Discriminant Functions In Distinguishing Beta Thalassemia ...

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ISPUB.COM The Internet Journal of Hematology Volume 7 Number 2 1 of 6 Discriminant Functions In Distinguishing Beta Thalassemia Trait and Iron Deficiency Anemia: The value of the RDW-SD T P, S A Citation T P, S A. Discriminant Functions In Distinguishing Beta Thalassemia Trait and Iron Deficiency Anemia: The value of the RDW-SD. The Internet Journal of Hematology. 2010 Volume 7 Number 2. Abstract The most commonly encountered disorders with mild microcytic anemia are iron deficiency anemia (IDA) and β-thalassemia trait (BTT). It is important to distinguish between IDA and BTT to avoid unnecessary iron therapy and the development of hemosiderosis. Red blood cell counts red blood cell distribution (RDW) and other discriminant functions calculated by blood cell analyzers have been helpful in distinguishing between the two disorders. In this study we evaluated ten discriminant functions to determine their ability to distinguish between IDA and BTT. A total of 216 cases of microcytic hypochromic anemia were studied. We were especially interested in comparing the effectiveness of the RDW-SD, a recently introduced function, and the RDW-CV, currently used in most clinical laboratories. The RBC count and E & F Index were found to be very useful functions while the RDW-CV was ineffective. However, the study revealed that the RDW-SD was the most useful discriminant function for distinguishing IDA from BTT. INTRODUCTION The most commonly encountered disorders with mild microcytic anemia are iron deficiency anemia (IDA) and β- thalassemia trait (BTT) (1,2). The gene frequency of β- 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. The incidence of β- thalassemia trait is particularly high in Jamnagar district, Gujarat, India with over 250 cases of β- thalassemia major registered in our hospital. The differential diagnosis between IDA and BTT is important to avoid unnecessary iron therapy. Iron therapy would be indicated in IDA but usually contraindicated in BTT. Standard tests used for the differentiation between IDA and β-thalassemia trait include: complete blood count (CBC), serum iron, serum ferritin, total iron binding capacity (TIBC), bone marrow iron stores, HbA 2 levels, free erythrocyte protoporphyrin and zinc protoporphyrin levels (3-8). Despite their usefulness, these standard tests are often expensive and time consuming. Routine electronic red blood cell counts and other discriminant functions derived from modern blood cell analyzers can be rapidly obtained, are inexpensive, and can help distinguish between IDA and BTT. There is controversy about which discriminant functions are best in identifying IDA and BTT. Also, there are reasonable questions as to whether discriminant functions are sufficiently accurate to warrant not ordering serum ferritins and other standard tests to detect iron deficiency (5, 6). . In this study we evaluated ten discriminant functions for their ability to distinguish between IDA and BTT in 216 cases of microcytic hypochromic anemia. We were especially interested in determining the value of a recently introduced discriminate function, RDW-SD. MATERIALS AND METHODS The study was carried out at Outdoor Patient Hematology Laboratory, Pathology Department, M P Shah Medical College, Jamnagar in co-operation with two voluntary organizations named Indian Red Cross Society, Jamnagar Branch and Jamnagar Thalassemic Society. The study groups included were: 1) The relatives of known thalassemia Major Patients (as a part of family screening); 2) Antenatal patients coming to Gynecology and Obstetrics Department of our Hospital. Screening for thalassemia is compulsory for antenatal cases in our hospital; 3) Thalassemia screening camps held in high risk communities in our area. A total of 216 subjects (age<30 years) with confirmed microcytic anemia were included in the study. Anemia was

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ISPUB.COM The Internet Journal of HematologyVolume 7 Number 2

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Discriminant Functions In Distinguishing Beta ThalassemiaTrait and Iron Deficiency Anemia: The value of the RDW-SDT P, S A

Citation

T P, S A. Discriminant Functions In Distinguishing Beta Thalassemia Trait and Iron Deficiency Anemia: The value of theRDW-SD. The Internet Journal of Hematology. 2010 Volume 7 Number 2.

Abstract

The most commonly encountered disorders with mild microcytic anemia are iron deficiency anemia (IDA) and β-thalassemia trait(BTT). It is important to distinguish between IDA and BTT to avoid unnecessary iron therapy and the development ofhemosiderosis. Red blood cell counts red blood cell distribution (RDW) and other discriminant functions calculated by blood cellanalyzers have been helpful in distinguishing between the two disorders. In this study we evaluated ten discriminant functions todetermine their ability to distinguish between IDA and BTT. A total of 216 cases of microcytic hypochromic anemia were studied.We were especially interested in comparing the effectiveness of the RDW-SD, a recently introduced function, and the RDW-CV,currently used in most clinical laboratories. The RBC count and E & F Index were found to be very useful functions while theRDW-CV was ineffective. However, the study revealed that the RDW-SD was the most useful discriminant function fordistinguishing IDA from BTT.

INTRODUCTION

The most commonly encountered disorders with mildmicrocytic anemia are iron deficiency anemia (IDA) and β-thalassemia trait (BTT) (1,2). The gene frequency of β-thalassemia, however, is high and varies considerably fromarea to area, having its highest rate of more than 10% aroundthe Caspian Sea and Persian Gulf. The incidence of β-thalassemia trait is particularly high in Jamnagar district,Gujarat, India with over 250 cases of β- thalassemia majorregistered in our hospital.

