Diagnostic Techniques in Haematology PDF

Diagnostic Techniques in Haematology 1st Block Posting in Pathology/ Pharmacology Lecturer: Dr. Adeyemi  Outline Introduction Basic haematologic tests – Quantitative measurement of cells in the blood (Manual) – Automated blood count anal...

Diagnostic Techniques in Haematology 1st Block Posting in Pathology/ Pharmacology Lecturer: Dr. Adeyemi  Outline Introduction Basic haematologic tests – Quantitative measurement of cells in the blood (Manual) – Automated blood count analysis – Erythrocyte sedimentation rate – Peripheral Blood Smear – Bone marrow examination Special Hematology Techniques Principles of Flowcytometry Molecular and cytogenetic techniques Conclusion  Learning Goals To be able to enumerate and describe basic techniques required for diagnosis of haematologic conditions To be able to enumerate and describe complex and sophisticated techniques for the diagnosis of haematologic diseases  Introduction The care of a patient with a suspected haematologic abnormality begins with a systematic attempt to determine the nature of the illness by eliciting an in-depth medical history and performing a thorough physical examination. Further evaluation is necessary in establishing a definite diagnosis and this is achieved by utilizing several diagnostic tools that are relevant to substantiate the clinician's suspicion. Range of haematological tests are used daily in the management of patients, from basic tests such as the complete blood count (CBC) to complex molecular tests such as Florescence Insitu Hybridization (FISH). Choosing the right test is necessary.  Basic Haematologic Tests QUANTITATIVE MEASURES OF CELLS IN THE BLOOD This can be done manually, or by semi-automated or automated techniques in order to determine the various components of the full blood count (FBC). Manual techniques are generally low cost with regard to equipment and reagents but are labour intensive; automated techniques entail high capital costs but permit rapid performance of a large number of blood counts by a smaller number of laboratory workers. Automated techniques are more precise, but their accuracy depends on correct calibration and the use of reagents that are usually specific for the particular analyser.  Measurement of Hemoglobin (Hb) Haemoglobin is intensely colored, and this property has been used in methods for estimating its concentration in blood. Erythrocytes contain a mixture of Hb, oxyhaemoglobin, carboxyhaemoglobin, methaemoglobin, and minor amounts of other forms of Hb. To determine Hb concentration in the blood, red cells are lysed and Hb variants are converted to the stable compound cyanmethaemoglobin for quantification by absorption at 540 nm. All forms of hemoglobin are readily converted to cyanmethaemoglobin except sulfhaemoglobin, which is rarely present in significant amounts.  Two methods are in common use, both are analysed by spectrometry: – Haemiglobincyanide (HiCN; cyanmethaemoglobin) method – Oxyhaemoglobin (HbO2) method. Range of Hb concentration in health – Men: 15.0+/- 2.0 g/dL – Women: 13.5+/-1.5 g/dL  Packed Cell Volume or Haematocrit This is the fractional volume of blood occupied by erythrocytes Uses – Simple screening test for anaemia – Used as a reference method for calibrating automated blood count systems – Used as a rough guide to the accuracy of haemoglobin measurements.  Microhaematocrit Method – Blood is subjected to sufficient centrifugal force to pack the cells while minimizing trapped extracellular fluid. Range of PCV in Health – Adult males: 0.41–0.51 – Adult females: 0.36–0.45 A value below an individual’s normal value or below the reference interval for age and sex indicates anaemia, and a higher value, polycythemia.  MANUAL CELL COUNTS AND RED CELL INDICES Leukocyte Counts (Manual) The blood specimen is collected in EDTA bottle. A diluting fluid is used to lyse the erythrocytes so that they will not obscure the leukocytes. The WBCs are then counted using a haemocytomer or Neubauer counting chamber. Normal Reference range in health: For an adult is 4.5–11.0 × 109/L. Manual Differential Leucocyte Count Are usually performed by visual examination of blood films that are prepared on slides by the spread or ‘wedge’ technique. The differential count, expressed as the percentage of each type of cell, should be related to the total leucocyte count and the results should be reported in absolute numbers ( 109/l). PLATELET COUNT (Manual) Hemocytometer Method—Phase-Contrast Microscope Venous blood is collected with EDTA as the anticoagulant. A portion of the blood is mixed with 1% ammonium oxalate. Platelets are then counted with the Neubauer counting chamber under a phase-contrast microscope. Reference values for platelet counts are 150–450 × 109/L.  