Hematopathology / FLOW CYTOMETRY IN HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

Flow Cytometric Measurement of Hemoglobin F in RBCs Diagnostic Usefulness in the Distinction of Hereditary Persistence of Fetal Hemoglobin (HPFH) and Hemoglobin S–HPFH From Other Conditions With Elevated Levels of Hemoglobin F
James D. Hoyer, MD, Connie S. Penz, Virgil F. Fairbanks, MD, Curtis A. Hanson, MD, and Jerry A. Katzmann, MD
Key Words: Flow cytometry; Hereditary persistence of fetal hemoglobin; HPFH; Kleihauer-Betke; Hemoglobin F

Abstract
The cellular distribution of hemoglobin F is important for evaluating persistently elevated hemoglobin F levels, such as in hereditary persistence of fetal hemoglobin (HPFH) or delta/beta-thalassemia, and for differentiating homozygous hemoglobin S (or hemoglobin S–beta0-thalassemia) from hemoglobin S–HPFH, traditionally done by using the KleihauerBetke (K-B) acid elution test. We evaluated a flow cytometric method using an anti–hemoglobin F antibody as a replacement for the K-B test. We used 172 specimens representing a variety of conditions: HPFH trait, 19 cases; delta/beta-thalassemia trait, 8 cases; hemoglobin S–HPFH, 10 cases. By flow cytometry, all cases of HPFH trait gave a hemoglobin F pattern comparable to the homocellular pattern obtained by the K-B test; all cases of delta/beta-thalassemia tested gave a pattern comparable to a K-B heterocellular pattern. Most cases of hemoglobin S–HPFH gave a homocellular distribution of hemoglobin F, whereas all cases of homozygous hemoglobin S with elevated hemoglobin F levels gave a heterocellular pattern. Flow cytometry provides a more rapid and objective method for assessing cellular distribution of hemoglobin F and is useful for patient evaluation when HPFH trait, delta/beta-thalassemia trait, or hemoglobin S–HPFH trait is suspected.

Hereditary persistence of fetal hemoglobin (HPFH) and delta/beta-thalassemia are a heterogeneous group of disorders characterized by elevated fetal hemoglobin concentrations persisting into adult life.1 In the common forms of HPFH trait (for example, those seen in Africans or African Americans), the hemoglobin F level is approximately 20% to 35%, and the hemoglobin concentration, RBC count, and mean corpuscular volume (MCV) usually are normal.2-4 In contrast, in delta/beta-thalassemia trait, hemoglobin F is in the range of 10% to 15%, and the patients have thalassemic indices, such as a low MCV and a high RBC count. At the molecular level, multiple mutations have been described in many different ethnic groups. Most mutations for both HPFH (such as those in African Americans) and delta/beta-thalassemia are large deletions spanning both the delta- and beta-globin genes on chromosome 11, although some nondeletional HPFH mutations involve a single nucleotide substitution in the promoter region of one of the gamma chains.5-7 A wide range of hemoglobin F levels is seen in the nondeletional types of HPFH, ranging from 3% to 30% hemoglobin F. HPFH can be classified in several ways: by the molecular mutation, the gamma chain subtype produced (G-gamma vs Agamma), or by the cellular distribution of hemoglobin F.1 The cellular distribution of hemoglobin F (homocellular vs heterocellular) traditionally has been evaluated by using the Kleihauer-Betke (K-B) acid elution test.8,9 This test is slide-based and takes advantage of the fact that hemoglobin A is eluted from RBCs on a fixed blood smear using a citric acid–sodium phosphate buffer, but hemoglobin F remains in cells. The slides then are stained with H&E and examined microscopically. The deletional forms of HPFH show a uniform (homocellular or pancellular) distribution of hemoglobin F, whereas
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❚Table 1❚ Conditions Associated With Elevation of Hemoglobin F Levels
Condition beta-Thalassemia trait delta/beta-Thalassemia trait Deletional HPFH trait Nondeletional HPFH trait Homozygous delta/beta-thalassemia Homozygous HPFH (deletional) Homozygous beta-thalassemia Homozygous hemoglobin S or hemoglobin S–beta0-thalassemia Hemoglobin S–HPFH Hemoglobin E–beta0-thalassemia Hemoglobin C–beta0-thalassemia Percentage of Hemoglobin F Hemoglobin F Distribution <10 5-20 20-40 3-30 100 100 30-95 5-30* 20-40 10-50 10-30 Heterocellular Heterocellular Homocellular Heterocellular or homocellular Homocellular Homocellular Heterocellular or homocellular Heterocellular Homocellular Heterocellular Heterocellular

HPFH, hereditary persistence of fetal hemoglobin. * Higher hemoglobin F levels are seen with hydroxyurea therapy.

