patients with Hb value <8.7 g/dl were excluded in order to not confuse more severe IDA with [beta]-TT. In the remaining 59 patients younger than 10 years and 94 patients older than 10 years, the diagnosis of TT was based on presence of microcytosis and elevated levels of Hb[A.sub.2] (>3.5%). RBC count and red blood cell distribution width (RDW) [14,15] were obtained with Coulter Counter and the other discrimination indices were calculated by using RBC indices as defined below:

Mentzer Index (MI): MCV/RBC [16],

Shine and Lal Index (S&L): MC[V.sup.2] x mean corpuscular hemoglobin (MCH) x 0.01[17],

England and Fraser (E&F): MCV-RBC-(5 x Hb)-k [18], k is calculated to be 4.54 in our counter as described in the published report [19],

Srivastava Index (SI): MCH/RBC [20],

Green and King Index (G&K): MCV2 x RDW/100 x Hb [21],

RDW Index (RDWI): MCV x RDW/RBC [22],

Mean Cell Hb Density (MCHD) Index: MCH/MCV [23],

Mean Density of Hb/Liter of Blood (MDHL) Index: MCHD x RBC [23].

Differential value for each of above indices was applied as given in the original published reports [6-22].

We calculated two hematological parameters including MDHL and MCHD in healthy male and female subjects and patients separately using data from Shafa Hospital based on the method described by Telmissani et al. [23]. The diagnostic method was that patients with values equal to or below the mean of healthy subjects were most likely to have IDA, and patients with values higher than the mean of healthy subjects were most likely to have [beta]-TT. We computed mean and median of MCHD and MDHL for those 323 patients and compared the obtained values to the normal values, which are shown in Table 6. We attempted to compare 10 different discrimination indices with respect to five parameters include sensitivity, specificity, positive predictive value, negative predictive value, and Youden's index.

Sensitivity and specificity were calculated according to standard formulas, namely: Sensitivity = (TP) / (TP + FN) and specificity = (TN) / (TN + FP), where TP = true positives, FN = false negatives, TN = true negatives and FP = false positives. The predictive values (PV), whether positive (+) or negative (-), were similarly calculated, with +PV being (TP) / (TP + FP) and -PV being (TN) / (TN + FN). Finally, Youden's index was calculated as follows: (Sensitivity + specificity)-100.

Results

Of the 323 patients, 170 (114 children, 56 adults) were diagnosed to have IDA and 153 (59 children, 94 adults) were diagnosed to have [beta]-TT. The hematological data of the groups are shown in Table 1. The different indices for patients younger or older than 10 years were calculated individually. The differential value for each index in differentiation between [beta]-TT and IDA and the number and proportion of correctly identified patients (true positives) calculated using these indices are shown in Table 2 (those younger than 10 years) and Table 4 (those older than 10 years). The sensitivity, specificity, positive and negative predictive values and Youden's index of each index in differentiation between [beta]-TT and IDA are shown in Table 3 (those younger than 10 years) and Table 5 (those older than 10 years). All of the indices showed overlapping in the patients with [beta]-TT and IDA. The overlaps were between 12.5 and 21.1% in RDW and between 4.3 and 5.49 x 1012 in RBC for patients younger than 10 years, while these respective values were between 11.4 and 25.1% and between 4.38 and 5.94 x 1012 in those older than 10 years. None of the indices was completely sensitive or specific in differentiation between [beta]-TT and IDA. Youden's indices in decreasing order in patients younger than 10 years were as follows: S&L > RBC > SI > MI > E&F > MDHL > G&K > RDWI > RDW > MCHL, and in patients older than 10 years were: RDWI > RBC > MI > E&F > SI > MDHL > RDW > S&L > G&K > MCHL. The scatter-gram of the hematological data of all patients showed significant differences between these two disorders (Figure 1).

However, MDHL, which showed that patients with values equal to or below the mean of healthy subjects (for younger than 10 years: 1.71 in males and 1.98 in females; for older than 10 years: 1.80 in males and 1.76 in females) were most likely to have IDA and those whose MDHL value was greater than the mean were more likely to have [beta]-TT. We computed mean and median of MCHD and MDHL for these 323 patients and compared results with the normal values (Table 6). We found that mean and median MCHD values were very close to normal in both IDA and [beta]-TT patients, while mean and median MDHL values were significantly more than normal values in [beta]-TT and lower than normal in IDA patients (Table 7).

