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Do you know which disease fits this month’s case? Then test your knowledge in the quiz below!

What was the origin of this extreme anaemia? Haemolytic transfusion incident due to ABO incompatibility
Haemolytic uraemic syndrome (HUS)
Mycoplasma-induced sepsis with autoimmune haemolytic anaemia (AIHA)
Megaloblastic anaemia

Online Version of this month´s case:

The correct answer to April´s Quiz is:

Mycoplasma-induced sepsis with autoimmune haemolytic anaemia (AIHA)

Scattergrams and microscopy

Patient history: after undergoing surgery, a middle-aged woman was submitted to the intensive care unit in a state of shock.

 

Table

Interpretation and Differential Diagnosis

The answer can be inferred from…

  • Normocytic, normochromic anaemia: low HGB and RBC, normal RBC-He (substitute of MCH)
  • Reticulocytosis
  • Equal values for total haemoglobin (HGB) and cellular haemoglobin (HGB-O)
  • Suspected RBC agglutination
  • Absence of fragmented RBC
  • No increase of HYPER-He

 

 

Case history

 

Case results

RBC agglutination was suspected based on an abnormal RBC histogram, very low RBC count and highly elevated MCH and MCHC values. This triggered the appearance of the ‘RBC Agglutination?’ flag. In addition, the RBC count from the impedance channel (RBC = 0.67 x 1012/L) was much lower than the count from the RET channel (RBC-O = 2.38 x 1012/L). The presence of RBC aggregates was confirmed by microscopy (not shown) indicating that the RBC count and HCT measurement from the impedance channel were incorrect and that, consequently, all parameters derived from these values were also incorrect.

 

Correct values were determined as follows:

- A correct RBC count could be derived from the RET channel (RBC-O) because the higher temperature (>37°C) in the RET reaction chamber dissolves RBC aggregates: RBC-O = 2.38 x 1012/L

- The RBC most frequent volume (R-MFV), a service parameter derived from the impedance channel, could replace MCV in this sample’s context: R-MFV = 97.5 fL (therefore, the Macrocytosis flag was wrong here)

- HCT could be corrected based on R-MFV and RBC-O: HCT-CALC = R-MFV x RBC-O = 0.232 L/L

- The RBC haemoglobin equivalent (RBC-He) from the RET channel equals the mean cellular haemoglobin content (MCH): RBC-He = 32.6 pg

- MCHC could be corrected based on RBC-He and R-MFV: MCHC-O = HGB-O / HCT-CALC = (RBC-He x RBC-O) / (R-MFV x RBC-O) = RBC-He / R-MFV = 334 g/L

- The absolute reticulocyte concentration could be corrected based on RET% and RBC-O: RET-CALC# = RET% x RBC-O = 321.3 x 109/L

 

The resulting values indicated that the patient had a normocytic, normochromic anaemia with a reticulocytosis, suggesting a proper bone marrow response. This indicated loss or destruction of RBC, which can be the result of either blood loss or haemolysis.

 

An eosinophilia and atypical lymphocytes were also observed, indicating an infection (the WPC channel was not available so a WPC reflex measurement was not performed; it would have resulted in the removal of the ‘Blasts/Abn Lympho?’ flag). The high amounts of immature granulocytes (IG% = 8.6%) and NRBC (37.5%) indicated severe bone marrow stress. A thrombocytopenia was also found. 

 

In summary, the normocytic normochromic anaemia with effective erythrocytosis, equal values for cellular haemoglobin and total haemoglobin and the absence of RBC fragments suggested an extravascular haemolytic anaemia. Therefore, a megaloblastic anaemia (no effective bone marrow response), transfusion incident (intravascular haemolysis) and haemolytic uraemic syndrome (presence of fragments and intravascular haemolysis) could be excluded.

 

 

The following answers are incorrect for the described reasons

 

Haemolytic uraemic syndrome (HUS)

HUS belongs to the microangiopathic haemolytic anaemia (MAHA) subgroup of haemolytic anaemias and is caused by bacterial toxins that damage the endothelial layer of small blood vessels in multiple organs resulting in fibrin deposition and platelet aggregation. RBC are shredded as they travel through these damaged vessels resulting in red cell fragmentation and intravascular haemolysis. The outcome would be a normocytic, normochromic anaemia and a consequent reticulocytosis due to increased erythropoiesis by the bone marrow, as observed here. However, fragmented RBC were not found in the presented patient. In addition, intravascular haemolysis would have resulted in extracellular haemoglobin in the blood and therefore different HGB and HGB-O values: extracellular haemoglobin would have only been measured as part of HGB, because the cells are destroyed before optical density measurement, but not as part of HGB-O, because only intracellular haemoglobin is measured. In addition, unlike autoimmune haemolytic anaemia, HUS is not typically associated with cold antibody-induced RBC aggregation so RBC-I and RBC-O would be the same. Therefore, a HUS diagnosis is unlikely.

