Experiences With Sickle Hemoglobin Testing in the Texas Newborn Screening Program

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I

American Academy of Pediatrics.

Experiences

With Sickle

Hemoglobin

Testing

in

the Texas

Newborn

Screening

Program

Bradford

L. Therrell,

Jr, PhD,

Jennifer

L. Simmank,

BS, and

Mae

Wilbom,

RN, MAHS

From the Texas Department of Health, Austin

In 1965, the Texas Department of Health became

responsible for “. . . a program designed to combat

mental retardation in children suffering from a

genetic defect which causes phenylketonuria.”

Similar programs were instituted in many other

states throughout the 1960s. These state screening

programs developed in response to demonstrations

by Dr. R. Guthrie of the ability to detect the

pres-ence or absence of phenylalanine in dried blood

spotted on filter paper.2 Throughout the years,

pro-cedures for detecting various other metabolic

dis-orders from filter paper specimens were developed;

however, low incidences combined with economic

factors limit their inclusion in most screening pro-grams.

During the 1970s, automated procedures for

punching from filter paper were advanced. This

advancement, coupled with improved micro

meth-ods for detection of thyroxine and thyrotropin and

with reported incidences of congenital

hypothyroid-ism in newborns of one in 5,000, led most

screen-ing programs to seriously consider expanding to

include testing for hypothyroidism. Thus, in 1979,

the Texas law’ was changed to include other

herit-able diseases in addition to phenylketonuria.

Ad-ditionally, the Texas Board of Health was

empow-ered to “. . . adopt a rule specifying the heritable

testing for hypothyroidism, galactosemia, and

hom-ocystinuria.” The limit of four screening

proce-dures was defined on economic and logistical

grounds because automated punching limited the

technician to four 0.3175-cm (0.125-in) diameter

specimens from a single 1.3-cm (0.5-in) blood spot.

Punching a second time from another spot raises

the limit to eight tests: however, many additional

considerations (including personnel and

equip-ment) complicate this change.

Because only one patient with homocystinuria

was detected during the initial 5-year testing period

(approximately 1 million births), the Board of

Health decided to discontinue screening for this

disorder, electing instead to screen for sickle

he-moglobin. This decision was made only after

tech-nical considerations indicated that such screening

was feasible from blood spot specimens. Thus, on

Nov 1, 1983, screening for sickle hemoglobin began

at the Texas Department of Health.

PROCEDURE

The department’s newborn screening program

operates according to published rules provided for

in the enabling legislation. As one of the heritable

diseases included in the newborn screening

pro-gram, sickling hemoglobinopathy testing is required on all initial specimens (ie, first specimens collected

from babies younger than seven days of age).

Sic-kling hemoglobinopathies are defined as inherited

conditions (including sickle cell disease), which, if not known at the time a physician treats an already

ill child, may lead to a fatal outcome. The rules

require that all newborns be tested unless the

par-ent or guardian objects on the basis of religious

conflict or certain medical considerations indicate

the need to delay testing. Additionally, no

physi-cian, technician, or person who provides testing can

be liable because of the failure or refusal of the

parent to give permission for the test. Ensuring

that testing is performed is the primary responsi-bility of the physician or nonphysician attending

the newborn. The blood specimen is to be taken

from a peripheral location onto filter paper accord-ing to instructions set forth by the department.

Currently, it is recommended that all newborns be

tested a second time when 1 to 4 weeks of age. This second testing is required if the initial screening

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SUPPLEMENT 865 protein for 24 hours or before the infant attained

36 hours of age.

Approximately 2,500 specimens are analyzed daily by the laboratory. Specimens arrive through the mail and are screened for acceptability and are assigned laboratory identification numbers. For hemoglobinopathy testing, 0.3175-cm diameter spots are punched into dimpled trays. Initial he-moglobinopathy screening is performed by using a standard cellulose acetate and citrate agar electro-phoretic protocol similar to that used by the New

York Department of Health5 and detailed earlier by Garrick et al.6 Despite more than 10 years of

experience with hemoglobinopathy testing on high

volumes of liquid whole blood specimens, initial attempts at analysis and interpretation of results from dried blood specimens on filter paper met with

extreme difficulty. The critical change in procedure

necessary for quality results involved the use of backlighted-viewing boxes.

