Aplasia versus pancytopenia, including the pure red cell variant

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 protected])

the supporting architecture derived largely This is the first in our third series of clinical vignettes that are centred on everyday from mesenchyme and its derivatives with a presentations. Each emphasises practical aspects of team-based care, having relevance wide range of locally secreted growth factors for general practitioners, specialists and paramedical professionals alike. Disorders (Fig. 2). arising in blood and result in some of the commonest symptoms and signs for which patients seek medical advice. A carefully taken history and meticulous With appropriate stimuli the first division physical examination, complemented by thoughtful and judicious use of laboratory tests, leads to replenishment of this stem cell pool provide the clinical basis for a working diagnosis. Advanced haematology may be needed as a unique and defining characteristic. The to extend evaluation to the bone marrow and plasma while supplementary imaging daughter cell undergoes commitment to or radionuclide technology further links primary care practitioner or specialist to the blood formation via the haemangiocytoblast experienced haematologist. Such a multidisciplinary and fully interactive approach offers that can give rise to vascular endothelium the preferred way to problem solving and optimum management. and the full range of haematopoietic cells, collectively known as the haematon. Differentiation separates lineages – one will Aplasia Physiology result in both B and T lymphocytes that A frequent clinical and haematological The bone marrow continuously produces, fulfil immunological function. The second, question is what pathological process has and delivers daily, to the circulating blood collectively described as myeloid, generates caused the full blood count result, also 2.5 billion new red cells, 100 - 500 million red cells or the erythron, granulocytes and sometimes called the haemogram, to become granulocytes and a further vast number monocytes as well as megakaryocytes from abnormal? This is particularly important when of lymphocytes, together with 2.5 billion which will ultimately be derived one or other peripheral value is significantly platelets at a rate to exactly match loss by (Fig. 3). Within each lineage the earliest raised or, conversely, reduced. In the latter the body through utilisation and natural progeny are known as progenitors and instance involvement of a single lineage, ageing or death in the process, described not morphologically identifiable, needing giving rise to anaemia or thrombocytopenia, as apoptosis. These balanced processes the use of in vitro laboratory culture has vastly different implications than when dynamically and rapidly respond to any techniques. These give rise to precursors all three cell types are below normal – the changes in the internal environment where that are microscopically recognisable as entity of pancytopenia. The cardinal first step individual lineages selectively accelerate normoblasts comprising the red cells series is to separate global bone marrow failure or singly or in any appropriate combination, that include erythroid islands (Fig. 4) aplasia from hypercellularity with ineffective to maintain levels within narrow ranges production or intramedullary destruction, and so protect tissue or organ functional also known as shunting, that occurs with integrity. vitamin B12 and folate deficiency. This needs to be distinguished from the myelodysplastic The spongy medullary cavity, sandwiched (preleukaemic) syndromes. The variability between layers of dense supporting cortical in presentation can be misleading and, to bone, is where the immunohaematopoietic rapidly arrive at the correct interpretation stem cells home to receptors through of the patient’s problem, it is prudent to seek particular patterns of adhesion molecules, explanation in how normal (i.e. physiological) grow and mature in specialised endosteal structure and particularly function are areas called niches (Fig. 1). In this specialised disturbed – causation or pathophysiology. microenvironment they are nurtured by Fig. 1. The bone marrow niche.

339 CME September 2012 Vol. 30 No. 9 Practical haematology

central to regulation of iron traffic between also formed during myelopoiesis and these referred to as the fixed components of the the stores in macrophages and haemoglobin ultimately liberate cytoplasmic contents into microenvironment. Maturation occurs in synthesis in this population. the circulation as platelets. a post-mitotic compartment, and is made up of metamyelocytes, band forms and At the same time, and occurring Proliferation is best examined by mature neutrophils, and allows the cells approximately 3 times more frequently, focusing on granulocytes. Consecutive to reach full functional state prior to the a range of granulocytes and monocytes phases vary, giving rise to a kinetically release into the circulation: also called the develop that acquire quite different distinct mitotic pool in which numbers labile compartment or pool. Apoptosis, cytomorphological characteristics during expand to include the myeloblasts, intramedullary death that is sometimes their growth (Fig. 5). Megakaryocytes are progranulocytes and myelocytes – loosely known as shunting, provides a system