 

Haemolytic transfusion incident due to ABO incompatibility

The most common cause of a haemolytic transfusion incident is ABO incompatibility after imprudent transfusion of mismatched blood to a patient. It results in a medical emergency because donor erythrocytes are rapidly destroyed by the recipient’s immune system. As in HUS, the outcome would be a normocytic, normochromic anaemia and a reticulocytosis. As donor RBC are destroyed inside the recipient’s blood vessels, free haemoglobin would have been present in the blood and HGB would be higher than HGB-O, as explained above. In addition, RBC aggregation would not be expected after a haemolytic transfusion incident so RBC-I and RBC-O would be the same. Therefore, this diagnosis could also be excluded.

 

Megaloblastic anaemia

The ‘classical’ RBC parameters appear to indicate a macrocytic, hyperchromic anaemia (large MCV, very high MCH), as seen in megaloblastic anaemia. In addition, the reduced platelet count was observed here, which could the consequence of an ineffective thrombopoiesis, caused by impaired DNA synthesis and assembly, often associated with megaloblastic anaemias. However, it also leads to ineffective erythropoiesis and therefore normal or reduced RET%/# values. Even before correction based on RBC-O, RET% and RET# are both elevated indicating an adequate bone marrow response to the anaemia. In addition, megaloblastic anaemia is associated with the presence of large RBC containing large amounts of haemoglobin, and resulting in a large fraction of hyperchromic RBC. Therefore, the normal HYPER-He and high RET%/# can be used to exclude a megaloblastic anaemia in this patient. In addition, unlike autoimmune haemolytic anaemia, megaloblastic anaemia is not typically associated with cold antibody-induced RBC aggregation so RBC-I and RBC-O would be the same.

Underlying Disease

Sepsis

The term ‘sepsis’ describes a spectrum of clinical conditions resulting from the patient’s immune response to an infection; it is characterised by systemic inflammation and disturbances of haemostasis (1). The severity of sepsis ranges from an infection producing a systemic inflammatory response to acute organ dysfunction, multiple organ failure and ultimately death in a large proportion of patients (2). Sepsis is a major cause of morbidity and death in intensive care units worldwide, particularly in elderly, immunocompromised and critically ill patients (3, 4). The main predisposing causes are cytotoxic chemotherapy, malignant disease, severe trauma, burns, diabetes mellitus, kidney failure and liver failure. Mortality rates vary from 40% for uncomplicated sepsis to 80% for septic shock and multi-organ dysfunction (5). It has been estimated that as many as 500,000 patients are affected each year in both the USA and Europe. Sepsis therefore consumes a significant proportion of intensive care resources and remains an ever-present problem (6).

 

The principal definitions of systemic inflammatory response syndrome (SIRS) and sepsis are as follows:

1. SIRS is defined as a clinical response to a variety of insults including (but not exclusively) infection; the traditional 4 SIRS criteria are:

 a) Temperature > 38°C or < 36°C;

 b) Heart rate > 90 beats / minute;

 c) Respiratory rate > 20 breaths / minute or PaCO2 < 32 mm Hg;

 d) WBC > 12 x 109 /L or < 4 x 109 /L or granulopoiesis left shift.

A number of additional clinical features indicating physiological decompensation (altered mental status, ileus) and changes in laboratory parameters (procalcitonin, C-reactive protein, creatinine and various cytokines) have recently been added (7-9).

 

2. Sepsis is present when an infection exists in addition to at least 3 SIRS criteria. The key transition from SIRS to sepsis is the presence of an identified pathogen.

 

3. Severe sepsis exists when there is evidence of organ hypoperfusion with signs of organ dysfunction affecting one or more of the following organs and organ systems: cardiovascular system, respiratory system, central nervous system, kidney, liver. Haemostasis may also be dysfunctional.

 

4. Septic shock exists when severe sepsis is accompanied by hypotension (defined as a systolic blood pressure below 90 mm Hg) despite adequate fluid resuscitation. In acutely ill patients, sepsis and SIRS can lead to a state known as multiple organ dysfunction syndrome (MODS). When this occurs, homeostasis cannot be maintained without intervention. Evidence indicates that haemostatic changes play a significant role in many SIRS-linked disorders.

