Iron Deficiency Anemia â€“ Needs Attention and Proper Management Pramod Kumar Sinha
Iron Deficiency in Heart Failure K Sarat Chandra
Anaemia of Chronic Disease: Current Update Ananda Bagchi, Aradhya Sekhar Bagchi
Clinical Utility of Erythrocyte Sedimentation Rate in Modern Era Pratap Singh, Sanjay Kumar
Pancytopenia: Clinical approach Ajai Kumar Garg, AK Agarwal, GD Sharma
Current Management of Hemophilia S Usha
Hemophilia: Prophylactic Therapy with Conventional and Newer Agents Tarun Kumar Dutta, Shailendra Prasad Verma, Deepak Charles
Newer Therapies in Hemophilia Sandeep Garg, Naresh Gupta, Sunita Aggarwal
Artificial Blood: An Update on Current Red Cell and Platelet Substitutes AS Mohan
Blood Component Therapy Mathew Thomas
Chronic Myeloid Leukaemia â€“ An Overview Sunita Aggarwal, Jahnvi Dhar, Sandeep Garg, Naresh Gupta
Iron Deficiency Anemia â€“ Needs Attention and Proper Management
C H A P T E R
Pramod Kumar Sinha
Iron deficiency is the most prevalent nutritional deficiency and the commonest cause of anaemia. It is a major health problem not only in India but world wide. 30% to 70% population in the developing world is iron deficient. As per WHO, over two billion people globally that is 30% of world population suffers from anaemia and more than half of them are due to iron deficiency. In spite of many national health programs, the situation in India continues to be bad with 56% women and 70% of children suffering from anaemia as estimated by the third National Family Health Survey (India) and more than half of these is because of iron deficiency. Iron deficiency anaemia impairs cognitive ability and reduces physical capacity in general, affects proper growth of children and is an important contributor to maternal and peri-natal mortality, adds to the severity of many chronic diseases and often represents serious underlying cause so needs to be recognized and managed properly on individual basis together with population based National strategy of preventing iron deficiency.
Human body on an average contains 3gm to 4 gm total iron in male(50 mg/kg ) and 2gm to 3 gm in women (40gm/ kg) of which nearly 71% is present in haem compounds: 65% haemoglobin, 4% myoglobin and about 2% incorporated in enzymes like cytochromes, peroxidases and others subserving important and critical role in body metabolism. About 29% is in non-haem form stored as ferritin and haemosiderin in bone marrow, spleen and liver. Nearly 0.1% of total iron is bound to carrier protein transferrin flowing in plasma.
Iron is absorbed in the duodenum and upper jejunum by a very carefully regulated process. At the brush Transferrin
Storage Iron : RE system /liver -1500 mg
Dietary iron absorption 1-2 mg / day
4 mg iron Supply = Demand
Bone marrow erythroblastic demand-30-40 mg iron /day
Spleen 120 day life span of erythrocyte releases 30-40 mg iron / day Iron Losses : 1-2 mg/day
Erythrocytes 2500 mg iron
Fig. 1: Iron cycle : normal situation (Hb = 14 g/dl)
border of the absorptive cell, the ferric iron is converted into ferrous form by a ferrireductase, then transported across the membrane by divalent metal transporter type 1 (DMT-1). Once inside the cell, iron is stored as ferritin or transported to plasma transferrin by ion exporter â€“ ferroportin, during the process iron is reconverted to ferric form by a ferroxidase- hephaestin. The function of ferroportin is negatively regulated by hepcidin, the principal iron regulatory hormone whose level gets lower where demand is increased as in iron deficiency states.
THE IRON CYCLE
Iron absorbed from diet or released from stores circulates in plasma bound to transferrin until it interacts with specific transferrin receptor on the surface of marrow erythroid cells, where then iron gets internalized and haem synthesis takes place, the excess iron in the cell gets stored as ferritin. This iron exchange mechanism also takes place in other cells of body expressing transferrin receptors specially hepatocytes and reticulo-endothelial cells where iron can be incorporated into haem containing enzymes or stored. The iron incorporated into haemoglobin subsequently enters the circulation as new red cell mass. 0.8 to 1% of red cells are replaced each day by reticuloendothelial(RE ) system and the iron released is recycled through the circulating transferrin. There is no regulated excretory pathway for iron, the only mechanism of iron loss is through the loss of epithelial cells and blood loss (Figure 1).
NUTRITIONAL IRON BALANCE
Most of the iron in body is recycled. The daily loss normally is approximately 1 mg in men and post menopausal women and about 2 mg in menstruating women which gets replaced by dietary iron normally absorbed.. Dietary iron usually amounts to 7mg/1000Kcal that is about 12 to 15 mg /day of which less than10% is from haem iron and more than 90% from non-haem iron. 30% iron of haem source and less than 5% of nonhaem source is available for absorption. Normally only 10 to 15% of ingested iron is absorbed so when demand increases, dietary intake has to be increased. Pregnancy, anaemia, hypoxia and reduced iron stores increases the demand as also the growing infant, children and adolescent. However absorption can be raised only to the maximum of about 3.5 mg / day. Presence of amino acids and vitamin C increases the absorption of non-haem iron whereas phytates and phosphates in vegetarian diet, tannins in tea and alkali intake decreases the absorption implying the importance of rationalizing the timing of different food and iron tablets.
Table 1: Causes of Iron Deficiency
haematocrit remains normal.
Increased Iron Loss / Blood Loss –
Asymptomatic - diagnosed incidentally on routine laboratory examination, or may present with symptoms attributed to the anaemia or the iron deficiency proper beside to that of the underlying cause.
Gastrointestinal blood loss Occult gastric or colorectal malignancy Gastritis Peptic ulceration Chronic use of aspirin or NSAIDS Varices Inflammatory bowel disease Diverticulitis Polyp Hemorrhoids Angiodysplastic lesions Hookworm, Schistosomiasis, Trichuriasis
2. Menstrual blood loss 3. Reapetd blood donation 4. Alveolar hemorrhage 5. Haemoglobinuria
Related to Anemia: Fatigue, Breathlessness, Palpitation, Dizziness, Headache, Irritability
Related to Iron Deficiency:
Impaired Growth and cognitive function, Poor concentration
Skin and mucosal changes Fingernails may become brittle, fragile, or longitudinally ridged and then spooning (koilonychia) develop. Tongue may show soreness, mild papillary atrophy and absence of filiform papillae Angular cheilosis Dysphagia (Plummer-Vinson syndrome)
Miscellaneous: disturbed thermoregulation, impaired immune function, increase in infection, decreased physical performance and exercise tolerance, Pica –craving for specific food (ice, clay, starch etc), Restless leg syndrome
6. Massive hemorrhage II
Decreased Iron Intake or Absorption 1. Inadequate diet 2. Malabsorption from disease (Coeliac disease), 3. Malabsorptin from surgery (gastrectomy, some forms of bariatric surgery) 4. Acute or chronic inflammation - H. pylori associated gastritis etc 5. Achlorhydria
III Increased Requirements For Iron 1. Rapid growth in infancy, childhood and adolescence
Aggravates complication in pregnancy for both mother and fetus. Mother has increased likelihood to develop unpleasant symptoms, antepartum and postpartum hemorrhage, reduced quality and quantity of lactation and delayed wound healing etc. Fetus/ baby suffer low uterine growth, premature delivery, low birth weight etc.
Aggravates the severity with increased morbidity and mortality of cardiovascular instability
2. Pregnancy, lactation 3. Erythropoetin therapy
CAUSES OF IRON DEFICIENCY
Iron deficiency results when iron losses or physiological requirements exceed absorption (Table 1).
STAGES OF IRON DEFICIENCY
Iron deficiency passes through pre latent (negative iron balance) and latent (iron deficient erythropoiesis) phase to Iron deficiency anaemia. 1.
Pre-latent Phase: When demands for or losses of iron exceeds the body capacity to absorb iron from diet resulting in iron deficiency, iron is mobilized from reticulo-endothelial storage to maintain erythropoiesis. Thus iron store decreases and is reflected by reduced – stainable marrow iron and serum Ferritin. Other parameters remains normal. Latent Phase: Once the iron stores gets exhausted (serum Ferritin < 15ug/L ), serum iron starts falling and Total iron binding capacity (TIBC) and erythrocytes Protoporphyrin begins to rise, and when transferrin saturation falls to 15 – 20%, hemoglobin synthesis gets impaired but RBC and
Iron Deficiency Anemia: Once the transferrin saturation falls to 10-15%, hemoglobin and haematocrit falls with the appearance of microcytic and hypochromic RBC.
Erythrocytes: Blood hemoglobin falls below the lower limit of normal, RBC count and PCV decreased, mean corpuscular volume (MCV) decreased to less than 80 fl, mean corpuscular haemoglobin (MCH) < 28 pg/ cell, and mean corpuscular haemoglobin concentration (MCHC)<30g/dl, increased red cell distribution (RDW) with low or inappropriately normal reticulocyte count. Peripheral Smear: Characterized by microcytic and hypochromic RBC along with Anisocytosis (cells of varying size) and poikilocytosis (cells of varying shape – pencil/cigar shaped)
Value less than 12ng/cc is a highly reliable indicator of depleted iron stores. However higher level are the rule in
Table 2: Differential Diagnosis of Microcytic Anaemia Tests
Micro/hypo + Anisocytosis
Normal or micro/ hypo
Serum iron (ug/dl)
<50 or norm
Nor. To high
Normal to high
Free RBC Protoporphyrin
Haemoglobin pattern on electrophoresis
Low or absent
Normal or increased
Ring sideroblasts >15%
the presence of infection or inflammation.
Serum Iron and TIBC
Serum iron level (normal 30 ug/dl to 160 ug /dl ) represents the amount of circulating iron bound to transferrin and is decreased in iron deficiency. TIBC (normal 220ug/dl to 420 ug/dl), an indirect measure of circulating transferrin is increased. Transferrin saturation which is normally 2550% is always reduced to less than 16% in IDA.
Soluble Transferrin Receptor
Soluble transferrin receptor (sTfR – normal 4-9 ug/L) is markedly increased in IDA and helps to distinguish IDA from anaemia of chronic disease(ACD).
Red Cell Protoporphyrin Level
Free erythrocyte Protoporphyrin (FEP-normal <30ug/ dl of RBC) gets increased with impaired haem synthesis in IDA and sideroblastic anaemia and helps distinguish these from thalassaemia where it is normal.
Decreased or absent stainable iron in marrow macrophages is indicative of iron deficiency.
IDA needs to be differentiated from three other important causes of microcytic hypochromic anaemia- 1. Thalassaemia resulting from inherited defect of globin synthesis, 2. Anaemia of chronic disease resulting from impaired iron utilization and 3. Sideroblastic anaemia resulting from impaired haem synthesis because of impaired iron incorporation in haem. Body iron stores are increased in all these three situation where as it is decreased in IDA (Table 2).
Management of IDA needs not only the correction of anaemia but also requires replenishment of depleted iron stores. It also warrants careful search of the underlying cause and their proper treatment. The common and usual sufferers –pregnant women, growing children and adolescents must always be looked and properly managed for IDA to prevent untoward complications. Treating
hookworm infection empirically is justified in Indian context. Cardinal rule of considering gastrointestinal blood loss as a cause of unexplained IDA until otherwise proved in adult male and post-menopausal women should be observed. Patients with recurrent or not responding IDA should be screened for celiac disease or other causes of mal-absorption. There are three major therapeutic approaches for treating iron deficiency-
Reserved for patients with severe symptomatic anaemia, anaemia with cardio-vascular instability, and those with continued and excessive blood loss.
Usually treatment with oral iron is adequate. Ferrous sulphate 200 mg 8-hourly (120 mg of elemental iron/day) is usually more than sufficient. Usual adult dose is 150mg to 200mg of elemental iron per day and the dose for children is 3 mg /kg/day. Absorption is better when taken on empty stomach. If not tolerated, dose reduction to 12 hourly and taking after meal may help or shift to other ferrous salt. Haemoglobin should rise by 1g/dl every 7 to 10 days and a reticulocyte response will be evident by 1 week. After correction of anaemia, sustained treatment should be continued for 4 to 6 months to replenish the iron store.
PARENTERAL IRON THERAPY
Needed in patients who are non-compliant or intolerant to oral iron, who suffers mal-absorption states, whose need is relatively acute as in pregnancy or cardiovascular instability or in conjunction with erythropoietin in renal failure and other causes of qualitative iron deficiency. Iron dextran requires test dose and may cause anaphylaxis but the newer Parenteral preparations (iron sucrose, ferric carboxymaltose, ferumoxytol) are relatively safe as reactions are very rare and is given intravenous. Parenteral iron excepting iron sucrose can be given to deliver total dose. Total iron (mg) needed = Body weight (kg) x 2.3 x (15 –
Serum transferrin receptor
patientâ€™s haemoglobin, g/dl) + 500 or 1000 mg (for stores). Iron sucrose (100mg in 5cc)- only iv use, maximum single dose 100mg can be given as slow iv injection over 5 minutes or as infusion in 100 cc saline over 30 minutes. Maximum 600 mg/week.
meticulous evaluation and proper management to prevent mortality and morbidity and use of IV iron where indicated.
World Health Organization. Iron deficiency anaemia: assessment, prevention and control: A guide for program managers. WHO, UNICEF, UNU, Geneva, Switerzerland.2001;3 6:314-62.
Reddy Raakrishna et al. Prevalence of iron deficiency anaemia and malnutrition in India. Social and economic change monograph series Jan 2004; 3:01-115
Umbreit J. Iron deficiency: A concise review. Am j Hematol 2005; 78:225-31.
Ferric carboxymaltose â€“ 1000mg /week as iv infusion over 6 to 15 minutes, (not > 15 mg/kg).
Comparative clinical trials showed a faster, better tolerated & more prolonged effect with better quality of life on IV iron than with oral iron in the indicated situation.
National program to prevent iron deficiency needs to be instituted more effectively. Iron deficiency warrants
C H A P T E R
Iron Deficiency in Heart Failure K Sarat Chandra
Iron deficiency in heart failure has attracted considerable attention in recent years. It is agreeable to treat a patient of heart failure with iron in case the patient has iron deficiency anaemia. However, several trials which were recently published have indicated that patients of heart failure will benefit significantly when infused with iron, even in the absence of anaemia, if there is iron deficiency. The following questions need to be answered before concluding the above hypothesis.
prognosis. There is not enough data regarding oral iron therapy in this setting possibly due to intolerance of oral Iron as well as a number of drug interactions. Most of the trials of treating iron deficiency in heart failure used intravenous iron. There are several iron preparations for IV use, iron dextran, iron gluconate, iron sucrose and ferric carboxymaltose (FCM). The iron dextran is given up due to enaphylactic reactions. The newer preparations do not seem to have these issues.
WHAT IS THE SCOPE OF THE PROBLEM?
An initial study has compared IV sucrose in 24 patients with CHF and iron deficiency to 11 patients with CHF and without iron deficiency (ferric HF Study)8. It showed that 16 weeks of iron therapy was well tolerated and led to improvement in exercise capacity and symptoms in patients with iron deficiency.
Iron deficiency is an independent risk factor for mortality in HF.1,2,3 Half of all patients with HF have either absolute iron deficiency or functional Iron deficiency defined as ferritin less than 100 µg/L or transferrin saturation less than 20% and serum ferritin between 100-300 µg/L.4,5 This may or may not be associated with anaemia.
HOW DOES HEART FAILURE LEAD TO IRON DEFICIENCY?
This could be due to reduced absorption of iron from the duodenum secondary to edema. There is another interesting mechanism. Increased levels of pro inflammatory cytokines such as interleukin-6 enhance production and release of hepcidin, which is a protein synthesized in the liver. Hepcidin regulates the release of stored iron from enterocytes and hepatocytes by leading to degradation of iron transporter protein, ferroportin. Thus high levels of hepcidin can “trap” iron in storage cells.
HOW DOES IRON DEFICIENCY LEAD TO WORSENED SYMPTOMS IN HEART FAILURE PATIENTS WITHOUT ANEMIA?
It is understandable that iron deficiency could lead to worsened symptoms in heart failure patients with anaemia. However the mechanism of increased symptoms in the absence of anaemia is not fully understood. Iron could be acting as a co-factor in skeletal and cardiac muscle function.6,7 Experimental evidence suggests that iron therapy improves muscle function and exercise capacity without increase in the haemoglobin. In addition iron is an important constituent of myoglobin which binds and releases oxygen. Mitochondrial function also needs iron as a co-factor for heme proteins.
THE DATA FROM DIFFERENT TRIALS
It is natural that if iron deficiency can lead to increased symptoms and worse prognosis in patients of heart failure, administration of iron could lead to improved
A later study called FAIR-HF used FCM in a multicentre, double blind, placebo controlled fashion.9 The investigators showed that FCM treatment in both Iron deficient anaemic and non-anaemic patients with CHF increased the following: a.
Distance walked during 6 minutes walk test
Overall quality of life
The study enrolled 459 patients with CHF (Hb ranging from 9.5 to 13.5 gm%). The definition for Iron deficiency in this trial was ferritin between 100 to 300 ng/ml with TSAT less than 20%. The improvement in QoL occurred in both anaemic and non-anaemic patients suggesting that Iron deficiency is an important comorbidity in HF that can be treated. A rise in ferritin level and TSAT is there for all the patients. CONFIRM-HF is a multi-centre, double-blind, placebocontrolled trial that enrolled 304 symptomatic HF patients with left ventricular ejection fraction (LVEF) less than or equal to 45%, elevated natriuretic peptides, and Iron deficiency (ferritin, 100 ng/ml or 100-300 ng/ml, if TSAT, 20%).10 Patients were randomized 1:1 to treatment with ferric carboxymaltose or placebo for 52 weeks. The primary end point was the change in 6MWT distance from baseline to week 24. Secondary end points included changes in NYHA class, patient global assessment (PGA), 6MWT distance, health-related QoL Fatique score at weeks 6,12,24,36 and 52 and the effect of FCM on the rate of hospitalization for worsening HF. Treatment with FCM significantly prolonged 6MWT distance at week 24. The treatment effect of FCM was consistent in all subgroups
and was sustained to week 52. Throughout the study, an improvement in NYHA class, PGA, QoL, and Fatigue score in patients treated with FCM was detected with statistical significance observed from week 24 onwards. Treatment with FCM was associated with a significant reduction in the risk of hospitalization for worsening HF. The number of deaths (FCM:12, placebo:14 deaths) and the incidence of adverse events were comparable between both groups (Figures 1 and 2). The IRON-HF study analysed the effects of intravenous as well as oral Iron on functional capacity in patients of heart failure with Iron deficiency and anaemia.11 The results showed that IV iron is superior to oral therapy. In a recent meta-analysis published in European Journal of Heart Failure, all the randomized trials of Iron therapy in HF have been analysed.12 Data from a total of 851 patients from 5 trials was studied. The results confirm that intravenous Iron therapy reduces the need for hospital admissions for worsening HF. There is a reduction in NYHA class, increase in 6 min walking distance and an improvement in quality of life by different scoring systems (Table 1). There was no effect on all cause and cardiovascular mortality which may be due to a low number of reported deaths (4% vs.6% for those treated vs. not treated with I.V Iron). Also the benefits were there for both anaemic and non-anaemic patients.
Okonko DO, Mandal AK, Missouris CG, et al. Disordered Iron homeostasis in chronic heart failure: Prevalence, predictors, and relation to anemia, exercise capacity, and survival. J Am Coll Cardiol 2011; 58:1241-51.
Klip IT, Comin-Collet J. Voors AA et al. Iron deficiency in chronic heart failure. An international pooled analysis. Am Heart J 2013; 165:575-82.
Jankowska EA, von Haehling S, Anker SD, et al. Iron deficiency and heart failure: Diagnostic dilemmas and therapeutic perspectives. Eur Heart J 2013; 34:816-29.
Van Veldhuisen D J, Anker SD, Ponikowski P, et al. Anemia and iron deficiency in heart failure: Mechanisms and therapeutic approaches. Nat Rev Cardiol 2011; 8:485-93.
Kapoor M, Schleinitz M D, Gemignani A, et al. Outcomes of patients with chronic heart failure and iron deficiency treated with intravenous Iron: A Meta-analysis. Cardivasc Hematol Disord Drug Targets 2012; 13:35-44.
Willis WT, Gohil K, Brooks GA, et al. Iron Deficiency: Improved exercise performance within 15 hours of Iron treatments in rats. J Nutr 1990; 120:909-16.
Tobin BW, Beard JL. Interactions of Iron deficiency and exercise training relative to tissue norepinephrine turnover, triodothyronine production and metabolic rate in rats. J Nutr 1990; 120:900-8.