Pluripotential stem cell

Bone marrow Early acting molecules, primarily interleukins

Commitment to progenitors, recognisable only in culture

Lymphoid and myeloid precursors morphologically identifiable Blood

Antigen drive Erythropoietin Thrombopoietin GSCF

Mature lymphocytes Granulocytes Red cells Platelets B T Monocytes

Fig. 2. Growth or stimulatory factors. The orderly development, and then delivery of mature cells to the peripheral blood is moderated by changing patterns of migration through the marrow. An array of molecules, or peptides, has a distinct pattern of expression during the various stages of differentiation, proliferation and maturation. These steps are coupled with alterations in adhesion factors that govern release from the niche and balance this exactly to intramedullary destruction or apoptosis, as dictated by physiological requirements. The focused activity of these cytokines can be recapitulated with commercially available equivalents to selectively drive each line in therapy: e.g. erythropoietic stimulating factors for erythrocytes and granulocyte or monocyte stimulating agents (G:CSF) in the case of these two lineages.

340 CME September 2012 Vol. 30 No. 9 Practical haematology

Pluripotential stem cell Myeloid Progenitors Myeloblast Monoblasts Megakaryocyte Erythroid Lymphoid Pre-B Pre-B Pro-T Pro-T Mitotic

Promelocytes Normoblasts Myelocytes precursors Marrow pool Marrow

Metamyelocytes Promonocytes bands Maturing megakaryocytes Maturing Post-mitotic Lymphoblast Reticulocytes B lymphoblast

Neutrophils, eosinophils and cells basophils Monocytes plasma cells Erythrocytes B lymphocytes and B lymphocytes T-lymphocytes and NK and T-lymphocytes Blood and tissue function tissue Innate immunologic Haemostasis Platelets Platelets Haemostasis function function Adaptive Macrophage Macrophage immunologic Oxygen transport Oxygen

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 -megakaryocyte system in parallel with earliest monocytes and granulocytes. of quality control in which about 15% of Pathophysiology from cuts and less often accompanied by the daily production is removed by the Clinical1 epistaxis while oozing from the gums is phagocytic cells of the reticuloendothelial Many individuals with aplasia, particularly in an early warning sign.3 Once levels drop system and never circulate – a similar the early phases or during disease evolution, below 10 or 15 x 109/l mucocutaneous pattern is seen in the other cell lines. are asymptomatic. Presentation is typically bruising, particularly at sites of relatively fatigue, with decreasing effort tolerance minor injury, is characteristic and may be These stages can be disordered at a number and palpitations that correlates roughly aggravated by concurrent use of antiplatelet of different levels and may affect one or more with the rate as well as fall in haemoglobin drugs such as aspirin or non-steroidal anti- lines. Global production failure defines level.2 Less commonly pale skin, headache inflammatory agents. aplasia while a single lineage is described as and dizziness are the first warning signs. cytopenia. This is a fundamental distinction Recurrent bacterial infections occur due to Haematological upon which is based classification that neutropenia that may be compounded by The first step is meticulous examination of will, in turn, dictate further investigation leucocyte dysfunction. peripheral blood count values to make the en route to individualised comprehensive initial crucial distinction between single management. Low platelet counts result in a primary cytopenia such as anaemia, low white haemostatic defect with prolonged bleeding count or reduced platelets, in contrast to