Discussion

The most frequently encountered diseases with mild microcytic anemia are TT and IDA. However, in addition to genetic counseling for identification of thalassemia carriers in order to prevent the birth of thalassemia patients, differentiating TT from IDA is warranted because the thalassemia heterozygote should not be given iron in a vain attempt to normalize MCV. The diagnosis of [beta]-TT is established by the presence of characteristic RBC microcytosis and elevated levels of Hb[A.sub.2] [24]. Decreased levels of SI, TS and ferritin with increased levels of SIBC are the main diagnostic criteria for IDA [25]. Less time-consuming methods are based on the calculation of discrimination indices from RBC indices obtained during routine complete blood count. However, later studies have shown that the different reported indices correctly identify only 61-91% of the patients with microcytic anemia due to TT or IDA. As each index showed overlapping values in patients with TT and IDA, none of them was entirely satisfactory in discriminating between these conditions [26]. We calculated each discrimination index in the same patient groups with [beta]-TT and IDA. In addition to calculating the sensitivity, specificity, positive predictive value and negative predictive value of each discrimination index, we also calculated Youden's index in their differentiation between [beta]-TT and IDA.

In 2007, Suad et al. [10] in their research work applied and compared nine well-documented discriminant functions in a population of 153 confirmed cases of microcytic anemias and measured validity using Youden's index. They showed that the E&F index had the highest Youden's index value (98.2%) in correctly differentiating between IDA and [alpha]--and [beta]-thalassemia minor, while the S&L index was found ineffective in differentiating between microcytic anemias in their study. Finally, they concluded that the E&F index showed with great sensitivity and specificity to be the best discriminant function to differentiate between IDA and thalassemia minor cases. Our data showed that the S&L index had the highest Youden's index, with a value of 89%, in correctly differentiating between IDA and [beta]-thalassemia minor among patients aged younger than 10 years, while RDW and RBC count indices showed the highest Youden's index, with values of 93% and 90%, respectively, in the patients older than 10 years.

[FIGURE 1 OMITTED]

In another research work, Ntaios et al. [11] examined the diagnostic accuracy of six discrimination indices in the differentiation between IDA and [beta]-TT. He calculated these indices in 373 patients (205 men, 168 women) with [beta]-TT and 120 patients (50 men, 70 women) with IDA, as well as their sensitivity, specificity, positive and negative prognostic value, efficiency, and Youden's index. The G&K index showed the highest reliability, followed by E&F, RBC count, MI, and RDWI. On the contrary, RDW completely failed to differentiate between IDA and [beta]-TT. G&K proved to be the most reliable index, as it had the highest sensitivity (75.06%), efficiency (80.12%), and Youden's index (70.86%) for the detection of [beta]-TT. In comparison, our data showed Youden's indices in decreasing order in patients younger than 10 years to be as follows: S&L > RBC > SI > MI > E&F > MDHL > G&K > RDWI > RDW > MCHL, and in patients older than 10 years as: RDWI > RBC > MI > E&F > SI > MDHL > RDW > S&L > G&K > MCHL. S&L, with the highest efficiency (92%), sensitivity (100%) and YI (89%) in the former group, and RDWI, with highest efficiency (93%), sensitivity (95%) and YI (86%) in latter group, proved to be the most reliable indices.

In recent research work published by Beyan et al. [9] in 2007, they attempted to evaluate the predictive value of these indices in the differential diagnosis of IDA and [beta]-TT in adult cases. The study consisted of 45 IDA cases (36 women and 9 men, age range 17-57 years) and 66 [beta]-TT cases (41 women and 25 men, age range 14-74 years). Patient groups were evaluated according to RBC, RDW, MI, S&L, E&F, SI, G&K, RDWI, and Ricerca index. Sensitivity, specificity, positive and negative predictive values, and Youden's index were calculated. They concluded that none of these formulations was superior to RBC value obtained from automated analyzers in adult cases with IDA and [beta]-TT, and that total body iron status and Hb[A.sub.2] level should be obtained for accurate differential diagnosis of IDA and [beta]-TT until more efficient tools are developed. Our data showed that some of those indices could be used as efficient tools for differentiating between these two disorders.