Okonko DO, Grzeslo A, Witkowski T, et al. Effect of intravenous iron sucrose on exercise tolerance in anemic and nonanemic patients with symptomatic chronic heart failure and Iron deficiency FERRIC-HF: A randomized, controlled, observer-blinded trial. J Am Coll Cardiol 2008; 51:103-12.
Anker SD, Comin Colet J, Filippatos G, et al. Ferric Carboxymaltose in patients with heart failure and Iron deficiency. NEJM 2009; 361:2436-48.
The following conclusions can be drawn at this stage: 1.
Iron deficiency is common in heart failure patients.
It may or may not be associated with anaemia.
Iron replacement therapy improves quality of life, 6MWT and NYHA class in patients with and without anaemia.
There is also a reduction in the need for hospitalization for worsening heart failure.
Mortality however is not improved.
These patients should be investigated for source of bleeding like peptic ulcer.
Based on the above data the European Society of Cardiology guidelines in 2012 have recommended Iron replacement therapy for symptomatic patients of heart failure with systolic dysfunction and Iron deficiency (Class II a, level of evidence A).
10. Ponikowski P, Van Veldhuisen DJ, Comin Colet J, et al. Beneficial effects of long term intravenous Iron therapy with Ferric Carboxymaltose in patients with symptomatic heart failure and Iron deficiency. Eur Heart J 2015; 36:65768. 11. Beck-Da-Silva L, Piardi D, Soder S, et al. IRON- HF study: A randomized trial to access the effects of Iron in heart failure patients with anemia. Int J Cardiol 2013; 168:3439-42. 12. Ewa A. Jankowska, MichaĹ‚ Tkaczyszyn, Tomasz Suchocki, et al. Effects of intravenous Iron therapy in Iron-deficient patients with systolic heart failure: A meta-analysis of randomized controlled trials. European Journal of Heart Failure 2016; 18:786â€“795.
C H A P T E R
Anaemia of Chronic Disease: Current Update
Anaemia of chronic disease (ACD) or anaemia of chronic inflammation may be secondary to infections, auto immune disorders or malignancies. It is characterized by an immune activation with an increase in inflammatory cytokines and resultant increase in hepcidin levels. In addition, inappropriate erythropoietin levels or hyporesponiveness to erythropoietin and reduced red blood cell survival contribute to the anaemia. Hepcidin being the central regulator of Iron Metabolism plays a key role in the Patho physiology of ACD. Hepcidin binds to the Iron export protein, ferroportin, present on macrophages, hepatocytes and enterocytes, causing degradation of the later. This leads to iron trapping within the macrophages and hepatocytes, resulting in functional Iron deficiency. Production of hepcidin in turn regulated by iron stores, inflammation and erythropoiesis via the BMP-SMAD and JAK-STAT signalling pathways. Treatment of anaemia should primarily be directed at the underlying disease, and conventional therapy such as red blood cell trans--fusions, iron, erythropoietin and novel agents targeting the hepcidin ferroportin axis and signalling pathways (BMP6-HJV-SMAD and IL-6-JAK-STAT) involved in hepcidin production also may be considered.
Anaemia of chronic disease (ACD) or Anaemia of chronic inflammation is the term used to describe the hypoprolifeative anaemia seen in response to chronic infection, chronic immune activation and malignancy. It is the second most prevalent form of anaemia after iron deficiency anaemia (IDA).1 The anaemia is typically normochromic,normocytic and mild in degree. However other observations have shown that ACD can be seen in a variety of other Conditions including severe trauma, diabetes mellitus and anaemia of older adults. All these condition produce massive elevation of interleukin-6 (IL-6), which stimulates Hepcidin production & release from the liver which in turn reduces the Iron Carrier protein, ferroportin,2 so that access of Iron to the circulation is reduced as well as iron absorption in the small intestine, iron transport across the placenta and release from the macrophages are also reduced. Also there is impaired production of erythropoietin (EPO), blunted marrow erythroid response to EPO, Iron restricted erythropoiesis and a diminished pool of EPO- responsive cells.3
Ananda Bagchi, Aradhya Sekhar Bagchi
ACD is considered the second most common cause of anaemia world wide however detailed statistics on its prevalence are not available. Often the anaemia in individuals with inflammatory diseases is complex and multifactorial4 and it may be challenging to separate out the component due to ACD. This is especially true in patients with diabetes. Examples of the prevalence of ACD in various inflammatory states include the following : •
Anaemia is observed in 33 to 60 % of patients with rheumatoid arthritis.5
ACD accounts for about 1/3rd of the cases of anaemia of the elderly because of concomitant inflammatory conditions or chronic kidney diseases.
Cancer elated anaemia accurs in more than 30% of the cases at diagnosis,6 the rate reached 63% in an observational study on 888 consecutive cancer patients. however cancer related anaemia is multifactorial and includes types of anaemia other than ACD (eg IDA). Anaemia is even more common in haematologic malignancies like lymphoma and multiple myeloma.
ACUTE VARIANT OF ACD
Acute event related anaemia, such as that occruing after surgery, major trauma, my ocardial infarction or sepsis, a condition called the Anaemia of Critical illness, shows many of the features of ACD (i.e. low serum Iron, high ferritin, blunted response to EPO), presumably secondary to tissue damage and acute inflammatory changes.7 It appears to be an acute variant of ACD and is also characterized by shortened RBC survival.8,9
Anaemia with Chronic Obstructive Pulmonary Disease
A subset of patients with chronic obstructive pulmonary disease,estimated at approximately 50%, have laboratory findings consistent with ACD (eg, anaemia, elevated levels of CRP, IL-6, IFN γ and low serum erythropoietin) suggesting the presence of back ground infection or inflammation.
Anaemia with chronic kidney disease
Anaemia with chronic kidney disease shares some of the characteristics of ACD, although the decrease in the production of erythropoietin, mediated by renal insufficiency and the anti proliferative effects of accumulating uremic toxins, contribute importantly.10 In addition, in patients with ESRD, chronic immune
Table 1: Diseases associated with ACD Associated diseases
concert with hepcidin in inducing the changes found in ACD.12
Causes and Pathogenesis A.
Altered iron homeostasis with Inflammation and Hepcidin including role of cytokines
It has been suggested that the underlying inflammatory medical condition causes the release of cytokines such as the interleukins (IL-1 & IL6) and tumor necrosis factor-alpha by activated monocytes, these cytokines unleash a cascade which include the secretion of interferon (IFN-B and IFN-γ) by T-lymphocytes 11. As an example, IFN gamma h when given to experimental animals, can produce picture of ACD.
The decreased bone marrow responsiveness to erythropoietin is mediated by inflammatory cytokines.
Especially IL-1 beta and TNF-α which may induce apoptosis of red cell precursors as well as down regulation of erythropoietin receptors on progenitor cells. Cytokines may also decrease erythropoietin expression by renal cells.13 In vitro treatment of cultured cells with proinflammatory cytokines can also alter ferritin and transferrin receptor expression and Iron-responsive protein activity in macrophages.
Studies suggest that IL-6 is required for the induction of hepcidin and hypoferremia during inflammation in both animals and humans, although hepcidin can also be upregulated by the cytokine IL -1, while the molecular mechanisms responsible for this activation are only partially understood, IL-6 appears to be involved in the regulation of hepcidin levels through the JAK-stat 3 signalling pathway.
Reduced EPO production: Under normal physiologic conditions, levels of EPO are inversely correlated with haemoglobin levels and tissue oxygenation, but in chronic inflammatory conditions the EPO response is blunted, leading to inadequate levels of EPO.
Reduced Erythroid Responsiveness(Impaired proliferation of EP cells): In ACD, the proliferation and differentiation of Erythroid Progenitor (EP) cells is reduced.Hepcidin itself has an inhibitory effect on erythropoiesis in vitro at low EPO concentrations.
Systemic lupus erythematosusand related conditions
Inflammatory bowel disease
Chronic renal failure
Chronic heart failure
activation can arise from contact activation of immune cells, by dialysis membranes, from frequent episodes of infection or from both factors, and such patients present with changes in the homeostasis of body iron that is typical of anaemia of chronic disease.
Diseases associated with ACD
Conditions associated with ACD are listed in Table 1. These diseases all share features of acute or chronic immune activation.
Anemia of chronic disease is immune driven; cytokines and cells of the reticuloendothelial system induce changes in iron homeostasis, the proliferation of erythroid progenitor cells, the production of erythropoietin, and the shortened life span of red cells, all of which contribute to the pathogenesis of anemia.11 Erythropoiesis can be affected by disease underlying anemia of chronic disease through the infiltration of tumor cells into bone marrow or of microorganisms, as seen in human immunodeficiency virus (HIV) infection, hepatitis C, and malaria. Moreover, tumor cells can produce proinflammatory cytokines and free radicals that damage erythroid progenitor cells. Bleeding episodes, vitamin deficiencies (e.g., cobalamin, folic acid and vitD), hypersplenism, autoimmune hemolysis, renal dysfunction, and radioand chemotherapeutic interventions themselves can also aggravate anemia. There are sufficient datas and findings that suggest that hepciin may be central to the anaemia of chronic disease. A recently identified gene ‘HEMOJUVELIN’ may act in
In ACD both groups of erythroid precursors erythroid burst forming units and erythroid colony forming units are impaired and are linked to inhibitory effects of IFNalpha,beta and gamma,TNF-alpha and interleukin -1 which influence the growth of erythroid burst forming units and erythroid colony forming units. IFN-gamma appears to be most potent inhibitor.14 The underlying mechanisms may involve cytokine
Table 2: Serum levels that differentiates Anemia of Chronic disease from iron deficiency anemia Anemia of Chronic Disease
Reduced to normal
Normal to increased
Reduced to normal
Soluble transferrin receptor
Normal to increased
Ratio of soluble transferrin receptor to log ferritin
*Relative changes are given in relation to the respective normal values. †Patients with both conditions include those with anemia of chronic disease and true iron deficiency.
mediated induction of apoptosis. Moreover, cytokines exert direct toxic effects on progenitor cells by inducing the formation of labile free radicals such as nitric oxide or superoxide anion by neighbouring macrophage like cells.
REDUCED RED CELL SURVIVAL
Red cell survival is modestly shortened in patients of Rheumatoid Arthritis and other conditions, may be a contributory factor in ACD, but there have been no direct studies of the mechanisms involved: these may include increased Erythrophagocytosis induced by inflammatory cytokines.
Diagnostic issues in ACD
The widespread settings in which ACD may be seen can make diagnosis difficult. Typically the anaemia is mild to moderate, Hb Conc. 10 to 11 gm/d l normochromic and normocytic (although anaemia may become microcytic as disease progresses) and the reticulocyte count is low, the absolute reticulocyte count is <25000/micro L &reticulocyte Hb content is <28 pg/dl. The anaemia may be accompanied by an elevation of cytokines (IL-6) as well as acute phase reactants (eg fibrinogen, ESR, CRP, ferritin, haptoglobin factor, viii) reflecting the hypoproliferative nature of the anaemia. Exclusion of IDA is very important in the work-up of patients with ACD, although the two conditions frequently co-exist. Typically, serum iron and transferrin saturation are both decreased in ACD and iron deficiency, indicating limited iron supply to the erythron, but transferrin levels are increased in IDA, whereas in ACD they are normal or decreased.
If the distinction between ACD and ACD/IDA cannot be made by laboratory tests alone, one may monitor the response to a short trial (4-6 wks) of oral iron supplementation. Measurement of erythropoietin levels is useful only for anemic patients with hemoglobin levels of less than 10 g per deciliter, since erythropoietin levels at higher hemoglobin concentrations remains well in the normal range. The measurement of Serum transferrin receptor (sTFR), the truncated fragment of the membrane receptor, has been suggested as a possible tool for differentiating between ACD and IDA. The ratio of sTFR to the log of the serum ferritin (sTFR/ferritin ratio) has been proposed to be a useful tool in thediagnosis of ACD, and particularly in differentiating ACD from IDA. This ratio is effective in making this distinction since STfr is increased in IDA and normal in ACD while ferritin or log ferritin is decreased in IDA and normal to increased in ACD. Specifically a sTfr/ log ferritin ratio < 1 suggests ACD while a ratio >2 suggests IDA. Those with combination or ACD & IDA will also have a Tfr-ferritin index >2.13 Hepcidin Assays-Assays to measure serum Hepcidin are not yet routinely available for clinical use. In one study measurement of Hepcidin-25 level by mass spectrometry was proposed as a potential tool for differentiating ACD from IDA.14 The use of a Hepcidin-25 cut off ≤ 4nmol/L allowed the differentiation of IDA from ACD. Bone marrow picture-Examination of bone marrow for its content and distribution of Iron is instructive, although this examination is not routinely performed in all patients with suspected ACD. In the most classical presentation of ACD, bone marrow macrophages contain normal or increased amounts of storage Iron, reflecting Reduced export of iron from macrophapes due to the action of hepcidin. In addition,erythroid precursors show decreased or absent Staining for Iron (ie, decreased no. of sideroblasts) reflecting reduced availability of Iron for Redcell production.15
Differential Diagnosis by Bone marrow study
While bone marrow examination is not required for most patients in whom ACD is suspected, in difficult cases the diagnosis can often be established by bone marrow examination. Findings in the most common disorders include: •
ACD - Bone marrow macrophages contain normal to increased Iron,while erythroid precursors show
Measurement of serum ferritin is frequently of little value, as ferritin is an acute phase protein as well as an indicator of iron stores, and levels will be increased in the presence of inflammation. The gold standard for assessment of iron stores remains a Perl’s stained bone marrow aspirate, but a bone marrow biopsy is otherwise of limited value in the diagnosis of ACD, so other non-invasive tools for measurement of iron supply are needed.
Table 3: Therapeutic Options for the Treatment of Patients with Anemia of Chronic Disease
Anemia of Anemia of Chronic Disease Chronic Disease with True Iron Deficiency
Treatment of underlying disease
Yes, in patients who do not have a response to iron therapy
* This treatment is for the short-term correction of severe or lifethreatening anemia. Potentially adverse immunomodulatory effects of blood transfusions are controversial. †Although iron therapy is indicated for the correction of anemia of chronic disease in association with absolute iron deficiency, no data from prospective studies are available on the effects of iron therapy on the course of underlying chronic disease. ‡ Overcorrection of anemia (hemoglobin >12 g per deciliter) may be potentially harmful to patients: the clinical significance of erythropoietin-receptor expression on certain tumor cells needs to be investigated.
decreased to absent amounts of Iron (ie decreased to absent sideroblasts). •
IDA- stainable Iron is absent from both macrophages and erythroid precursors.
Single or multilineage dysplastic changes with or without increased number of sideroblasts, including ring forms, are commonly seen in myelodysplasia.
The diagnostic hallmark of the sideroblastic anaemias is the presence of ring sideroblasts on bone marrow examination. The amount of Iron in bone marrow macrophages is strikingly increased due to the presence of ineffective erythropoesis. Single or multilineage dysplastic changes are not seen.
Management of ACD
The anaemia observed in ACD is frequently mild, and correction may not always be necessary. Treatment of the underlying inflammatory or malignant process associated with ACD will often result in improvement in the degree of anaemia. Reasons for attempting to correct the anaemia present; firstly, anaemia may be deleterious in itself, with effects on the cardiovascular system needed to maintain tissue oxygen supply. Secondly, anaemia may be associated with a poorer prognosis in many chronic disease states. Thirdly, treatment may improve the quality of life for patients living with chronic conditions.
In cases in which treating the underlying disease is not feasible, alternative strategies are necessary (Table 3).
Blood transfusion is a simple means of treating patients with moderate to severe anaemia, but blood remains a precious and expensive resource, and transfusion therapy carries long-term risks of viral transmission, iron overload and alloimmunization. Transfusion should therefore be reserved for patients with severe or life-threatening anaemia in the context of ACD, and is not an appropriate treatment for patients with this form of chronic anaemia.
The rationale for the use of erythropoiesis-stimulating agents (ESA) in ACD is based on the blunted EPO response seen in ACD,16 with lower serum levels of EPO detected than would be expected for the observed degree of anaemia, together with the reduced sensitivity of erythroid progenitors to endogenous EPO seen in ACD. Measurement of serum EPO concentration may be helpful in patients with ACD who have symptomatic anaemia. and/or who have not responded to treatment of their underlying disorder and continue to have symptomatic anaemia requiring treatment. Recombinant human EPO (rHuEPO) and its derivatives are widely used in patients with chronic renal failure, patients with cancer undergoing chemotherapy and in patients infected with HIV on myelosuppressive antiretroviral medication. Several different rHuEPOs are currently available or in development: Haematology 2011, 154, 289–300].epoetin-α, epoetin-β, epoetin-δ, biosimilar epoetins, darbepoietin- α. The percentage of patients with anemia of chronic disease who respond to therapy with erythropoietic agents is 25 percent in myelodysplastic syndromes, 80 percent in multiple myeloma, and up to 95 percent in rheumatoid arthritis and chronic kidney disease. The therapeutic effect involves counteracting the antiproliferative effects of cytokines, along with the stimulation of iron uptake and heme biosynthesis in erythroid progenitor cells. Dosage of EPO- Although one of the hallmarks of ACD is a reduced erythropoietic response to both endogenous as well as exogenous EPO, high doses of EPO may overcome this hyporesponsiveness.17
TWO TREATMENT OPTIONS ARE AVAILABLE
standard dosing of EPO is a start up dose of 100 to 150 units /kg, subcutan- eously three times weekly along with Supplemental iron. Responders may show a rise in the haemoglobin concentration of at least 0.5 gm/dl by two to four wks. If there is no elevation in the Hb concentration by six to eight weeks.Then the regimen can be intensified to daily therapy or 300 units /kg three times weekly. It is not worth while to continue EPO in patients who
do not have clinically meaningful response by 12 weeks. B.
An alternative treatment schedule is to employ 30000 to 40000 units of EPO given subcutaneously once per week,18 a single dose that is numerically equivalent to a dose of 140 to 190 units/kg three times per week for a 70 kg person. the dose can be increased to 60000 units if there is no response (ie Hb rise <1 gm/dl) at four weeks. For ease of use and to minimize inconvienience to the patient, the second schedule is preferred.
Potential adverse effects of EPO therapy can be minimised by initiating treatment when the patients Hb% is <10 gm/dl and stopping treatment when Hb% reaches 12gm/dl.
Darbepoietin: Although darbepoietin has had limited use in the treatment of ACD cases in humans, it is capable of reversing anaemia due to chronic Inflammatory disease in experimental animals. A dose of darbepoietin equivalent to the above noted dose of EPO is in the range of 60 to 100 microgm/wk or 300 ugm every 3 weeks. However,if we are looking for a rapid, short term response of the anaemia, darbepoietin with its prolonged half life is less preferred.
Oral iron supplements are often poorly tolerated, and patients frequently exhibit poor compliance: in addition, patients with ACD will usually have raised hepcidin levels, which would be expected to inhibit intestinal iron absorption. However, oral iron is cheap, widely available, and easy to administer, and given the difficulties in ruling out concomitant IDA in many patients with ACD, a therapeutic trial of oral iron will be undertaken by many clinicians treating ACD. It must however be recognized that failure to respond to oral iron rules out neither true, nor functional iron deficiency.
Novel Agents for the Future
The increased production of hepcidin in ACD Serves as the rationale for development of inhibitors targeting the BMP-HJV-SMAD and IL-6-JAK-STAT pathway that are involved in hepcidin synthesis. • Dorsomrphin, is a small molecular inhibitor of BMP receptor and LDN-193189,19 a more selective BMP inhibitor has been tried. • Other agents include anti- BMP- 6 monoclonal antibody and soluble HJV. In addition heparin has also been shown to decrease BMP-SMAD signalling20 and inhibit hepcidin transcription. • Iron chelation therapy has also been advocated to induce endogenous formation of EPO. • Also in the development are hepcidin antagonists20 (monoclonal antibodies, small interfering RNA [SiRNA], antisense oligonucleotides, hepcidin binding proteins and aptamers). Anti hepcidin monoclonal antibodies bind to hepcidin and prevent it from binding to ferroportin.21 • In addition hepcidin binding proteins, anticalines and spiegelmers are also being developed22 the anti hepcidin spieglmers NOX-H94 is in a phase II-A clinical trial for the treatment of ACD with a very promising result. • In regards to the IL- 6- JAK-STAT pathway, monoclonal IL-6 and IL-6R23 antibodies (siltuximab and tocilizumab) and JAK-2 and STAT-3 inhibitors, all have been shown to down regulate hepcidin expression.
