Regenerative vs Non-regenerative Anemia

Regenerative vs Non-regenerative Anemia

Regenerative Anemia

1. Haemorrhage

Haemorrhage may occur from any site and it may be external (often due to trauma) or internal. Any form of spontaneous haemorrhage with no apparent cause may suggest the presence of an underlying coagulopathy. The most common haemorrhagic presentations are:

• Epistaxis due to disruption or erosion of blood vessels of the nasal cavity by trauma, neoplasia, fungal infection or a foreign body.
• Haematuria which may arise due to haemorrhage from any part of the urinary tract, especially the kidney (due to trauma, neoplasia or idiopathic haematuria) and bladder (due to trauma, cystitis, urolithiasis and neoplasia).
• Melaena, haematochezia or haematemesis due to gastro-intestinal haemorrhage. Meleana refers to the production of black tarry faeces with digested blood whereas haematochezia refers to the production of fresh blood with the faeces. Classically, haematemesis is described as resembling 'coffee grounds' as blood is denatured by a low gastric pH but, as the gastric pH of the dog may vary widely between 2 and 6, vomited blood may also appear as fresh red blood.
• Haemoptysis refers to the production of blood from the respiratory tract. It may occur with severe forms of pneumonia and with pulmonary haemorrhage.
• Haemoabdomen, haemothorax and haemopericardium are all forms of haemorrhagic effusion that occur in body cavities.

2. Hemolysis

Haemolysis may occur in the following processes:

• Immune-mediated disease including Immune Mediated Haemolytic Anaemia and Neonatal Isoerythrolysis.
• Infectious agents including Babesia spp. in dogs and cattle, Mycoplasma haemofelis in cats, Leptospira spp. in various species and Clostridium haemolyticum causing redwater fever in cattle.
• Inherited defects of red blood cells enzymes including pyruvate kinase (which is occur most commonly in West Highland white terriers) and phosphofructokinase (PFK).
• Hypophosphataemia which occurs in post-parturient cattle (causing post-parturient haemoglobinuria), with refeeding syndrome and when animals with diabetes mellitus are stabilised with insulin.
• Exposure to toxins including rape and kale (which contain SMCO radicals) in cattle, onions and garlic in dogs and paracetamol in cats.
• Microangiopathic anaemia which occurs when red blood cells are forced through small meshworks of fibrin as with haemangiosarcomas, disseminated intravascular coagulation (DIC) or bacterial endocarditis.

Haemolysis usually results in a more strongly regenerative response than haemorrhage and can be differentiated by plasma protein concentrations; these will fall with haemorrhage, but not with haemolysis.

Non-Regenerative Anemia

The failure to regenerate indicates that there is a failure to produce red blood cells in the bone marrow. Erythrocytes are produced from stem cells in the bone marrow and they then undergo sequential stages of maturation before and after they are released into the circulation.

1. Failure of the bone marrow stem cells to produce erythroid cells

This occurs in the following conditions:

• Pure red cell aplasia
• Aplastic anaemia
• Bone marrow suppression
• Myelophthisis
• Myelodysplasia

2. Failure of erythrocyte maturation

This can occur with:

• Iron deficiency
• Vitamin B12/folate deficiency

Pernicious Anemia (Vit B12 Deficiency Anemia)

Pernicious Anemia (Vit B12 Deficiency Anemia)

Vitamin B12 (Vit B12)
 

Vitamin B12 is an essential nutrient that the body needs to make healthy red blood cells and to maintain a healthy functional nervous system.  It is found in animal foods, including meat, fish, eggs, milk, and other dairy products - but not in fruit or vegetables. Hence vegetarians have a high potential health risk towards this hematological disorder.

Normally, after a meal the body digestive enzymes will induce enzymatic breakdown of the food to release Vit B12 and allow its combination with an intrinsic factor in the stomach. The combined Vit B12-intrinsic factor complex is then absorbed into the body further down the gut at the end of the small intestine. (Intrinsic factor is produced by cells in the lining of the stomach and is essential for the absorption of Vit B12).