AUTOMATED BLOOD COUNT ANALYSIS This is the cornerstone of the modern haematology lab, allowing rapid, cost-effective, and accurate analysis of the cells of the blood, including new parameters with diagnostic utility. Principle: Light scatter at various angles: This yields information about cell size (using scatter at low-incident angles), nuclear lobulation, and cytoplasmic granularity (using high-angle light scatter) and refractive index, with polarization of the scattered light as an additional parameter. Electrical impedance and conductivity: This also determines cell size as estimated by measuring change in electrical resistance, which is proportional to cell size as cells enter a narrow orifice through which a direct current is maintained Fluorescence or light absorption of cells stained in flow: Differential lysis with detergents of varying strength or pH is used to separate certain leukocyte types, such as basophils and immature granulocytic cells, from the major normal blood cell types  Standard Red Cell Indices Mean Cell Volume Automated blood counters measure the MCV directly by either electrical impedance or light scatter measurements of individual red cells. But in semi-automated counters MCV is calculated by dividing the Hct by RBC. The MCV has been used to guide the diagnostic workup in patients with anaemia e.g. testing patients with microcytic anemia for iron deficiency or thalassemia, and those with macrocytic anemia for folate or vitamin B12 deficiency. Mean Cell Haemoglobin (MCH) and Mean Cell Haemoglobin Concentration (MCHC) MCH is derived from the Hb divided by RBC. The amount of hemoglobin per red cell, increases or decreases in parallel with the red cell volume (i.e., MCV) and generally provides similar diagnostic information The MCHC is derived in the traditional manner from the Hb and the Hct with instruments that measure the Hct and calculate the MCV. The MCHC is not used much diagnostically, and is primarily useful for quality control purposes, such as detecting sample turbidity. Red Cell Distribution Width (RDW) The is an estimate of the variance in volume within the population of red cells, expressed as 1 SD of red cell volume measurements divided by the MCV The normal reference range is in the order of 12.8 +/- 1.2% as CV and 42.5 +/- 3.5 fl as SD. It has some value in distinguishing between iron deficiency (RDW usually increased) and thalassaemia trait (RDW usually normal) and between megaloblastic anaemia (RDW often increased) and other causes of macrocytosis (RDW more often normal).  Other functions of the automated cell analyser include: Automated differential and total/ absolute leucocyte count. Increasingly, automated blood cell counters have a differential counting capacity, providing either a three-part or a five- to seven-part differential count. Platelet count and platelet indices Automated reticulocyte counts and reticulocyte index  ERYTHROCYTE SEDIMENTATION RATE Erythrocyte sedimentation rate (ESR) is a useful but nonspecific marker of underlying inflammation It is influenced by changes in fibrinogen, α-macroglobulins, and immunoglobulins which enhance red cell aggregation in vitro. When well-mixed venous blood is placed in a vertical tube, erythrocytes will tend to fall toward the bottom. The length of fall of the top of the column of erythrocytes over a given interval of time is called the ESR. Westergren Method – Manual – Semi- automated Normal ranges – 0–10mm/h for ♂ 18–65 years. – 1–20mm/h for ♀ 18–65 years. – Upper limits of normal i by 5–10mm/h for patients >65 years. Westergren Method  Causes of Raised ESR Condition Causes Pregnancy Increase in pregnancy; maximal in 3rd trimester Infections Acute and chronic infections, including TB Note: increased ESR also occurs in HIV infection Collagen disorders Rheumatoid, SLE, polymyalgia rheumatica, vasculitides, etc. (including temporal arteritis); ESR useful as non-specific monitor of disease activity Other inflammatory Inflammatory bowel disease, sarcoidosis, processes post-Myocardial infarction Neoplastic conditions Carcinomatosis, NHL, Hodgkin lymphoma and paraproteinaemias (benign and malignant)  Peripheral Blood Smear Examination of peripheral blood with bone marrow smear/ biopsy represents the cornerstone of hematologic diagnosis. Microscopic examination of the blood spread on a glass slide or cover-slip yields useful information regarding all formed elements of the blood. The reliability of the information obtained depends heavily on well-made and well-stained films that are systematically examined. Blood films should be prepared immediately if possible.  Peripheral Blood Smear Blood Stains Romanowsky Dyes – May–Grunwald Stain – Jenner’s Stain – Giemsa’s Stain – Azure B–Eosin Y Stock Solution – Leishman’s Stain Colour Responses of Blood Cells to Romanowsky Staining Blood films made on slides A A well-made film. B An irregular patchy film on a dusty slide. C A film that is too thick. D A film that has been spread with inconsistent pressure and using an irregularly edged spreader, resulting in long tails. E A film made on a very greasy slide.  BONE MARROW EXAMINATION This provides a semi-quantitative and qualitative assessment of the state of haematopoiesis, and aids in the diagnosis of several hereditary and acquired benign and malignant diseases. The study provides marrow cells for immunophenotyping, cytogenetic, molecular and genomic studies, culture of infectious organisms, and storage of marrow cells for further analysis. An aspirate and biopsy of the marrow can be obtained with minimal risk and only minor discomfort and are quickly and easily processed for examination. The marrow should be examined when the clinical history, blood cell counts, blood film, or laboratory test results suggest the possibility of a primary or secondary haematologic disorder for which morphologic analysis or special studies of the marrow would aid in the diagnosis. Indications for bone marrow aspiration and trephine biopsy. BONE MARROW EXAMINATION (a) The bone marrow aspiration needle and a smear made from a bone marrow aspirate. (b) The bone marrow trephine (biopsy) needle and normal