Much of the literature concerning intravenous iron supplementation has come from the field of renal medicine, where the superiority of parenteral over oral iron supplementation is now well established. There is now evidence that intravenous iron can enhance the effects of ESAs in patients with other forms of ACD, particularly cancer-related anaemia. Auerbach et al (2004) randomized 155 patients being treated with ESAs for chemotherapy-related anaemia to no iron, oral iron or intravenous iron: there were significant improvements in haematological responses in patients receiving intravenous iron compared with those receiving either no iron or oral iron.
• Vit D deficiency is associated with an increased prevalence of ACD, and vit D replacement lowers hepcidin levels.
It is not yet known how intravenous iron might overcome the reticuloendothelial blockade on iron utilization thought to be fundamental to the pathogenesis of ACD, but it is possible that the infused iron may become bound directly to transferrin rather than being taken up by macrophages, and is thus available to the erythron.
For monitoring the response to erythropoietic agents, hemoglobin levels should be determined after four weeks of therapy and at intervals of two to four weeks thereafter. If the hemoglobin level increases by less than 1 g per deciliter, the iron status should be re -evaluated and iron supplementation considered. If iron-restricted
• Since hepcidin excess blocks ferroportin by causing degradation of the latter, agents that stabilize ferroportin or inhibit the interaction with hepcidin are an attractive target which will pave the way for the development of antiferroportin monoclonal antibody that blocks hepcidin ferroportin interaction.
Before the initiation of therapy with an erythropoietic agent, iron deficiency should be ruled out.
Recent developments have led to the release of several new iron formulations including low molecular weight iron dextran, iron sucrose, ferric carboxymaltose and sodium ferric gluconate In the trials, no excess of adverse effects was observed with these newer intravenous iron preparations.
erythropoiesis is not present, a 50 percent escalation in the dose of the erythropoietic agent is indicated. The dose of the erythropoietic agent should be adjusted once the hemoglobin concentration reaches 12 g per deciliter. If no response is achieved after eight weeks of optimal dosage in the absence of iron deficiency, a patient is considered nonresponsive to erythropoietic agents.
The anaemia in ACD contributes hugely to the morbidity experienced by millions of patients worldwide suffering from a large variety of inflammatory, infective and malignant conditions. Recent years have seen a marked expansion in our understanding of the pathogenesis of ACD, particularly in the key role played by hepcidin in mediating the functional iron deficiency that is the hallmark of this very common form of anaemia. Future strategies may include the use of iron-chelation therapy to induce the endogenous formation of erythropoietin, Hepcidin antagonists that overcome the retention of iron within the reticuloendothelial system, and hormones or cytokines that might effectively stimulate erythropoiesis under inflammatory conditions.
Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J Med 2005; 352:1011-23.
Cullis JO. Diagnosis and management of anaemia of chronic disease: current status. British Journal of Haematology 2011; 154:289–300.
Gangat N and Wolanskyj AP. Anemia of Chronic Disease. Semin Hematol 2013; 50:232–238.
Sun C C, Vaja V,Babitt JL etal, targeting the hepcidin Ferroportin axis to develop new treatment strategies for anaemia of chronic disease and anaemia of inflammation. Am J Haemato 2012; 87:392-400.
Weiss G, Schett G. Anaemia in Inflammatory Rheumatic deiseases. Nat Rev Rheumatol 2013; 9:205.
Ludwig H, Van Belle S, Barett –Lee P, etal. The European cancer anaemia survey (ECAS). Eur J Cancer 2004; 40:2293.
Maccio A, Madeddu C etal. The role of inflammation, iron and nutritional status in cancer.related anaemia; result of a large, prospective, observational study. Haematologica 2015; 100:124.
Van Iperen CE, Van de Weil A etal. Acute event related anaemia. Br J Haematology 2001; 115:739.
Rodriguez RM, Corwin HL, Gettinger A, et al. Nutritional deficiencies and blunted erythropoietin response as causes of the anaemia of critical illness. J Crit Care 2001; 16:36
10. Corwin HL, Gettinger A, et al. Efficacy of recombinant human erythropoietin in critically ill patients- a randomized controlled trial. JAMA 2002; 288:2827. 11. Raj DS. Role of Interleukin -6 in the anaemia of chronic disease. Semin Arthritis Rheum 2009; 38:382. 12. Means RT jr. Recent developments in the anaemia of chronic disease. Curr Haematol Rep 2003: 2:H6. 13. Punnone NK, Irjala K, Rajamaki A. Serum transferrin receptor and its ratio to serum ferrition in the diagnosis of iron deficiency. Blood 1997; 89:1052. 14. Thomas C, Kobold U, Thomas L. Serum hepcidin-25 in comparison to biochemical markers and hematological indices for the differentiation of Iron restricted erythroporesis. Clin Chem Lab Med 2011; 49:207. 15. Gardner Dl,Roy LM. Tissue Iron and the Reticuloendothelial system in Rheumatoid Arthritis. Ann Rheum Dis 1961; 20:258. 16.
Theurl I, Mattle V etal. Dysregulated monocyte Iron homeostasis and erythropoietin formation in patients with anaemia of chronic disease. Blood 2006; 107:4142
17. Ludwig H,Fritz E etal.Prediction of response to erythropoietin treatment in chronic anaemia of cancer. Blood 1994; 84:1056. 18. Soignet S. Management of cancer related anaemia :epoietin –alpha and quality of life. Semin Haematol 2000; 37:9 19. Theurl I, Schroll A, et al. pharmacologic inhibition of hepcidin expression reverses anaemia of chronic inflammation in rats. Blood 2011; 118:4977-84. 20. Andriopoulos B Jr.,Corradini E etal. BMP -6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nat Genet 2009; 41:482-87. 21. Sasu B J, Cooke KS etal. Antihepcidin antibody treatment modulates Iron metabolism and is effective in a mouse model of inflammation Induced anaemia. Blood 2010; 115:3616-24. 22. Vater A, Klussmann S etal. Toward third generation Aptamers :Spiegelmers and their therapeutic prospects. Curr Opin Drug Discov Dev 2003; 10:23-33. 23. Van Rhee F,Fayad L etal. Siltuximab, a novel anti – interleukin-6 receptor antibody, for castlemanns disease. J Clin Oncol 2010; 28:3701-08.
C H A P T E R
Clinical Utility of Erythrocyte Sedimentation Rate in Modern Era
Erythrocyte sedimentation rate (ESR) is one of the most frequently ordered test in clinical medicine. In the present era ESR is losing its glory, because of availability of more specific and sensitive markers like CRP, fibrinogen and ferritin. The method of ESR estimation was first described in 1921 by R Fahraeus and A Westergren in pulmonary tuberculosis, and it rapidly became a common screening test worldwide.1-2 Despite its limitations, the ESR remains a cost effective and widely used test for the screening and monitoring of infectious, autoimmune, malignant and other chronic inflammatory diseases, especially in developing country like India.
PATHOPHYSIOLOGY OF ESR
ESR is the rate at which the erythrocytes fall down by their own weight when anticoagulated blood is held in a straight column, in first hour. ESR is measured by Westergren and Wintrobe method. The International Committee for Standardization in Hematology (ICSH) recommended Westergren method as standard for measuring the ESR, which has now been accepted worldwide.3 In addition to these methods there are now automated methods available which give readings in less than one hour but the results correlates to the standard 1hr Westergren reference method. The ESR process in all can be divided into 3 phases. The first phase is the phase of rouleaux formation. During this phase the red cells owing to their discoid shape stack over each other. However due to a negative charge on their surface they repel each other and do not form rouleaux. The plasma proteins that neutralize this charge and enhance rouleaux formation are principally gammaglobulins and fibrinogen. Thus any factor that increases these proteins is going to increase the rate of rouleaux formation and thus the ESR. The common example to this is the increased ESR in chronic inflammation due to increase in fibrinogen as an acute phase reactant and increased gammaglobulins in multiple myeloma. In addition to this another factor that determines this initial stage of ESR process is the shape of red cells. As described earlier the red cells stack over each other in a straight axis due to discoid shape. The conditions like sickle cell anemia is going to adversely affect this process as this new shape does not allow stacking and causes reduced ESR. The second phase in the process of ESR is the stage of sedimentation where the rouleaux formed fall through
Pratap Singh, Sanjay Kumar
the plasma. However during the fall of each red cell group they form a negative retarding force of plasma. Thus if more red cells are present there will be more retarding force and thus reduced ESR (polycythemia) and the conditions where red cell mass is low there will be less retarding force causing increased ESR (anemia). The third phase in ESR process is the stage of packing where the sedimented red cells pack within the space to give final readings. This component is principally affected by the abnormal shapes of the red cells causing the plasma to get trapped between the red cells and increase the ESR. This happens principally in anemia where there is a lot of anisopoikilocytosis, classic example of which is iron deficiency anemia. Interpretation of ESR should be done carefully as it depends on many physiological and pathological factors as given in table 1 and 2.4
ESR IN RHEUMATOLOGIC DISORDERS
Rheumatoid arthritis is one of the most common disease in which ESR and/or CRP are used to measure disease activity. The DAS and DAS28 are the major clinical scoring systems used to measure the disease activity in RA wherein the ESR is an essential component.5 The patients with SLE relapse have raised ESR but normal CRP level, while with infection both are increased. Thus measuring both together is helpful in differentiating between infection and relapse. In giant cell arteritis (GCA) increased ESR is a hallmark of undiagnosed disease and returns to normal following therapy. ESR of more than 50 mm 1st hr, is an essential component of this disease classification. Levels of ESR and/or CRP are also a useful marker of response to prednisone in GCA. Patients who have lower ESR are expected to show better response to prednisone.
ESR IN MALIGNANT DISORDERS
A high ESR has been found to correlate with overall poor prognosis for various types of cancer, including Hodgkinâ€™s
Table 1: Normal values of ESR by Westergren method Age (Years)
Females (mm at 1st Hr)
Males (mm at 1st Hr)
Table 2: Factors that influence the ESR Increased ESR
No clinically significant effect or questionable effect
Physiological Old age
Sickle cell disease
Elevated fibrinogen level
Hypogammaglobinemia Dysproteinemia with hyperviscosity state
disease, gastric carcinoma, renal cell carcinoma, breast cancer, colorectal cancer and prostate cancer. In patients with breast carcinoma a combination of biochemical markers including CA15.5, CEA and raised ESR can be used to screen the metastatic disease in more than 90% patients. Also this combination with biochemical markers has shown to decrease the cost of treatment by up to 50% by preventing costly radiological workup. It has also been observed that no obvious cause is found in fewer than 2 percent of patients with a markedly elevated ESR.
ESR IN CARDIAC DISORDERS & COPD
It has been shown that raised ESR is an important marker of atherosclerosis and a strong predictor of mortality from coronary artery disease (CAD). It is also a minor criterion for diagnosis of rheumatic fever by Jones criterion. In COPD patients the ESR is seen as a cheaper alternative to CRP in monitoring the disease activity. It increases in COPD as a response to rising levels of fibrinogen, gammaglobulins and in response to developing anemia.6
ESR IN INFECTIONS
ESR is mostly raised in conditions like active tuberculosis and infective endocarditis. It also has been used as a marker of response to treatment in tuberculosis. However it may be normal in infections like typhoid fever and malaria. Increased ESR can be an early predictive marker of HIV seropositive progression towards AIDS. It also has been proposed by some authors that the ESR can be used as an inexpensive “sickness index” in the elderly.7
disease activity and response to treatment in these disorders.
ESR IN ASYMPTOMATIC PATIENTS
ESR should never be used as a screening tool in an asymptomatic patient. A normal ESR reassures the patient as well as the clinician that no underlying serious condition is present, especially when clinical suspicion of disease is low. A mild to moderate elevated ESR without obvious underlying disease should prompt repeat testing after several months rather than an expensive search for hidden diseases. However, an extremely high ESR even in asymptomatic patients should prompt the clinician to search for occult infections or inflammatory disease as stated above.
The ESR is an old, inexpensive yet still widely used test; although it’s a non-specific but frequently ordered for diagnosis and monitoring of certain diseases.
High ESR can be incidental finding in asymptomatic persons but it should be repeated after some months and to be correlated with clinical examination and other laboratory tests.
Patient who have a markedly (>100 mm 1st hr) raised ESR are the one where probability of finding a serious disorder is quite high.
The ESR also has important prognostic role in conditions such as malignancy, stroke, CAD and tuberculosis.
The clinician should be aware that ESR is only one parameter that should be supported by other acute phase markers like CRP, fibrinogen or ferritin.
It will always remain an important marker in developing country like India.
EXTREME ELEVATION OF ESR
An extreme elevation of the ESR (defined as > 100 mm in 1st hour) is associated with a low false-positive rate for a serious underlying disease.8 Most common causes are inflammatory disease like polymyalgia rheumatica, giant cell arteritis, multiple myeloma, JRA and metastatic malignant tumors. It can also be used to monitor the
Fåhraeus R. The suspension-stability of the blood. Acta Med Scand 1921; 55:1-228.
Toshihiro M, Yoshiaki K, Jinju N, Atsushi K, Yoshito E, Shigeto T. Comparison of composite disease activity indices for rheumatoid arthritis. Mod rheumatol 2011; 21:134-143.
Westergren A. Studies of the suspension stability of the blood in pulmonary tuberculosis. Acta Med Scand 1921; 54:247-82.
Smith EM, Samadian S. Use of the erythrocyte sedimentation rate in the elderly. Br J Hosp Med 1994; 51:394-397.
ICSH recommendations for measurement of erythrocyte sedimentation rate. International Council for Standardization in Haematology (Expert Panel on Blood Rheology). J ClinPathol 1993; 46:198-203. Practical hematology. Dacie and Lewis: eleventh edition.
7. Wolfe F, Michaud K. The clinical and research significance of the erythrocyte sedimentation rate. J Rheumatol 1994; 21:1227-37. 8.
Fincher RM, Page MI. Clinical significance of extreme elevation of the erythrocyte sedimentation rate. Arch Intern Med 1986; 146:1581–3.
C H A P T E R
Pancytopenia: Clinical approach
Pancytopenia is a common haematological condition often encountered in day to day clinical practice. It is defined as a decrease in all the three cell lines of blood viz., red blood cells, leucocytes, and platelets. Many diseases affect production of these cells by bone marrow resulting into pancytopenia i.e., simultaneous presence of anaemia, leucopenia, and thrombocytopenia. Pancytopenia is defined as haemoglobin of < 9 gm/dl, WBC < 4,000/cmm, and platelets < 100,000/cmm. Severe pancytopenia is defined as absolute neutrophil count < 500/cmm, platelet count < 20,000/cmm, and corrected reticulocyte count < 1%. Presenting symptoms of pancytopenia may be attributable to anaemia, leucopenia, and/or thrombocytopenia. Anaemia may present with fatigue, breathlessness, and cardiac symptoms. Neutropenia may present with febrile illness due to increased susceptibility to infections. Patients with thrombocytopenia may present with mucocutaneous bleed or bruising. Pancytopenia should be suspected on clinical grounds in any patient presenting with unexplained anaemia, prolonged fever and bleeding tendency. The severity of pancytopenia and underlying aetiology determine the management and prognosis. Pancytopenia usually presents with the clinical sign and symptoms of bone marrow failure such as pallor, easy fatigability, dyspnoea, bleeding or bruising, and increased tendency to infection. As platelets have shortest half life, platelet count is first to be affected leading to thrombocytopenia. Mucocutaneous bleed is typical manifestation of decreased platelet count with petechial haemorrhages in the skin and mucous membrane. Epistaxis, haematuria, gastrointestinal bleeding, menorrhagia, and rarely intracranial bleeding are the presenting features of thrombocytopenia. Anaemia develops slowly because RBC has longest half life. Early manifestation of neutropenia is often a sore throat, or chest or soft tissue infection with poor response to antibiotics. Patients with pancytopenia may develop overwhelming sepsis without any focal sign of infection, with malaise and fever being the only clinical features.
AETIOLOGY OF PANCYTOPENIA
Normal marrow has tremendous capacity to increase the output of peripheral blood cells whenever necessary with the help of growth factors and cytokines. All the peripheral cells arise from common progenitor pluripotent cells having enormous capacity of self renewal. The
Ajai Kumar Garg, AK Agarwal, GD Sharma
normal adult marrow produces about 170x109 RBC, 100x109 neutrophils, and 200x109 platelets daily. Defects in the stem cells or in the stroma or microenvironment of bone marrow can lead to bone marrow failure and pancytopenia. Pancytopenia is not a disease by itself but a triad of haematological finding that can result from a number of disease processes (Tables 1 & 2). It can be a feature of many serious and life threatening illnesses like drug induced bone marrow hypoplasia, fatal bone marrow aplasia, and leukaemias. It can result from failure of production of stem cells in bone marrow, infiltration of bone marrow by malignant cells or fibrosis, immune mediated bone marrow suppression, ineffective erythropoiesis and dysplasia, peripheral sequestration of blood cells by overactive reticuloendothelial system, and immune or non-immune mediated increased destruction of blood cells. Marrow damage may be caused by infiltration of marrow with tumour or fibrosis that crowds normal marrow cells. Tumour or fibrosis that infiltrates the marrow may originate in the marrow as in leukaemia or myelofibrosis or be secondary to process originating outside marrow as in metastatic cancer or myelophthisis. Incidence of various disorders causing pancytopenia varies according to geographical distribution and genetic mutations. Main causes of pancytopenia in our country are megaloblastic anaemia due to nutritional deficiencies, hypersplenism (congestive splenomegaly, malaria, and leishmaniasis), aplastic anaemia, myelodysplastic syndrome, subleukaemic leukaemias, military tuberculosis, multiple myeloma, paroxysmal nocturnal haemoglobinuria. According to a study of 200 cases of pancytopenia conducted by Khunger et al at a large general hospital in North India, the commonest cause found was megaloblastic anaemia seen in 72% cases, followed by aplastic anaemia (14%). The other causes included subleukaemic leukaemia (10 cases), myelodysplastic syndrome (4 cases), hypersplenism due to kala azar (4 cases), hypersplenism due to malaria (2 cases), NonHodgkinâ€™s lymphoma, myelofibrosis, multiple myeloma (2 cases each), Waldenstrom macroglobulinaemia and disseminated tuberculosis (1 case each). In another prospective study of 104 pancytopenic patients conducted by Gayatri and Rao at a teaching institute in South India for a period of two years, commonest cause of pancytopenia was megaloblastosis (74%) followed by aplastic anaemia (18%).
OVERVIEW OF COMMON CAUSES OF PANCYTOPENIA
Megaloblastic anaemia (Tables 3 & 4)
Megaloblastic haematopoiesis is a hypercellular bone marrow failure due to deficiency of vitamin B12 (cobalamin) and folate. These nutrients have important role in synthesis of DNA. Megaloblastic anaemia is a predominant cause of pancytopenia in India because of high prevalence of nutritional anaemia in Indian
Table 1: Causes of pancytopenia with cellular bone marrow Primary bone marrow Secondary to systemic disease disease Myelodysplasia
Vitamin B12 deficiency, folate deficiency
Paroxysmal nocturnal haemoglobinuria
Sepsis, enteric fever
HIV infection, hepatitis B, hepatitis C, Ebstein-Barr virus, cytomegalovirus
Bone marrow lymphoma
Malaria, leishmaniasis, filariasis
Hairy cell leukaemia
Systemic lupus erythematosus, sarcoidosis
subcontinent. Vitamin B12 deficiency may also cause subacute combined degeneration of cord and psychiatric illness (megaloblastic madness). The degree of bone marrow suppression is inversely related to presence and severity of neurological dysfunction. The coexistence of significant anaemia and neurological deficit is thought to be rare.
Hypersplenism is characterized by splenomegaly, cytopenia(s), normal or hyperplastic bone marrow, and a response to splenectomy. In hypersplenism there is peripheral pooling and destruction of cells in enlarged spleen resulting in pancytopenia. Causes of hypersplenism include congestive splenomegaly (cirrhosis, congestive heart failure), malaria, hyperreactive malarial splenomegaly, leishmaniasis, thalassaemia, and Hodgkin’s disease. Hypersplenism can rarely be idiopathic.