Specimens are initially prepared by adding com-mercially obtained hemolysate reagent (Helena

Laboratories, Beaumont, TX) to each specimen well, and elution occurs overnight. Cellulose acetate electrophoresis is performed at 460 V for 17 minutes

using two rows of applications on Mylar-backed cellulose acetate cards (Gelman Sciences, Ann Ar-bor, MI). Voltage and times vary with commercial products in use. Reading and interpretation of the

Ponceau S-stained cards is performed

independ-ently by at least two technicians using homemade,

backlighted-viewing boxes. Specimens exhibiting

abnormal or questionable results are reaccessed,

repunched, and reanalyzed by both cellulose acetate and citrate agar electrophoresis. Citrate agar-im-pregnated cellulose acetate cards are routinely used for this confirmation.7 There are occasional

migra-tion problems apparently associated with different lots of agar, which may require the use of both commercial and homemade agar for final determi-nations. Recently, isoelectric focusing procedures used by the Maryland Department of Health8 have

been adopted for those instances in which definitive

interpretations with standard techniques are not

possible. Isoelectric focusing appears to give reliable results, and specimen degradation with time does not present the difficulties in band evaluation

en-countered with cellulose acetate.

Computer-generated mailers are used, and

labo-ratory results are reported to the address indicated

by the submitting office. The Bureau of Maternal and Child Health is also notified of all abnormal results for follow-up and tracking. If a clinically significant disorder is detected, the patient’s phy-sician is notified of the findings by telephone and

by follow-up letter. He or she is requested to con-firm the hemoglobin disorder as soon as possible

and additionally is advised of the department’s

ability to provide testing for other family members.

According to a specific protocol,9 developed in

co-operation with an external advisory committee, fur-ther physician contact occurs at 4 weeks and again at 4 months (the time at which confirmational electrophoresis is recommended). Upon confirma-tion of a significant disorder, the patient’s physician is asked to complete a questionnaire from which

the Bureau of Maternal and Child Health obtains

program assessment data. This material is updated

annually through the use of follow-up

question-naires.

Since July 1, 1985, patient demographic and re-sult information has been captured by computer for all newborn screening specimens. These data have been surveyed relative to result findings according to race as illustrated in Tables 1 and 2. All racial

assignments were taken from specimen submittal

forms, and no correlation has been made between

this information and that recorded officially on birth certificates. Because approximately 85% of all infants receive a second screening test in Texas,

duplication of the data analyzed was limited by

considering only specimens denoted as “initial” by

the submitter. Specimens having no indication as to “initial” or “follow-up” status were assumed to be “initial” if the collection date was before 2 weeks

after the birth date. Such specimens were included

in this analysis.

The raw data relative to laboratory result

inter-TABLE 1. Abnormal Laboratory Findings by Racial Assignments of Submitter: July 1985 to February 1987

Race Hb S Hb C Hb D Hb E Other Sickle Hb 5, C Homozygous Homozygous Homozygous Homozygous Total

Trait Trait or G Trait Hemoglobin Cell Disease Hb C Hb D or G Hb E Hb F Screened

Trait Trait* Disease

120 231 25 411 66 95 5 27 6 2 6 18 27 47 51 10 37 177 2 114 23

White 426 110 3 6 0 53 0 15 298,748

Black 5301 1687 175 59 17 12 0 13 75,007

Hispanic 769 88 4 0 0 21 0 8 154,000

Asian 14 2 0 0 0 2 1 0 5,951

Indian 2 0 0 0 0 1 0 0 1,236

Other 35 6 1 0 0 1 0 0 5,514

No race given 292 73 5 1 0 4 0 2 41,537

* Refers to hemoglobins other than A, F, 5, C, G, E, 0, or D.

