Transfusion Associated Peak in Hb HPLC Chromatogram – a Case Report

High performance liquid chromatography (HPLC) and electrophoresis are commonly used to diagnose various hemoglobinopathies. However, insufficient information about the transfusion history can lead to unexpected and confusing results. We are reporting a case of Juvenile myelomonocytic leukemia (JMML) in which HbHPLC was done to quantify fetal hemoglobin (HbF). The chromatogram showed elevated HbF along with a peak in the HbD window. A transfusion acquired peak was suspected based on the unexpectedly low percentage of HbD and was subsequently confirmed using parental HbHPLC.


Introduction.
It is well known that incomplete history on test request forms sent to laboratories and inappropriate patient samples can lead to wrong diagnosis and hazardous consequences. Hemoglobin electrophoresis and High Performance Chromatography (HPLC) are routinely done to diagnose and classify hemoglobinopathies. Acquired and inherited conditions in which abnormal HPLC result can be seen include high fetal hemoglobin in Juvenile Myelomonocytic Leukemia (JMML), Diamond Blackfan Anemia (DBA) and Fanconi Anemia. 1 Blood transfusion from donors with hemoglobinopathies which are clinically silent (e.g HbE, HbD, HbS) may lead to abnormal peaks or altered percentages of abnormal hemoglobins.

Case.
A 7 year old female presented to the outpatient department with complaints of hepatosplenomegaly and lymphadenopathy for 3 months. Hemogram showed a hemoglobin of 9.5 gm/dl, total leukocyte High performance liquid chromatography (HPLC) and electrophoresis are commonly used to diagnose various hemoglobinopathies. However, insufficient information about the transfusion history can lead to unexpected and confusing results. We are reporting a case of Juvenile myelomonocytic leukemia (JMML) in which HbHPLC was done to quantify fetal hemoglobin (HbF). The chromatogram showed elevated HbF along with a peak in the HbD window. A transfusion acquired peak was suspected based on the unexpectedly low percentage of HbD and was subsequently confirmed using parental HbHPLC.
It is well known that incomplete history on test request forms sent to laboratories and inappropriate patient samples can lead to wrong diagnosis and hazardous consequences. Hemoglobin erformance Liquid hromatography (HPLC) are routinely done to diagnose and classify hemoglobinopathies. Acquired and inherited conditions in which abnormal HPLC result can be seen include high fetal hemoglobin in eukemia (JMML), nemia (DBA) and Fanconi Blood transfusion from donors with hemoglobinopathies which are clinically silent (e.g HbE, HbD, HbS) may lead to abnormal peaks or altered percentages of abnormal hemoglobins.
A 7 year old female presented to the outpatient department with complaints of hepatosplenomegaly and lymphadenopathy for 3 months. Hemogram showed a hemoglobin of 9.5 gm/dl, total leukocyte count of 57.24 X10 9 /L and platelet count of 16 X10 Peripheral smear showed 35% monocytes with an absolute monocyte count of 20 X10 Juvenile myelomonocytic leukemia was suggested and Hb HPLC was advised to look for increased fetal haemoglobin.
High Performance Liquid Chromatography (HPLC) was don Variant II instrument with beta thalassemia short program. Hb HPLC chromatogram showed a raised HbF (11%) with HbA 0 69.2%, HbA in D-window of 10.2% with a retention time of 4.06 minutes (Figure 1). The expected percentage of HbD in heterozygotes is ~40% but in our case it was significantly low. Hence, a possibility of transfusion acquired HbD was considered and Hb HPLC was done in both the parents. Both mother and father showed normal Hb HPLC ( High performance liquid chromatography (HPLC) and electrophoresis are commonly . However, insufficient information about the transfusion history can lead to unexpected and confusing results. We are reporting a case of Juvenile myelomonocytic leukemia (JMML) in which HbHPLC was done to quantify fetal showed elevated HbF along with a peak in the HbD window. A transfusion acquired peak was suspected based on the unexpectedly low percentage of /L and platelet count of 16 X10 9 /L. ral smear showed 35% monocytes with an absolute monocyte count of 20 X10 9 /L. Diagnosis of Juvenile myelomonocytic leukemia was suggested and Hb HPLC was advised to look for increased fetal haemoglobin.
High Performance Liquid Chromatography (HPLC) was done using BioRad Variant II instrument with beta thalassemia short program. Hb HPLC chromatogram showed a raised 69.2%, HbA 2 1.8% and a peak window of 10.2% with a retention time of 4.06 ). The expected percentage of HbD in heterozygotes is ~40% but in our case it was significantly low. Hence, a possibility of transfusion acquired HbD was considered and Hb HPLC was done in both the parents. Both mother and father showed ure 2A and 2B). The transfusion history was taken and it was discovered that the patient had received 3 units of PRBC one week prior to HPLC. Thus a final diagnosis of transfusion associated peak in D window was made.
There are only a few reports of abnormal