Haematologic abnormalities have been frequently observed in patients with HIV infection. Pancytopenia is usually seen in advanced stage of HIV infection. Aetiology of pancytopenia in advanced HIV stage is multi-factorial
Table 2: Causes of pancytopenia with hypocellular marrow Acquired aplastic anemia Congenital aplastic anaemia (Fanconi’s anaemia) Some myelodysplasias Acute myeloid leukaemia Acute lymphoid leukaemia Lymphoma of bone marrow
Table 3: Causes of Vitamin B12 deficiency Food
Decreased consumption-vegan diet, cobalamin malabsorption (common in elderly)
Pernicious anaemia, atrophic gastritis, gastrectomy, gastric bypass, H. pylori infection, Zollinger-Ellison syndrome
Chronic pancreatitis, tropical sprue, celiac disease, ileal resection, fish tapeworm infestation, bacterial overgrowth syndrome, HIV infection
Metformin, Proton pump inhibitors, H 2 blockers
Table 4: Causes of folate deficiency Nutritional deficiency
Infancy and childhood, pregnancy, malignancy, chronic haemolytic anaemia, chronic exfoliative dermatitis, chronic inflammatory disorders (tuberculosis, rheumatoid arthritis, Crohn’s disease), CHF, chronic liver disease, haemodialysis, homocystinuria
Tropical and non-tropical sprue, gluten sensitive enteropathy
Antiepileptic drugs, methotrexate, pyrimethamine, alcohol
Abnormalities of folate metabolism
In another prospective study of 250 cases of pancytopenia conducted by Jain and Naniwadekar at a tertiary care hospital in Maharashtra hypersplenism (29.2%), infections (25.6%), myelosuppression (16.8%), megaloblastosis (13.2%), and hypoplastic/aplastic anaemia (4.8%) were found to be the common causes of pancytopenia. In this study a male preponderance was observed, male to female ratio being 2.6:1 and majority of cases were encountered in third and fourth decade of life. In an analysis of 166 cases of pancytopenia conducted by Kumar et al at two tertiary care hematology centers where patients receiving myelotoxic chemotherapy or those with leukaemic cells in peripheral blood smears were excluded from the study, it was observed that aplastic anaemia (49 cases) was most common cause followed by megaloblastic anaemia (37cases), aleukaemic leukaemia or lymphoma (30 cases), and hypersplenism (19 cases). Megaloblastosis was not commonest cause of pancytopenia in these series probably because many cases of megaloblastic anaemia need not be referred to a tertiary care centre and could easily be treated at general hospitals.
in nature and includes high viral load, use of antiretroviral therapy, and presence of acute or chronic opportunistic infection. Viral hepatitis has been known to cause transient pancytopenia during the course of illness and has also been associated with aplastic anaemia. Hepatitis associated with pancytopenia and aplastic anaemia is usually fatal. Whereas hepatitis B and hepatitis C are common causes; Epstein-Barr virus, cytomegalovirus, and rarely hepatitis A and dengue virus can also cause pancytopenia. Mild blood count depression is common in course of many viral and bacterial infections but resolves with the resolution of infection. Sepsis and enteric fever continue to be important public health problems in India and have been associated with pancytopenia which has been attributed to bone marrow suppression, disseminated intravascular coagulation, and infection associated haemophagocytic syndrome. Tuberculosis is a common disease in India. Disseminated miliary tuberculosis is known to cause pancytopenia. Although pancytopenia is a rare presentation of tuberculosis, it should always be considered in patients presenting with pancytopenia, unexplained pyrexia and weight loss. Both tuberculous bacilli and anti-tuberculous therapy have been implicated in pathogenesis of pancytopenia. Wuchereria bancrofti is an endemic filarial nematode spread by a mosquito vector. The clinical manifestations vary from asymptomatic microfilaraemeia to lymphoedema. Cases of microfilaria in bone marrow aspirate presenting as pancytopenia in peripheral blood have been reported. Though, aetiopathological correlation of pancytopenia with microfilaria infection is not clear.
Haematologic abnormalities such as anaemia, leucopenia, and thrombocytopenia secondary to peripheral destruction are commonly seen in SLE. Most frequent haematologic manifestation of SLE is normocytic and normochromic anaemia. Leukopenia is also common and almost always consists of lymphopenia and not granulocytopenia. Thrombocytopenia may be a recurring problem in SLE. Cytopenias from autoimmune myelofibrosis are also uncommonly seen in SLE.
Aplastic anaemia is defined as pancytopenia with hypocellular marrow in absence of abnormal infiltrate or increased fibrosis. It is a normocytic normochromic anaemia that results from a loss of blood cell precursors, causing hypoplasia of bone marrow leading to pancytopenia. Severe aplastic anaemia is defined as a bone marrow cellularity < 25% with at least two of the three criteria i.e., neutrophils < 500/mcL, platelets < 20,000/mcL, and reticulocyte count < 20,000/mcL. It is a potentially life threatening failure of bone marrow. Most cases of aplastic anaemia are acquired and T-cell mediated autoimmune disease. Triggering factors may include drugs, viruses, and toxins but most cases are idiopathic. In some cases radiation, drugs, toxic chemicals and viruses induce depletion of haematopoietic stem cells by direct toxicity.
Paroxysmal nocturnal haemoglobinuria (PNH)
PNH is an acquired chronic haemolytic anaemia characterized by persistent intravascular haemolysis with recurrent exacerbations due to activation of complement C. In addition to haemolysis, there is often pancytopenia and a risk of venous thrombosis. Haemolysis in PNH is due to an intrinsic abnormality of the red cell, which makes it sensitive to activated complement C. Diagnostic gold standard of PNH is flow cytometry which can be carried out on granulocytes as well as on RBC. A bimodal distribution of cells with a discrete population that is CD59 and CD55 negative is diagnostic of PNH.
Acute myeloid leukaemia occurs in all age group but predominantly in older adults. Acute lymphoblastic leukaemia is the most common acute leukaemia in childhood. Clinical history and symptoms usually indicate bone marrow failure. These include fatigue, dyspnoea, dizziness, bleeding, easy bruising, and recurrent infections. Cytogenetic abnormalities are prognostically important and affect patient management. In all cases of severe pancytopenia (symptomatic anaemia, WBC < 500/mcL, and platelets < 20,000/mcL) investigations are mandatory within first 24-48 hrs. Supportive therapy with RBC, platelets, and broad spectrum antibiotics may be initiated before underlying cause has been ascertained.
Myelodysplastic syndromes (MDS) are the common haematological diseases characterized by cytopenias associated with abnormal appearing cellular marrow producing ineffective red blood cells. The incidence of MDS increases with advancing age. Median age at disease onset is 70 years with only about 10% of the patients below 50 years. MDS are diseases of haematopoietic stem cells. They are characterized by disturbance of differentiation and maturation, and by changes in the bone marrow stroma. MDS are accompanied not only by reducing blood cell counts but also with an increased risk (20-25%) of developing acute myeloid leukaemia. The disease course varies greatly from patient to patient, with median survival time from few months to years.
Systemic lupus erythematosus (SLE)
Idiopathic cytopenia of undetermined significance
Idiopathic cytopenia of undetermined significance (ICUS) is a recently proposed provisional diagnosis that recognizes patients who present with cytopenias of undetermined aetiology. Diagnostic criteria for ICUS include: i) persistent cytopenia for 6 months (Hb < 11mg/ dl, neutrophil < 1.5x109 /L, and platelets < 100x109/L); ii) No morphologic feature of myelodysplasia; iii) normal chromosome analysis; and iv) A detailed clinical history and investigation that excludes other secondary causes of cytopenias. The diagnosis of ICUS should only be established when all possible differential diagnosis have been excluded. The term Idiopathic fatal pancytopenia has been suggested for ICUS because it is a fatal disease with no definite cause.
Drug Induced pancytopenia (Table 5)
Drugs are the common cause of pancytopenia. Drug induced pancytopenia can be dose dependent or immune
Table 5: Common drugs causing pancytopenia a. By bone marrow suppression: Cytotoxic drugs b. By dose dependent effect: Chloramphenicol c. By immune mediated idiosyncratic reaction
NSAIDS, chloremphenicol, sulphonamide, phenothiazines, thiazides, anti-thyroid drugs, anti-epileptics, anti-diabetic drugs, colchicine, azathioprine
CONGENITAL CAUSES OF PANCYTOPENIA
Fanconi’s anaemia is an autosomal recessive disorder and manifests as congenital developmental anomaly, progressive pancytopenia, and an increased risk of malignancy. Patients typically have short stature, café au lait spots, and anomalies involving thumb, radius, and genitourinary tract. Dyskeratosis congenita is characterized by mucous membrane leukoplakia, dystrophic nails, reticular hyperpigmentation, and development of aplastic anaemia during childhood.
APPROACH TO A CASE OF PANCYTOPENIA
Evaluation of pancytopenia requires a careful history and physical examination. The causes of pancytopenia are diverse. Attention must be paid to history of the patient and the family. Nutritional history, drug history and history of alcohol intake should always be assessed. History suggestive of previous pancytopenia, aplastic anaemia, inherited bone marrow failure syndrome, repeated early foetal loss, cancer, liver disease, metabolic disorders, or connective tissue disorder is important. Cytotoxic chemotherapy and radiotherapy are important cause of transient pancytopenia. History of weight loss and anorexia may suggest underlying infection or malignancy. Recurrent oral ulcers and chronic diarrhoea may point towards HIV infection. Recurrent oral ulcers, malar rash and joint pain may suggest SLE. Bone pain and loss of height indicate multiple myeloma. A thorough physical examination is of paramount importance in evaluation of pancytopenia. It should include assessment of jaundice, clubbing of fingers, lymphadenopathy and splenomegaly (underlying infection, infectious mononucelosis, lymphoproliferative disorder, and malignancy), loss of height (suggestive of multiple myeloma), malar rash, retinal haemorrhage, oral petechiae, gingival hyperplasia, stomatitis or cheilitis, oropharyngeal candidiasis, RUQ abdominal tenderness, signs of chronic liver disease. Laboratory evaluation: A routine complete blood count (CBC) is required as a part of initial evaluation of pancytopenia. CBC should include red cell indices, peripheral blood film, reticulocytes count and absolute reticulocyte count. A very high MCV (>110fl) indicates
Peripheral blood smear provides important information in pancytopenia and it should always be done prior to transfusion of blood. Blood smear may reveal polychromasia—red cells that are slightly larger than normal and greyish blue in colour. These cells are reticulocytes that have been prematurely released from the bone marrow. These cells may appear in circulation due to architectural damage of the bone marrow caused by fibrosis or malignant cell infiltration. Bone marrow examination is almost always indicated in cases of pancytopenia unless the cause is otherwise apparent (e.g., chronic liver disease with portal hypertension, deficiency of vitamin B12 or folate). In megaloblastic anaemia bone marrow shows megaloblastic erythroid hyperplasia, sieved nuclear chromatin, asynchronous nuclear maturation, bluish cytoplasm with cytoplasmic blebs. Giant metamyelocytes and band forms are predominant in granulocyte series. Bone marrow in aplastic anaemia is hypocellular with suppression of erythropoiesis, myelopoiesis, and megakaryopoiesis with relative lymphoplasmacytosis. In acute leukaemias, bone marrow is hypercellular with reduced erythroid and megakaryocytic series and majority of cells are myeloblast or lymphoblast. Bone marrow aspiration in AML shows myeloblast with Auer rods.
ABSOLUTE RETICULOCYTE COUNT
An accurate reticulocyte count is the key to initial evaluation of pancytopenia. Normally, reticulocytes are the red cells that have been recently released from the bone marrow. Normal reticulocyte count ranges from 1-2% and reflects the daily replacement of 0.8-1% of the circulating RBC population. Reticulocyte count provides a reliable measure of RBC production. Reticulocyte count and absolute reticulocyte count (ARC) should be done on day one along with CBC in order to avoid therapy related changes in reticulocyte count particularly with nutritional anaemia. Absolute reticulocyte count (ARC) is a calculated index derived from the product of reticulocyte count percentage and RBC count (Normal; Male: 4.32-5.72 million/cmm, Female: 3.90-5.03 million/cmm). ARC is a marker of red cell production by bone marrow. It plays important role in establishing the cause of pancytopenia and helps in distinguishing between hypoproliferative and hyperproliferative anaemias. Normal range of absolute reticulocyte count is 50,000-100,000/cmm. All cases of pancytopenia with very low ARC (< 25,000/ cmm) should be examined by bone marrow aspiration for aplastic anaemia. All cases of pancytopenia with high ARC (> 100,000/cmm) should also be evaluated by bone marrow aspiration unless there is a history suggestive of sepsis or malaria. Pancytopenia with ARC 25,000-50,000/
mediated (idiosyncratic). Chloramphenicol can cause pancytopenia by both the mechanisms. Azathioprine, an immunosuppressive drug used for treatment of various diseases, usually causes leucopenia and rarely pancytopenia.
megaloblastic anaemia. In addition liver function test, viral markers for hepatitis, coagulation profile, fibrinogen, D-dimer, serum B12, folate levels, HIV serology, antinuclear antibodies (ANA) should be done. Serum ferritin levels should also be assessed. Low levels of serum ferritin along with low serum B12 and/or folate levels may indicate mixed anaemia/pancytopenia.
Flow diagram for evaluation of pancytopenia 454
Peripheral blood smear and reticulocyte count
Bone marrow aspiration,
Liver function test, coagulation proﬁle
Vitamin B12, folate levels Viral serology (HIV, HBV, HCV, EBV, CMV) Autoimmune proﬁle
Bone marrow cytogenetics and immunophenotyping
Fig. 1: Flow diagram for Evaluation of Pancytopenia cmm should initially be evaluated with serum B12, folate and ferritin assays and if any one of these is found to be low, bone marrow aspiration is not needed.
Lymphoproliferative disorders: immunophenotyping, cytogenetics, lymph node biopsy Multiple myeloma: serum electrophoresis, bone marrow aspiration
Madhuchanda Kar, Alokendu Ghosh. Pancytopenia. J Ind Acad of Clinical Medicine 2002; 3:29-34.
BN Gayatri, Kadam Satyanarayan Rao. Pancytopenia: A Clinico Haematological Study. Journal of Laboratory Physicians 2011; 3:15-20.
Kumar R, Kalra SP, Kumar H, Anand AC, Madan H. J Assoc Physician India 2001; 49:1078-81.
Arvind Jain, Manjari Naniwadekar. An etiological reappraisal of pancytopenia-largest series reported to date from a tertiary care teaching hospital. BMChematology. biomedcentral.com/articles/10.1186/2052-1839-13-10.
Jitendra Mohan Khunger, S. Arulselvi, Uma Sharma et al. Pancytopenia- A Clinico Haematological study of 200 cases. Indian journal of Pathology and Microbiology 2002; 45:375-79.
Poorana Priya P, Subhashree AR. Role of absolute reticulocyte count in evaluation of Pancytopenia: A Hospital based study. Journal of Clinical and Diagnostic Research 2014;8: FC01-03.
Eduardo J, Santiago Rodriguez, Angel M mayer et al. Profile of HIV infected Hispanics with Pancytopenia. Int J Environ Res Public Health 2016; 13:38
Martinez Faci C, Ros Arnal I, Martines de Zabarte Fernandes JM et al. Azathioprine induced pancytopenia: case series. Arch Argent Pediatr 2016; 114:e252-5.
Jain M, Shukla A, Kumar A et al. Wuchereria bancrofti: Unusaual presentation as Pancytopenia. Journal of Clinical and Diagnostic Research 2016; 10:ED05-6.
Paroxysmal nocturnal haemoblobinuria: flow cytometry (CD55, CD59) Cytomegalovirus infection: serology for CMV (IgG, IgM) Epstein-Barr virus: Serum monospot, viral capsid antigen, EB nuclear antibody Leishmaniasis: Blood and bone marrow culture, LD bodies Carcinoma prostate: Serum PSA Fanconi’s anaemia: diepoxybutane test for chromosomal breakage in peripheral blood lymphocyte
CONCLUSION (FIGURE 1)
Pancytopenia is not a disease itself. It is a haematological feature of varying aetiology with slight male preponderance. Megaloblastic anaemia along with mixed nutritional anaemia is leading cause of pancytopenia in India followed by hypersplenism and aplastic anaemia being second and third common causes respectively. Absolute reticulocyte count is an important indicator for determining the cause of pancytopenia and should be done along with peripheral smear in the very beginning. Serum B12, folate and ferritin assays should be done for evaluation of mixed nutritional anaemia. Bone marrow aspiration is an important and safe invasive procedure for evaluation of cause of pancytopenia.
10. Ungprasert P, Chowdhary VR, Davis M, Makol A. Autoimmunefibrosis with pancytopenia as a presenting manifestation of SLE responsive to mycophenolate mofetil. Lupus 2016; 25:427-30 11. Mohammad Arphan Azad, Yongping Le, Quirong Zhang, Haixia Wang. Detection of Pancytopenia associated with Clinical Manifestation and their final Diagnosis. Open journal of blood diseases 2015; 5:17-30.
Current Management of Hemophilia
C H A P T E R
Haemophilia is an X-linked congenital bleeding disorder caused by a deficiency of clotting factor VIII (Haemophilia A) or factor IX (Haemophilia B). In haemophilia, the FIXa/ VIIIa complex fails to form on the platelet surface and does not activate sufficient FX for a large burst of thrombin. Without this thrombin burst, the subsequent conversion of fibrinogen to fibrin, the haemostatic plug is unstable, leading to prolonged bleeding (Figure 1).
The worldwide incidence of hemophilia A is 1 in 5000 male live births, and that of hemophilia B is 1 in 30,000 males. The number of people with haemophilia in the world, based on WFH’s annual global survey of 2014 is approximately 1, 78,500. India has the highest disease burden of 17,470 patients including 14,450 with
II X TF
VIII vVVF IIa
Haemophilia is an X-linked recessive disorder and is more likely to manifest in males. A female who carries the defective gene on one X chromosome will be a carrier (heterozygous) and on both X chromosomes will have Haemophilia (homozygous). Some carriers may have clotting factor level so low that they fall within the moderate-to-severe range of haemophilia due to a phenomenon called ‘extreme lyonisation’. One-third of cases may arise from spontaneous mutation of the F8 and F9 genes.
Typical history includes recurrent bleeding symptoms that may occur spontaneously or due to any trivial injury. Depending on the site and severity of bleeding, bleeds are classified as mild to moderate and severe/major/ life threatening in nature.
Mild to Moderate Bleeds
Haemophilia A, which constitutes only about 23% of the actual number of cases; due to under diagnosis.
Muscle bleed (Figures 3 & 4)
Major or Life Threatening or Severe Bleeds
Fig. 1: FactorVIII function
Intracranial Factor VIII
AD AP 1
AI distal tel
AP AD Factor VIII 1
Fig. 2: intron 22 inversion-most common genetic abnormality in Haemophilia A
COMPLICATIONS OF HAEMOPHILIA
Bleeding from neck, throat, chest
Pseudotumor( muscle hematoma)
Chronic hemophilic arthropathy
The difference betweeen platelet and coagulation disorders are enumerated in Table 1. Common sites of bleeding are listed in Table 2.
Screening tests reveal isolated prolongation of aPTT in haemophilia (Table 3). However, a normal aPTT does not exclude haemophilia because of its relative insensitivity. A FVIII (normal 50-150%) and or FIX assay should be requested to confirm the diagnosis of haemophilia.
Joint bleeds ( haemarthrosis) and disability
MANAGEMENT OF HAEMOPHILIA
The primary goal of haemophilia therapy is to prevent
Table 1: Difference between platelet and coagulation disorders Platelet Disorders
Coagulation Factor Disorder
Site of bleeding
Skin, mucous membrane and soft tissue
Deep in soft tissues (joints and muscles)
Bleeding after cuts and scratches
Bleeding after surgery and trauma
Immediate, usually mild
Delayed (1-2 days), often severe
Bleeding starts within the joint
As blood accumulates in the joint, it swells, become warm to the touch and may be painful. Appropriate treatment at this state will stop further bleeding in to the joint
The synovium breaks down the bleed and absorbs it from the joint
After about a week, all the blood is absorbed and the joint returns to its pre-bleeds state
Fig. 3: Short term impact of joint bleeds
A Single bleeding event may be suﬃcient to provoke inﬂammation (synovitis)
Recurrent bleeding leads to swelling of the joint and ongoing synovitis
Growth of the joint lining (synovium) leads to an inﬂammed, vascular, and fragile tissue that is more likely to bleed. Further bleeds can destroy the cartilage
Destruction of cartilage leads to long - lasting joint damage, resulting in arthritis and stiﬀened joints
Fig. 4: Long term impact of joint bleeds
In later stages, there is complete loss of cartilage and the bone may become deformed, changing the shape of the joint
and treat bleeding with the deficient clotting factor; and whenever possible should be treated with specific factor concentrate. Acute bleeds should be treated as quickly as possible, preferably within 2 hours. If in doubt, treat.