Youden's index takes into account both sensitivity and specificity and gives an appropriate measure of validity of a particular question or technique. In our study, Youden's index of RBC count and RDWI were the highest and they were the most reliable discrimination indices in differentiating [beta]-TT from IDA in patients older than 10 years. For patients younger than 10 years, the most reliable discrimination indices were RBC and S&L. These indices are not very important today given the availability of Hb electrophoresis. As physicians, how many of us memorize these formulas and use them in our daily practice in crowded outpatient settings? However, we do consider MCV and RBC and MI, which is easy to calculate. How many of us would be brave enough to not study Hb electrophoresis in a woman with mild hypochromic anemia unresponsive to iron therapy who was planning a pregnancy? Our calculated normal values of MCHD and MDHL were very close to those published by Telmissani et al. [23]. Although we found that mean and median values of MDHL in IDA were significantly lower than those in [beta]-TT and normal values, Youden's index of MDHL was not found to be adequately high in either age group.

In conclusion, in patients with microcytic anemia, if [beta]-TT or IDA is shown in a patient younger than 10 years with correct measures on S&L and RBC and in a patient older than 10 years with correct measures on RBC and RDW indices, the diagnoses are likely to be correct. However, in a small number of patients, it would still be necessary to study body iron status or Hb[A.sub.2] for accurate diagnosis.

Acknowledgement

This work was supported by the Research Centers of Thalassemia and Hemoglobinopathies and Physiology and the directors of these centers, Professors Mohammad Pedram and Alireza Sarkaki.

Received: November 10, 2007 Accepted: June 10, 2009

Gelis tarihi: 10 Kasim 2007 Kabul tarihi: 10 Haziran 2009

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Address for Correspondence: Dr. Fakher Rahim, Golsetan 1366, Ahwaz Jondishapour University of Medical Sciences, Ahwaz,Iran Phone: 00986113362411 E-mail: fakherraheem@yahoo.com

Fakher Rahim (1), Bijan Keikhaei (2)

(1) Research Center of Thalassemia & Hemoglobinopathies, Ahwaz Jondishapour University of Medical Sciences and Apadana Clinical Research Center, Apadana Private Hospital, Ahwaz, Iran

(2) Physiology Research Center, Ahwaz Jondishapour University of Medical Sciences, Ahwaz, Iran
Table 1. Hematological data of study groups

                                IDA
                               n = 170

                         Younger than 10 years
                               (n=114)

Hematological        Range      Mean [[+ or -] SD]
data

Hb (g/dl)           8.7-11.4      10.12 (0.72)
RBC                 3.7-5.49       4.45 (0.56)
MCV (n)              51-80.7      69.70 (0.36)
MCH (pg)           14.7-26.7      22.12 (3.31)
MCHC (g/dL)          22-35.1      31.14 (3.46)
RDW (%)            12.5-24.2      14.20 (4.21)
SI ([micro]g/dl)    3.8-31        20.03 (8.01)
SIBC                275-480      378.60 (70.01)
([micro]g/dl)
Ferritin (ng/ml)    0.9-9.2        4.23 (2.01)
TS (%)             0.68-8.1        3.81 (2.10)

                               IDA
                              n = 170

                         Older than 10 years
                              (n=56)

Hematological         Range     Mean [[+ or -] SD]
data

Hb (g/dl)           8.7-12.9      10.25 (1.30)
RBC                3.57-5.94       4.48 (0.97)
MCV (n)            57.1-84.8      71.64 (8.56)
MCH (pg)             16-27.6      22.16 (3.47)
MCHC (g/dL)          24-40.4      30.23 (9.19)
RDW (%)            11.4-30.2      15.60 (6.11)
SI ([micro]g/dl)    4.8-37        21.09 (8.42)
SIBC                315-570      429.70 (72.1)
([micro]g/dl)
Ferritin (ng/ml)    1.2-9.9        4.57 (1.99)
TS (%)             0.85-10.05      4.42 (2.28)

                            [beta]--TT
                              n=153

                        Younger than 10 years
                                (n=59)