The K-B test, however, is subject to many pitfalls. It is cumbersome, time-consuming, and subject to errors owing to seemingly trivial variations in the pH of the citric acid–sodium phosphate buffer, time of incubation, or other subtle variables. Microscopic evaluation of these cases is highly subjective and often difficult. A flow cytometric method has been reported that uses a monoclonal anti–hemoglobin F antibody for detecting fetal RBCs. The RBCs are permeabilized so the antibody can enter the interior of the RBC. This method counts 50,000 cells, is highly linear, and is sensitive to as little as 0.02% of hemoglobin F cells.14 Previously, this flow cytometry method was used as a replacement for the K-B test only in the obstetric situation of fetal-maternal bleeding. We evaluated this flow cytometric method as an alternative to the K-B test to determine the cellular distribution of hemoglobin F in the evaluation of hemoglobin disorders.

❚Table 2❚ Breakdown of Specimens Tested by Flow Cytometry

Materials and Methods
Category No. of Specimens 38 13 4 14 7 4 1 6 8 6 2 1 19 27 2 2 10 1 7 172 Normal adult Neonate Neonate after transfusion Infant, 1-5 mo Hemoglobin S trait Hemoglobin E trait Hemoglobin E–beta0-thalassemia beta-Thalassemia trait delta/beta-Thalassemia trait beta+ Thalassemia–HPFH or beta+/beta+ thalassemia beta-Thalassemia major Homozygous HPFH HPFH trait Homozygous S or hemoglobin S–beta0-thalassemia Hemoglobin S–beta+ thalassemia Hemoglobin S/C disease Hemoglobin S–HPFH Hemoglobin C–HPFH Miscellaneous elevated hemoglobin F Total
HPFH, hereditary persistence of fetal hemoglobin.

other conditions with elevated hemoglobin F, such as betathalassemia or delta/beta-thalassemia, show an unequal (heterocellular) distribution. Nondeletional forms of HPFH can show either a homocellular or a heterocellular distribution of hemoglobin F. The K-B test also has been used to differentiate homozygous hemoglobin S from hemoglobin S in combination with HPFH. Patients with homozygous hemoglobin S show a heterocellular pattern, whereas patients with hemoglobin S–HPFH show a homocellular pattern similar to that seen with HPFH trait. This distinction is important because hemoglobin S–HPFH is a clinically benign condition.10-13 Conditions with elevated hemoglobin F levels are summarized in ❚Table 1❚.
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We analyzed 172 specimens by flow cytometry; diagnoses are listed in ❚Table 2❚. All specimens were evaluated by cellulose acetate electrophoresis (pH 8.6) and citrate acid electrophoresis (pH 6.2) (Helena Laboratories, Beaumont, TX). Hemoglobin A2 and hemoglobin F quantitation was obtained using the Bio-Rad Classic Variant High Performance Liquid Chromatography System (Bio-Rad Laboratories, Hercules, CA). Classification of the specimens into the various subgroups was based on several variables, including the RBC count, hemoglobin, MCV, and percentages of hemoglobin F and hemoglobin A2. Patients with sickle cell anemia who had received recent blood transfusions were excluded from the study. All specimens tested were peripheral blood using either sodium EDTA or acid citrate dextrose as the anticoagulant. Molecular analysis was not performed in any case. All specimens were analyzed by the flow cytometry method outlined by Davis et al.14 In short, RBCs are briefly fixed with glutaraldehyde and permeabilized by Triton X100 (Sigma, St Louis, MO). This is a 1-color flow cytometry assay using a murine monoclonal antibody against hemoglobin F conjugated to fluorescein isothiocyanate (Caltag Laboratories, Burlingame, CA). Results are displayed plotting fluorescence intensity vs the number of events. In the initial group of specimens, the flow cytometry assay was compared with the standard K-B test, as previously described,9,10 for validation of the assay. This included 10 cases of HPFH trait, 4 cases of hemoglobin S–HPFH, and 3 cases of delta/beta-thalassemia trait. In a few cases (1 normal, 3 HPFH trait), a 2-color analysis was performed using both anti–hemoglobin F (fluorescein
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isothiocyanate) and anti–hemoglobin A (phycoerythrin). The anti–hemoglobin A antibody (Cortex Biochem, San Leandro, CA) was unconjugated and used a 2-step staining process. The secondary antibody was antimouse phycoerythrin (Sigma, St Louis, MO).