FIRST AID MEASURES
The patient’s factor level should be measured 15 minutes after the infusion, to verify the calculated dose. Factor concentrates should be infused by slow IV injection at a rate not exceeding 3mL/min in adults. Dose Required = Body Weight x Desired Factor Level (IU/dL) x 0.5 (Haemophilia A)
P - Protection (Splint)
R - Rest - avoid weight bearing / restrict movement
I – Ice (crushed, indirect, 3-4x/day)
C – Compression
E – Elevation
Prothrombin complex concentrates which contain factor IX along with activated clotting factors such as II VII & X may predispose to thromboembolism and so not preferred in treatment of Factor IX deficiency.
REPLACEMENT THERAPY AND DIFFERENT TYPES OF CLOTTING FACTORS (FIGURE 5)
Replacement therapy is done by providing concentrates of deficient clotting factors and is preferred over the use of cryoprecipitate and fresh frozen plasma.
CLOTTING FACTOR CONCENTRATES Plasma Derived
Recombinant Therapy (Figure 6)
1 unit/kg factor concentrate increases factor VIII level by 2IU/dL, and factor IX level by 0.8 IU/dL in the absence of an inhibitor.
Table 2: Common sites of bleeding in haemophilia Site of Bleeding
• more common into hinged joints: ankles, knees and elbows • less common into multi-axial joints: shoulder, wrists, hips
OTHER PLASMA PRODUCTS
Fresh Frozen Plasma
It contains all coagulations factors but is poor in factor VII. 1 ml of FFP contains 1 unit of factor activity. Acceptable starting dose is 15-20ml/kg.
They are of two types•
Dose Required = Body Weight x Desired Factor Level (IU/dL) x 1.25 (Haemophilia B)
It is rich in Factor VIII, XIII, vWF and fibrinogen. It is preferable to FFP for the treatment of haemophilia A in situations where clotting factor concentrates are not available
The goal of prophylaxis is to prevent bleeding events and joint degeneration to preserve normal musculoskeletal function. The World Health Organisation (WHO) and WHF guidelines recommend prophylactic therapy as the gold standard of management. Two prophylaxis protocol are supported by long-term data (Tables 5 & 6).
25-40 IU/kg as per MALMO protocol per dose
15-30 IU/kg as per UTRECHT protocol per dose
Other major bleeds
Central nervous system (CNS)
administered three times a week for those with
Table 3: Interpretation of screening test PT
Hemophilia A or B**
Normal or prolonged*
Normal or prolonged
Normal or reduced
Normal or prolonged
Normal or reduced
Table 4: Category of Bleeding Tendency for People with Haemophilia A Category
FVIII concentration (Relative %)
<0.01 IU/mL (<1% of normal)
Spontaneous joint and muscle bleeding; bleeding after injuries, accidents, and surgeries
0.01 – 0.05 IU/mL (1-5% of normal)
Bleeding into joint and muscles after minor injuries; excessive bleeding after surgery and dental extraction
>0.05 – 0.40 IU/mL (5-40% of normal)
Spontaneous bleeding does not occur; bleeding after surgery, dental extraction, and trauma
haemophilia A, and twice a week for those with haemophilia B
Epsilon aminocaproic acid (EACA)
ON-DEMAND TREATMENT OTHER PHARMACOLOGICAL OPTIONS
oral tablet 3 to 4 times daily. It may be given alone or together with factor VII concentrates.
Desmopressin, a synthetic analogue of the vasopressin may be the treatment of choice in mild or moderate haemophilia. It enhances the interaction of FVIII and vWF and elevates FVIII three to six times the baseline levels to control bleeding. A single dose of 0.3ug/kg body weight, either by intravenous or subcutaneous route, may be given.
It is an antifibrinolytic agent and is usually given as an
It is similar to tranexamic acid and is given to adults orally or intravenously every 4 to 6 hours up to a maximum of 24 g/day in an adult.
Other Management Options
Adjuvant treatment and other supportive strategies used in the management of haemophilia are shown in Tables 7 & 8 below.
COMPLICATIONS OF HAEMOPHILIA TREATMENT
Inhibitors are allogenic IgG antibodies that neutralise clotting factors. Inhibitor development occurs in 20-30% of patients with severe haemophilia A and 2-3% with haemophilia B who are on prophylaxis. Confirmation and quantification of an inhibitor is performed in the laboratory using the Nijmegan-Modified Bethesda assay.
When to screen for Inhibitors
Fig. 5: Strategies for clotting factor replacement at different ages and impact on outcomes First generation
Once every 5 exposure days until 20 exposure days and every 10 exposure days between 21 & 50 exposure days, then 2/year
Intensively treated for >5 days, within 4 wks of the last infusion
Prior to surgery
Recovery assays are not as expected
Clinical response is sub-optimal in the postoperative period
Final product Human-or animal-derived protein used
No human-or animal-derived protein used
Fig. 6: Types of recombinant therapy
Table 5: Plasma Factor Replacement Target for Haemophilia A and B (when there is no significant resource constraint) Hemophilia A Types of Hemorrhage
Desired Level (IU/DL)
1-2, may be longer if response is inadequate
1-2, may be longer if response is inadequate
Superficial muscle/no NV compromise (except iliopsoas)
2-3, sometimes longer if response is inadequate
2-3, sometimes longer if response is inadequate
3-5, sometimes longer as secondary prophylaxis during physiotherapy
3-5, sometimes longer as secondary prophylaxis during physiotherapy
Iliopsoas and deep muscle with NV injury, or substantial blood loss
CNS/head • maintenance Throat and neck • initial • maintenance Gastrointestinal • initial • maintenance
Surgery (major) • Pre-op
Surgery (minor) • Pre-op
50-80 1-5, depending on type of procedure
Management of Bleeding in Patients with Inhibitors
Patients with low-titre inhibitors (<5 Bethesda Units (BU)) may be treated with specific factor replacement at a much higher dose (50-200 IU/kg/dose) to overcome inhibitors and control bleeding. Options for management of hightitre inhibitors (>5BU) are summarised below in Table 9.
BLOOD-BORNE INFECTIONS (INCLUDING HEPATITIS C AND HIV)
Viral inactivation procedures have virtually eliminated the risk of transmission of enveloped viruses (i.e. hepatitis B and C viruses and HIV) in plasma-derived concentrates. There is still possibility of transmission of non-lipid
1-5, depending on type of procedure
enveloped viruses (e.g. parvovirus B19), prions, and undetectable pathogens in plasma-derived concentrates.
RECOMBINANT FACTOR CONCENTRATES
First- generation (Recombinate)
Animal and human derived proteins are used in cell culture medium and human albumin is used to stabilise the final product.
Second- generation (Kogenate FS)
Animal and human derived proteins are used in cell culture medium and sucrose instead of human albumin is used to stabilise the final product.
Desired Level (IU/DL)
Table 6: Plasma Factor Replacement Target for Haemophilia A and B (when there is significant resource constraint)
Types of Hemorrhage
Desired Level (IU/DL)
Desired Level (IU/DL)
1-2 may be longer if response is inadequate
1-2, may be longer if response is inadequate
Superficial muscle/no NV compromise (except iliopsoas)
2-3, sometimes longer if response is inadequate
2-3, sometimes longer if response is inadequate
Iliopsoas and deep muscle with NV injury, or substantial blood loss • initial
3-5, sometimes longer as secondary prophylaxis during physiotherapy
3-5, sometimes longer as secondary prophylaxis during physiotherapy
Throat and neck
Surgery (major) • Pre-op
Surgery (minor) • Pre-op
40-80 1-5, depending on type of procedure
1-5, depending on type of procedure
Third- generation (Turoctocog Alfa)
Novo Eight undergoes a 5 step purification process that includes solvent detergent treatment and nanofiltration with 20 nm membranes as recommended by WFH.
TUROCTOCOG ALFA (FIGURE 7)
Management of haemophilia is a multimodality comprehensive health care approach which includes first aid measures, factor replacement preferably recombinant factor concentrates, pharmacotherapy, pain management prevention of disabilities, rehabilitation by physiotherapy and psychological counselling for patients as well as family. Prophylactic factor replacement therapy is advisable in the long term because it eliminates high cost associated with management of diseased joints and
No animal or human plasma- derived proteins are used during production in cell culture or during stabilisation process. It is a B domain truncated third generation recombinant analogue of factor VIII(rDNA) prepared without the addition of any human or animal derived protein in the cell culture process, purification or final formulation thereby bringing down the risk of infection to the greatest extent. It has been approved for use in the treatment of bleeding in all age groups and for long term prophylaxis in severe haemophilia A.
Table 7: Supportive Strategies for Haemophilia Management Adjunctive treatment for mucosal bleeds and dental extractions. Contraindications to the use of antifibrinolytic drugs include upper urinary tract bleeding (due to risk of clot retention in the ureter and bladder) and subarachnoid bleeding (due to risk of vasospasm and ischaemic stroke)
Certain COX-2 inhibitors1
Used judiciously for joint inflammation after an acute bleed and in chronic arthritis
First aid measures1
Protection (splint), rest, ice, compression and elevation (PRICE) as adjunctive management for bleeding in muscles and joints
To manage recovery after a muscle or joint bleed; prevent future bleeding episodes or support recovery from surgical procedures
To control arthropathic pain
Conservative management: serial casting, bracing, orthotics1
To correct deformities or support painful and unstable joints
Elective orthopaedic surgery1
Synovectomy to reduce bleeding frequency Extra-articular soft tissue release to correct contractures Arthroscopy to diagnose and correct adhesions or impingements within joint Arthrodesis for surgical immobilization of a damaged joint Prosthetic joint replacement for treatment of severely affected major joints
Table 8: Strategies for pain management 1
Paracetamol/acetaminophen If not effective ďƒ”
COX-2 inhibitor (e.g. celecoxib, meloxicam, nimesulide, and others) OR Paracetamol/acetaminophen plus codeine (3-4 times/day) OR Paracetamol/acetaminophen plus tramadol (3-4 times/day)
Morphine: Use a slow release product with an escape of a rapid release. Increase the slow release product if the rapid release product is used more than 4 times/day
Fig. 7: Structural Comparison between Full Length FVIII and Turoctocog Alfa
Antifibrinolytic drug (e.g. tranexamic acid, epsilon aminocaproic acid)1
Table 9: Management of Patients with High-Titre Inhibitors Bypassing agents
Immune tolerance induction to eradicate the inhibitor
• Examples are activated prothrombin complex concentrates (aPCCs) and recombinant activated Factor VII (rFVIIa)
• Immune tolerance induction (ITI) is an immune • Immunosuppressive system “desensitization” technique to eradicate agents can be given as an alloantibody inhibitor to FVIII co-adjuvants during ITI
• Studies suggest improved efficacy for rFVIIa (81-91%) than for aPCC (64-80%) when used for on demand treatment of bleeding episodes in haemophilia patients with inhibitors.34
• High doses of FVIII concentrates (recombinant or plasma-derived) are administered regularly per protocol over months • The success of ITI approaches 90% usually over approximately 6-12 months for allo-FVIII antibody inhibitors.35 • The success of ITI is eradicating FIX inhibitors in haemophilia B is reported to be as low as 1331%.36-38
improves the quality of life. Gene therapy may offer a potential cure for haemophilia in the near future.
Definitions in haemophilia. Recommendations of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Hemostasis. JTH 2012 (in press).
Massimo Franchini, Pier Mannuccio Mannucci. Past, present and future of hemophilia: a narrative review. Orphanet Journal of Rare Diseases 2012; 7:241-6
Kavakli K, Aydogdu S, Taner M, et al. Radioisotope synovectomy with rhenium186 in haemophilic synovitis for elbows, ankles and shoulders. Haemophilia 2008; 14:51823.
Other treatment modalities1,33
Haemophilia of Georgia. Protocols for the treatment of haemophilia and von willebrand disease. Haemophilia of Georgia, 2012.http://www.hog.org/publications/ page/protocols-for-the-treatment-of-hemophilia-andvonwillebrand-disease-2 (Accessed June 6 2012). Haemophilia of Georgia. Protocols for the Treatment of
Haemophilia and Von WillebrandDisease, February 2012. Available at 6.
Franchini M, Zaffanello M, Lippi G. The use of desmopressin in mild haemophilia A. Blood Coagul Fibrinolysis 2010; 21:615-9.
Trigg DE, Stergiotou I, Peitsidis P, KadirRA. A Systematic Review: the use of desmopressin for treatment and prophylaxis of bleeding disorders in pregnancy. Haemophilia2012;18:2533.http://www.hog.org/ docLib/20090820_HoG Protocol2009.pdf (Accessed June 6, 2012).
Castaman G. Desmopressin for the haemophilia. Haemophilia 2008; 14:15–20.
Kouides PA, Byams VR, Philipp CS, et al. Multisite management study of menorrhagia with abnormal laboratory haemostasis: a prospective crossover study of intranasal desmopressin and oral tranexamic acid. Br J Haematol 2009; 145:212-20.
10. Franchini M, Zaffanello M, Lippi G. The use of desmopressin in mild haemophilia A. Blood Coagul Fibrinolysis 2010; 21:615-9.
C H A P T E R
Hemophilia: Prophylactic Therapy with Conventional and Newer Agents Tarun Kumar Dutta, Shailendra Prasad Verma, Deepak Charles
Hemophilia is an x-linked congenital bleeding disorder caused by deficiency of coagulation factors VIII and IX in hemophilia A and B respectively, which affects males with females being asymptomatic carriers. Underlying cause is a mutation of the respective clotting factor genes. According to WFHâ€™s annual global survey, number of hemophilia patients world-wide is approximately 400,000. Reported prevalence of hemophilia in India is approximately 1.1/100000 male population which is remarkably less in comparison to western countries due to underreporting of cases. Only 10% of total cases are registered with Hemophilia federations in India. Given that incidence of Hemophilia A is one in 5000 and hemophilia B is one in 30000, the expected number of people with hemophilia (PWH) in India should be close to 100,000. Typical phenotypic presentation of hemophilia patient is life-long bleeding tendency. Children with hemophilia start bleeding when they begin crawling, walking and running. Hemophilia patients are categorized into three categories of mild, moderate and severe hemophilia based on severity of factor deficiency. Patients with severe hemophilia have FVIII level <1% and tend to bleed spontaneously into joints and muscles, while those with moderate hemophilia have FVIII level 1-5% and rarely have spontaneous bleed. The most common haemorrhagic manifestations in patients with haemophilia are recurrent joint bleeds followed by muscle bleeds. Commonly affected joints are elbows, knees and ankles, also known as index
joints. Recurrent bleeding in these joints in due course of time leads to progressive joint destruction, irreversible arthropathy and chronic pain. This in turn leads to loss of school days, multiple emergency visits, poor quality of life, loss of job opportunities and psychological stress among patients with haemophilia. The current practice in India and most other developing countries is replacement of the deficient coagulation factor on episodic (demand) basis after joint bleed has already occurred (Table 1). Episodic therapy may delay progression to arthropathy but cannot prevent it. Prophylactic therapy with factor concentrates administered 2 to 3 times a week on a regular basis is the best way to prevent joint bleeds. Purpose of regular prophylactic factor replacement is to convert severe forms to milder forms. The Swedish experience demonstrates a decreasing need for orthopedic surgery in hemophilic patients who are managed from the age of 1 to 2 years (before onset of arthropathy) with a prophylactic regimen of clotting factor concentrate replacements. The doses and frequency of replacement therapy are adjusted to prevent the factor VIII levels from falling below 1% to 2% of normal, thereby converting severe hemophilia into mild or moderate disease (thus preventing spontaneous bleed). Improvements translate into decreased absence from school or work, fewer bleeds and days spent in the hospital, increased personal and professional productivity, improved overall performance status, and a healthier self-image. There is no evidence that prophylaxis begun early in childhood increases the incidence of inhibitor antibody formation.
Table 1: Definitions of regimens of factor replacement therapy in haemophilia Episodic (on-demand treatment) CONVENTIONAL*
Treatment given at the time of clinically evident bleeding (widely practiced in India and other developing countries)
In the absence of documented osteochondral joint disease, determined by physical examination and/or imaging studies, started before the second clinically evident large joint* bleed and age <3 years
After 2 or more bleeds into large joints* and before the onset of joint disease documented by physical examination and imaging studies
After the onset of joint disease documented by physical examination and plain radiographs of the affected joints
Intermittent (periodic) prophylaxis
Treatment given to prevent bleeding for periods not exceeding 45 weeks in a year
Adapted from the recommendations of the Subcommittee on factor VIII and factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis, reported in the Guidelines for the management of hemophilia from the Working Group of the World Federation of Haemophilia. *Practised in developing countries; **Practised in developed countries; ***Large joints: ankles, knees, hips, elbows and shoulders.
Prophylactic therapy with factor concentrates administered 2 to 3 times a week on regular basis is the best way to prevent joint bleeds. Prophylactic replacement of clotting factor has been shown to be useful even when factor levels are not maintained above 1% at all times. Between late 1970s and 1990, children with haemophilia have been treated with prophylactic therapy using incremental doses (5–10 to 20–40 IU/kg body weight) of intermediate purity clotting factor concentrates or cryoprecipitate two to three times a week. Recently much stronger evidence on efficacy of prophylaxis was provided by randomized control trial by Manco-Johnson et al. and the ESPIRIT study by Gringeri A et al. In these studies factor VIII was used in doses of 25 IU/ kg body weight on alternate days, which involves a high amount of factor consumption and leads to high cost, resulting in unaffordability of such treatment in developing countries like India and even in some of the western countries. Following protocols are practiced currently in developed countries: •
Malmo protocol : 25-40iu/kg/dose thrice weekly
Utrecht protocol : 15-30iu/kg/dose thrice weekly
But the optimal regimen remains to be defined. Resource-strained countries need to have their own factor prophylaxis strategy. Very low-dose FVIII prophylaxis (10 IU/ kg body weight twice a week) may be a feasible option for prophylaxis in resource constraint countries like India as proven in one recent single-centre short-term pilot study from China.
INDIAN EXPERIENCE AT JIPMER, PUDUCHERRY
The aim of the study was to find out the efficacy, cost effectiveness and safety of very low-dose factor prophylaxis in patients with haemophilia A in Indian scenario. This study conducted in 2013 on 21 children concluded, even very low-dose FVIII prophylaxis with 10 IU/kg body weight twice a week was effective in preventing joint bleeds and overall bleeds and also was cost effective. Significantly less emergency visits, lesser days of school absenteeism were found in children. Even if the factor trough levels were not maintained above 1% in all cases, bleeding events were still prevented in all children.
ONCE WEEKLY FACTOR VIII PROPHYLAXIS IN CHILDREN WITH SEVERE HEMOPHILIA – A NEW CONCEPT
However, compliance with twice a week schedule is a limiting factor in the long run. Thus, another option for the treatment of very young children is to start prophylaxis once a week and escalate dose depending on bleeding. Low dose factor VIII prophylaxis once weekly has not been practiced earlier. Once weekly therapy is likely to have better patient compliance in long run because of significant reduction in injection pricks. In a Canadian study based on tailored dose approach, the use of once weekly prophylaxis has been found with 40% of children remaining without major bleeds for a median period of four years. However, 18% required an escalation of dose.
The same investigators from JIPMER undertook a second study hoping once weekly prophylaxis would ensure better patient compliance and thus, reduce the cost of treatment as compared to twice/thrice weekly regime (making it a very valuable option in a resource poor setting like ours). Very low-dose FVIII prophylaxis in a dosage of 20 IU kg/body weight once a week (instead of 10 IU/ kg body eight twice a week in the previous study) was given to the same group of patients belonging to the previous study. Overall bleed, joint bleed and school absenteeism were significantly less in this once weekly low dose factor group also as compared to on-demand group (and differences were statistically significant). Further, result was not inferior to the result of twice a week schedule studied earlier.
Advantage – longer half life
IMPACT OF LONGER ACTING FACTOR CONCENTRATES ON INDIVIDUALIZATION OF PROPHYLAXIS
In last few years, there has been tremendous progress in developing new bioengineered factor concentrates with prolonged t1/2. In comparison to currently available FIX and FVIII, longer acting FIXs have shown greatly improved PKs (3- to 5.8-fold longer t1/2),while longer acting FVIIIs have shown more modest t1/2 prolongation (1.4- to 1.7-fold longer). The lower increase in FVIII t1/2 prolongation when compared with FIX is related to the dependence of FVIII clearance on the clearance of VWF, which is not altered by using the longer acting FVIII concentrates in development.
