'Components are listed in order of decreasing concentration. FSA means F>S>A. HPFH hereditary persistence of fetal hemoglobin.

=

2These are only examples; a parent could also be a homozygote or a compound heterozygote. For example, an SS child could have parents who are AS and SC; AS and SS; or SS and SS, etc. Similar relationships hold for hemoglobins C and E. CC and EE diseases cause mild anemia, as does C thalassemia. E thalassemia is a serious illness, but it is rare in this country, except in persons of Southeast Asian heritage.

kept and assessed regularly to assure that all infants identified with possible disease are located and provided with followup that includes confirmation of the screening result and assignment of a definitive diagnosis. Since followup may be difficult (Miller, Stilerman, Rao, et al., 1990), lines of responsibility must be clearly drawn.

Whether the infant's parents should be notified directly depends largely on local circumstances or law. Sending such a report assures that the parents are informed, but if prior education about screening was inadequate or absent, unnecessary anxiety may be created by the report. Reports to parents should be delivered by mail. In those instances where disease is suspected, a telephone call to parents may be used. There are advantages to making the call either before or after the estimated arrival of the letter. The person making the telephone call must be specially trained. Reports to providers and/or parents should include a preliminary interpretation of the hemoglobin phenotype and stress the need for immediate medical followup. The reports should indicate where additional information can be obtained or list State, community, and other agencies that can provide such information. Followup and definitive diagnosis of infants suspected of having disease, however, is the responsibility of the infant's physician. Definitive diagnosis requires repeat testing to confirm the initial hemoglobin phenotype and additional studies to assign a probable (or definitive) genotype. Either venous or capillary blood can be used, but the larger sample obtained from venipuncture is recommended. Tables 11 and 12 compare various methods for sample collection and testing.

Definitive diagnosis should be established by a two-tier testing routine, immunoassays, or DNA analysis. Additional tests must include examination of the infant's blood smear and measurement of the infant's hemoglobin concentration and red cell indices. When both parents are

Table 11. Comparison of sample types for confirmation tests of

available for testing and agree to be tested, characterization of the parents' hemoglobin phenotype and their levels of Hb A, and Hb F can facilitate a definitive diagnosis for the infant. When one or both parents are unavailable for study, analysis of the infant's ẞ globin gene complex by DNA/RNA methods may yield a definitive diagnosis.

The parent should be given an identification card bearing the child's definitive diagnosis, the facility that made the diagnosis, and sufficient information to permit rechecking of the laboratory records at a later date.

Results of all confirmatory tests should be reported to the administrative office of the screening program to ensure that program objectives are met. As a component of laboratory quality assurance, it is essential that confirmatory test results be reported to the screening laboratory.

The procedures for establishing a definitive diagnosis must be comprehensive enough to include not only infants with sickle cell disease, but also those who may have other hemoglobin disorders (Hb CC, Hb EE, or E ẞ-thalassemia) and those with other less common hemoglobin variants.

Quality Assurance and Quality Control

The laboratory must participate in a proficiency testing program and, when feasible, should retest at least a sample of all newborns screened to determine the sensitivity and specificity of its screening

methodology.

There are multiple sources of error in screening for sickle cell disease. These include:

Sampling errors, including incorrect labeling of sample, contamination of sample with maternal blood or transfusion, and improper collection. ■ Transportation errors, including delays and exposure to high

temperature.

Testing errors, including poor technique, improper methods, or incorrect interpretation.

■ Reporting errors secondary to patient misidentification or clerical errors. ■ Errors in retesting, similar to the above.

All testing must be done by laboratories licensed by their respective States and must meet the requirements of the Clinical Laboratory Improvement Act of 1988 (CLIA 88). As part of the CLIA requirements, laboratories must meet standards for quality control and must participate in an extramural proficiency testing program (Therrell, Panny, Davidson, et al., 1990). Such a program (ISQAL) for neonatal hemoglobinopathy testing has been established recently by the Centers for Disease Control. Of 37 States that participated, only 21 performed a two-tier test (Adam and Bell, 1992). Of the 36 laboratories that participated in the study, 35 (97%) recognized that a sample from a carrier of hemoglobin D did not contain hemoglobin S; but 2 of 36 (5.6%) laboratories misdiagnosed a sample from an infant with sickle cell anemia. Filter paper samples that had been in transit for 3 to 10 days showed the same percentages of hemoglobins F, A, D, C, and S as they had contained initially, confirming earlier reports of filter paper sample stability for this length of time.

Results of followup testing must be considered a part of the proficiency testing process and be included with the results of other proficiency tests. Based on the evidence presented, it is suggested that both

false-positive and false-negative reports should each be less than 0.01 percent of total samples.

The sensitivity and specificity of laboratory methods that detect sickle cell trait, other trait conditions, and the normal hemoglobin phenotype have not been defined for newborns because no studies have been done where infants with these conditions were retested at a later date. As part of the laboratory's quality assurance program, consideration should be given to developing a plan where statistically valid random samples from infants with all phenotypes would be retested to establish rates of sensitivity and specificity for the testing methods. The infants could be located through well-baby clinics. Careful explanations to the parents would be a requirement for such a program. While the cost of this type of program may be prohibitive, it would be desirable to ensure the accuracy of the screening methodology.

The evidence presented in this section suggests that cellulose acetate followed by citrate agar hemoglobin electrophoresis, isoelectric focusing, or high performance liquid chromatography are acceptable laboratory methods for screening newborns. The selection of a specific method should be based upon the personnel and financial resources available to the laboratory. The laboratory must function as a component of a comprehensive newborn screening program.