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Results
When specimens from adults with normal hemoglobin values were stained with anti–hemoglobin F, a single peak with minimal to no fluorescence was seen representing hemoglobin A ❚Figure 1A❚. In neonates with normal hemoglobin values, a single peak with bright fluorescence was seen, representing hemoglobin F ❚Figure 1B❚. In neonates after transfusion and infants 1 to 5 months old, both peaks were seen, indicating the presence of both hemoglobin F and hemoglobin A in 2 separate cell populations ❚Figure 1C❚. Results obtained from cases of HPFH trait and delta/beta-thalassemia trait are compared in ❚Table 3❚ . Patients with HPFH trait had higher median levels of hemoglobin F and a higher median MCV than did patients with delta/beta-thalassemia trait. In all cases of HPFH trait, flow cytometry showed a single peak with fluorescence intermediate between the normal hemoglobin A and hemoglobin F peaks. This uniform cell distribution is consistent with the presence of hemoglobin A and hemoglobin F within the same cell ❚Figure 2A❚ and represents the equivalent of the homocellular (pancellular) distribution of hemoglobin F by the K-B test. The 3 cases of HPFH trait that were analyzed by dual-color flow cytometry (ie, both anti–hemoglobin A and anti–hemoglobin F) confirmed the presence of both hemoglobin A and hemoglobin F in the same cell. In contrast, all cases of delta/beta-thalassemia trait showed 2 separate peaks corresponding to hemoglobin A and hemoglobin F in separate cell populations ❚Figure 2B❚. This dual distribution represents the heterocellular distribution of hemoglobin F by the K-B test. Other conditions with elevated hemoglobin F levels, such as cases of betathalassemia trait, hemoglobin S/C disease, and hemoglobin E–beta0-thalassemia, also exhibited 2 peaks (heterocellular distribution) by flow cytometric analysis. In 3 cases of delta/beta-thalassemia trait and 10 cases of HPFH trait, the flow cytometric method was compared with the traditional K-B test. All 3 cases of delta/betathalassemia trait had a heterocellular distribution by both methods. Eight of 10 cases of HPFH trait were homocellular by both methods. Two cases of HPFH trait had a homocellular pattern by flow cytometry but were heterocellular by the K-B test. One of these latter cases of HPFH deserves further comment. This patient was pregnant at the time of the study,
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B

200 160 120 80 40 0 100 101 102 HbF 103 104

C

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❚Figure 1❚ Flow cytometry results using anti–hemoglobin F A, . Normal adult specimen. A single peak is obtained with minimal fluorescence, corresponding to hemoglobin A (HbA). B, Newborn specimen. A single peak is obtained with bright fluorescence, corresponding to hemoglobin F (HbF). C, Specimen from a 5month-old child with a slight elevation of HbF for age (14.5%). There are two distinct peaks obtained corresponding to RBCs containing primarily HbA and HbF respectively. ,

and there was clinical concern about fetal-maternal bleeding. One-color flow cytometry showed a single peak of intermediate fluorescence. However, 2-color flow cytometry (using anti–hemoglobin F and anti–hemoglobin
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❚Table 3❚ Comparison of HPFH Trait With delta/beta-Thalassemia Trait
Distribution of Hemoglobin F Median (Range) Hemoglobin F (%) 31.8 (21.7-40.5) 13.2 (6.9-20.5) Hemoglobin A2 (%)* 2.2 (1.9-2.7) 2.5 (1.9-3.0) Hemoglobin (g/dL)* 12.7 (10.2-14.5) 11.4 (9.4-14.6) MCV (fL)† 83.5 (70.2-95.3) 72.1 (65.9-77 .2) Flow Cytometry Homocellular 19 0 Heterocellular 0 8 Kleihauer-Betke Test No. Tested 10 3 Homocellular 8 0 Heterocellular 2 3