POTENTIAL BENEFITS OF LONGER ACTING FACTOR CONCENTRATES
Lower infusion frequency •
Fewer patient clinic visits or home care nurse visits when commencing patients on prophylaxis, possibly leading to earlier start of prophylaxis
Less need for central venous line leading to some cost savings and reduced morbidity
Allows for more convenient dosing days and times (which might improve adherence)
Less relevance of morning administration of factor
May allow treatment on non-work or non-school days
Increased uptake of prophylaxis among patients not currently on prophylaxis (e.g., those with moderate hemophilia) leading to better bleed protection
In March 2014, FDA approved long awaited first longacting factor – a long-acting factor IX concentrate, i.e. recombinant factor IX Fc fusion protein, eftrenonacog (Alprolix).
LONG-ACTING CLOTTING FACTORS (TABLE 2)
Technologies used to produce longer action
Table 2: Newer Products (Normal and Long-Acting) Name
Type of product
Mean Half-life and dose frequency
Factor IX Alprolix Biogen Idec (Eftrenonacogα)
Recombinant, FC fusion protein
Half-life 107-111 hrs, once in 7-10 days
1st FDA approved long acting factor (March 2014), No inhibitor develops
Recombinant, albumin fusion protein
Very long half-life (90 hrs), once in 2-3 weeks
FDA approved (March 2016)
Advate (octocog α)
Half-life normal, three times a week
Launched in India in July 2016.
Half-life 1.4 times normal, twice a week
FDA approved (Nov 2015)
Recombinant, Fc fusion protein
19 hours, 20 IU/kg body weight twice a week
1st FDA approved long acting factor VIII (June 2014), Available in India through World Federation of Hemophilia
Recombinant, 3rd generation full length FVIII
Slightly extended, 2-3 times a week
FDA approved (March 2016)
Novoeight Novo (Turoctocog α) Nordisk
Recombinant, 3rd generation full length FVIII
Half life normal, 3 times a week
Launched in India in April 2016. No inhibitor develops
Nuwig Octapharma (simoctocog α)
Human cell line Half-life slightly recombinant FVIII prolonged (17 hours)
Factor IX – Normal half-life 20 hours; Factor VIII – Normal half-life 12 hours; All factors have prolonged half-life (except Advate and Novoeight)
Morfini M, Coppola A, Franchini M, Minno GD. Clinical use of factor VIII and factor IX concentrates. Blood Transfus 2013; 11 Suppl 4: s55-63 DOI 10.2450/2013.010s
Wu R, Luke KH, Poon MC et al. Low dose secondary prophylaxis reduces joint bleeding in severe and moderate hemophiliac children: a pilot study in China. Haemophilia 2011; 17: 70-4.
Manco-Johnson MJ, Abshire TC, Shapiro AD et al. Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. N Engl J Med 2007; 357: 535-44.
Gringeri A, Lundin B, Von Mackensen S, Mantovani L, Mannucci PM. A randomized clinical trial of prophylaxis in children with hemophilia A (the ESPRIT Study). J Thromb Haemost 2011; 9:700-10.
Verma SP, Dutta TK, Mahadevan S, Nalini P, Basu D,
Biswal N, Ramesh A, Charles D, Vinod KV, Harichandra Kumar KT. A randomized study of very low-dose factor VIII prophylaxis in severe haemophilia. Haemophilia 2016; 1-7, Doi:10.1111/hae.12838 6.
Feldman BN, Pai M, Rivard GE, et al. Tailored prophylaxis in severe hemophilia A: interim results from the first 5 years of the Canadian hemophilia Primary prophylaxis study. J Thromb Haemost 2006; 4:1228-36
Charles D, Dutta TK. Efficacy of once weekly factor VIII prophylaxis in the prevention of clinically significant bleeds in patients with severe haemophilia A. A dissertation submitted to JIPMER, Puducherry in partial fulfillment of the rquirements for the award of the degree of D.M. Clinical Haematology, July 2016
Carcao MD, Lorio A. Individualizing Factor Replacement Therapy in Severe Hemophilia. Semin Thromb Hemost 2015; 41: 864-871.
Laffan M. New products for the treatment of haemophilia. British Journal of Haematology 2016; 172: 23-31
10. Product pipeline | Factor VIII & VWF (February 29, 2016). www.hemophilia.ca/files/Pipeline%20-%20Factor%20 VIII%20and%20VWF.pdf
Newer Therapies in Hemophilia
C H A P T E R
Sandeep Garg, Naresh Gupta, Sunita Aggarwal
Hemophilia A and B are X-linked recessive hemorrhagic disorder due to deficiency of factor VIII and IX respectively. However, 1/3rd cases are due to spontaneous mutations. These patients present with bleeding. The platelet count, BT and PT are normal but APPT is deranged. Correction of APTT on mixing with normal plasma suggests intrinsic pathway defect. Normal factor levels are between 50% -150%. Hemophilia is defined as mild (factor levels >5% to 40%), moderate (1-5%) and severe (<1%).
therapy are need for daily infusions, risk of TTI’s and development of inhibitors.
NEWER THERAPEUTICS FOR PATIENTS WITH INHIBITORS
Role of factor VIII bypassing agent- Presently patients with inhibitors are treated with either Factor VII or factor eight inhibitor bypassing agents (FEIBA).
OBIZUR- Due to variations in the amino acid sequence between human and porcine FVIII, there is reduced reaction between inhibitors and porcine FVIII. As a consequence, porcine FVIII can be used to treat bleeding episodes in patients with inhibitors. However, it was found to transmit parvo virus in one of the studies. This lead to development of a recombinant B-domain truncated porcine FVIII (OBI-1). This was found to be well tolerated in trials, however it is not yet approved for the treatment.
Alb-rFVII a-FP- Alb-rFVIIa-FP is a fusion protein (FP) linking human coagulation Factor VIIa and human albumin. A single recombinant gene
PRESENT STANDARD OF CARE
Over the year’s treatment changed from giving whole blood, plasma transfusion, cryoprecipitate and later factors plasma derived followed by recombinant ones. This lead to decrease in transfusion transmitted infections and increase in inhibitors. Initial treatment begins with PRICE therapy- protection (splint), rest, ice, compression, and elevation. For mild bleeds desmopressin and antifibrinolytics are used for moderate to severe disease each unit of FVIII/ FIX per kg iv will raise the plasma FVIII and FIX level to 2 IU/dl and 1IU/dl respectively. Depending upon the type of bleed the factor level can be raised to desired level. Problems with existing factor
Tissue factor (extrinsic) pathway
Contact activation (intrinsic) pathway Damaged surface
X Prothombin (II)
VII VIIa Tissue factor X
Thrombin (IIa) Fibrinogen (I)
Fibrin (Ia) XIIIa
Active Protein C
Cross-linked ﬁbrin clot
Protein S Protein C + Thrombomodulin Fig. 1: Coagulation cascade
TheraPEG-Factor VII a-TheraPEG is a molecule in which PEG is conjugated to recombinant human clotting factors at a site remote from the active immunogenic site so as to decrease immune response The goal is to retain clotting activity while reducing immunogenicity, prolonging the plasma half-life and allowing small-volume s/c administration of clotting factors. It is still under development.
NEWER THERAPEUTIC TARGETS FOR HEMOPHILA
Longer Acting Factor VIII and IX – Once Weekly
Protein modifications are done to increase the halflife of factors by decreasing their clearance. This includes linkage of recombinant clotting factor to various molecules such as fragment crystallizable (Fc) region of human antibodies, to recombinant albumin and to polyethylene glycols (PEGs) of various sizes (Pegylation). Some of the extended half-life (EHL)factorIX clotting factor products are as follows:
EHL-FVIII Agents - Similar to the lines of FIX some of the EHL FVII are peglayted factor VIII (PEGylated rFVIII), GlycoPEGylation of rFVIII and TheraPEG(given s/c)
Factor 8 Mimetics
Monoclonal humanized bispecific antibody Emicizumab (ACE 910) has been developed recently as a conformational replica of factor VIII. It binds to Factor IX and X and helps in progression of the coagulation cascade leading to thrombin generation. Its greatest advantage is that it is given subcutaneously once a week. In one study5 by Naoki Uchida et al. involving64 subjects with hemophilia A who had bleeding despite prophylactic or on-demand therapy, once-weekly subcutaneous administration of emicizumab (1mg/ kg) markedly decreased bleeding rates. There were no serious adverse events, anaphylactic reactions and thrombotic complications. No neutralizing anti-emicizumab antibodies developed in any of the patient. The estimated FVIII equivalent activity of emicizumab is around 10–30 IU/dL at dose of 1–3 mg/Kg/wk.
Targeting Tissue Factor Pathway Inhibitor (TFPI)
It has been seen that patients with factor VIII and IX deficiency or with inhibitors to both can maintain their hemostasis if Factor VII is given i.e. maintaining the activated factor X (FXa) by extrinsic pathway. TFPI is single-chain polypeptides which can reversibly inhibit FXa on the membrane, the formed FXa-TFPI complex can subsequently also inhibit TF/FVIIa and thus controlling the excess coagulation. A close interaction between the TFPI KPI-2 (Kunitz type protease inhibitor) domain and the FXa active site is essential for this inhibitory action of TFPI and further FXa generation. Scientists have now invented Anti -TFPI agents such as:
NN-7415 (concizumab/mAB2021) (anti-TFPI). Concizumab is an anti-TFPI antibody that binds to the K2 domain of TFPI. The aim is to decrease the inhibitor activity of TFPI against the tissue
EHL-FIX Agents 1.
rFIX:Albumin-Fusion to albumin prolongs drug half-life and reduces renal clearance of large molecules. rFIX:Albumin is produced in Chinese hamster ovary cells with albumin linked to the cleavage site i.e. C-terminus of rFIX . The linker between the albumin and the cleavage site is designed in such a way that it allows the albumin removal once the FIX gets activated and does not prolong coagulation. This rFIX:Albumin is approved by the US FDA in 2016 for prophylaxis or treatment of bleeding in individuals with hemophilia B. The half-life is five to six fold longer than unmodified factor IX products.
Eftrenonacogalfa:It is a newer agent in which one molecule of rFIX is covalently fused directly to the dimeric Fc of IgG1 (rFIX:Fc). It is currently approved for use in the USA. This fusion shields FIX from intravascular degradation proteases. In one study the annualized bleeding rates (ABRs) of prophylaxis regimens once weekly (Group 1) or initially once every 10 days (Group 2) were compared with ondemand therapy (Group 3, episodic). Low ABRs were achieved in both prophylaxis regimens. No bleeding was seen in 23% and 42.3% of group I and II respectively. Efficacy of acute bleed treatment was 90.4% with a single dose and 97.3% with more than one dose. No inhibitors or vascular thrombotic events were detected. However, 10.9% had at least one serious adverse event.
(rFIX:PEG). Here rFIX is produced in Chinese hamster ovary cells and glycoPEGylation is performed at the activation peptide site. PEGylation within the activation peptide domain maintains the catalytic activity and increases the half-life to fivefold. In a recent Phase III study rFIX:PEG weekly IV prophylaxis at 10 IU/kg or 40 IU/kg was compared with on-demand dosing. Lower ABRs was seen in both the prophylaxis arm than on-demand with the 40 IU/kg having the lowest ABR. In the 40 IU/kg group the bleeding episodes, 99% resolved with one dose as compare to 84% with 10 IU/kg dose. No inhibitors, deaths and thromboembolic events were seen. However adverse events were experienced by 81% of patients.
construct is expressed in Chinese hamster ovary cells yielding a recombinant human albumin attached to the C-terminus of rFVIIa via a flexible glycine–serine linker. It has a half-life of 8.5 hrs and can be used in patients with inhibitors.
factor pathway and thus increase the ability of this pathway to support hemostasis. By doing so, the need for intrinsic pathway support of activated Factor X (FXa) production is reduced. In a first-inhuman, Phase I, multicenter, randomized, doubleblind, placebo-controlled trial, escalating single IV (0.5–9,000 μg/kg) or SC (50–3,000 μg/kg) doses of concizumab were given to healthy subjects (n=28) and hemophilia patients (n=24). No serious adverse events and antibodies were seen.
Non-anticoagulant sulfated polysaccharides (NASP) including Fucoidanare derived from seaweed. These NASP interact with the highly positively charged C-terminal of TFPI and inhibit it, however actual mode of action is unclear. Studies on Fucoidan AV513 are going on in hemophilic animals models.
A PEGylated version of the aptamer BAX499 blocked the inhibition by TFPI of the TF/FVIIa activity. BAX499 markedly improved clotting in hemophilia A and B plasmas and was able to reduce bleeding in a nonhuman primate model.
Specific peptides with high affinity for TFPI obtained by phage display technique represent yet another type of antagonist developed for the treatment of hemophilia patients. A PEGylated anti-human TFPI 20-mer peptide (JBT2329) was shown to improve survival of hemophilic mice.
RNAi Therapeutic Targeting Antithrombin
ALN-AT3 (Alnylam). It is an RNAi therapeutic agent (small interfering RNAs) which reduces the production of antithrombin-3proteins (AT3) thus preventing the inhibition of FXa.Since AT3 controls FXI, FIX, FII, and to some degree FVII, therefore inhibiting AT3 will have the potential to modify a variety of clotting factor deficiencies. In animal models, Alnylam yields potent (up to 100%), dosedependent (1–30 mg/kg), and durable (30 days) knockdown of AT3.This silencing of antithrombin results in fourfold increase in thrombin generation. In a Phase I multiple ascending S/C dosing study in normal subjects (low dose only) and patients with hemophilia A or B, around 70% decrease in AT3 with concurrent 334% increase in thrombin generation was noted at a dose of 45 μg/kg given weekly for 3 weeks.
Apparently safe adeno-associated viral (AAV) vectors have been used for delivery of the FIX gene
to hematocicytes in hemophilia B. Factor IX activity levels sufficient to reduce spontaneous bleeding have been obtained in patients with severe disease for more than 4 years in some patients. The use of high-activity variants of FIX may allow achieving higher factor levels. However this can have a problem in patients with preexisting antibodies to vectors which are capable of eradicating transduced cells. The progress in gene therapy for hemophilia A has been hampered to a significant degree by the size of the gene. While the FIX gene can be placed easily in AAV vectors, the FVIII gene barely fits. Therefore there is a need to modify the FVIII gene cassette to allow accommodation in AAV vectors or use of some other viral vectors such as lentiviral vectors that are capable of carrying the FVIII gene. The advantage of the lentivirus is that they integrate into the target genome, even if the cells are in quiescent phase and are not actively dividing. Recently use of stem cells have been tried in delivering the gene therapy. Gene therapy for hemophilia B is on the horizon, and gene therapy for hemophilia A is becoming feasible.
Uchida n, Sambe T, yoneyama K Fakazawa N et al. A first in -human phase 1 study of ACE910, a novel factor VIIImimetic bispecific antibody, in healthy subjects. Blood 2016; 27:1633-1641
Powell JS, Apte S, Chambost H, et al. Long-acting recombinant factor IX Fc fusion protein (rFIXFc) for perioperative management of subjects with hemophilia B in the phase 3 B-LONG study. Br J Haematol 2015; 168:124134.
Collins PW, Young G, Knobe K, et al; Paradigm 2 Investigators. Recombinant long-acting glycoPEGylated factor IX in hemophilia B: a multinational randomized phase 3 trial. Blood 2014; 124:3880-3886.
Chowdary P, Lethagen S, Friedrich U, et al; The Explorerâ1 Investigators. Safety and pharmacokinetics of anti-TFPI antibody (concizumab) in healthy volunteers and patients with hemophilia: a randomized first human dose trial. J Thromb Haemost. 2015; 13:743-754.
Sorensen B, Mant T, Akinc A, et al; Aln-AT3 Investigators, International Multicenter Study. A subcutaneously administered RNAi therapeutic (ALN-AT3) targeting antithrombin for treatment of hemophilia: interim phase 1 study results in healthy volunteers and patients with hemophilia A or B. Blood 2014; 124:693.
Nathwani AC, Reiss UM, Tuddenham EG, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 2014; 371:1994-2004.
C H A P T E R
Artificial Blood: An Update on Current Red Cell and Platelet Substitutes
The complexity of blood is far too great to allow for absolute duplication in a laboratory. Instead, researchers have focused their efforts on creating artificial substitutes for two important functions of blood: oxygen transport by red blood cells and hemostasis by platelets. A number of driving forces have led to the development of artificial blood substitutes. One major force is the military, which requires a large volume of blood products that can be easily stored and readily shipped to the site of casualties. Another force is HIV; with the advent of this virus, the medical community and the public suddenly became aware of the significance of transfusiontransmitted diseases and became concerned about the safety of the national blood supply. A third force is the growing shortage of blood donors. Approximately 60% of the population is eligible to donate blood, but fewer than 5% are regular blood donors. The number of units transfused each year has been increasing at twice the rate of donor collection. Artificial blood products offer many important benefits. First, they are readily available and have a long shelf life, allowing them to be stocked in emergency rooms and ambulances and easily shipped to areas of need. Second, they can undergo filtration and pasteurization processes to virtually eliminate microbial contamination. No product can claim to be 100% risk-free for infectious agents, but these substitutes have a greatly increased level of safety. Third, they do not require blood typing, so they can be infused immediately and for all patient blood types. Fourth, they do not appear to cause immunosuppression in the recipient.
RED CELL SUBSTITUTES
Two major types of red cell substitutes are under development: hemoglobin based and per fluorocarbon (PFC) based. PFCs are completely synthetic hydrocarbonbased compounds. The hemoglobin-based substitutes use hemoglobin from several different sources: human, animal, and recombinant. Human hemoglobin is obtained from donated blood that has reached its expiration date and from the small amount of red cells collected as a byproduct during plasma donation. One unit of hemoglobin solution can be produced for every 2 units of discarded blood. There is a concern that the worsening shortage of blood donors will eventually limit the availability of human hemoglobin for processing. The companies that use human hemoglobin are confident in their supply,
especially from the plasma centers that use paid donors. Animal hemoglobin is obtained from cows. This source creates some apprehension regarding the possible transmission of animal pathogens, specifically bovine spongiform encephalopathy. The Biopure Corporation, which uses bovine hemoglobin, has an affiliation with a local breeding farm, allowing close monitoring of the health and diet of the animals. The company is very confident about the safety of its product. Forty units of hemoglobin solution can be obtained per slaughtered cow. Recombinant hemoglobin is obtained by inserting the gene for human hemoglobin into bacteria and then isolating the hemoglobin from the culture. This process allows for the manipulation of the gene itself to create variant forms of hemoglobin. One unit of hemoglobin solution can be produced from 750 L ofÂ Escherichia coliÂ culture. Once obtained from any of these sources, the hemoglobin must be purified and modified to decrease its toxicity and increase its effectiveness. This task has not proven to be very easy. Research with hemoglobin-based substitutes has actually been under way for over a century. In the 1930s, scientists collected free hemoglobin by lysing red blood cells and then transfused the unmodified product into animals after their blood had been drained. Shortterm survival rates were good, but the animals eventually experienced renal failure, intravascular coagulopathy, and vasoconstriction. Much of the toxicity was later attributed to the presence of residual red cell stroma in the product. Hemoglobin also has been determined to have a strong affinity for a relaxing factor derived from endothelial cells (i.e., nitric oxide). By binding to nitric oxide, the free hemoglobin produces unopposed vasoconstriction with subsequent hypertension and bradycardia. Hemoglobin normally circulates within red blood cells as a tetramer. When free hemoglobin is transfused, the tetramers rapidly break down into dimers and monomers. These small molecules then freely diffuse into the renal tubules and the sub-endothelium. To decrease the toxicity of hemoglobin solutions, manufacturers have had to develop methods to stabilize the hemoglobin tetrameric structure and increase its size. Several such methods now exist: larger molecules are added to the surface, the dimers are cross-linked with sugar molecules, or polymers of several tetramers are formed. Additional modifications of the hemoglobin, such as pyridoxylation, will create a product with near-normal oxygen-binding affinity.
Surface-modified hemoglobin is created by attaching large molecules, such as polyethylene glycol, to surface lysine groups. This modification also increases the viscosity and oncotic pressure of the solution. Two companies, Enzon and Apex Bioscience, have developed surfacemodified hemoglobin solutions. Both of the companies’ products, however, triggered moderate vasoconstriction after infusion. The companies have now positioned their products for specialized markets. Enzon is targeting its polyethylene glycol–conjugated hemoglobin product for treatment of patients with stroke and cancer; the small size of the hemoglobin molecules allows them to pass through constrictions and oxygenate areas that cannot be reached by red blood cells. For patients with cancer, the solution can deliver oxygen to tumor cells to increase susceptibility to radiation or chemotherapy. Apex Bioscience is developing its product for treatment of hypotension induced by septic shock.