Background Information

Pernicious anemia is a type of anemia characterized by the body's inability to produce sufficient healthy RBCs due to the deficiency in Vit B12. In the circumstances of Vit B12 deficiency, RBCs are unable to divide normally resulting in large sized RBCs known as Macrocytes. Hence, structurally unable to squeeze out of the bone marrow into the blood stream. While, when they do squeeze through, most will develop an Ovalocytic morphology.

Theoretically, with insufficient functional healthy RBC to transport oxygen throughout the body, one will feel lethargic. Severe or prolonged pernicious anemia can damage the heart, brain, and other organs in the body. Additionally, nerve damage, neurological problems (such as memory loss), and digestive tract problems may occur too. Patients who suffer pernicious anemia also may be at higher risk for weakened bone strength and stomach cancer.

Causes of Pernicious Anemia

1. Autoimmunity  

In a regulated manner, a host's immune system normally produces antibodies to attack bacteria, viruses and other germs. A patient suffering from autoimmune disease, infers that the immune system is producing antibodies against specific host's own body cells - self-antibodies. In the case of pernicious anaemia, self-antibodies are either specifically produced against the host intrinsic factor or against the cells in the stomach that synthesize intrinsic factor. This then prevent the downstream absorption processes of Vit B12. Unfortunately, in most cases the trigger of this condition is idiopathic.

Pernicious anaemia usually develops over the age of 50. Women are more commonly affected than men, and it tends to run in families. It occurs more commonly in people who have other autoimmune diseases. For example, thyroid diseases, Addison's disease and vitiligo (a condition where white patches develop on skin). Fortunately, there are diagnostic tests to confirm the presence of such antibodies which cause pernicious anaemia.

2. Stomach or gut problems  

Patients who had undergone surgery to remove the stomach or the end of the small intestines will mean absorption of vitamin B12 may be disrupted

Some diseases like Crohn's disease affect the end of the small intestines where Vit B12 is absorbed and thus disrupts its absorption.

There are some clinical disorders such as atrophic gastritis (thining of the lining of the stomach) that may disrupt the synthesis and/or secretion of intrinsic factor leading to malabsorption of the vitamin .

3. Side-effects of Medication

Certain medicines used for other conditions may disrupt the absorption of vitamin B12. The most common example is metformin which is a medicine often used for diabetes. Other medicines include colchicine, neomycin, and some anticonvulsants used to treat epilepsy.

Long-term use of medicines that disrupts stomach acid synthesis and/or secretion, such as H2 blockers and proton pump inhibitors, can deteriorate the Vit B12 deficiency condition. This is because stomach acid is needed to release Vit B12 bound to proteins in food. However, they are not considered critical root causes of vitamin B12 deficiency.

4. Dietary Deficiency

In this modern society and the wealth of food sources, it is unusual to suffer from dietary  Vit B12 deficiency under a normal balanced diet regime. Though, strict vegans on the other hand who consume no animal or dairy produce may suffer from vitamin B12 deficiency. 

 

Diagnosis

• Low hemoglobin level 
• Evaluated MCV
• Elevated/normal MCHC
• Ovalocytes are observed on the PBF
• Hypersegmented neutrophils are observed on the PBF.

Flow Cytometry-based Hematology Analyzers

Hematology Analyzers

Background Information of Hematological Analyzers

Hematology analyzers are computerized, highly specialized machines that count the number of different types of red and white blood cells, blood platelets, haemoglobin, and haematocrit levels in a blood sample. They include programmable automated alarm systems for indicating results outside the reference range. Each hematological anaylzer has their own testing principles to derive with their cell counts and parameters. One of the most common principle is flow cytometry. 

Flow Cytometry

 

Background Information of Flow Cytometry

The term ‘cytometry’ is defined as a measurement of the physico-chemical properties of cells. ‘Flow cytometry’ is the measurement of cells and other particles in a flow.Traditional flow cytometry has always been considered the best method to distinguish and differentiate the various cell populations. However, the costly need to use fluorescence-labelled antigen-antibody reagents and the cumbersome tedious preparation procedures is a major disadvantage. In order to meet the clinical demands, some companies (Eg Sysmex) have professionally refined basic techniques of flow cytometry and uses highly specific polymethine dyes to adapt this sophisticated technology to fit the high volume and automation demands of clinical laboratories.