341 CME September 2012 Vol. 30 No. 9 Practical haematology

pancytopenia in which all the levels are sub- film or smear. Cytomorphologically, the Diagnostic algorithm normal or pancytopenia is a problem. distribution of mature cells in the peripheral It cannot be over-emphasised that this blood can be enumerated in a differential possibility requires immediate referral It is difficult to overemphasise the leucocyte count. Particularly relevant at this to an appropriate academic centre, so importance of supplementing instrument- point is establishing total marrow activity avoiding problems with both under- generated figures in the haemogram by as reflected in the reticulocyte count, and over-diagnosis and preventing careful scrutiny of a well-prepared blood corrected for the haemoglobin level, to inappropriate treatment or side-effects. 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. Fig. 4. Erythroblastic island. Iron traffic to maturing red cell population, the Once a production defect due to global normoblasts, broadly described as the marrow failure or aplasia is confirmed, a erythron, are in part furnished by recovery number of different explanations arise for from the diet via enterocytes in the small loss of precursor cells in the marrow (Fig. bowel. Simultaneously, recycling of this trace 7). Here the experienced haematologist is metal from effete red cells in the macrophage needed to direct confirmatory investigations system is exemplified by those in the bone as a basis for treatment. marrow. The regulation is genetically determined and modulated by expression of Causation Fig. 6. The trephine biopsy. Normally between the iron exporter known as ferroportin. The Of the inherited disorders, Fanconi 60% and 80% of the marrow is occupied by latter is under surveillance and regulation anaemia may present late and with blood-forming tissue with the architectural by the master regulator – the hepatic peptide minimal anatomical abnormalities which arrangement best appreciated from trephine known as hepcidin. can be overlooked, making recognition biopsy shown in the top panel. In contrast, loss of the syndrome difficult. With the of haematopoiesis depletes these precursors, advent of molecular testing telomerase leaving residual stroma comprising delicate mutations and other genetic defects are reticulin, lymphocytes, monocytes and becoming diagnosable. Acquired injuries abundant fat spaces. include irradiation and a wide spectrum of cytotoxic drugs and industrial toxins, Ineffective haematopoiesis among which are benzene and a number B or folate deficiency of relatively commonly used medications. 12 Myelodysplasia A surprising range of viral infections have * been incriminated, including , Severe infections parvovirus and, in sub-Saharan Africa of Extensive infiltration Fig. 5. Bone marrow precursors – aspirated particular significance, HIV (Fig. 8). Malignancy sternal sample. The ratio of myeloid to Fibrosis erythroid cells (shown by arrow), or the Once these possibilities have been M-E ratio is physiologically 3 to 1. A range excluded immune-mediated mechanisms, Aplasia of granulocytes are recognisable from supported by experimental and clinical Congenital neutrophils to the earlier myelocytes (shown observations, become the basis for Acquired by *) distinguished by azurophilic granules selection of suppressive interventions. in the cytoplasm. Megakaryocytes are large These range from antithymocyte Fig. 7. Pancytopenia due to marrow failure. and prominent (not present). Both erythroid globulin, with or without cyclosporin, Diagnosis may be reflected in the peripheral and myeloid lines undergo varying degrees determined by severity of disease. Other blood smear. The cardinal investigation is of maturation prior to release into the choices are mycophenolate mofetil and examination of the bone marrow aspiration circulation. cyclophosphamide.5 and trephine biopsy.

342 CME September 2012 Vol. 30 No. 9 Practical haematology

Cytotoxic drugs and radiation Moderate aplasia Idiosyncratic drug reaction Bone marrow cellularity <30% Chloramphenicol Absence of severe pancytopenia Non-steroidal anti-inflammatory Depression of at least two of three blood elements below normal Sulphonamides Severe aplastic aplasia Industrial toxins A bone marrow biopsy showing <25% of normal cellularity Benzene or Viral infections A bone marrow biopsy showing <50% normal cellularity in which fewer than 30% of the cells are haematopoietic Hepatitis And at least two of the following are present: HIV Absolute reticulocyte count <40 000/microliter Epstein-Barr virus Absolute neutrophil count (ANC) <500/microliter Immune disorders Platelet count <20 000/microliter Systemic lupus erythematosus Miscellaneous Very severe aplasia Paroxysmal nocturnal haemoglobinuria The criteria for severe aplastic anaemia are met and the ANC is <200/microliter Thymic tumours Fig. 9. Diagnostic criteria and grading for aplasia. The correlation of survival is with absolute Pregnancy neutrophil count at diagnosis. Where this is under 0.2 x 109 /l outcome is substantially and Idiopathic or primary significantly inferior predicating a more aggressive approach to interventions and moving therapeutic options towards early immunohaematopoietic stem cell allogeneic transplantation. Fig. 8. Causes of bone marrow aplasia. ANC = absolute neutrophil count; 500 ANC = 0.5 x 109/l. In some cases the cause is apparent and predictably reversible – only careful paroxysmal nocturnal haemoglobinuria maintain haemostasis. The local practice of observation is needed. In others it may be or myelodysplasia, the impact of HIV or replacement without documenting response obscure and have an immunological basis. infiltrative lesions causing hypersplenism should be avoided, as such practices may The relevance of accurate diagnosis is choice on the basis of congestive splenomegaly. accelerate isoimmunisation. The major of therapy and options extend from support However, in these cases the marrow is not emphasis is to restrict this period and and monitoring to active intervention ranging devoid of haematopoietic tissue. proceed to specific curative interventions as from immunosuppression to allogeneic bone rapidly as necessary. marrow transplantation. Therapeutic decisions and management Allogeneic immunohaematopoietic stem While symptomatic anaemia or thrombo- Supportive care is often adequate in mild cell transplantation from a fully matched cytopenic mucocutaneous bleeding may be and stable to improving forms, anticipating sibling is preferable in severe forms of the presenting symptoms, the most ominous repopulation of the marrow where recovery the disease. Where this is not possible a association is with recurrent bacterial is variable and independence may occur matched unrelated volunteer donor is the infections as a result of profound neutropenia from transfusion. This approach requires next choice. Depending on conditioning and dysfunctional innate immune system. detailed record-keeping with antigen- programmes and other variations in these It is impossible to sufficiently stress the reduced packed red cells sufficient for procedures, complications range from importance of a thorough history and clinical symptomatic relief given with chelation rejection through acute and possibly examination preceding laboratory studies.6 therapy to avoid iron overload. subsequent chronic graft-versus-host Disease severity is characterised after disease to even graft failure. Nevertheless, confirmation on bone marrow aspiration Granulocyte stimulatory peptides have in younger individuals cure rates have and scrutiny of the trephine biopsy (Fig. 9). limited value since the precursor cells are continually improved over the years and This also excludes inherited bone marrow generally absent. However, be aware of currently exceed 90%.2,3,6 failure syndromes, hairy cell and chronic the local prevalence of specific bacterial lymphocytic leukaemia, collagen vascular pathogens in order to treat infections Immunosuppressive regimens have emerged diseases and particularly systemic lupus empirically where necessary. An infectious over the years with varying responses to erythematosus. disease or microbiology consultant on the regimens that include antithymocyte globu- team is invaluable. Thrombocytopenia is lin through high-dose corticosteroids and Do not overlook the presence of also challenging and replacement should be cyclosporin or novel agents. Preliminary abnormal populations or clones due to limited to single units given only to data suggest that all three products in com-