To produce cross-linked hemoglobin, small bridges of sugar molecules are covalently attached to the dimers to create a stable tetramer. The US Army had partnered with Baxter Corporation to develop a cross-linked product, HemAssist. However, after increased mortality was noted in phase III trials, product development was discontinued. Baxter had also partnered with Somatogen to produce Optro, a recombinant product produced byE. coli. This product is also no longer under development.
To polymerize hemoglobin, surface amino acid groups are linked by reagents such as glutaraldehyde. Polymerized hemoglobin is the only product to date that has not triggered significant vasoconstriction after infusion. Three companies currently have products in phase III clinical trials. Hemolink, which is being developed by a Canadian company, Hemosol, is created from human hemoglobin polymerized with oraffinose. The solution has a 12- to 18-month shelf life and a 24-hour half-life. PolyHeme is produced by Northfield Laboratories, another Canadian company. It is created from human hemoglobin and has a 1-year shelf life and a 24-hour half-life. During phase III trials, trauma patients received up to 10 units of PolyHeme with minimal side effects. Presently, Northfield can produce about 10,000 units of PolyHeme per year. Hemopure, produced by Biopure, is a bovine-based hemoglobin. It has a 3-year shelf life and a 24-to 36-hour half-life. Biopure can currently produce about 100,000 units of Hemopure per year; plans to open another plant will allow production to increase to 900,000 units per year. Hemopure is the only hemoglobin solution that has received FDA approval for use in dogs. A recent blinded multicenter trial in patients undergoing infra-renal aortic reconstruction showed that 27% of patients receiving Hemopure were able to avoid transfusion of allogeneic blood. This product has also been transfused several
times on a compassionate-use basis. Minimal toxic side effects have been noted.
PFC-based solutions have been in development for several decades. An article by Clark and Gollan in the 1960s contained the famous photo of a mouse submerged in a container and “breathing” liquid. The liquid was an oxygen-saturated PFC solution. PFCs are synthetic hydrocarbons with halide substitutions and are about 1/100th the size of a red blood cell. These solutions have the capacity to dissolve up to 50 times more oxygen than plasma. Because PFC solutions are modified hydrocarbons, however, they do not mix well with blood and must be emulsified with lipids or oils. Moreover, the best results are obtained if the patient is breathing 100% oxygen at the time of infusion (PaO2 ≥350 mm Hg). The PFCs are inert products. After infusion, the molecules vaporize and are then exhaled over several days. After halting development of its hemoglobin-based substitutes, Baxter Corporation joined with Alliance Pharmaceutical Corporation to create a new company, PFC Therapeutics, which will market Alliance’s Oxygent product. Oxygent has a 2-year shelf life and a 12- to 48hour half-life. The product is currently in phase III clinical trials for use in cardiac and general surgical patients. In addition, the company has patented a procedure for use of its product in augmented acute normovolemic hemodilution: before surgery, approximately one third of the patient’s red cells are removed and stored, and Oxygent and saline are infused to maintain normovolemia and adequate oxygenation during surgery. The stored blood is then infused during or at the end of the surgical procedure. Because the blood lost by patients during surgery is of a lower hematocrit, they lose less of their red cell mass. Some patients have been able to completely avoid transfusion of allergenic blood with this procedure. Other clinical uses being investigated for PFC solutions in general include replacing red blood cells during acute blood loss, increasing oxygenation of localized areas of hypoxia, increasing oxygenation of solid tumors to improve radio sensitivity, removing gas micro emboli during cardiopulmonary bypass, preserving organs used for transplantation, and allowing liquid breathing for treatment of respiratory distress in premature infants. In addition, PFC-based substitutes would be acceptable to Jehovah’s Witnesses, who refuse all human and animal forms of hemoglobin.
Adverse reactions and limitations in the use of oxygen-carrying solutions
Adverse reactions associated with hemoglobinbased products include elevations in blood pressure, gastrointestinal dysmotility, and mild, temporary increases in pancreatic enzymes. Patients also develop jaundice due to the infusion of free hemoglobin. Treatment with PFCbased products can cause mild thrombocytopenia (10% to 15% decrease) and a flulike syndrome. Because patients need to be on high concentrations of oxygen when PFCs
are used, the risk of oxygen toxicity exists with prolonged administration. Since both types of products are taken up by human macrophages, there is also the theoretical risk that macrophage function will be altered.
The greatest progress in the field of blood substitutes has been with the oxygen-carrying solutions. However, research on platelet substitutes has been under way since the 1950s. One of the biggest factors pushing the need for platelet alternatives is the 5-day shelf life of the current blood product. This rapid outdate adds additional constraints to an already limited supply. The platelets are also stored at room temperature, thus increasing the risk of bacterial overgrowth. The risk of bacterial contamination of random donor platelets has been estimated to be 1:1500. Ideally, a platelet substitute would have the following properties: effective hemostasis with a significant duration of action, no associated thrombogenicity, no immunogenicity, sterility, long shelf life with simple storage requirements, and easy preparation and administration. Several different forms of platelet substitute are now under development: infusible platelet membranes (IPM), thrombospheres, and lyophilized human platelets. Only one product, IPM, is currently in clinical trials in the USA.
Infusible platelet membranes
Infusible platelet membranes are produced from outdated human platelets. The source platelets are fragmented, virally inactivated, and lyophilized; they can then be stored up to 2 years. Although the platelet membranes still express some blood group and platelet antigens, they appear to be resistant to immune destruction. One company, Cypress Bioscience Incorporated, manufactures an IPM product that is currently in phase II trials. The company is focusing its product for use in patients who have become refractory to platelet transfusions because of the formation of antibodies to HLA antigen or platelet antigens. The product has successfully stopped bleeding in about 60% of such patients. Overall, the product appears to be safe. No adverse effects have been noted, and there is no evidence that those who receive this product have an increased risk of thrombosis.
Thrombospheres (Hemosphere, Irvine, Calif) are not platelets; they are composed of cross-linked human albumin with human fibrinogen bound to the surface.
Lyophilized human platelets
A lyophilized platelet product has been under development since the late 1950s. The current process involves briefly fixing human platelets in para formaldehyde prior to freeze-drying in an albumin solution. The fixation step kills microbial organisms, and the freeze-drying greatly increases the shelf life. The adhesive properties of the platelets appear to be maintained. This product is currently in animal trials.
Once available, artificial blood substitutes will allow for rapid treatment of anemic patients. Unfortunately, the effects thus far are short lived, so many patients will eventually require allogeneic blood transfusions. With the ongoing shortage of blood donors, which is worsening each year, it has become increasingly important to learn and practice blood conservation measures. For example, in the past, a minimum order for a red cell transfusion consisted of 2 units. Physicians were questioned if they ordered anything less. Now physicians are being educated about transfusing judiciously. It may be possible to maintain the patient by transfusing just 1 unit of blood. That single unit may successfully ameliorate patients’ symptoms until their endogenous red cell production increases adequately. Folate, iron, and erythropoietin can also be given to help the bone marrow respond and thus avoid additional transfusions. Limiting laboratory testing and using smaller collection tubes will also conserve patients’ blood and prevent worsening of their anemia. Other techniques that enable the practice of “bloodless” medicine and surgery are available but too numerous to detail in this discussion. With progress, red cell and platelet substitutes may be able to diminish our dependency on donor blood. Until then, it will be exciting to explore the possibilities of the current products once they reach the market.
1. Winslow R. Red cell substitutes. In: Anderson K, Ness P, editors. Scientific Basis of Transfusion Medicine: Implications for Clinical Practice. 2nd ed. Philadelphia: WB Saunders; 2000. pp. 588–597. 2. Nucci ML, Abuchowski A. The search for blood substitutes. Sci Am 1998; 278:72–77. [PubMed] 3. Cohn SM. Is blood obsolete? J Trauma 1997; 42:730– 732. [PubMed] 4. Cohn SM. Blood substitutes in surgery. Surgery 2000; 127:599–602. [PubMed] 5. Gould SA, Moss GS. Clinical development of human polymerized hemoglobin as a blood substitute. World J Surg 1996; 20:1200–1207. [PubMed]
All current red cell substitutes have a short duration of action—lasting only about 24 hours in the circulation and are very expensive, with estimates at $500 per unit in US. Finally, use of these products can interfere with clinical laboratory testing. Hemoglobin solutions will make the patient’s blood specimens appear hemolyzed, and PFC solutions can produce lipemia. Both factors can affect the results obtained by some test systems. Close communication between the clinicians using the products and the laboratory will have to occur if reliable test results are to be reported.
The mechanism of action has not yet been elucidated. Experimentally, the thrombospheres appear to enhance platelet aggregation but do not themselves activate platelets. Thus far there has been no evidence of thrombogenicity. A similar product, Synthocytes (Andaris Group Ltd, Nottingham, UK), has just entered into clinical trials in Europe.
6. White C. Blood substitute makers to cash in on blood shortage, Technology Investor, September 2000; 33–35
Cobas Integra, Chiron blood gas analyzer 840, Sysmex SE9000 and BCT. Clin Chem Lab Med 1999; 37:71–76. [PubMed]
7. LaMuraglia GM, O’Hara PJ, Baker WH, Naslund TC, Norris EJ, Li J, Vandermeersch E. The reduction of the allogeneic transfusion requirement in aortic surgery with a hemoglobin-based solution. J Vasc Surg 2000; 31:299– 308. [PubMed]
12. Ma Z, Monk TG, Goodnough LT, McClellan A, Gawryl M, Clark T, Moreira P, Keipert PE, Scott MG. Effect of hemoglobin-and Perflubron-based oxygen carriers on common clinical laboratory tests. Clin Chem1997; 43:1732– 1737. [PubMed]
8. Mullon J, Giacoppe G, Clagett C, McCune D, Dillard T. Transfusions of polymerized bovine hemoglobin in a patient with severe autoimmune hemolytic anemia. N Engl J Med 2000; 342:1638–1643. [PubMed]
13. Danielson C, Ryder K, Glick M, Limiac A. Hemoglobinbased blood substitutes in the blood bank. Lab Med 1997; 28:131–134.
9. Clark L, Gollan R. Survival of mammals breathing organic liquids equilibrated with oxygen at atmospheric pressure. Science 1966; 152:1755–1756. [PubMed]
14. Lee DH, Blajchman MA. Novel platelet products and substitutes. Transfus Med Rev 1998; 12:175–187.[PubMed]
10. Lee R, Neya K, Svizzero TA, Vlahakes GJ. Limitations of the efficacy of hemoglobin-based oxygen-carrying solutions. J Appl Physiol 1995; 79:236–242. [PubMed]
15. Galan AM, Bozzo J, Hernandez MR, Pino M, Reverter JC, Mazzara R, Escolar G, Ordinas A. Infusible platelet membranes improve hemostasis in thrombocytopenic blood: experimental studies under flow conditions. Transfusion 2000; 40:1074–1080. [PubMed]
11. Wolthuis A, Peek D, Scholten R, Moreira P, Gawryl M, Clark T, Westerhuis L. Effect of the hemoglobin-based oxygen carrier HBOC-201 on laboratory instrumentation:
16. Vostal JG, Reid TJ, Mondoro TH. Summary of a workshop on in vivo efficacy of transfused platelet components and platelet substitutes. Transfusion 2000; 40:742–750. [PubMed]
C H A P T E R
Blood Component Therapy
Blood component therapy has advanced over the past few decades. Its application has found roots in almost all specialties. So a doctor (whichever specialty he practices) should know about the basic principles of blood component therapy. He should know the various indications in his specialty, and should be familiar with the type and dose of the components which he should order for these various indications. Red cell transfusions are given to increase the hemoglobin level in patients with anemia. Whole blood transfusion is restricted to patients who need an additional volume replacement e.g. an adult who has bled acutely and massively. Current practice is restrictive red cell transfusion policy. Transfusion is not indicated if the HB level is more than 10 g/dL and usually indicated if the HB level is less than 7 g/dL. The only important exception to this rule is ongoing symptomatic myocardial ischemia where the threshold is 10 g/dL. Plasma components which are commonly ordered are FFP and cryoprecipitate. They contain all the requisite plasma coagulation factors in a slightly reduced quantity than plasma. Cryoprecipitate contains Factor VIII, Factor XIII, fibrinogen, fibronectin and VWF. FFP is used to replace coagulation factors in case of major bleeding associated with warfarin anticoagulation and or with vitamin K deficiency. It is also used for treating bleeding episodes associated with liver disease, DIC, as a component in massive transfusion protocols and as a source of factor replacement in rare inherited coagulation disorders. Cryoprecipitate is indicated in congenital and acquired deficiency of fibrinogen and factor XIII and in uremic bleeding. For platelet transfusions, platelets are collected either by isolation from a unit of donated blood (Random donor platelet- RDP) or by apheresis from a donor in blood bank (Single donor platelets-SDP). SDP is always better than RDP because the recipient is exposed only to one donor and one unit of SDP will yield platelet number equal to platelet number in six units of RDP. Platelet transfusion is given 1) Therapeutically (to treat active bleeding or in preparation for an invasive procedure that would cause bleeding) or 2) prophylactically (to prevent spontaneous bleeding when the platelet count is low). Platelets are stored in room temperature and transfused quickly in patients who are bleeding to keep the platelet count above 50,000/microL in most situations and above 100,000 in case of nervous system bleeding. Guidelines are available
Mathew Thomas for preparation of an invasive procedure. Prophylactic platelet transfusions are usually given when the platelet count is less than 10,000/microL. In ITP and Dengue fever with thrombocytopenia, platelets are given only if there is bleeding. There is renewed interest in granulocytic transfusion after overcoming the difficulties in collecting adequate amount of viable functional granulocytes. Granulocyte transfusion is indicated when the absolute neutrophil count (ANC) is less than 500/microL, evidence of bacterial or fungal infections and unresponsiveness to antimicrobial treatment. Indications are neutropenia from chemotherapy or transplantation, aplastic anemia, chronic granulomatous disease and neonatal sepsis. The most important and fatal complication of component therapy is transfusion associated GVHD (ta-GVHD). Complications can be reduced by leuko reduction; irradiation and giving ABO and HLA matched components.
Blood components used in clinical practice are whole blood, red blood cells, plasma components, platelets, lymphocytes and granulocytes which are derived from whole blood or platelet rich plasma. (Figure 1) These components are collected from normal donors by phlebotomy or hemapheresis using the technique of differential centrifugation. Blood components should be distinguished from plasma derivatives which are fractionated from large volumes of (thousands of litres) plasma in large industrial units e.g. for manufacturing albumin, intravenous gamma globulin, Factor VIII etc. Major blood components which are going to be discussed in this chapter are red cells, platelets concentrates, fresh frozen plasma and cryoprecipitate.
RED CELL TRANSFUSIONS
Red cell transfusions are given to increase the hemoglobin level in patients with anemia or to replace losses after acute bleeding episodes. Whole blood should be considered as the choice only when treating an adult who has bled acutely and massively, where volume replacement is necessary. Patients with chronic anemia should be transfused with RBCs (Packed RBCS â€“ PRBC) as volume replacement is not required. With the current use of CPD- Adenine as the anticoagulant preservative (AP) solution and use of the new generation additive solutions, the red cells can be stored for 35 â€“ 42 days.
Fig. 1: The Components of Human Blood Indications – Since anemia is associated with adverse clinical outcomes, red cell transfusion is indicated in conditions of anemia. The current consensus is a restrictive policy for transfusion. Red cell transfusion is not indicated if HB is more than 10 g/dL. It is generally indicated if the value is less than 7 g/dL. It is found that in various clinical trials, restrictive policy is safe, improves clinical outcomes and reduces unnecessary transfusion even in ICU setting. The exceptions to this rule are 1.
Patients with symptomatic myocardial ischemia with HB < 10g/dL should be transfused to improve hemodynamic instability.
In patients with acute coronary syndrome, an individualistic approach should be followed. In general it is desirable to have the HB equal to or more than 10g/dL.
In patients requiring massive transfusion (e.g. trauma or ongoing bleeding) a strict HB threshold may not be possible.
The volume of one unit of RBCs with citrate-phosphatedextrose adenine (CPD-A1) anticoagulant is between 225 – 350 ml. Several complications of RBC transfusion seem to be due to contamination with leukocytes. The complications are febrile non hemolytic transfusion reactions, HLA alloimmunization, post-operative infection and cardiac reperfusion injury. These complications can be reduced by leukocyte reduction filters. Transfusion associated graft versus host disease which is a fatal complication, cannot be reliably prevented by leuko reduction and requires irradiation of the blood products.
FFP - The plasma that is separated and frozen at -18°C to -30°C within 8 hours of collection is called FFP.
PF24 – Plasma that is separated and frozen at -18°C - -30°C within 24 hours of collection is called PF 24.
These can be stored for one year. Both these components contain all the requisite plasma coagulation factors in a slightly reduced quantity than plasma. As the clotting factors are not in a concentrate form, FFP should not be used as a source of specific clotting factors.
Thawed plasma – Thawed plasma is plasma that was frozen (FFP & PF24), thawed and kept at refrigerator temperature for up to 5 days.
Liquid plasma – Liquid plasma is plasma that has never been frozen.
Solvent/Detergent plasma (S/D plasma) – S/D plasma is plasma treated with viral inactivating agents prior to freezing.
Plasma cryoprecipitate reduced - Plasma cryoprecipitate reduced is plasma from which cryoprecipitate has been removed. This is also called cryo-poor plasma.
FRESH FROZEN PLASMA
This can be taken as a prototype of many of the plasma
components and will be discussed in detail. This can be prepared from single units of whole blood or from plasma collected by apheresis techniques. Standard FFP unit collected from single unit has a volume of approximately 200 – 250 mL.
Coagulation factor replacement in the management of major bleeding associated with warfarin anticoagulation and or Vitamin K deficiency.
To treat a deficiency of multiple coagulation factors e.g. liver disease Disseminated intravascular coagulation.
As a component of massive transfusion protocols.
FFP is also used as source of factor replacements for rare inherited coagulation factor disorders.
For healthy adult 2-3 mL/kg/hour approximately one unit in 1.5 hours).
For patients with volume overload or heart failure 1mL/kg/hour (approximately one unit in 4 hours).
For patients undergoing plasmapheresis, 60mL/ minute; this can be increased to 100mL/minute if the patient tolerates well.
FFP may be used in the unlikely circumstance when a specific factor concentrate or recombinant product is not available for managing bleeding in a patient with a coagulation factor deficiency (e.g. Factor VIII, IX, and XIII). This circumstance is very common in India. Liver disease leads to a form of “rebalanced” hemostasis, in which diminished hepatic function leads to both procoagulant and anticoagulant effects. It is now clearly established that in CLD there is a decrease in the anticoagulant factors as Protein C and Protein S together with a decrease in Vitamin K dependant clotting factors. The decreased Protein C and Protein S will lead to a prothrombotic state in CLD. If this prothrombotic state is dominant, patients with CLD and prolonged INR may not bleed but may actually go on for thrombosis: e.g. Portal vein thrombosis and lower extremity DVT. So an increased INR may not truly indicate the actual balance of bleeding and the prothrombotic state in CLD. For these reasons FFP is now given only when there is bleeding associated with prolonged INR in CLD. There is also no indication for FFP for treating bleeding or as prophylaxis for invasive procedures in patients with INR<2.
The potential risks of plasma exchange include infection, volume overload, febrile and allergic reaction, anaphylactic reaction, TRALI (Transfusion-related acute lung injury).
Cryoprecipitate: (Cryoprecipitated Anti Hemophilic Factor – AHG)
This is the precipitate that forms when FFP is thawed at 4°C. This precipitate (cryo) is separated from the thawed plasma by centrifugation. Cryo is a concentrated preparation that contains all of the factor VIII, Fibrinogen, Fibronectin, factor XIII and vonWillebrand factor (VWF) from the FFP. It is reduced from an initial volume of 250mL to a final volume of 10mL. The remaining portion can be refrozen and used as Plasma Cryoprecipitate Reduced (or Cryo-Poor Plasma). Cryo cannot be made from PF24 because the level of factor VIII is very low. Cryo contains approximately 200mg of fibrinogen and 100 units of Factor VIII per unit. It carries an equivalent infectious risk as a unit of plasma.
Congenital and acquired deficiency of Fibrinogen e.g. CLD is an acquired deficiency.
Congenital and acquired deficiencies of Factor XIII.
Rarely it is used in uremic bleeding that does not respond to other measures.
Platelets are collected either by isolation from a unit of donated blood or by apheresis from a donor in the blood bank. 1.
Pooled platelets – One unit of platelet can be isolated from every unit of donated blood by centrifuging the blood within the closed collection system to separate the platelets from the red cells (RBC). The average platelet yield from one unit is 7 X 1010 platelets. This number is inadequate to increase the platelet count in an adult recipient. Four to six units are pooled to allow transfusion of 3 to 4 X 1011 platelets per transfusion. These are called random donor pooled platelets (RDP). The lower cost and the ease of collection and processing are the advantages of RDP. The major disadvantage is the recipient exposure to multiple donors in a single transfusion and issues regarding bacterial testing.