 

Clinical and laboratory features of sepsis

 

An infection must be present before a diagnosis of sepsis can be made. The most common infections involve the respiratory tract, abdomen and blood stream. More than 90% are caused by bacteria, with Gram-negative and Gram-positive bacteria being responsible in approximately equal proportions. Fungi, especially Candida species, are occasionally responsible. Accurate microbiological diagnosis is mandatory so that appropriate and effective antibacterial treatment can be given.

 

Biological markers of infection include the neutrophil count, C-reactive protein (CRP) and procalcitonin (PCT). The neutrophil count is not sensitive enough in a hospital population to distinguish sepsis from other causes of neutrophil elevation. CRP and PCT assays correlate well with the degree of inflammatory response and are particularly useful for following response to treatment (10, 11). Studies in ICU patients showed that a CRP cutoff of 50 mg/L or higher had a sensitivity of 98.5% and specificity of 75% (12) and a PCT cutoff of 1.5 ng/mL or higher had a sensitivity of 100% and specificity of 73% in identifying sepsis (13). The low specificity indicates that such markers alone cannot distinguish sepsis from other causes of SIRS.

 

A prominent component of the systemic inflammatory response is the release of cytokines (14). However, both cytokines and their soluble receptors are also greatly elevated in patients with malaria, burn injuries, trauma, pancreatitis, heart failure, renal allograft rejection and following surgery so their increase is non-specific. In addition, a complex relationship exists between cytokines and their inhibitors and antagonists, so evaluation of inflammatory pathways must take into account the pro- and anti-inflammatory factors that affect each pathway. It would therefore seem more appropriate to consider the cytokine balance at the onset of sepsis and then follow it over time.

 

Following a sepsis diagnosis, the clinician must assess the patient for the presence of acute organ dysfunction (severe sepsis), which is often recognized based on a patient’s clinical symptoms but in some instances laboratory data or results from invasive monitoring will confirm the diagnosis. Cardiovascular dysfunction is indicated by the presence of tachycardia, hypotension, increased central venous pressure and increased pulmonary artery occlusive pressure. Respiratory dysfunction is indicated by tachypnea, hypoxaemia, reduced oxygen saturation and decreased ratio of arterial oxygen to inspired oxygen. Jaundice, increased liver enzymes, hypoalbuminaemia and increased prothrombin time test indicate hepatic dysfunction. Altered consciousness, confusion, psychosis, delirium indicate central nervous system dysfunction. Renal dysfunction manifests itself by oliguria, anuria, and increased creatinine. Haematological dysfunction is indicated by thrombocytopenia, abnormal coagulation tests, decreased levels of Protein C and increased D-dimers. Although the severity of sepsis is related to the degree of organ dysfunction, progression is often unpredictable.

 

Pathophysiology of sepsis

The pathophysiology of sepsis is complex and results from interactions between immune, inflammatory and haemostasis mediators. Early in the sepsis cascade the vascular endothelium is damaged directly by the host immune response: an excess of pro-inflammatory mediators is released which results in vascular endothelial dysfunction producing the so-called ‘capillary leak’ syndrome. This interferes with normal tissue function, progressing to tissue damage and organ dysfunction (15-17). In addition, damage to the vascular endothelium results in exposure of subendothelial structures with the release of Tissue Factor that triggers the formation of blood clots (18, 19). As sepsis increases in severity, the coagulopathy becomes more severe and when septic shock exists, laboratory changes include evidence of profound Protein C deficiency, prolonged prothrombin time and activated partial thromboplastin time, elevated fibrin monomers, reduced fibrinogen and, of course, elevated D-dimer levels. Disseminated intravascular coagulation in sepsis is commonly associated with multiple organ failure and carries a very poor prognosis.

 

Acquired haemolytic anaemia

Haemolysis is defined as premature death of RBC. Normally, RBC destruction is compensated by the production of new RBC. However, if the rate of destruction exceeds the erythropoietic capacity of the bone marrow, the patient will develop anaemia.

 

Haemolytic anaemia can be classified by location:

 

1. Intravascular haemolytic anaemia occurs within the blood vessels:

 

2. Extravascular haemolytic anaemia occurs in other organs, especially those involved in regular breakdown/removal of senescent RBC (i.e.: the reticuloendothelial system including the spleen and liver).