343 CME September 2012 Vol. 30 No. 9 Practical haematology

bination may have a better outcome. Late relapse and clonal malignant disease arise Primary in a quarter of those treated. Additionally, Associated with in 10 - 15% of cases in comparison to transplanted individuals, Idiopathic causes while a good life is possible, cure remains infrequent despite prolonged cyclosporin Secondary and steroid maintenance.5 Neoplasia Chronic leukaemia, leukaemia, lymphomas; large granular lymphocytic proliferative These realities should be fully understood disorders; prodrome to myelodysplastic syndromes by referring doctors and mandate access to Systemic lupus erythematosus or rheumatoid arthritis experienced multidisciplinary clinics who Associated with pregnancy and autoimmune diseases regularly audit and report outcome. Drugs Phenytoin, chlorpropamide, zidovudine (AZT), trimethoprimsulfamethoxazole, isoniazid Alternative or innovative options include unconfirmed observations that a single Infections Infectious mononucleosis, viral hepatitis, parvovirus infection, HIV course of cyclophosphamide may prolong survival in about half the patients. Attempts to combine this with other Fig. 10. Causation of red cell aplasia. The majority of cases have no demonstrable associated immunosuppressive regimens, particularly pathology and are therefore momentarily accepted as idiopathic or primary. It is nevertheless antithymocyte globulin, were unsuccessful important to search carefully for possible causes – particularly if they are reversible. and further evaluation of this approach is awaited. Another promising approach, currently unconfirmed, is response to a Pure red cell aplasia Haematology monoclonal antibody that binds to the In adults this is acquired, with profound Bone marrow aspiration and particularly interleukin-2 receptor, thereby modulating anaemia, characteristically without reticu- trephine biopsy are diagnostic. Interestingly T-cell-mediated immunosuppression. locytes in the blood and erythroid precur- there may be giant pro-normoblasts in the sors absent from the marrow while the other marrow that many consider pathognomonic Current practice elements are preserved. (Fig. 11). The treatment of choice, when a suitable donor is available, remains a fully matched Pathophysiology immunohaematopoietic stem cell transplant The total loss of erythropoiesis is believed from a sibling as an early priority. When this to be immune-mediated by both antibody is not possible alternative options include and T-cells at a point of development prior timeous use of matched unrelated volunteer to the pro-erythroblasts. Additionally there donors. Success depends upon performance may be humorally directed inhibition status and absence of significant co- of erythropoietin but the alternative morbidity. explanation is that erythropoietin- stimulating activity leads to the loss of Conditioning is immunosuppressive rather these precursors. In many instances there than myeloablative. Cyclophosphamide with is a specific attack on the precursors by antithymocyte globulin and fludarabine and increasing concern of Fig. 11. Bone marrow failure due to produces a survival rate of approximately an underlying early myelodysplasia. parvovirus B19 infection. This particular 70% at 15 years in a cohort under 20 years and unusual morphological feature is of age. In older patients, certainly up to 60, The aetiology may be primary or secondary distinctive and regarded by many as unique, good results are possible even following (Fig. 10). signalling the underlying causation as that of previous treatment. this particular infection. Diagnosis depends upon usually severe One major point is the need to blunt po- degrees of anaemia that is, initially at least, In evaluating associated and possibly tential graft-versus-host disease. Campath asymptomatic but typically preceded by causative mechanisms, use tomography of the monoclonal antibodies, particularly ex vivo fall in exercise tolerance and relentlessly chest to exclude thymoma, flow cytometry or using in-the-bag technique, have shown progressive fatigue. Reticulocytes are absent to identify large granular lymphocyte success.7 These results were from centres but the remainder of the peripheral blood proliferation and cytogenetics together with with experienced multidisciplinary teams. findings are normal. a fluorescent in situ hybridisation panel to