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TABLE 2. Abnorm al Lab orato ry Findings by Rac ial Assi gnments of Submitte r: July 1985 to February 1987*

Race Hb S

Trait Hb C Trait Hb D or G Trait Hb E Trait Other He-moglobin Trait Sickle Cell Disease

Hb 5, C Disease

Homozygous Hb C

Homozygous Hb D or G

Homozygous Hb E

Homozygous Hb F

White 1.4 0.4 0.4 0.2 0.8 0.2 <0.1

Black 70.7 22.5 0.3 0.1 5.5 2.3 0.8 0.2 0.2 0.2

Hispanic 5.0 0.6 0.4 0.2 0.6

Asian 2.4 18.7t 4.5

Indian

Other 6.3

No race given 7.0 1.8 0.7

20.7 0.6

3.3 1.1

* Results are numbers of samples per 1,000 samples tested. No incidence was calculated if less than ten findings were reported.

t Value recalculates to 29.7 if assuming all specimens marked “other” for race are Asian. Preliminary data suggest that this is the case.

pretation are given in Table 1. These data have

been analyzed for incidence, as shown in Table 2, in those instances in which at least ten specimens

were observed to exhibit the same result. The

mci-dences of apparent hemoglobin disorders in blacks correlate well with various US data summarized

elsewhere.1#{176} Data comparisons must recognize the

limitations of the laboratory screening protocol with regard to detecting possible instances of S-/3-thalassemia, S with high F, etc. Although three

instances of apparent sickle cell disease were noted

in whites, subsequent follow-up has determined the

whites to be more properly defined as black.

mci-dences of apparent sickle cell disease among His-panics closely parallel those reported in the Los Angeles area” on a much smaller population. The

apparent incidence of C trait, however, is slightly

lower (0.6% v 0.9%). A significant number of Asian

refugees relocated in Texas following the

Vietnam-ese conflict and, of the almost 6,000 screened thus far whose race was noted as Asian, evidence of Hb

E trait has been noted in about 1.9%. Of the Hb E

trait specimens reported with no racial identifica-tion, program follow-up has found those contacted to be exclusively of Asian descent. Thus, the actual

percentage will likely approach the 2.9% to 3.0%

range (2.9% is the incidence obtained when

assum-ing 100% of those whose race was marked “other”

were actually of Asian descent).

To further assess the validity of the analytical

procedures on dried blood collected on filter paper,

we are currently involved in the “blind” analysis of

approximately 1,000 specimens in cooperation with

Drs Honig and Rosenblum at the University of

Illinois College of Medicine in Chicago. Preliminary results of that study based on the first 395

speci-mens analyzed show 97% agreement in interpreta-tion between our filter paper results and theirs

obtained on liquid cord blood. If the liquid results are assumed to be correct, filter paper analysis has

thus far yielded a false-negative rate of 0.5% and

false-positive rate of 2.5%. There has been 13.6%

agreement in observing Hb Bart’s. Preliminary

in-dications are that successful identification of Hb

Bart’s routinely occurred only if the initially

ob-served concentration was in excess of 7%. Detailed

analyses of data from this study have not yet been

made, but the results thus far favor validity of the filter paper analytical procedures used. Although it

would be preferable to detect all instances of the

presence of Hb Bart’s, it must be reemphasized that

our newborn screening program is primarily for the

detection of sickle hemoglobin, and the procedures

were designed accordingly. This cooperative study

has not currently addressed the isoelectric focusing

procedure, but its inclusion is anticipated.

CONCLUSION

Since Nov 1, 1983, we have tested more than 1

million infants for sickling hemoglobinopathies.

The laboratory procedures used for analysis of the

filter paper blood spots include a combination of

cellulose acetate and citrate agar electrophoresis. Isoelectric focusing offers an alternative screening procedure. Satisfactory interpretations of

electro-phoretic patterns are generally acceptable on

spec-imens analyzed within five days of collection.