Test Principle of Flow Cytometry

 
Once a cell/object comes into the path of a ray of light (Laser), the light changes its orientation. This phenomenon is called light scatter and occurs under all angles between 0 and 360°. 

Detection of this scattered light provides information on the size and the quality of the object. Especially light scattered in forward direction (‘forward scattered light’) or, respectively, low-angle scattered light allows statements on the size of an object. Low-angle scattered light is decisively influenced by the object’s form and refraction index, aside from its size.

On the other hand, light scattered from the side (‘side scattered light’) or wide-angle forward scattered light provides information on the internal granularity content of a cell/object. That way, blood cells can be analysed with regard to their size and their intracellular, structural complexity (form, size and density of the nucleus, cytoplasmic granularity). Wide-angle forward scattered light is decisively influenced by the presence or absence of granules.

A particularly sensitive detector is required for the detection of side scattered light – as
a rule a photo multiplier – since, compared with forward scattered light, the signal intensity is weaker by some powers of ten. A fast photo diode can be used for the forward scattered light. 

Under specific prerequisites, the exact size of particles can be determined by means of the light scatter method. Furthermore successful will be the excellent separation of different particle populations based on additional properties such as the surface of the cell.

Myeloblastic Auer Rods

Myeloblastic Auer Rods

Background Information of Myeloblastic Auer Rods

Auer rods are clumps of azurophilic lysosomal granular material that form elongated needles seen in the cytoplasm of leukemic blasts. They can be seen in the leukemic blasts of acute myeloid leukemia with maturation and acute promyelocytic leukemia (known as acute myeloid leukemia M2 and M3, in the FAB classification, respectively) and in high grade myelodysplastic syndromes and myeloproliferative syndromes. They are composed of fused lysosomes/primary neutrophilic granules and contain peroxidase,lysosomal enzymes, and large crystalline inclusions. Morphologically, the Auer "rods" come in all sizes and shapes. They have been typically described as needle-shapes with pointed ends (most common), but may appear in comma-shapes and diamond-shapes; others were long and rectangula more appropriately referred as Auer bodies.

They are also used to distinguish the pre-leukemia myelodysplastic syndromes: refractory anemia with excess blasts 2 (which has Auer rods) from RAEB 1 (which does not), it is noteworthy, though, that rare cases of RAEB1 show rare auer rods, and when they do, they have a worse prognosis.

Cellular Description

Presence of purplish- stained rod-like shaped granular materials in the cytoplasm of myeloblasts or promyelocytes.

Eosinophils

Eosinophils

Background Information of Eosinophils

Eosinophils are WBCs and are one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. Additionally they also control mechanisms associated with allergy and asthma. They are granulocytes that develop during hematopoiesis in the bone marrow before migrating into blood.

These cells are eosinophilic (ie. acid-loving) as shown by their affinity to coal tar dyes.The staining is concentrated in small granules within the cellular cytoplasm, which contain many chemical mediators, such as histamines and proteins such as eosinophil peroxidase, ribonuclease (RNase), deoxyribonucleases, lipase, plasminogen, and major basic protein.These mediators are released by a process called degranulation following activation of the eosinophil, and are toxic to both parasite and host tissues.

In normal individuals, eosinophils make up about 1-6% of WBCs.

Clinical Significance

Eosinophilia is a condition to describe an abnormal increased in eosinophils, i.e., the presence of more than 500 eosinophils/microlitre of blood and is typically seen in people with a parasitic infestation of the intestines, collagen vascular disease (such as rheumatoid arthritis), malignant diseases such as Hodgkin's disease, extensive skin diseases (such as exfoliative dermatitis), Addison's disease, reflux esophagitis (in which eosinophils will be found in the squamous epithelium of the esophagus), eosinophilic esophagitis, and with the use of certain drugs such as penicillin. 