344 CME September 2012 Vol. 30 No. 9 Practical haematology

avoid overlooking myelodysplasia as well as the offending virus. In refractory cases importance of protocol management, antibody tests for parvovirus. Ferrokinetic interventions parallel those for aplastic preferably on clinical trial by an accredited studies demonstrate selective absence of anaemia, with significant response rates multidisciplinary group in an academic erythropoiesis but these investigations are following immunosuppression, particularly centre of excellence, is regarded worldwide still largely in a research setting. with antithymocyte globulin and as standard of care. prednisone, and possibly in combination Treatment with anti-CD 20 monoclonal antibody, Acknowledgements. Supported by the Treatment requires antigen-reduced packed among other therapeutic possibilities. Haematological Research Trust, with grants red cells to control symptoms, and iron over- from the Louis Shill Foundation. Christine load should be diminished by concurrent use Conclusion Dölling helped with the bibliographic of chelation therapy. With an accurate diag- As for aplasia, this latter entity is also review and Natasha Gordon typed the nosis the course may be self-limiting, includ- relatively uncommon but requires manuscript: appreciation is expressed to our ing parvovirus B19 infections, and patients consideration in patients whose anaemia research assistants. can be managed expectantly, although intra- is otherwise not readily explained. venous gammaglobulin remains an option, Distinction is necessary from the more particularly in those associated with HIV. common nutritional deficiencies or the anaemia of chronic disorders where Pathophysiologically the explanation is marrow findings are usually diagnostic. an inability of the immune system to clear Once the diagnosis is established, the References available at www.cmej.org.za

Single suture Fingertip tingle enhances a surgeon’s sense of touch

Our fingers are precision instruments, but there are plenty of things they are not sensitive enough to detect. Now we can augment their talents – using wearable electronic fingertips that provide tingling feedback about whatever we touch.

John Rogers of the University of Illinois at Urbana-Champaign and colleagues have designed a flexible circuit that can be worn over the fingertips. It contains layers of gold electrodes just a few hundred nanometres thick, sandwiched between layers of polyimide plastic to form a ‘nanomembrane’. This is mounted on a finger-shaped tube of silicone rubber, allowing one side of the circuit to be in direct contact with the fingertips. On the other side, sensors can be added to measure pressure, temperature or electrical properties such as resistance.

People wearing the device receive electrotactile stimulation – a tingling sensation caused by a small voltage applied to the skin. The size of the voltage is controlled by the sensor and varies depending on the properties of the object being touched.

Surgical gloves are one potential application. Rogers, who worked with colleagues at Northwestern University in Evanston, Illinois, and Dalian University of Technology in China, says gloves fitted with the nanomembrane could sense the thickness or composition of tissue via its electrical properties. A surgeon could also whittle away at the tissue using a high-frequency alternating current supplied by a battery attached at the wrist and delivered via the nanomembrane itself, says Rogers.

Fiorenzo Omenetto at Tufts University in Medford, Massachusetts, is impressed. ‘The work sets the stage for a new generation of devices,’ he says.

There are applications beyond surgery, too. MC10, the company commercialising the technology, is running animal trials of a nanomembrane ‘sock’ that can be wrapped around the heart. This provides a 3D map of its electrical activity, useful in treating irregular heartbeat.

MC10 is also working with medical device company Medtronic to use the membrane inside the heart, sending it in on a limp balloon, which is then inflated to push the membrane onto the heart’s interior walls.

Rogers says MC10 is also collaborating with sportswear firm Reebok on a product to be launched by the end of this year. The aim is to build a ‘body-worn piece of electronics’ designed for contact sports, although Rogers declined to say exactly how it will be used.

New Scientist, 10 August 2012, online

345 CME September 2012 Vol. 30 No. 9