 

In an alternative classification, by aetiology, two main groups are distinguished: inherited and acquired haemolytic anaemia. Acquired haemolytic anaemia can be further subdivided into immune-mediated haemolytic anaemia, incorporating both autoimmune- and alloimmune-mediated causes, and non-immune causes:

 

1. Warm antibody autoimmune haemolytic anaemia (AIHA):

a) Idiopathic (most common);

b) Associated with autoimmune diseases (e.g., lupus erythematosus);

c) Lymphoproliferative disorders;

d) Infections.

 

2. Cold antibody AIHA:

a) Idiopathic cold haemagglutinin syndrome;

b) Infections (e.g., infectious mononucleosis);

c) Paroxysmal cold haemoglobinuria.

 

3. Alloimmune haemolytic anaemia:

a) Haemolytic anaemia of the newborn;

b) Alloimmune haemolytic blood transfusion reaction after non-compatible transfusion.

 

4. Drug-induced immune haemolytic anaemia: for example, high doses of penicillin may provoke an immune response against RBC coated with the drug.

 

5. Non-immune mediated acquired haemolytic anaemia:

a) Direct haemolytic action mediated by drugs or toxins;

b) Mechanical haemolysis by artificial heart valves or extensive surgery on the blood vessels;

c) Microangiopathic haemolytic anaemia causing RBC shredding by fibrin strands deposited in the microvasculature (e.g., disseminated intravascular coagulopathy, haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura);

d) Haemolytic anaemia resulting from an infection, for example, parasites targeting RBC (malaria, babesiosis) or as a direct or indirect effect of other pathogenic organisms in the blood stream;

e) Haemolytic anaemia resulting from acquired membrane disorders such as paroxysmal nocturnal haemoglobinuria or some forms of liver disease.

Literature

  1. Mesters et al. (1996): Increase of plasminogen activator inhibitor levels predicts outcome of leukocytopenic patients and sepsis. Thromb Haemost 75(6): 902-907
  2. Brun-Buisson et al. (1995): Incidence, risk factors, and outcome of severe septic shock in adults. A multicenter prospective study in intensive care units. JAMA 274(12): 968-974
  3. Friedman et al. (1998): Has the mortality of septic shock changed with time? Crit Care Med 26(12): 2078-2086
  4. Sands et al. (1997): Epidemiology of sepsis syndrome in 8 academic medical centers. JAMA 278(3): 234-240
  5. Salvo et al. (1995): The Italian SEPSIS study: preliminary results on the incidence and evolution of SIRS, sepsis, severe sepsis and septic shock. Intensive Care Med 21(Suppl 2): S244-249
  6. Angus et al. (2001): Epidemiology of severe sepsis in the united States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 29(7): 1303-1310
  7. Levy et al. (2003): 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med 31(4): 1250-1256
  8. Society of Critical Care Medicine (2012): International Guidelines for Management of Severe Sepsis and Septic Shock: 2012. www.sccm.org
  9. Angus et al. (2013): Severe Sepsis and Septic Shock. N Engl J Med 369 (9): 840-851
  10. Yentis et al. (1995): C-reactive protein as an indicator of resolution of sepsis in the intensive care unit. Intensive Care Med 21(7): 602-605
  11. Oberhoffer et al. (1999): Sensitivity and specificity of various markers of inflammation for the prediction of tumor necrosis factor-alpha and interleukin-6 in patients with sepsis. Crit Care Med 27(9): 1814-1818
  12. Povoa et al. (1998): C-reactive protein as an indicator of sepsis. Intensive care Med 24(10): 1052-1056
  13. De Werra et al. (1997): Cytokines, nitrite/nitrate, soluble tumor necrosis factor receptors, and procalcitonin concentrations: comparisons in patients with septic shock, cardiogenic shock, and bacterial pneumonia. Crit Care Med 25(4): 607-613
  14. Matot and Sprung (2001): Definition of sepsis. Intensive Care Med 27(Suppl 1): S3-9
  15. Lorente et al. (1993): Time course of hemostatic abnormalities in sepsis and its relation to outcome. Chest 103(5): 1536-1542
  16. Astiz and Rackow (1998): Septic shock. Lancet 351(9114): 1501-1505
  17. Wheeler and Bernard (1999): Treating patients with severe sepsis. N Engl J Med 340(3): 207-214
  18. Carvalho and Freeman (1994): How coagulation defects alter outcome in sepsis: survival may depend on reversing procoagulant conditions. I Crit Illness 9: 51-75
  19. Esmon (2001) The normal role of Activated Protein C in maintaining homeostasis and its relevance to critical illness. Crit Care 5(Suppl 2): S7-S12.

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