The differential diagnosis between IDA and BTT isimportant to avoid unnecessary iron therapy. Iron therapywould be indicated in IDA but usually contraindicated inBTT. Standard tests used for the differentiation betweenIDA and β-thalassemia trait include: complete blood count(CBC), serum iron, serum ferritin, total iron binding capacity(TIBC), bone marrow iron stores, HbA2 levels, free

erythrocyte protoporphyrin and zinc protoporphyrin levels(3-8). Despite their usefulness, these standard tests are oftenexpensive and time consuming.

Routine electronic red blood cell counts and otherdiscriminant functions derived from modern blood cellanalyzers can be rapidly obtained, are inexpensive, and canhelp distinguish between IDA and BTT. There iscontroversy about which discriminant functions are best in

identifying IDA and BTT. Also, there are reasonablequestions as to whether discriminant functions aresufficiently accurate to warrant not ordering serum ferritinsand other standard tests to detect iron deficiency (5, 6). .

In this study we evaluated ten discriminant functions fortheir ability to distinguish between IDA and BTT in 216cases of microcytic hypochromic anemia. We wereespecially interested in determining the value of a recentlyintroduced discriminate function, RDW-SD.

MATERIALS AND METHODS

The study was carried out at Outdoor Patient HematologyLaboratory, Pathology Department, M P Shah MedicalCollege, Jamnagar in co-operation with two voluntaryorganizations named Indian Red Cross Society, JamnagarBranch and Jamnagar Thalassemic Society.

The study groups included were: 1) The relatives of knownthalassemia Major Patients (as a part of family screening); 2)Antenatal patients coming to Gynecology and ObstetricsDepartment of our Hospital. Screening for thalassemia iscompulsory for antenatal cases in our hospital; 3)Thalassemia screening camps held in high risk communitiesin our area.

A total of 216 subjects (age<30 years) with confirmedmicrocytic anemia were included in the study. Anemia was

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defined as a hemoglobin concentration of at least 2 standarddeviations lower than age- and sex-specific average. IDAand BTT were diagnosed by the following tests: serum ironlevels, serum TIBC levels and HbA2 levels performed by

chromatography.

We did not test for serum ferritin levels since we do not havefacilities to perform this assay. We retested the patients

having borderline HbA2 values and low serum iron levelsafter giving them iron replacement therapy.

The following hematological data were obtained:Hemoglobin, Red cell count, Hematocrit, MCV, MCH,

MCHC, RDW-CV, RDW-SD, Serum iron, HbA2.

Figure 1

Figure 1: RDW-CV and RDW-CV (From Sysmex -Operating Manual)

Figure 1 demonstrates how RDW-CV and RDW-SD werecalculated (CV = coefficient of variance. SD = one standarddeviation). A Sysmex KX-21, three-part differentialanalyzer, (India, Pvt, Ltd., Mumbai, India) was used toobtain data

The data were analyzed individually using the tenmathematical discriminant functions: Mentzer Index, MI,Shine and Lal (S & L) Index, England and Fraser (E & F)Index, Green and King (G & K) Index, RDW, Index(RDWI), Srivastava Index (SI), Ricerca Index (RI)

The sensitivity, specificity, positive predictive values,negative predictive values and the Youden’s indices for eachof the 10 discriminant functions were calculated. The

Youden's index provides an appropriate measure of validityof a particular technique or question by taking into accountboth sensitivity and specificity (7,8).

RESULTS

The mean and standard deviations of various hematologicaland biochemical parameters of all patients are shown inTable 1a. Table 1b displays the cut-offs for RBC Indices andRDW. All parameters for IDA and BBT except serum ironwere significantly different as determined by an independentt test analysis as shown in Table 2. A serum ferritin mighthave been a more reliable parameter.

Eighty one patients were diagnosed IDA based low serumiron levels, high serum TIBC levels, response to iron therapyand absence of HbA2 elevation (<3.5%). One hundred and

thirty five patients were diagnosed to have beta thalassemiatrait based on absence of evidence of iron deficiency andelevated

Figure 2

Figure 3

HbA2 (>3.5 %). Alpha thalassemia cases could be missed

since HbA2 not elevated in this condition. However, we do

not believe that any of the patients in the study had alphathalassemia.

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The ability of 10 discriminant functions to identify IDA andBTT is shown in Table 2. Formulas for discriminantfunction that were used are shown in Table 3. The numberand percent of IDA and BBT identified by each discriminantfunction is shown. The percent of IDA and BBT identifiedby discriminant functions was based on the numberidentified by the discriminant function/ the total number ofIDA and BBT. Only 4 of the 10 discriminant functionsdetected more that 90% of IDA (E&F, RBC count, RDW-CFand RDW-SD). Only 3 discriminant functions detected morethat 90% BBT (S&K, SI and RDW-SD). .