Generally the most common cause for eosinophilia is an allergic condition such as asthma.

 
Cellular Description

Eosinophils are typically slightly larger than neutrophils and the presence of coarse large orange-red granules are their signature identity. The nuclear chromatin structure (nucleus) are quite synonymous to the ones found in neutrophils (ie. segmented), but is usually segmented into two swollen oval lobes.

Basophils

Basophils

Background Information of Basophils  

 

Basophils are typically the least commonly seen granulocytes, representing about 0.01% to 0.5% of circulating WBCs seen in the PBFs. The name comes from the fact that they are basophilic, i.e., they are susceptible to staining by basic dyes. They contain an abundance of large cytoplasmic granules which obscure the cell nucleus under the microscope when stained.

Clinical Significance

Basophilia is also uncommon but may be seen in some forms of leukaemia or lymphoma.  More commonly, excessive appearance of basophils is highly suggestive in various inflammatory reactions, particularly those that cause allergic symptoms. Basophils contain anticoagulant heparin, which prevents blood from clotting too quickly. When activated, basophils degranulate to release vasodilator histamine, which promotes blood flow to tissues. They can be found in unusually high numbers at sites of ectoparasite infection, e.g., ticks. Basophils play a role in both parasitic infections and allergies. They are found in tissues where allergic reactions are occurring and probably contribute to the severity of these reactions. Basophils have protein receptors on their cell surface that bind IgE, an immunoglobulin involved in macroparasite defense and allergy. It is the bound IgE antibody that confers a selective response of these cells to environmental substances, such as pollen proteins or helminth antigens. Recent studies in mice suggest that basophils may also regulate the behavior of T cells and mediate the magnitude of the secondary immune response.

 

Basopenia (low basophil count) is difficult to demonstrate as the basophils are generally extremely low/rare in count; it has been reported in association with autoimmune urticaria (a chronic itching condition).

Cellular Description

The size of basophils are typically smaller than neutrophils and the abundance of coarse large dark purple staining granules in the cytoplasm over lying the nucleus are the signature hallmark.

Monocytes

Monocytes

Background Information of Monocyte 

 

Monocytes are the largest physiologically occurring leukocytes found in the peripheral blood film. This subset constitutes 2% to 10% of all leukocytes in the human body. They play multiple roles in immune function. Such roles include: (1) replenishing resident macrophages under normal states, and (2) in response to inflammation signals, monocytes can move quickly (approx. 8–12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. 

Monocytes originate from the bone marrow as precursors called monoblasts, bipotent cells that differentiated from hematopoietic stem cells. Monocytes circulate in the bloodstream for about one to three days and then typically move into various tissues in different antomical locations throughout the body. In the tissues, monocytes will then mature into tissue-specific resident macrophages or dendritic cells.

 

Clinical Significance

1. Monocytosis

Increased appearance of monocytes in PBF is typically indicative of monocytosis. Monocytosis is a clinical symptom of diseases: 

• Severe infection (sepsis)
• Chronic inflammation
• Stress response
• Cushing's syndrome (hyperadrenocorticism)
• Immune-mediated disease
• Pyogranulomatous disease
• Necrosis
• RBC regeneration
• Viral Fever
• Sarcoidosis

2. Monocytopenia

Reduced appearance of monocytes in PBF is typically indicative of monocytopenia. This is typically due to a general leukopenia condition associated with a deficiency in monocytes

Cellular Description

Being the largest WBC in the PBF, it appear to be significantly larger than all other WBCs (lymphocytes, neutrophils, basophils and eosinophils) in size. The cytoplasm is plentiful and stains slightly greyish-blue (synonymous to frosted glass). Their cytoplasm contains irregularly scattered fine purplish granules. They have an irregular kidney/bean-shaped dark purplish stained nucleus with deep indentation. *Note that the staining intensity of monocytes are weaker than lymphocytes. Vacuoles may also be observed in the cytoplasm.