Hematological       Range         Mean [[+ or -] SD]
data

Hb (g/dl)           9.1-12.25      10.45 (0.92)
RBC                 4.3-6.95        5.66 (0.58)
MCV (n)              50-75         58.25 (5.26)
MCH (pg)           16.4-24         18.59 (1.82)
MCHC (g/dL)          27-39.4       32.60 (2.9)
RDW (%)            10.5-21.1       11.79 (6.75)
SI ([micro]g/dl)     45-196        75.80 (29.1)
SIBC                224-425       321.70 (36.8)
([micro]g/dl)
Ferritin (ng/ml)     12-85.4       31.09 (6.79)
TS (%)             12.8-57         21.09 (1.15)

                            [beta]--TT

                        Older than 10 years
                             (n=94)

Hematological       Range       Mean [[+ or -] SD]
data

Hb (g/dl)           8.7-15.4       11.6 (1.42)
RBC                4.38-7.71        5.94 (0.7)
MCV (n)              54-80         61.41 (5.21)
MCH (pg)             15-26         19.45 (2.00)
MCHC (g/dL)        24.1-47.3       36.12 (4.37)
RDW (%)            10.1-25.1       15.12 (2.37)
SI ([micro]g/dl)     58-262        88.01 (33.6)
SIBC                257-478       345.50 (47.34)
([micro]g/dl)
Ferritin (ng/ml)     12-92         37.05 (7.34)
TS (%)               15-67         26.84 (1.23)

[beta]-TT: Beta thalassemia trait; Hb: Hemoglobin; IDA: Iron deficiency
anemia; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular
hemoglobin concentration; MCV: Mean corpuscular volume; RBC: Red blood
cells; RDW: Red blood cell distribution width; SI: Serum iron; SIBC:
Serum iron binding capacity; TS: transferrin saturation

Table 2. The differential values of each discrimination index and
correctly identified number of patients in the group younger than 10
years

Differential values          IDA           [beta]--TT
                           (n=114)           (n=59)

E & F
  IDA > 0                105 ([dagger])               17
  [beta]TT < 0             9               ([dagger]) 42
SI
  IDA > 3.8             ([dagger]) 105     ([dagger]) 11
  [beta]TT < 3.8                     9                48
RBC
  IDA < 5               ([dagger]) 101                 4
  [beta]TT > 5                      13     ([dagger]) 55
MI
  IDA > 13               ([dagger]) 97                 4
  [beta]TT < 13                     17     ([dagger]) 55
RDW
  IDA > 14                          99                29
  G & K< 14                         15                30
  IDA > 65               ([dagger]) 97                10
  [beta]TT < 65                     17     ([dagger]) 49
RDWI
  IDA > 220              ([dagger]) 82                 2
  [beta]TT < 220                    32     ([dagger]) 57
S&L
  IDA > 1530            ([dagger]) 101                 0
  [beta]TT < 1530                   13     ([dagger]) 59

Differential values   Total correctly    Percentage of correctly
                        identified       identified patients (%)
                      patients (n=173)
E & F
  IDA > 0                 147 (105+42)               85%
  [beta]TT < 0
  SI
  IDA > 3.8               153 (105+48)               89%
  [beta]TT < 3.8
RBC
  IDA < 5                 151 (101+50)               90%
  [beta]TT > 5
MI
  IDA > 13                152 (97+55)                88%
  [beta]TT < 13
RDW
  IDA > 14                129 (99+30)                74%
  G & K< 14
  IDA > 65                146 (97+49)                84%
  [beta]TT < 65
RDWI
  IDA > 220               139 (82+57)                81%
  [beta]TT < 220
S&L
  IDA > 1530              160 (101+59)               92%
[beta]TT < 1530

([dagger]) True positives; [beta]TT: Beta thalassemia trait; IDA: Iron
deficiency anemia; E & F: England and Fraser index; G & K: Green and
King index; MI: Mentzer index; RBC: Red blood cells; RDW: Red blood
cell distribution width; RDWI: Red blood cell distribution width index;
SI: Srivastave index; S & L: Shine and Lal index

Table 3. Sensitivity, specificity, positive predictive value (PPV),
negative predictive value (NPV) and Youden's index of each
discrimination index in prediction of IDA and 3TT groups younger than
10 years

Group        Sensitivity (%)   Specificity (%)   PPV (%)   NPV (%)

