Case Study

July 04, 2017

Patient History

A 23-year-old male from Bangladesh presents to the ER with complaints of generalized  fatigue and malaise.  Physical examination was normal and showed no signs of icterus, pallor, splenomegaly or lymphadenopathy.  Laboratory workup reveals a slightly decreased hemoglobin of 11.0 g/dL, decreased mean corpuscular volume (MCV) of 55.0 fL, decreased mean corpuscular hemoglobin (MCH) of 19.8 pg and a normal mean corpuscular hemoglobin concentration (MCHC) of 35.9 g/dL.

Laboratory Results

Hemogram

Test

Patient’s Results

Reference Range

Red Blood Cell Count (RBC)

5.56 x 10 x106/uL

4.2 – 5.4 x 10 x106/uL

Hemoglobin (HgB)

11.0 g/dL

12-16 g/ dL

Hematocrit (Hct)

30.6 %

37-47%

Mean Corpuscular Volume (MCV)

55.0  fL

80-100 fL

Heam Corpuscular Hemoglobin (MCH)

19.8  pg

27-31 pg

Mean Corpuscular Hemoglobin Concentration (MCHC)

35.9 g/dL

32-36 g/dL

Red Cell Distribution Width (RDW)

18.8 %

11.5-14.5 %

Platelets

162 x 103/uL

140-440 x 103/uL

White Blood Cell Count (WBC)

6.84 x103/uL

4.8-10.8 x 103/uL

Sysmex XE-2100 Interpretation/Flags

According to the hospital laboratory protocol on slide criteria, a blood smear was prepared based on the following:

RBC/RET IP Message(s): Microcytosis

PLT IP Message(s): PLT Abn Distribution  

Manual Differential

Test

Patient’s Results

Reference Range

Neutrophils

53.7%

3.7 x103/uL

50 – 70%

1.4-6.5 x103/uL

Lymphocytes

40.1%

2.7 x103/uL

20 – 40%

1.2 - 3.4x103/uL

Monocytes

2.5%

0.2 x103/uL

2-9%

0-0.7 x 103/uL

Eosinophils

3.7%

0.3 x103/uL

0-4 %

0-0.5 x 103/uL

Basophils

0.0%

0.0 x103/uL

0-2 %

0-0.2 x 103/uL

Peripheral Blood Smear

Microscopic Objective: 100x          

Microscopic Objective: 100x

Microscopic Objective: 100x

Red Blood Cell Morphology

Peripheral blood smears demonstrated a hypochromic microcytic anemia. Red blood cell morphology showed (1+) anisocytosis, rare polychromasia, as well as many (3+) microcytosis, many (3+) target cells(codocytes), and occasional poikilocytosis. A rare immature myeloid cell was seen on scanning.  

Hemoglobin Electrophoresis

https://lh5.googleusercontent.com/7KN081eVNzZbsUi_zMeipWWRra_Q8fBCsImbSLRD-mkTuOcO6cm_zje8BI6J3YIj_goWP6AzScSJLvJtMtmpU9lHZ97hlvGY1bWVFUUeq-q5LrP-UiYsdyAu6Gfz1LK5lJ-aac_-

 Name                                                                 %                                

 HbF                                                                    0.9

 Hb E                                                                   96.0

 Hb A2                                                                3.1

Electrophoretic Profile:  Hemoglobin electrophoresis shows the major hemoglobin to be HbE, with HbE plus HbA2 constituting 95-99% of total hemoglobin.  

Diagnosis

Hemoglobin E disease (homozygous, HbEE)

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Discussion

Hemoglobin E is a common β-chain hemoglobin variant caused by the substitution of lysine for glutamic acid at the amino acid position 26 (∞2β2 26Glu→Lys). .  It is prevalent among persons from Southeast Asia, Vietnam, Malaysia, northeastern India, Bangladesh, Pakistan, Nepal and Sri Lanka. Because highest incidence of the Hb E gene is in areas where malaria is most prevalent, it is thought that P.falciparum multiplies more slowly in Hb EE RBCs than in Hb AE or HbAA RBCs and that the mutation began as a response to the selective pressure of malaria. The studies to date have been inconclusive.

Hemoglobin E does not produce a positive hemoglobin solubility test result and must be confirmed using electrophoresis or HPLC.  In the homozygous state there is greater than 90% Hemoglobin E, hypoxemia due to reduced oxygen affinity of RBCs,  and a normal reticulocyte count.  On cellulose acetate electrophoresis at an alkaline pH, Hb E migrates with Hb C, Hb O, and Hb A2.  On citrate agar electrophoresis at an acid pH, Hb E can be separated from Hb C, but it co-migrates with Hb A and Hb O.

CBC/RBC Morphology typically show:

▪ Target shaped red blood cells (up to 75% on smear)

▪ Microcytic red blood cells (mean corpuscular volume [MCV] of 55-65 fL)

▪ Decreased hemoglobin concentration (Hgb approximately 12 g/dL)

Note: Because the red blood cell indices are abnormal in hemoglobin EE disease, iron deficiency may need to be assessed with additional testing (iron panel).

Hemoglobin E does not produce a positive hemoglobin solubility test result and must be confirmed using electrophoresis or HPLC.  

Hemoglobin Electrophoresis                                      

                                                                                                     http://path.upmc.edu/cases/case250.htmlHEM.jpg

On cellulose acetate electrophoresis at an alkaline pH, Hb E migrates with Hb C, Hb O, and Hb A2.   On citrate agar electrophoresis at an acid pH, Hb E can be separated from Hb C, but it co-migrates with Hb A and Hb O.

High Performance Liquid Chromatography

                                                                                 http://image3.slideserve.com/6656725/hb-ee-dd-n.jpg

On high performance liquid chromatography (HPLC), Hb E has the same retention time as HbA2 but is identified because of its quantity which is substantial in comparison to HbA2.

References

  1. Beckman Coulter. (2013). Beckman Coulter. Retrieved July 1, 2017, from http://www.beckmancoulter.com/ucm/idc/groups/public/documents/webasset/glb_bci_152233.pdf
  2. Melissa Halpern, MD UPMC. (2000, November). UPMC. Retrieved July 1, 2017, from http://path.upmc.edu/cases/case250.html
  3. Keohane, Elaine. (2016). Rodak's Hematology Clinical Principles and Applications. St. Louis: Elsevier.
  4. Retrieved July 1, 2017, from http://image3.slideserve.com/6656725/hb-ee-dd-n.jpg

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 Hafizul Islam

CAD877EB-2006-4F93-85D8-20AAD7C7E051.JPG Samantha Dewey, MLS(ASCP)SH

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