Aplasia versus pancytopenia, including the pure red cell variant.
A frequent clinical and haematological question is what pathological process has caused the full blood count result, also sometimes called the haemogram, to become abnormal? This is particularly important when one or other peripheral value is significantly raised or, conversely, reduced. In the latter instance involvement of a single lineage, giving rise to anaemia or thrombocytopenia, has vastly different implications than when all three cell types are below normal - the entity of pancytopenia. The cardinal first step is to separate global bone marrow failure or aplasia from hypercellularity with ineffective production or intramedullary destruction, also known as shunting, that occurs with vitamin B12 and folate deficiency. This needs to be distinguished from the myelodysplastic (preleukaemic) syndromes. The variability in presentation can be misleading and, to rapidly arrive at the correct interpretation of the patient's problem, it is prudent to seek explanation in how normal (i.e. physiological) structure and particularly function are disturbed - causation or pathophysiology.
The bone marrow continuously produces, and delivers daily, to the circulating blood 2.5 billion new red cells, 100 - 500 million granulocytes and a further vast number of lymphocytes, together with 2.5 billion platelets at a rate to exactly match loss by the body through utilisation and natural ageing or death in the process, described as apoptosis. These balanced processes dynamically and rapidly respond to any changes in the internal environment where individual lineages selectively accelerate singly or in any appropriate combination, to maintain levels within narrow ranges and so protect tissue or organ functional integrity.
The spongy medullary cavity, sandwiched between layers of dense supporting cortical bone, is where the immunohaematopoietic stem cells home to receptors through particular patterns of adhesion molecules, grow and mature in specialised endosteal areas called niches (Fig. 1). In this specialised microenvironment they are nurtured by the supporting architecture derived largely from mesenchyme and its derivatives with a wide range of locally secreted growth factors (Fig. 2).
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With appropriate stimuli the first division leads to replenishment of this stem cell pool as a unique and defining characteristic. The daughter cell undergoes commitment to blood formation via the haemangiocytoblast that can give rise to vascular endothelium and the full range of haematopoietic cells, collectively known as the haematon. Differentiation separates lineages - one will result in both B and T lymphocytes that fulfil immunological function. The second, collectively described as myeloid, generates red cells or the erythron, granulocytes and monocytes as well as megakaryocytes from which platelets will ultimately be derived (Fig. 3). Within each lineage the earliest progeny are known as progenitors and not morphologically identifiable, needing the use of in vitro laboratory culture techniques. These give rise to precursors that are microscopically recognisable as normoblasts comprising the red cells series that include erythroid islands (Fig. 4) central to regulation of iron traffic between the stores in macrophages and haemoglobin synthesis in this population.
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At the same time, and occurring approximately 3 times more frequently, a range of granulocytes and monocytes develop that acquire quite different cytomorphological characteristics during their growth (Fig. 5). Megakaryocytes are also formed during myelopoiesis and these ultimately liberate cytoplasmic contents into the circulation as platelets.
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Proliferation is best examined by focusing on granulocytes. Consecutive phases vary, giving rise to a kinetically distinct mitotic pool in which numbers expand to include the myeloblasts, progranulocytes and myelocytes - loosely referred to as the fixed components of the microenvironment. Maturation occurs in a post-mitotic compartment, and is made up of metamyelocytes, band forms and mature neutrophils, and allows the cells to reach full functional state prior to the release into the circulation: also called the labile compartment or pool. Apoptosis, intramedullary death that is sometimes known as shunting, provides a system of quality control in which about 15% of the daily production is removed by the phagocytic cells of the reticuloendothelial system and never circulate - a similar pattern is seen in the other cell lines.