Category HPFH trait (n = 19) delta/beta-Thalassemia trait (n = 8)

HPFH, hereditary persistence of fetal hemoglobin; MCV, mean corpuscular volume. * Values given are conventional. Conversions to Système International are as follows: hemoglobin A , multiply by 0.01 for a proportion of 1.0; hemoglobin, multiply by 10.0 and 2 express as g/L. † Values given are Système International. To convert to conventional units, multiply by 1.0 and express as µm3.

A

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B

200 160 120 80 40 0 100 101 102 HbF 103 104

❚Figure 2❚ Comparison between delta/beta-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) by flow cytometry. A, Specimen from a 13-year-old boy with HPFH trait (mean corpuscular volume [MCV], 95.3 µm3 [95.3 fL]; hemoglobin A2, 2.2% [0.02]; hemoglobin F [HbF], 40.5%). There is a single peak present corresponding to the homocellular pattern. B, Specimen from a 70-year-old woman with delta/beta-thalassemia (MCV, 70.9 µm3 [70.9 fL]; hemoglobin A2, 2.6% [0.03]; HbF 12.1%) showing 2 , peaks consistent with a heterocellular distribution of HbF . C, Specimen from a 23-year-old woman with beta+ thalassemia together with HPFH (MCV, 62.8 µm3 [62.8 fL]; hemoglobin A, 12.2%; hemoglobin A2, 6.0% [0.06]; HbF , 81.8%). There is a single peak present indicating a homocellular distribution of HbF The Kleihauer-Betke test on . this sample also showed a homocellular pattern.

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C

200 160 120 80 40 0 100 101 102 HbF 103 104

A) showed that in addition to the majority of cells containing both hemoglobin A and hemoglobin F, there was a small population of RBCs (4.7%) that contained only hemoglobin F, indicating the presence of fetal cells. This seems to be the reason for the heterocellular pattern obtained by the K-B test in this case ❚Figure 3❚. Six cases that were studied by hemoglobin electrophoresis seemed to be beta-thalassemia trait in combination with HPFH trait vs a combination of 2 mild thalassemia mutations (beta+/beta+). In these cases, the mean percentages were as follows: hemoglobin A, 32.92% (range, 5.5%39.1%); hemoglobin F, 60.1% (range, 54.1%-92.5%). A history of recent transfusion was not obtained in these cases. In 5 of 6 cases, a homocellular pattern was obtained by flow cytometry, most consistent with beta-thalassemia trait in combination with HPFH trait. An example is given in ❚Figure 2C❚. The results obtained from patients homozygous for hemoglobin S (or hemoglobin S–/beta0-thalassemia) were
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compared with those from patients with hemoglobin S–HPFH ❚Table 4❚. Patients with homozygous hemoglobin S had lower hemoglobin F levels and were anemic, in contrast with patients with hemoglobin S–HPFH. Flow cytometry studies on samples from patients with hemoglobin S–HPFH exhibited a single peak of intermediate fluorescence (homocellular pattern), similar to that seen with samples from patients with HPFH trait ❚Figure 4A❚. In contrast, samples from patients with homozygous hemoglobin S or hemoglobin S–beta0thalassemia patients exhibited 2 distinct peaks (heterocellular pattern) ❚Figure 4B❚. K-B testing was performed in 4 cases of hemoglobin S–HPFH and showed a homocellular pattern by both methods in 3 cases. One case showed a heterocellular pattern, both by the K-B test and by flow cytometry.