Apheresis (single donor) platelets SDP – In this method of collection, platelets are collected from volunteer donors in the blood bank in a one to
There is also little evidence to support the practice of administering FFP to correct the INR prior to performing an invasive procedure. Solvent detergent treatment of FFP (S/D plasma) is effective for use in liver transplantation.
Dose and infusion rate
The usual dose of plasma is approximately 10-15 mL/kg (i.e. approximately three to five units) in most adults. But this dose (750-1250 mL) represents a significant volume challenge. The infusion rate is:
Warfarin associated intracranial hemorrhage is a medical emergency with an extremely high morbidity and mortality. After stopping the anti-coagulant, the ideal treatment is Vitamin K 10mg given by slow intravenous infusion not faster than 1mg/min to minimize anaphylactic shock. The effect of Vitamin K is delayed and takes about 12 to 24 hours. So for immediate action, 4 units of FFP should be given along with vitamin K, if better options like PCC (Prothrombin complex concentrates) are not readily available for a rapid action. The same principle is used for treating other serious bleeding episodes associated with supra therapeutic range of INR. Rare inherited deficiencies of factors XIII, X, VII, V and II are treated with FFP when they develop bleeding and when these rare factor concentrates are not easily available. FFP is used as a source of factor V in severe cases of DIC with persistent bleeding. The persistent bleeding in this situation is thought to be due to a factor V deficiency rather than a global decrease in coagulation factors. So it is also given for congenital Factor V deficiency. PF 24 should not be given under these circumstances as it contains reduced levels of factor V.
preparation for an invasive procedure that would cause bleeding).
Prophylactic (To prevent spontaneous bleeding).
Therapeutic indications in an actively bleeding patient:
Platelets should be transfused quickly in patients with thrombocytopenia and active bleeding to keep the platelet count above 50,000/microL in most bleeding situations. It should be kept above 100,000/microL if there is DIC or central nervous system bleeding. Other factors contributing to bleeding like surgical or anatomical defect, fever, infection or inflammation, coagulopathy and acquired platelet function defect should be addressed.
Preparation for an invasive procedure
The typical thresholds for the platelet count for some common procedures are as follows. These are based on retrospective studies of patients who are afebrile and have thrombocytopenia but not coagulopathy.
Fig. 2: Process of Single Donor Platelet Formation two hour pheresis procedure. In the process, platelets and some white cells are removed while the red cells and plasma are returned to the donor. A typical apheresis platelet unit is equal to six of more units of RDPs. (3 to 6 X 1011 platelets) (Figure 2). Here the recipient is exposed only to a single donor. Further the recipient’s features such as HLA type matching, CMV status and blood type can be assessed. The pooled and apheresis platelets contain some amount of WBC, plasma and RBCs. These can give rise to febrile non hemolytic transfusion reactions (FNHTR), alloimmunization, transfusion associated graft-versus host disease (ta-GVHD) & transfusion related acute lung injury (TRALI) in some patients.
Platelet storage and pathogen reduction
Since cooling induces a clustering of VWF receptors on the surface of the platelets leading to morphological changes, platelets are stored at room temperature. The morphologically changed platelets due to the cooling effect are increasingly cleared by hepatic macrophages and result in reduced platelet survival in the recipient. A disadvantage of room temperature storage is the increased growth of bacteria compared with blood components stored in the refrigerator or freezer. This is reduced by various methods like donor screening for blood borne pathogens, proper skin sterilization, to screen for bacterial contamination etc. The shelf life of platelets stored at room temperature is 5 days. This is because longer storage increases the risk of bacterial infection.
Indication of platelet transfusion 1.
Therapeutic (To treat active bleeding or in
Neuro surgery or ocular surgery – 100,000/microL
Most other major surgery – 50,000
Endoscopic procedures – 50,000 for therapeutic procedures; 20,000 for low risk diagnostic procedures
Central line placement – 20,000
Lumbar puncture – 10,000 to 20,000 in patients with hematologic malignancies and greater than 40,000 to 50,000 in patients without hematologic malignancies but lower in patients with ITP
Epidural anaesthesia – 80,000
Bone marrow aspiration/biopsy – 20,000
Bone marrow studies can be done with lower counts if sufficient pressure (10 – 15 minutes) is applied at the site of the procedure.
Prevention of spontaneous bleeding
Prophylactic transfusion is used to prevent spontaneous bleeding in patients at high risk of bleeding. The threshold for prophylactic transfusion varies depending on the patient and on clinical scenario.
Predictors of spontaneous bleeding
Patients with platelet count more than 50,000 are less likely to bleed but they can bleed sometimes even with platelet counts greater than 50,000. •
The platelet count at which the patient bled previously can be a good predictor for bleeding.
Mucosal bleeding (wet bleeding) are more predictive than petechial bleeding and ecchymoses.
Coexisting inflammation, infection and fever increase the risk.
The underlying condition responsible for the patient’s thrombocytopenia may help in estimating the risk of bleeding. Patients with ITP often tolerate very low platelet count without bleeding. Patients
with leukemia and coagulopathy can have bleeding at higher counts (30,000 – 50,000). •
Children are likely to experience bleeding compared with adults with bone marrow suppression.
Tests for platelet dependent hemostasis like bleeding time, Thromboelastogram (TEG) are not useful in predicting bleeding in thrombocytopenic patients.
Higher thresholds (30,000) are used in patients who are febrile or septic. For patients with APL (acute promyelocytic leukemia) who have coexisting coagulopathy, the platelet transfusion threshold is 30,000 to 50,000. For patients with platelet consumption (ITP, DIC) or platelet function disorders, platelets are transfused only for bleeding or in some cases for invasive procedures. For patients with hematological malignancies, HCT (Hemopoietic cell transplantation) and cytotoxic chemotherapy, the threshold is 10,000 to 20,000. For patients with TTP and HIT, platelet transfusion should be given only if they bleed because prophylactic transfusion may cause a slightly increased risk of thrombosis.
There is a renewed interest and application of granulocyte transfusion. There were a lot of difficulties in collecting adequate number of viable, functional granulocytes. Currently granulocytes are harvested from properly selected donors by apheresis after they are stimulated by dexamethasone and G-CSF. Usually it is transfused within a few hours after the collection though it can be stored for 24 hours in room temperature. The criteria for transfusions are absolute neutrophil count-ANC <500 cells/microL, evidence of bacterial or fungal infections and unresponsiveness to antimicrobial treatment for at least 48 hours. The main indications are neutropenia from chemotherapy or transplantation, aplastic anemia, chronic granulomatous disease and neonatal sepsis. Prophylactic GTX (granulocyte transfusion) is controversial. Complications are pulmonary adverse reaction, transfusion associated GVHD, alloimmunization and infection.
AABB, American Red Cross, America’s Blood Centres, and Armed Services Blood Program. Circular of Information for the use of human blood and blood components. http:// www.aabb.org/resources/bct/Documents/coi0809r.pdf.
Guidelines for the Administration of Blood Products. Australian and New Zealand Society of Blood Transfusion Ltd and Royal College of Nursing Australia, 2nd ed, Sydney Australia, December 2011. http://www.anzsbt. org.au/publications/documents/ANZSBT_Guidelines_ Administration_Blood_Products_2ndEd_Dec_2011_ Hyperlinks.pdf (Accessed on January 22, 2013).
Adamson JW. New blood, old blood, or no blood? N Engl J Med 2008; 358:1295.
Roback JD, Caldwell S, Carson J, et al. Evidence-based practice guidelines for plasma transfusion. Transfusion 2010; 50:1227.
Triulzi DJ. The art of plasma transfusion therapy. Transfusion 2006; 46:1268.
Callum JL, Karkouti K, Lin Y. Cryoprecipitate: the current state of knowledge. Transfus Med Rev 2009; 23:177.
Slichter SJ. Platelet transfusion therapy. Hematol Oncol Clin North Am 2007; 21:697.
McCullough J. Overview of platelet transfusion. Semin Hematol 2010; 47:235.
Slichter SJ. Platelet transfusion therapy. Hematol Oncol Clin North Am 2007; 21:697.
Dosing of platelets
A standard dose for prophylactic platelet transfusion is one RDP per 10kg body weight which amounts to 4 – 6 RDP or one SDP. This platelet dosing is expected to increase the platelet count by approximately 30,000/mL within 10 minutes of the infusion. This gradually wanes off after 72 hours. The rate of platelet transfusion (4 – 6 RDP or one SDP) is approximately 20 – 30 minutes. Complications of platelet transfusion include – infection, TRALI, Transfusion associated circulatory over load (TACO), alloimmunization, allergic and anaphylactic reactions, FNHTR, Ta GVHD and post transfusion purpura. Platelets express ABO and HLA class 1 antigens. They do not express Rh or HLA class II antigens. ABO and HLA compatible platelets appear to cause a greater platelet count increment in the recipient and they can be used to improve responses in patients who have become refractory to platelet transfusion due to alloimmunization. Refractoriness to platelet transfusion therapy is said to be present when the desired increment does not occur after one or two transfusions. Platelets can be modified by leukoreduction and irradiation to reduce the complications associated with platelet transfusions.
10. McCullough J. Overview of platelet transfusion. Semin Hematol 2010; 47:235. 11. Slichter SJ, Kaufman RM, Assmann SF, et al. Dose of prophylactic platelet transfusions and prevention of hemorrhage. N Engl J Med 2010; 362:600. 12. Kaufman RM, Djulbegovic B, Gernsheimer T, et al. Platelet transfusion: a clinical practice guideline from the AABB. Ann Intern Med 2015; 162:205.
Prophylactic platelet transfusion is given to prevent spontaneous bleeding in most afebrile patients with platelet counts less than 10,000 due to bone marrow suppression.
Chronic Myeloid Leukaemia – An Overview
C H A P T E R
Sunita Aggarwal, Jahnvi Dhar, Sandeep Garg, Naresh Gupta
Chronic myeloid leukaemia (CML) is an acquired genetic defect in the pleuripotent stem cell that is characterised by leucocytosis with granulocytic immaturity, basophilia, splenomegaly and a distinct genetic abnormality BCRABL fusion gene, Philadelphia(Ph+) chromosome [t(9;22) (q34;q11.2)].
According to the western literature, the annual incidence of CML is around 1.8 per 100,000. In India, it accounts for 50 – 70% of leukaemias with an annual incidence of 1-2 per 100,000. The median age of presentation is around 40 years. CML accounts for 15 - 20% of all leukaemias affecting the adults with M:F ratio 1.6:1.
BONE MARROW FINDINGS: Markedly hypercellular with marked myeloid hyperplasia along with large histiocytes (pseudo gaucher cells) with blue granules and marrow fibrosis of variable degree.(reticulin strain)
CML has a triphasic or biphasic clinical course: a chronic phase(85%), an accelerated phase(<10%) and blast crisis(23 %).
Chronic phase •
Peripheral blasts <10% in the blood and bone marrow.
Accelerated phase •
Blasts 10-19% of white blood cells in peripheral and/or nucleated bone marrow cells.
The clinical findings of CML vary and depend upon the stage of disease at diagnosis. 20-50 % of patients are asymptomatic, diagnosed from routine blood tests.
Persistent thrombocytopenia (< 100 × 109/L) or thrombocytosis (> 1000 × 109/L).
Signs and symptoms
Spleen size unresponsive to therapy.
Cytogenetic evidence of clonal evolution
Fatigue, weight loss.
Low-grade fever, hypermetabolism.
Early satiety due to splenomegaly.
Left upper quadrant abdominal pain from spleen infarction.
Appearance of palpable lymph nodes during the chronic phase indicates change to blast phase of the disease.
Leucocytosis in all stages of maturation with myelocyte bulge (The presence of a greater percent of myelocytes than the more mature metamyelocytes (“leukemic hiatus” or “myelocyte bulge”) is one of the classic findings in CML along with basophilia.
Decreased LAP score– decreased (normal 40- 100). The low LAP score is useful in excluding a reactive leukocytosis or “leukemoid reaction,” typically due to infection, in which the score is typically elevated or normal.
Blast phase •
Peripheral blasts ≥ 20% of white blood cells or nucleated bone marrow cells.
Extramedullary blast proliferation.
Large foci or clusters of blasts on bone marrow biopsy
Genetic testing for the Philadelphia chromosome, the BCR-ABL1 fusion gene or the fusion mRNA gene product is done by karyotyping, FISH analysis, or RT-PCR. There are several distinct BCR-ABL1 fusion proteins depending upon the site of the breakpoint in the BCR gene on chromosome 22. •
The most common abnormal BCR-ABL1 fusion transcript is a BCR-ABL1 protein with 210 kilodalton molecular mass known as the p210 BCRABL1 protein.
An alternative e19a2 fusion transcript is p230 BCRABL1. This is seen in rare CML cases (<1 %).
A smaller e1a2 fusion transcript, which produces the p190 BCR-ABL1 protein is also seen in a very small number of CML patients, but is more
FIRST LINE TREATMENT
Clinical trial or HCT or Omacetaxine
SECOND LINE TREATMENT AND SUBSEQUENT THERAPY
Clinical trial or HCT or Omacetaxine
Clinical trial or HCT or Omacetaxine BOSUTINIB
Fig. 1: Treatment of CML
Table 1: Treatment of Accelerated Phase Tests before treatment
Treatment of relapse
BCR-ABL gene mutation analysis
1. TKI therapy (600 mg OD)
TYROSINE KINASE INHIBITOR THERAPY (TKI): (Figure 1)
First generation : IMATINIB(400mg/d)
Second generation : DASATINIB(100mg/d), NILOTINIB (300 mg BD), BOSUTINIB(500mg/d)
Third generation : PONATINIB(45mg/d) (multitargeted TKI). It is only approved for patients with a T315I mutation or CML that is resistant to all Treatment of relapse other TKIs.
2. Omacetaxine 3. Consider HCT based on response
Tests before treatment
TREATMENT OF ACCELERATED PHASE Treatment options
frequently associated with Ph-positive acute lymphoblastic leukemia (ALL).
Common side effects of TKI therapy : Cytopenia, fluid retention, nausea and vomiting, muscle • The point mutation in BCR-ABL1 due to substitution BCR-ABL gene mutation analysis 1. TKI therapy(600mgcramps, OD ) Clinical trial skin rash, diarrhea, headache, thyroid Omacetaxine of amino-acid at position 315 results in 2.T3151 dysfunction, QT interval prolongation, 3. Consider HCT based on response mutation which is implicated in the development hyperglycaemia (nilotinib), etc. of imatinib resistance. 2. IMMUNOTHERAPY: Pegylated interferon may DIFFERENCIAL DIAGNOSIS be used TREATMENT OF BLAST PHASEfor those who cannot tolerate TKI therapy • Leukemoid reaction. side effects. It is not used as an initial treatment option inoptions patients of CML.Treatment of relapse Tests before treatment. Treatment • Juvenile myelomonocytic leukemia. Cell lineage 3. CHEMOTHERAPY: Omacetaxine was FDA • Chronic myelomonocytic leukemia. approved in 2012 for treatment of CML for those • Chronic eosinophilic leukemia with resistance and/or intolerance to two or more TKI. It is a protein synthesis inhibitor that • Chronic neutrophilic leukemia has demonstrated activity in patients with CML • Other Philadelphia chromosome positive in chronic phase with a T315I mutation. The malignancies. recommended dose and schedule is 1.25 mg/m 2
Table 2: Treatment of Blast Phase Tests before treatment.
Treatment of relapse
1. BCR-ABL mutation analysis
1. ALL-type chemotherapy plus TKI (800 mg OD) followed by HCT if possible.
2. TKI followed by HCT if possible. Myeloid
1. AML-type chemotherapy plus TKI (800 mg) followed by HCT if possible.
2. TKI followed by HCT if possible.
Table 3 Type of Response
WBC <10,000/microL with no immature granulocytes and <5 % basophils on differential; platelet count <450,000/microL and spleen not palpable.
Cytogenetic Response a. Complete cytogenetic response (CCgR)
No Philadelphia chromosome positive cells.
b. Partial cytogenetic response (PCgR)
1-35 % Philadelphia chromosome positive cells.
c. Major cytogenetic response (MCgR)
0-35 % Philadelphia chromosome positive cells
d. Minor cytogenetic response (mCgR)
36-65 % Philadelphia chromosome positive cells.
e. Minimal cytogenetic response (minCgR)
66-95 % Philadelphia chromosome positive cells.
f. No cytogenetic response (noCgR)
>95 % Philadelphia chromosome positive cells.
Molecular Response a. Major molecular response (MMolR)
Ratio of BCR-ABL transcript to housekeeping genes â‰¤0.1 percent (â‰Ľ3 log reduction) on the international scale (IS).
b. Complete molecular response (CMolR)
BCR-ABL transcript nondetectable and not quantifiable in an assay that has at least 4 to 5 log range of detection on two consecutive blood samples.
1. At diagnosis. 2. Every 15 days till complete haematological response confirmed. 3. Atleast every 3 months or as needed thereafter.
1. At diagnosis. 2. At 3 and 6 months. 3. Every 6 months until complete cytogenetic response confirmed. 4. Every 12 months if uncertain about regular molecular monitoring. 5. At treatment failure or unexplained anaemia, leukopenia and/or thrombocytopenia.
1. Every 3 months until major molecular response confirmed. 2. Atleast every 6 months thereafter.
4.Molecular (mutation analysis)
1. Suboptimal response or failure. 2. Before switching treatment.
subcutaneous injection twice daily for 14 days of a 28-day cycle for the induction phase and 1.25 mg/m 2 subcutaneous injection twice daily for 7 days of a 28-day cycle for maintenance.
Hydroxyurea (20 to 40 mg/kg/day) can be used to reduce white blood cell counts while awaiting confirmation of a suspected diagnosis of CML in
a patient with significant leukocytosis (eg >100 x10 9 white cells/L) or in patients with systemic symptoms or with symptomatic splenomegaly.
The treatment of Accelerated phase in given in Table 1 and Blast phase in Table 2. The type of response is given in Table 3.
ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION (HCT): High dose chemotherapy or radiation therapy followed by HCT is a treatment option for certain group of patients like unresponsive to TKI therapy, presence of T3151 mutation
Combination therapy : TKI with chemotherapy or interferon or cancer vaccines.
Newer drugs :
Farnesyl transferase inhibitors such as lonafarnib and tipifarnib.
Histone deacetylase inhibitor panobinostat.
Proteasome inhibitor bortezomib (Velcade).
Cancer Vaccines : The studies are being conducted on vaccines against CML called CMLVAX100. This, given along with imatinib seemed to increase its effectiveness.
Best treatment options are TKI and HCT. HCT is the only curable option in CML. But TKI have demonstrated better long term disease control and good tolerability, hence they are preferred over HCT.
AIMS OF INITIAL THERAPY AND DURATION OF TREATMENT
Complete hematologic response by 3-6 months.
Any cytogenetic response by 6 months.
Major cytogenetic response by 12 months.
Complete cytogenetic response by 18 months
HCT is only treatment curable option in CML but TKI therapy has revolutionized the management of CML. The need to overcome emergence of imatinib resistance has led to the investigation of combination therapies.
Kantarjian H, Cortes J. Chronic Myeloid Leukemia. Kasper DL, Fauci AS, Hause SL, Longo DL, Jameson L, Loscalzo J. Harrison’s principles of Internal Medicine. 19th ed. New York Mc Graw Hill Publishers 2015;687-695.
Deininger M. Chronic Myeloid Leukemia. Wintrobe’s Clinical Hematology. 13th ed. Philadelphia: Wolters Kluwer/Lippincott Williams & Wilkins 2013;1705-1721.
Diseases of White Blood Cells, Lymph nodes, Spleen and Thymus. Kumar V, Abbas AK, Aster JC. Robbins & Cotran Pathological asis of Disease. 9th ed. Elsevier 2014;579-628.
Radich JP, Deininger M, Abboud CN, Altman JK, Barta SK, Berman E et al .Chronic Myeloid Leukemia. NCCN Guidelines for Patients. Version 1.2016;1-71.
DONOR LYMPHOCYTE INFUSION (DLI): It is procedure in which the patient receives lymphocytes from the same person who donated blood stem cells for HCT. The purpose of DLI is to stimulate an immune response called GVL (graft versus leukemia effect). It can be used after allogenic HCT for CML who didn’t respond to the treatment or relapse after initial response.
TREATMENT OF CHRONIC PHASE
Medicine Update 2017