Pluripotential stem cell Myeloid Progenitors Myeloblast Monoblasts Marrow pool Mitotic Promelocytes Promonocytes precursors Myelocytes Post-mitotic Metamyelocytes bands Blood and Neutrophils, Monocytes tissue eosinophils and function basophils Innate Macrophage immunologic function Progenitors Megakaryocyte Erythroid Lymphoid Marrow pool Mitotic Maturing Normoblasts Pre-B precursors megakaryocytes Post-mitotic Reticulocytes B lymphoblast Blood and Platelets Erythrocytes B tissue lymphocytes function and plasma cells Haemostasis Oxygen Adaptive transport immunologic function Progenitors Marrow pool Mitotic Pro-T precursors Post-mitotic Lymphoblast Blood and T-lymphocytes tissue and NK cells function Fig. 3. Kinetics of blood cell generation - the concept of pools or compartments. Totipotentiality is found only when sperm fuses with ovum to create a zygote. Immediately there is restriction described as pluripotentially governing all tissue and organ development, well-exemplified in immunohaematopoiesis. Here the different lineages diverge into lymphoid and myeloid with the latter further narrowed down to give rise to red cells, the platelet-megakaryocyte system in parallel with earliest monocytes and granulocytes.
These stages can be disordered at a number of different levels and may affect one or more lines. Global production failure defines aplasia while a single lineage is described as cytopenia. This is a fundamental distinction upon which is based classification that will, in turn, dictate further investigation en route to individualised comprehensive management.
Many individuals with aplasia, particularly in the early phases or during disease evolution, are asymptomatic. Presentation is typically fatigue, with decreasing effort tolerance and palpitations that correlates roughly with the rate as well as fall in haemoglobin level. (2) Less commonly pale skin, headache and dizziness are the first warning signs. Recurrent bacterial infections occur due to neutropenia that may be compounded by leucocyte dysfunction.
Low platelet counts result in a primary haemostatic defect with prolonged bleeding from cuts and less often accompanied by epistaxis while oozing from the gums is an early warning sign. (3) Once levels drop below 10 or 15 x [10.sup.9]/l mucocutaneous bruising, particularly at sites of relatively minor injury, is characteristic and may be aggravated by concurrent use of antiplatelet drugs such as aspirin or non-steroidal anti-inflammatory agents.
The first step is meticulous examination of peripheral blood count values to make the initial crucial distinction between single cytopenia such as anaemia, low white count or reduced platelets, in contrast to pancytopenia in which all the levels are subnormal or pancytopenia is a problem.
It is difficult to overemphasise the importance of supplementing instrumentgenerated figures in the haemogram by careful scrutiny of a well-prepared blood film or smear. Cytomorphologically, the distribution of mature cells in the peripheral blood can be enumerated in a differential leucocyte count. Particularly relevant at this point is establishing total marrow activity as reflected in the reticulocyte count, corrected for the haemoglobin level, to generate the production index that provides a ready clinical assessment to quantitate overall function of the haematon reflected primarily in the red cells series. (4)
With this information it is logical to directly examine the bone marrow by means of aspiration coupled with trephine biopsy (Fig. 6). This remains the central investigation to secure the initial working diagnosis.
Once a production defect due to global marrow failure or aplasia is confirmed, a number of different explanations arise for loss of precursor cells in the marrow (Fig. 7). Here the experienced haematologist is needed to direct confirmatory investigations as a basis for treatment.
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Of the inherited disorders, Fanconi anaemia may present late and with minimal anatomical abnormalities which can be overlooked, making recognition of the syndrome difficult. With the advent of molecular testing telomerase mutations and other genetic defects are becoming diagnosable. Acquired injuries include irradiation and a wide spectrum of cytotoxic drugs and industrial toxins, among which are benzene and a number of relatively commonly used medications. A surprising range of viral infections have been incriminated, including hepatitis, parvovirus and, in sub-Saharan Africa of particular significance, HIV (Fig. 8).