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Discussion
The diagnosis of HPFH trait usually is straightforward when characteristic findings are present. This includes elevation of the hemoglobin F level in the range of 20% to 35%, a normal hemoglobin concentration, and a normal MCV. These findings usually permit differentiation of HPFH traits from other conditions with increased hemoglobin F levels, such as delta/beta-thalassemia trait or beta-thalassemia trait. Similarly, the diagnosis of hemoglobin S in combination with HPFH also is usually straightforward, as patients are not anemic and have a normal MCV. The elevation of the hemoglobin F level is similar to that seen in HPFH trait alone. Distinction from homozygous hemoglobin S or hemoglobin S in combination with beta-thalassemia trait usually is possible based on hemoglobin electrophoresis results, clinical history, and CBC count data alone. In both of these instances, molecular analysis is not necessary, nor is it available to most major medical centers. However, there are certain situations in which the diagnosis is more difficult. For example, patients with HPFH trait who also are anemic, particularly due to iron deficiency, may have CBC count parameters that mimic the

❚Figure 3❚ Example of 2-color flow cytometry with both anti–hemoglobin F and anti–hemoglobin A. The sample is from a 27-year-old pregnant woman known to have the hereditary persistence of fetal hemoglobin trait. There was clinical suspicion of fetal-maternal bleeding. The major population of RBCs stained for both hemoglobin A (HbA) and hemoglobin F (HbF), indicating maternal blood; however, there also was a small population (4.7%) of RBCs that stained only with the HbF antibody, indicating the presence of fetal blood. The Kleihauer-Betke test revealed a heterocellular distribution of HbF .

delta/beta-thalassemia trait. Distinction between homozygous hemoglobin S and hemoglobin S–HPFH may be difficult if the patient’s history is unknown or when patients with hemoglobin S–HPFH are anemic for other reasons. Many patients with sickle cell anemia now are treated with hydroxyurea to increase hemoglobin F levels, and the clinical history may not be known to the laboratory. In some cases of homozygous hemoglobin S, the hemoglobin F level can be so high as to resemble hemoglobin S–HPFH. The distinction between homozygous hemoglobin S and hemoglobin S–HPFH in children around 1 year of age also

❚Table 4❚ Comparison of Homozygous Hemoglobin S With Hemoglobin S–HPFH
Distribution of Hemoglobin F Median (Range) Hemoglobin F (%) 23.6 (6.9-34.7) 37 (17 .5 .8-53.5) Hemoglobin S (%) 71.3 (61.9-88.2) 58.4 (41.1-73.3) Hemoglobin (g/dL)* 9.1 (6.0-13.5) 12.1 (8.9-15.5) Flow Cytometry Homocellular 0 9 Heterocellular 27 1 Kleihauer-Betke Test No. Tested ND 4 Homocellular ND 3 Heterocellular ND 1

Category Homozygous S or hemoglobin S–beta0-thalassemia (n = 27) Hemoglobin S–HPFH (n = 10)

HPFH, hereditary persistence of fetal hemoglobin; ND, not done. * Values given are conventional. To convert to Système International, multiply by 10.0 and express as g/L.

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200 160 120 80 40 0 100 101 102 HbF 103 104

B

200 160 120 80 40 0 100 101 102 HbF 103 104

situations described, the flow cytometry assay can be used in place of the K-B test. Flow cytometry also may be an aid in the distinction between beta+/beta+ thalassemia and beta-thalassemia trait in conjunction with HPFH trait. In 5 of 6 patients included in this study, a homocellular pattern was obtained, suggesting the latter possibility. However, it remains necessary to correlate these findings with the evaluation of these patients, possibly including family studies and molecular analysis to determine whether this observation is correct. Analysis of the cellular distribution of hemoglobin F by flow cytometry represents an improvement over the standard K-B test and is the preferred method for this analysis. The method is straightforward, and the assay is readily standardized and less subject to subtle variations in technique. The results are displayed in a graphic form that is more objective and easier to interpret than the microscopic K-B test. These data can be archived, which is not possible with the K-B test. Although reagent costs are higher owing to the monoclonal antibody, there are significant savings in technologist time. For laboratories that already use a flow cytometer for other purposes (such as immunophenotyping), instrumentation should be readily available. The results from this study strongly support the routine use of flow cytometric analysis in the diagnostic hemoglobinopathy laboratory.
From the Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN. Address reprint requests to Dr Hoyer: Mayo Clinic, 200 First St, SW, Rochester, MN 55905.