.Table 4 lists the effectiveness of each discriminant functionto distinguish BTT and IDA. The following were analyzed:sensitivity, specificity, positive and negative predictive valueand the Youden index. All of the functions showedoverlapping in the patients with BTT and IDA. None of thefunctions was completely sensitive or specific indifferentiation between BTT and IDA. Youden's index indecreasing order was as follows: RDW-SD > RBC COUNT> E & F > MI > RI > RDWI > G & K > SI > S & L > RDW-CV.

Figure 4

TABLE 2: Number of cases correctly identified by the tendiscriminant functions

Figure 5

Table 3: Formulas for Discriminant Functions

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TABLE 4: Sensitivity, specificity, positive and negativepredictive value, and Youden's Index of each discriminantfunction used in differentiation between IDA and BTTDiscussion

The most frequently encountered conditions presenting withmild microcytic anemia are BTT and IDA. It is important todistinguish between BTT and IDA to avoid unnecessary irontherapy and to offer genetic counseling to individuals withBTT. The diagnosis of BTT can be established by familyhistory of HbA2 levels (9). Decreased serum iron, increased

levels of TIBC and serum ferritin are the standard diagnosticcriteria for IDA (10).

Discriminant functions such as the RBC count and RDW can

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be used to help identify IDA and BTT. These functions thatare calculated when routine blood counts are done by bloodanalyzers are less time-consuming methods and lessexpensive that the standard tests for diagnosing thesedisorders.

In 2007, Suad et al. (11) compared nine well-documenteddiscriminant functions in a population of 153 confirmedcases of microcytic anemia. They concluded that the E&Findex was the best discriminant function to differentiatebetween IDA and thalassemia minor cases

In 2007, Beyan et al. (12). attempted to evaluate thepredictive value of the following discriminant functions inthe differential diagnosis of IDA and BTT in adults: RBCCounts, RDW, MI, S&L, E&F, SI, G&K, RDWI, andRicerca index.. They concluded that none of theseformulations was superior to RBC Count. The authorssuggested that total body iron status and HbA2 levels should

be obtained to diagnose of IDA and BTT until more efficienttools are developed.

We carried out an comprehensive evaluation of the ability ofdiscriminant functions to distinguish IDA and BTT. Inaddition to calculating the sensitivity, specificity, positivepredictive value and negative predictive value of eachdiscriminant function, we also calculated Youden's index.The Youden's index takes into account both sensitivity andspecificity and therefore provides a reliable measure todetermine that validity of a technique. The Youden's Indexintroduces the least bias to the measure of effect (13)..Positive and negative predictive values are independent ofthe Youden Index and depend strongly on the underlyingprevalence of the disease.

Our data indicates that the 3 discriminant functions were theextremely very useful for distinguishing IDA and BTT.RDW-SD had the highest rating, RBC Count was 2nd bestand E & F is 3rd best in identifying these disorders. This wasbased on the the Youden index results as well as positive andnegative predictive values. Our data was consistent with thestudy of Saud et al. (11 ) who found that the E & F and withthe study of Beyen et al. (12) who reported that the RBCCount was the best discriminate function for distinguishingIDA and BTT.

Our study is unique in that it found that RDW-SD was themost useful functions to distinguish between IDA and BTTwhile the RDW-CF was ineffective. When calculatingRDW-CV and RDW-SD, the same particle size distribution

of RBC sizes are analyzed, but the functions are calculatedby a different formulas as shown in Figure 1. The RDW-SDcalculation includes a wider range of red blood cell sizes asshown in Figure 1.

Our findings are consistent with the previously reportedobservation that there are differences in the sensitivities ofthe RDW-SD and the RDW-CV and that the RDW-SD moreaccurately reflects the actual blood cell size variation asmeasured by the red blood cell distribution curve (14).

Since RDW-CV has been used in virtually all previouslystudies, it is not surprising that that there is considerablecontroversy about the value of the RDW-CV as adiscriminate function for differentiating between IDA andthalassemia minor (7,8,15-20).

CONCLUSION

The RDW-SD appears to be the best discriminant functionfor distinguishing between IDA and BTT. The RBC countand E & F Index are useful functions for that purpose.However, the RDW-CV was ineffective for this purpose.The fact that RDW-SD is calculated differently than theRDW-CV most likely accounts for it apparent greater valuein distinguishing between IDA and BTT.

It would be important to continue studying the value of theRDW-SD not only in distinguishing IDA and BTT but alsoin characterizing other anemias. Ideally, our study shouldhave serum ferritin levels to identify IDA. Nevertheless, webelieve our findings are meaningful and should stimulate theevaluation of the RDW-SD in clinical and research setting.Most medical centers report RDW based on RDW-CVcalculations.

The decision to rely solely on discriminant functions todetect IDA and BTT is a clinical decision. However, as weobtain more experience with the RDW-SD and other newdiscriminant functions, they might have major roles in theseclinical decisions. Discriminant functions would be helpfulin distinguishing alpha-thalassemia for IDA since HbA2

levels are not elevated in this disorder.

References

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Author Information

Trivedi Dhara PAssociate Professor, Department of Pathology, M P Shah Medical College

Shah Harsh AResident Doctor, Department of Pathology, M P Shah Medical College