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Once these possibilities have been excluded immune-mediated mechanisms, supported by experimental and clinical observations, become the basis for selection of suppressive interventions. These range from antithymocyte globulin, with or without cyclosporin, determined by severity of disease. Other choices are mycophenolate mofetil and cyclophosphamide. (5)
It cannot be over-emphasised that this possibility requires immediate referral to an appropriate academic centre, so avoiding problems with both under-and over-diagnosis and preventing inappropriate treatment or side-effects. While symptomatic anaemia or thrombocytopenic mucocutaneous bleeding may be the presenting symptoms, the most ominous association is with recurrent bacterial infections as a result of profound neutropenia and dysfunctional innate immune system. It is impossible to sufficiently stress the importance of a thorough history and clinical examination preceding laboratory studies.6 Disease severity is characterised after confirmation on bone marrow aspiration and scrutiny of the trephine biopsy (Fig. 9). This also excludes inherited bone marrow failure syndromes, hairy cell and chronic lymphocytic leukaemia, collagen vascular diseases and particularly systemic lupus erythematosus.
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Do not overlook the presence of abnormal populations or clones due to paroxysmal nocturnal haemoglobinuria or myelodysplasia, the impact of HIV or infiltrative lesions causing hypersplenism on the basis of congestive splenomegaly. However, in these cases the marrow is not devoid of haematopoietic tissue.
Therapeutic decisions and management
Supportive care is often adequate in mild and stable to improving forms, anticipating repopulation of the marrow where recovery is variable and independence may occur from transfusion. This approach requires detailed record-keeping with antigenreduced packed red cells sufficient for symptomatic relief given with chelation therapy to avoid iron overload.
Granulocyte stimulatory peptides have limited value since the precursor cells are generally absent. However, be aware of the local prevalence of specific bacterial pathogens in order to treat infections empirically where necessary. An infectious disease or microbiology consultant on the team is invaluable. Thrombocytopenia is also challenging and replacement should be limited to single apheresis units given only to maintain haemostasis. The local practice of replacement without documenting response should be avoided, as such practices may accelerate isoimmunisation. The major emphasis is to restrict this period and proceed to specific curative interventions as rapidly as necessary.
Allogeneic immunohaematopoietic stem cell transplantation from a fully matched sibling is preferable in severe forms of the disease. Where this is not possible a matched unrelated volunteer donor is the next choice. Depending on conditioning programmes and other variations in these procedures, complications range from rejection through acute and possibly subsequent chronic graft-versus-host disease to even graft failure. Nevertheless, in younger individuals cure rates have continually improved over the years and currently exceed 90%.2'3" Immunosuppressive regimens have emerged over the years with varying responses to regimens that include antithymocyte globulin through high-dose corticosteroids and cyclosporin or novel agents. Preliminary data suggest that all three products in combination may have a better outcome. Late relapse and clonal malignant disease arise in a quarter of those treated. Additionally, in comparison to transplanted individuals, while a good life is possible, cure remains infrequent despite prolonged cyclosporin and steroid maintenance.'
These realities should be fully understood by referring doctors and mandate access to experienced multidisciplinary clinics who regularly audit and report outcome.
Alternative or innovative options include unconfirmed observations that a single course of cyclophosphamide may prolong survival in about half the patients. Attempts to combine this with other immunosuppressive regimens, particularly antithymocyte globulin, were unsuccessful and further evaluation of this approach is awaited. Another promising approach, currently unconfirmed, is response to a monoclonal antibody that binds to the interleukin-2 receptor, thereby modulating T-cell-mediated immunosuppression.
The treatment of choice, when a suitable donor is available, remains a fully matched immunohaematopoietic stem cell transplant from a sibling as an early priority. When this is not possible alternative options include timeous use of matched unrelated volunteer donors. Success depends upon performance status and absence of significant comorbidity.
Conditioning is immunosuppressive rather than myeloablative. Cyclophosphamide with antithymocyte globulin and fludarabine produces a survival rate of approximately 70% at 15 years in a cohort under 20 years of age. In older patients, certainly up to 60, good results are possible even following previous treatment.
One major point is the need to blunt potential graft-versus-host disease. Campath monoclonal antibodies, particularly ex vivo or using in-the-bag technique, have shown success.' These results were from centres with experienced multidisciplinary teams.
