June 2023
Monthly Digital Case Study
May 2023 QSP Slides
Hemoglobinopathies - An introduction
Quiz
(PDF for print)
May 2023 QSP Slide 1:
Anaemia and sickle-shaped red blood cells are consistent with the clinical and biological context.
FBC Results | |||
| WBC | 7.2 (10^3/mm3) | Neutrophils | 24.6% |
| RBC | 2.64 (10^6/mm3) | Lymphocytes | 55.2% |
| HGB | 8.2 (g/dL) | Monocytes | 10.1% |
| HCT | 25.4 (%) | Eosinophils | 8.7% |
| MCV | 96 (fL) | Basophils | 1.4% |
| MCH | 31.1 (pg) | ||
| MCHC | 32.3 (g/dL) | ||
| PLT | 354 (10^3/mm3) | ||
Clinical Details:
01 May 2023, Male (age not given).
Slide Information:
Clinical context: "Result of the study of hemoglobin by HPLC/Electrophoresis: Presence of S hemoglobin (31.2%) in favour of homozygous sickle cell disease, known transfused or SBeta/Thal-Beta composite hemoglobinopathy.
Expert Comment:
Aniso-poikilocytosis(++). Sickle cell(++). Echinocytes(++). Hypochromic (+) RBCs. Reticulocytosis (9%). Erythroblastosis (23%). The white blood cells seem +/- altered, - Apoptosis is clearly visible, especially on certain images of Polynuclear Neutrophils, - And the presence of Echinocytes(++). The whole is very evocative of an "old" sample. Anemia and sickle-shaped red blood cells are consistent with the clinical and biological context. Suggest family investigation and/or a molecular biology study is recommended."
Hemoglobinopathy is a clinical term that describes a group of blood disorders that affect red blood cells. Hemoglobinopathies are inherited disorders of globin, the protein component of hemoglobin (Hb). Mutations in genes coding for the globin proteins which alter protein output produce the thalassemia syndromes. Mutations in the globin genes that lead to abnormal proteins are called variant Hbs. Hemoglobinopathies are the commonest genetic defect worldwide with an estimated 269 million carriers. Certain populations are particularly at risk of having a hemoglobinopathy, for example, in south-east Asia, there are 90 million carriers, about 85 million in sub-Saharan Africa, and 48 million in the West Pacific.
•Sickle cell disease
•Thalassemia
•Hemoglobin C disease
•Hemoglobin E disease
The insufficient production of hemoglobin or the abnormal structure results in poor functioning of red blood cells. Consequently, anemia can occur.In very serious cases of a hemoglobinopathy, patients may experience the following symptoms:
• Weakness and fatigue due to anemia
• Shortness of breath
• In children, there can be problems relating to growth
The detection and characterization of a hemoglobinopathy involves 3 stages.
Starting with full blood count and film, special hematological tests (HPLC) and possible DNA testing.
Initial detection of Hemoglobinopathies, especially thalassemia begins with the FBC. The first indication for a thalassemia would be a low MCV/MCH. As iron deficiency would give the similar MCV/MCH results, ensure iron deficiency is excluded/treated. Subsequent FBC results giving the same low MCV/MCH would be suggestive of a thalassemia. Note that MCV can also be raised in some other conditions, such as B12 and folate deficiency. HIV patients using nucleoside analogues can also develop a low MCV. A HbS carrier could be missed if only an MCV/MCH is used at initial screening.
It is common practice to make a blood film on samples with a reduced MCV/MCH.
A blood film would give a clue as to the presence of sickle disease (HbS) or an unstable Hb. Observations may be stippling and target cells. Although these features are not exclusively associated with hemoglobinopathies, they are helpful to build the patient picture.
•The RDW (red cell distribution width) measures the coefficient of variation for the MCV. It tends to be higher in iron deficiency but not in thalassemia's and so can give an indication to which is more likely. However, particularly during pregnancy, it is not unusual to find both could be present.
•Some hemoglobinopathies, particularly HbS, will have a normal MCV and normal MCH and would be missed if the full blood count is used as a screening test.
•Since the foetal to adult β globin switch is not usually complete until about 6 months of life, it is difficult to detect β thalassemia in the neonate based on the full blood count. However, a pediatric hematologist experienced in looking at blood films and dealing with thalassemia might make a reasonable guess, based on the hematologic parameters and red blood cell changes in the blood film whether these changes are suggestive of thalassemia. The presence or absence of Beta Thalassemia in the parents of the child is very important In determining the probability of thalassemia.
•At risk pregnancies which have not been monitored by prenatal diagnosis (a late diagnosis or the couple is not interested in prenatal testing), it is important to keep some of the cord blood for DNA testing to be undertaken.
The hemoglobinopathy screen using HPLC is used in conjunction with clinical details and results of the FBC to identify Hemoglobin variants and thalassemia. All Hb variants detected are confirmed by a secondary method. This screen is commonly used in antenatal screening, pre-operative screening and to provide HbS% for transfused sickle cell patients.
HPLC can be used to detect levels of the normal constituents of Hb, HbA, HbA2 and HbF as an increase in HbA2 is indicative of Beta Thalassemia or the presence of HbH (tetramers of beta globulin). HbH can be seen in the cytoplasm of red cells after staining with Brilliant cresyl Blue with the affected red cells displaying a characteristic Golf Ball appearance. The Prescence of abnormal Hb variants are detected by HPLC due to their abnormal transition time.
Special hematology tests are requested once a hemoglobinopathy is suspected based on family history and/or full blood count. Often these tests are ordered by requesting a “thalassemia or hemoglobinopathy screen”.
| Test | What it detects | What it means |
|---|---|---|
| HbEPG | Electrophoresis of globin proteins. Different techniques possible from gel or membrane-based kits to HPLC. Abnormal bands apart from the usual HbA, HbF and HbA2 peaks can be detected. | Gives indication of the HbA2 level but more importantly identifies if there are any variant Hbs – particularly Hbs such as HbE and HbS. |
| HbA2 | Globin electrophoresis and quantitation of the HbA2 peak. Different techniques from membrane or column-based kits but more universally suited HPLC are used. | A raised HbA2 is the key parameter indicating the presence of β thalassemia. Hbs can raise the HbA2 but this must be a rare event. A borderline normal/raised HbA2 could indicate silent β thalassemia. A low HbA2 is also important to note as this might indicate δ thalassemia. |
| HbF | Globin electrophoresis and quantitation with different methods available. | A slightly raised HbF to 2–3% (normal is <1% in an adult) might indicate heterocellular HPFH or a subtle pointer to an underlying silent β thalassemia. HbF levels 5% and above are more likely to be due to δβ thalassemia or. In the case of δβ thalassemia you would expect the HbA2 level to be low. |
Other special hematological tests are available, particularly when investigating the more uncommon variant Hbs. These include tests for oxygen affinity, hemoglobin stability and detection of methaemoglobin. Mass spectrometry has been used to characterise various variant Hbs.
The third stage of testing would include DNA testing. Methods include mutation analysis (usually PCR based), DNA scanning and DNA sequencing. See next issue for more information on Hemoglobinopathies.
Name this cell and what can be seen inside it?
The neutrophil cell above contains Döhle bodies. These are small, round or oval, pale blue–grey structures usually found at the periphery of the neutrophil. They consist of ribosomes and endoplasmic reticulum.
Bibliography
https://medlineplus.gov/lab-tests/hemoglobin-electrophoresis/
Essential Haematology A.V .Hoffbrand & J.E.Pettit
Dacie & Lewis (10th Edition) Lewis, Bain & Bates
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1390791/
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