S&L
  IDA         89               100               100        82
  [beta]TT   100                89                82       100
RBC
  IDA         89                93                96        81
  [beta]TT    93                89                81        96
MI
  IDA         85                93                96        76
  [beta]TT    93                85                76        96
SI
  IDA         92                81                91        84
  [beta]TT    81                92                84        91
RDWI
  IDA         72                97                98        64
  [beta]TT    97                72                64        98
G&K
  IDA         85                83                90        74
  [beta]TT    83                85                74        90
E&F
  IDA         92                71                86        82
  [beta]TT    71                92                82        86
MDHL
  IDA         80                72                85        66
  [beta]TT    72                80                66        85
RDW
  IDA         87                51                77        67
  [beta]TT    51                87                67        77
MCHD
  IDA         96                 6                66        44
  [beta]TT     6                96                44        66

Group        Youden's Index

S&L
  IDA         89
  [beta]TT
RBC
  IDA         82
  [beta]TT
MI
  IDA         79
  [beta]TT
SI
  IDA         73
  [beta]TT
RDWI
  IDA         69
  [beta]TT
G&K
  IDA         68
  [beta]TT
E&F
  IDA         63
  [beta]TT
MDHL
  IDA         54
  [beta]TT
RDW
  IDA         38
  [beta]TT
MCHD
  IDA          2
  [beta]TT

[beta]TT: Beta thalassemia trait; E & F: England and Fraser index;
G & K: Green and King index; IDA: Iron deficiency anemia; MI: Mentzer
index; RBC: Red blood cells; RDW, Red blood cell distribution width;
RDWI: Red blood cell distribution width index; SI, Srivastave index;
S & L: Shine and Lal index

Table 4. The differential values of each discrimination index and
correctly identified number of patients in the group older than 11
years

Differential values     IDA        [beta]--TT     Total correctly
                       (n=56)         (n=94)    identified patients
                                                     (n=150)
RDWI
  IDA > 220           51 ([psi])    5              140 (51+89)
  [beta]TT < 220       5           89 ([psi])
RBC
  IDA < 5             47([psi])     6              135 (47+88)
  [beta]TT > 5         9           88 ([psi])
MI
  IDA > 13            45 ([psi])    5              134 (45+89)
  [beta]TT < 13       11           89 ([psi])
E&F
  IDA > 0             52 ([psi])   16              130 (52+78)
  [beta]TT < 0         4           78 ([psi])
SI
  IDA > 3.8           47 ([psi])   15              126 (47+79)
  [beta]TT < 3.8       9           79 ([psi])
RDW
  IDA > 14            50           26              118 (50+68)
  S<<14 6           68
IDA > 1530            13 ([psi])    1              106 (13+93)
  [beta]TT < 1530     43           93 ([psi])
G&K
  IDA > 65            52 ([psi])   45              101 (52+49)
  [beta]TT < 65        4           49 ([psi])

Differential values   Percentage of correctly
                      identified patients (%)

RDWI
  IDA > 220                    93%
  [beta]TT < 220
RBC
  IDA < 5                      90%
  [beta]TT > 5
MI
  IDA > 13                     89%
  [beta]TT < 13
E&F
  IDA > 0                      87%
  [beta]TT < 0
SI
  IDA > 3.8                    84%
  [beta]TT < 3.8
RDW
  IDA > 14                     79%
  S<<14 6
IDA > 1530                     70%
  [beta]TT < 1530
G&K
  IDA > 65                     67%
  [beta]TT < 65

[psi] True positives; [beta]TT: Beta thalassemia trait; IDA: Iron
deficiency anemia; E & F: England and Fraser index; G & K: Green and
King index; MI: Mentzer index; RBC: Red blood cells; RDW: Red blood
cell distribution width; RDWI: Red blood cell distribution width
index; SI: Srivastave index; S & L: Shine and Lal index

Table 5. Sensitivity, specificity, positive predictive value (PPV),
negative predictive value (NPV) and Youden's index of each
discrimination index in prediction of IDA and [beta]TT groups in
patients older than 11 years

Group        Sensitivity (%)   Specificity (%)   PPV (%)   NPV (%)