Pure red cell aplasia
In adults this is acquired, with profound anaemia, characteristically without reticulocytes in the blood and erythroid precursors absent from the marrow while the other elements are preserved.
The total loss of erythropoiesis is believed to be immune-mediated by both antibody and T-cells at a point of development prior to the pro-erythroblasts. Additionally there may be humorally directed inhibition of erythropoietin but the alternative explanation is that erythropoietinstimulating activity leads to the loss of these precursors. In many instances there is a specific attack on the precursors by parvovirus B 19 and increasing concern of an underlying early myelodysplasia.
The aetiology may be primary or secondary (Fig. 10).
Diagnosis depends upon usually severe degrees of anaemia that is, initially at least, asymptomatic but typically preceded by fall in exercise tolerance and relentlessly progressive fatigue. Reticulocytes are absent but the remainder of the peripheral blood findings are normal.
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Bone marrow aspiration and particularly trephine biopsy are diagnostic. Interestingly there may be giant pro-normoblasts in the marrow that many consider pathognomonic (Fig. 11).
In evaluating associated and possibly causative mechanisms, use tomography of the chest to exclude thymoma, flow cytometry to identify large granular lymphocyte proliferation and cytogenetics together with a fluorescent in situ hybridisation panel to avoid overlooking myelodysplasia as well as antibody tests for parvovirus. Ferrokinetic studies demonstrate selective absence of erythropoiesis but these investigations are still largely in a research setting.
Treatment requires antigen-reduced packed red cells to control symptoms, and iron overload should be diminished by concurrent use of chelation therapy. With an accurate diagnosis the course may be self-limiting, including parvovirus B19 infections, and patients can be managed expectantly, although intravenous gammaglobulin remains an option, particularly in those associated with HIV.
Pathophysiologically the explanation is an inability of the immune system to clear the offending virus. In refractory cases interventions parallel those for aplastic anaemia, with significant response rates following immunosuppression, particularly with antithymocyte globulin and prednisone, and possibly in combination with anti-CD 20 monoclonal antibody, among other therapeutic possibilities.
As for aplasia, this latter entity is also relatively uncommon but requires consideration in patients whose anaemia is otherwise not readily explained. Distinction is necessary from the more common nutritional deficiencies or the anaemia of chronic disorders where marrow findings are usually diagnostic. Once the diagnosis is established, the importance of protocol management, preferably on clinical trial by an accredited multidisciplinary group in an academic centre of excellence, is regarded worldwide as standard of care.
Acknowledgements. Supported by the Haematological Research Trust, with grants from the Louis Shill Foundation. Christine Dolling helped with the bibliographic review and Natasha Gordon typed the manuscript: appreciation is expressed to our research assistants.
References available at www.cmej.org.za
Peter Jacobs, MB BCh, MD, PhD (Med), DSc (Med Sci), FRCP (Edin), MACP, FCP (SA), FRCPath (UK), IFCAP, MASSAf, FRSSAf
Emeritus Professor of Haematology, University of Cape Town, Honorary Consultant Physician, Groote Schuur Hospital Teaching Group, Professor of Internal Medicine, College of Medicine, University of Nebraska Medical Centre, Professor Extraordinaire in Haematological Pathology, Stellenbosch University and Tygerberg Academic Hospital, and Haematology Research Group, PathCare
Lucille Wood, FRCP (Edin), BA (Nursing Science), MSc (Med)(Haematol), RN, RM, Diploma in Intensive Nursing Care, Ward Administration and Clinical Teaching
Medical Biological Scientist, Senior Lecturer Extraordinaire in Clinical Haematology, Stellenbosch University and Tygerberg Academic Hospital, and Haematology Research Group, PathCare
Correspondence to: Natasha Gordon (email@example.com)
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|Title Annotation:||Practical haematology|
|Author:||Jacobs, Peter; Wood, Lucille|
|Publication:||CME: Your SA Journal of CPD|
|Date:||Sep 1, 2012|
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