trephine section. Jamshidi trephine needle for bone marrow biopsy  Special Haematology Techniques Immuno-haematology Direct Antiglobulin Test is a simple but sophisticated test using monoclonal, or sometimes mixed monoclonal and polyclonal, antibody directed against IgG and complement components. The DAT detects in vivo binding of antibody and/or complement to the surface of erythrocytes. A positive DAT indicates the presence of antibody and/or complement on the surface of the red cells, but not its cause. Antibody screening Red cell antibody screening is performed with commercially prepared sets of group O red cells selected for their expression of the red cell antigens most commonly responsible for antibody formation from exposure to transfusions or pregnancy. Analysis of Haemoglobinopathies, Haemoglobin Variants and Thalassemias Sickle test The sickling test Uses intact red blood cells dropped in suspension of 3% sodium metabisulfite and placed on a glass slide. A cover slip is placed over the drop and sealed to allow deoxygenation. After approximately 30 minutes the slides are examined microscopically for sickle cells.  Analysis of Haemoglobinopathies, Haemoglobin Variants and Thalassemias Sickle test Haemoglobin Electrophoresis Electrophoresis can be performed at alkaline and acid pHs. At both pHs a control mixture of known haemoglobins (F, A, S, and C) is run in parallel with the samples for comparison. After electrophoretic separation the plates are stained, which can be read visually or the fractions may be determined by densitometry Analysis of Haemoglobinopathies, Haemoglobin Variants and Thalassemias Sickle test Cation-exchange high performance liquid chromatography Quantitation of haemoglobins A, A2, and F and identification of the haemoglobin variants provide a rapid and complete diagnostic workup for the majority of samples.  Investigation of Haemostasis The haemostatic mechanisms have several important functions: – (1) to maintain blood in a fluid state while it remains circulating within the vascular system – (2) to arrest bleeding at the site of injury or blood loss by formation of a haemostatic plug – (3) to limit this process to the vicinity of the damage – (4) to ensure the eventual removal of the plug when healing is complete. Normal physiology thus constitutes a delicate balance between these conflicting tendencies and a deficiency or exaggeration of any one may lead to either thrombosis or haemorrhage.  Coagulation Assays Are functional bioassays and rely on comparison with a control or standard preparation with a known level of activity. Theses include – Prothrombin Time (PT) – Activated Partial Thromboplastin Time (APTT) – Thrombin Time (TT) – fibrinogen assay These tests are important for the diagnosis of haemophilias, disseminated intravascular coagulopathy, monitoring of patients on anticoagulant therapy, etc  Investigation of the Vascular Disorders of Haemostasis Bleeding time The PFA-100 System Enzyme-Linked Immunosorbent Assay for Von Willebrand Factor Antigen  Immunological tests – Immuno-diffusion – immunoelectrophoresis – radioimmunometric assays – latex agglutination (immunoturbidimetric) tests – tests using enzyme-linked immunosorbent assays (ELISA) Fundamentally, all these tests rely on the recognition of the protein in question by polyclonal or monoclonal antibodies.  FLOWCYTOMETRY Flow cytometers are automated haematology analyzers that use principles of light scatter and fluorescence to define cellular populations in which to analyze expression of proteins typically identified by fluorescent tagged antibodies. A single-cell suspension is aspirated into a laminar flow of isotonic diluent that passes in front of one or more laser beams. Light scatter and fluorescence data are collected using specific photomultiplier tubes with appropriate filtration to collect scattered light (same wavelength as the incident laser light) or fluorescence emitted light (at a longer wavelength determined by the dye used). Multiple detectors with different filtration coupled with single or multiple lasers are used to collect highly multiplexed data.  Applications of Flowcytometry in Haematology Diagnosis and classification of hemato-lymphoid neoplasia by examining lineage-specific or maturation stage-specific markers. Determining the presence of minimal residual disease (MRD) thereby used as a prognostic marker. Quantitation of CD34 -positive (CD34 +) stem cells when evaluating the adequacy of blood stem cell collections Immune function evaluation They are considered a high complexity test, with high accuracy, precision, sensitivity, and specificity. Molecular and Cytogenetic Analysis Application The identification of genetic defects in haemoglobinopathies allowing the provision of early prenatal diagnosis the assessment of genetic risk factors in thrombophilia the diagnosis and characterization of leukaemias the monitoring of minimal residual disease the study of host–donor chimerism following bone marrow transplantation.  Conclusion Haematology is a rapidly evolving area of medical knowledge that has seen a virtual explosion of diagnostic laboratory testing and therapeutics in the past decade. Common diagnostic methods range from visual peripheral blood and bone marrow morphology to flow cytometric analysis of erythrocytes, leukocytes and platelets as well as molecular techniques. The choice of test should be tailored to assist in diagnosis of haematologic diseases.