Spec-imen age does not seem to be as critical a factor

with isoelectric focusing. Consumable supply costs

are slightly more with isoelectric focusing protocol

(approximately $0.50 u $0.25), however, and the

technical complexity and time involved are also

somewhat higher.

We have found the incidences to be as expected.

Annual births in Texas number approximately

42,000 blacks, more than 90,000 Hispanics, and

more than 175,000 whites. The newborn screening

program is detecting approximately 100 cases of

sickle cell anemia, 40 cases of SC disease, and 4,200

sickle hemoglobin carriers annually. Although the

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SUPPLEMENT 867

effectiveness of this program will take some time

to accumulate, support for its continuation from

the physician community and the general public

appears widespread. Follow-up of disease

condi-tions is an integral part of the protocol, and the

involvement and recommendations of an advisory

committee, including qualified pediatric

he-matologists, have proven extremely beneficial. Both educational literature and treatment protocols have been addressed by the department and its advisers.

Although the program still lacks a strong genetic

counseling effort, and there are considerable

com-munication and transportation problems associated

with the state’s geography, the Texas Department

of Health remains dedicated to improving the

pub-lic health of infants throughout the state with its

quality newborn screening program.

ACKNOWLEDGMENT

The authors thank Virginia Enriquez and Dr Barnett B. Rosenblum for help with the preparation of this man-uscript.

REFERENCES

1. Tex Rev Civ Stat Ann, article 4447e

2. Guthrie R, Susi A: A simple phenylalanine method for

detecting phenylketonuria in large populations of newborn infants. Pediatrics 1963;32:338-843

3. Dussault JH, Coulombs P, Laberge C: Preliminary report on a mass screening program for neonatal hypothyroidism.

J Pediatr 1975;86:670-674

4. Fisher DA, Dussault JH, Foley TP Jr, et al: Screening for congenital hypothyroidism: Results of screening one million North American infants. J Pediatr 1979;94:700-705

5. Pass KA, Gauvreau AC, Schedlbauer L, et al: Newborn screening for sickle cell disease in New York state: The first decade, in Carter TP, Willey AM (eds): Genetic Disease: Screening and Management. New York, Alan R. Liss, Inc, 1986, pp 359-372

6. Garrick MD, Dembure P, Guthrie R: Sickle cell anemia and other hemoglobinopathies: Procedures and strategy for screening employing spots of blood on filter paper as speci-mens. N EngI J Med 1973;288:1265-1268

7. Schneider RG, Hosty TS, Tomlin et al: Identification of hemoglobins and hemoglobinopathies by electrophoresis on cellulose acetate plates impregnated by citrate agar. Clin Chem 1974;20:74-77

8. Corcoran L, Patel J, Panney SR, et al: Neonatal sickle-cell hemoglobinopathies screening from blood spots on filter paper, in Therrell BL (ed): Advances in Neonatal Screening,

Excerpta Medica ICS 741. Amsterdam, Elsevier Science Publishers, 1987, pp 435-436

9. Wilborn M, Pierson E, Therrell BL Jr, et al: Newborn screening for sickle hemoglobin in Texas, in Therrell BL

(ed): Advances in Neonatal Screening, Exerpta Medica ICS

741. Amsterdam, Elsevier Science Publishers, 1987, pp 429-432

10. Winter WP: Hemoglobin variants in the United States, in Winter WP (ed): Hemoglobin Variants in Human

Popula-tions. Boca Raton, CRC Press, 1986, vol 1, pp 49-69 11. Ewing N, Powars D, Hilburn J, et a!: Newborn diagnosis of

abnormal hemoglobin from a large municipal hospital in Los Angeles. Am J Public Health 1981;71:629-631

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1989;83;864

Pediatrics

Bradford L. Therrell, Jr, Jennifer L. Simmank and Mae Wilborn

Program