❚Figure 4❚ Comparison of homozygous hemoglobin S with hemoglobin S–hereditary persistence of fetal hemoglobin (HPFH) by flow cytometry. A, Sample from a 2-year-old girl with hemoglobin S–HPFH (hemoglobin S, 61.7%; hemoglobin F [HbF], 34.8%; hemoglobin A2, 3.3% [0.03]). There is a single peak with intermediate fluorescence consistent with a homocellular distribution of hemoglobin F . The Kleihauer-Betke test showed a homocellular pattern. B, Specimen from 15-year-old girl with homozygous hemoglobin S (hemoglobin S, 78.5%; HbF 17 , .6%; hemoglobin A2, 3.9% [0.04]). There are 2 peaks present, indicating a heterocellular distribution of HbF .

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References
1. Bollekens JA, Forget BG. deltabeta thalassemia and hereditary persistence of fetal hemoglobin. Hematol Oncol Clin North Am. 1991;5:399-422. 2. Conley CL, Weatherall DJ, Richardson SN, et al. Hereditary persistence of fetal hemoglobin: a study of 79 affected persons in 15 Negro families in Baltimore. Blood. 1963;21:261-280. 3. Charache S, Clegg JB, Weatherall DJ. The Negro variety of hereditary persistence of fetal haemoglobin is a mild form of thalassaemia. Br J Haematol. 1976;34:527-534. 4. Collins FS, Cole JC, Lockwood WK, et al. The deletion in both common types of hereditary persistence of fetal hemoglobin is approximately 105 kilobases. Blood. 1987;70:1797-1803. 5. Huismann THJ, Miller A, Schroeder WA. A 0gamma type of hereditary persistence of fetal hemoglobin with beta chain production in cis. Am J Hum Genet. 1975;27:765-777. 6. Collins FS, Stoeckert CJ Jr, Serjeant CR, et al. 0gamma beta+ hereditary persistence of fetal hemoglobin: cosmid cloning and identification of a specific mutation 5' to the 0gamma gene. Proc Natl Acad Sci U S A. 1984;81:4894-4898. 7. Craig JE, Rochette J, Sampietro M, et al. Genetic heterogeneity in heterocellular hereditary persistence of fetal hemoglobin. Blood. 1997;90:428-454.

can be problematic, as in many cases of homozygous hemoglobin S, the hemoglobin F levels are elevated much higher than those of a normal child. In all of these situations, analysis of the cellular distribution of hemoglobin F may be a useful additional avenue of investigation. In the past, the cellular distribution of hemoglobin F was evaluated by using the K-B acid elution test. Our study has shown that the homocellular (pancellular) pattern by the K-B test corresponds to a single peak with intermediate fluorescence by flow cytometry. The heterocellular pattern seen by the K-B test corresponds to 2 separate peaks of low and bright intensity by the flow cytometry assay. Thus, in the
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8. Kleihauer E, Braun H, Betke K. Demonstration von fetalem Hämoglobin in der Erythrocyten eines Blutausstrichs. Klin Wochenschr. 1957;35:637-638. 9. Shepard MK, Weatherall DJ, Conley CL. Semi-quantitative estimation of the distribution of fetal hemoglobin in red cell populations. Bull Johns Hopkins Hosp. 1962;110:293-310. 10. Jacob GJ, Raper AB. Hereditary persistence of foetal hemoglobin production, and its interaction with the sicklecell trait. Br J Haematol. 1958;4:138-149. 11. Stamatoyannopoulous G, Wood WG, Papayannopoulou T, et al. A new form of hereditary persistence of fetal hemoglobin and its association with sickle cell trait. Blood. 1975;46:683-692.

12. Rubin EM, Rowley PT. Sickle cell trait/hereditary persistence of fetal hemoglobin trait: misdiagnosis as sickle cell anemia by newborn screening. Am J Dis Child. 1979;133:1248-1250. 13. Murray N, Serjeant BE, Serjeant GR. Sickle cell–hereditary persistence of fetal haemoglobin and its differentiation from other sickle cell syndromes. Br J Haematol. 1988;69:89-92. 14. Davis BH, Olsen S, Bigelow NC, et al. Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry. Transfusion. 1998;38:749-756.

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