RDWI
  IDA        91                95                91        95
  [beta]TT   95                91                95        91
RBC
  IDA        84                94                87        91
  [beta]TT   94                84                91        87
E&F
  IDA        93                83                76        95
  [beta]TT   83                93                95        76
MI
  IDA        80                95                90        89
  [beta]TT   95                80                89        90
SI
  IDA        93                83                76        95
  [beta]TT   83                93                95        76
MDHL
  IDA        79                87                79        87
  [beta]TT   87                79                87        79
RDW
  IDA        89                72                66        92
  [beta]TT   72                89                92        66
G&K
  IDA        93                52                54        92
  [beta]TT   52                93                92        54
S&L
  IDA        23                99                93        68
  [beta]TT   99                23                68        93
MCHD
  IDA        96                 6                38        75
  [beta]TT    6                96                75        38

Group        Youden's Index

RDWI
  IDA        86
  [beta]TT
RBC
  IDA        78
  [beta]TT
E&F
  IDA        76
  [beta]TT
MI
  IDA        75
  [beta]TT
SI
  IDA        68
  [beta]TT
MDHL
  IDA        66
  [beta]TT
RDW
  IDA        62
  [beta]TT
G&K
  IDA        45
  [beta]TT
S&L
  IDA        22
  [beta]TT
MCHD
  IDA         2
  [beta]TT

[beta]TT: Beta thalassemia trait; E & F: England and Fraser index; G &
K: Green and King index; IDA: Iron deficiency anemia; MI: Mentzer
index; RBC: Red blood cells; RDW, Red blood cell distribution width;
RDWI: Red blood cell distribution width index; SI: Srivastave index;
S & L: Shine and Lal index

Table 6. The normal values of Mean Cell Hemoglobin Density (MCHD) and
Mean Density of Hemoglobin per Liter of blood (MDHL)

Variables                     Male
                     Group 1        Group 2

MCHD
  Median             0.32           0.32
Mean [[+ or -] SD]   0.32 (0.01)    0.319 (0.02)
MDHL
  Median             1.71           1.98
Mean [[+ or -] SD]   1.76 (0.22)    1.99 (0.28)

Variables                     Female
                     Group 1       Group 2

MCHD
  Median             0.32          0.31
Mean [[+ or -] SD]   0.32 (0.01)   0.31 (0.01)
MDHL
  Median             1.8           1.76
Mean [[+ or -] SD]   1.8 (0.19)    1.78 (0.17)

Group 1: Normal subjects who were younger than 10 years; Group 2:
Normal subjects who were older than 10 years

Table 7. Mean Cell Hemoglobin Density (MCHD) and Mean Density of
Hemoglobin per Liter of blood (MDHL) according to age groups and sex in
IDA and [beta]TT

                       Patients younger than 10 years

                                Males
Differential
values         IDA   [beta]TT   Total        Percentage
                                correctly    of correctly
                                identified   identified
                                patients     patients (%)
MCHD
  IDA          38    27
  [beta]TT      1     2         40 (38+2)    58%
  Total        39    29

MDHL
  IDA          39    13
  [beta]TT      0    16         55 (39+16)   80%
  Total        39    29

                       Patients younger than 10 years

                                  Females
Differential
values           IDA   [beta]TT   Total         Percentage
                                  correctly     of correctly
                                  identified    identified
                                  patients      patients (%)
MCHD
  IDA           72     28
  [beta]TT       4      2         74 (72+2)     69%
  Total         76     30

MDHL
  IDA            4      3
  [beta]TT      22     27      81 (54+27)       77%
  Total         76     30

                       Patients older than 10 year

                                 Males
Differential
values         IDA    [beta]TT   Total        Percentage
                                 correctly    of correctly
                                 identified   identified
                                 patients     patients (%)
MCHD
  IDA          4      41
  [beta]TT     0       5          9 (5+4)     18%
  Total        4      46

MDHL
  IDA          3       9
  [beta]TT     1      37         40 (3+37)    80%
  Total        4      46

                       Patients older than 10 year

                                 Females
Differential
values         IDA   [beta]TT   Total         Percentage
                                correctly     of correctly
                                identified    identified
                                patients      patients (%)
MCHD
  IDA          50    47
  [beta]TT      2     1         51 (50+1)     51
  Total        52    48

MDHL
  IDA          41     3
  [beta]TT     11    45         86 (41 +46)   86%
  Total        52    48
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