Laboratory Haematology Investigations Summary PDF

LABORATORY HAEMATOLOGY INVESTIGATIONS SUMMARY Contents Quality Control.................................................................................................................................................................................... 3 Facilities, Work Environment & Safety.............

LABORATORY HAEMATOLOGY INVESTIGATIONS SUMMARY Contents Quality Control.................................................................................................................................................................................... 3 Facilities, Work Environment & Safety........................................................................................................................................... 6 Process Management................................................................................................................................................................... 12 Documents & Records.................................................................................................................................................................. 13 Management Of Nonconforming Events...................................................................................................................................... 14 Monitoring & Assessment............................................................................................................................................................ 15 Process Improvement................................................................................................................................................................... 16 Techniques/Equipment..................................................................................................................................................................... 18 Morphology...................................................................................................................................................................................... 20 Stains............................................................................................................................................................................................ 26 Malarial Parasite Studies.............................................................................................................................................................. 30 Immunophenotyping........................................................................................................................................................................ 32 CD34 Cells..................................................................................................................................................................................... 35 Paroxysmal Nocturnal Haemoglobinuria...................................................................................................................................... 36 Platelet Antibody Studies............................................................................................................................................................. 36 DNA Ploidy Studies....................................................................................................................................................................... 36 Feto-Maternal Haemorrhage....................................................................................................................................................... 36 Red Cell Membrane Disorders...................................................................................................................................................... 37 Genotype Studies.............................................................................................................................................................................. 38 Cytogenetics................................................................................................................................................................................. 38 Molecular Genetics....................................................................................................................................................................... 40 Erythrocyte Studies........................................................................................................................................................................... 54 Iron Studies................................................................................................................................................................................... 54 Vitamin B12 Studies...................................................................................................................................................................... 56 Folate Studies............................................................................................................................................................................... 58 Other............................................................................................................................................................................................ 59 Haemolysis Studies........................................................................................................................................................................... 60 Tests For Thrombotic Thrombocytopenic Purpura (TTP)............................................................................................................. 63 Red Cell Enzyme Tests.................................................................................................................................................................. 64 Red Cell Membrane Tests............................................................................................................................................................. 65 Haemoglobin Studies.................................................................................................................................................................... 65 Coagulation Studies.......................................................................................................................................................................... 71 Coagulation Screen....................................................................................................................................................................... 71 Fibrinogen..................................................................................................................................................................................... 76 Coagulation Factor Assays & Inhibitor Studies............................................................................................................................. 79 Platelets........................................................................................................................................................................................ 82 Von Willebrand Factor Studies..................................................................................................................................................... 85 Thrombophilia Testing.................................................................................................................................................................. 87 Antiphospholipid Antibody Testing.............................................................................................................................................. 91 Tests For Heparin Associated Thrombocytopenia........................................................................................................................ 92 Anticoagulant Testing................................................................................................................................................................... 94 Viscoelastic Testing....................................................................................................................................................................... 97 Blood Transfusion Studies................................................................................................................................................................. 98 Blood Grouping And Antibody Screening By Manual, Semi-Automated & Automated Techniques............................................ 98 Red Cell Phenotyping.................................................................................................................................................................... 98 Antibody Detection, Identification & Titre................................................................................................................................... 99 Direct & Indirect Antiglobulin Test Using Polyspecific & Monospecific Reagents........................................................................ 99 Elution Of Antibodies From Red Cells........................................................................................................................................... 99 Auto & Allo-Antibody Absorption............................................................................................................................................... 100 Cross-Matching Procedures........................................................................................................................................................ 100 Antenatal Serology..................................................................................................................................................................... 100 Methods For Detection Of White Cell & Platelet Antibodies..................................................................................................... 105 Histocompatibility Testing, Including Tests For Selection Of Donors For Transplantation........................................................ 107 Tests For Transfusion Transmissible Diseases............................................................................................................................ 108 Selection & Preparation Of Blood Components For Transfusion Purposes............................................................................... 111 Transfusion Reaction Studies..................................................................................................................................................... 114 Paediatric Studies............................................................................................................................................................................ 117 Obstetric Studies............................................................................................................................................................................. 118 Laboratory Haematology Investigations Summary – Liesl Butler Page | 1 Other Studies.................................................................................................................................................................................. 119 Immunoelectrophoresis And Immunofixation Of Serum & Urine Proteins................................................................................ 119 Cryoglobulin & Cryofibrinogen Detection.................................................................................................................................. 119 Infectious Mononucleosis (Epstein Barr Virus) & Other Common Viral Testing........................................................................ 120 Miscellaneous............................................................................................................................................................................. 120 Laboratory Haematology Investigations Summary – Liesl Butler Page | 2 Quality Control Components Of A Quality Management System Laboratory Haematology Investigations Summary – Liesl Butler Page | 3 Laboratory Haematology Investigations Summary – Liesl Butler Page | 4 Definitions Used In Total Quality Management Terms Relating To Quality Control In Laboratory Practice Laboratory Haematology Investigations Summary – Liesl Butler Page | 5 ISO 9001&2: 15189: 2003 – General Requirements For The Competence Of Testing & Calibration Laboratories International equivalent AS 4633 – Australian Standards General Requirements For The Competence Of Testing & Calibration Laboratories Management requirements o Organisation o Quality system o Document control o Review of requests, tenders and contracts o Subcontracting of tests and calibrations o Purchasing services and supplies o Service to the client o Complaints o Control of nonconforming testing and/or calibration work o Corrective action o Preventive action o Control of records o Internal audits o Management reviews Technical requirements o General o Personnel o Accommodation and environmental conditions o Test and calibration methods and method validation o Equipment o Measurement traceability o Sampling o Handling of test and calibration items o Assuring the quality of test and calibration results o Reporting the results Facilities, Work Environment & Safety Topics To Cover In A Work Safety Training Program Laboratory Haematology Investigations Summary – Liesl Butler Page | 6 Items To Be Included In Laboratory Safety Policy Document Comparison Of Classes I, II & III Biological Safety Cabinets Laboratory Haematology Investigations Summary – Liesl Butler Page | 7 Blood Spill Cleanup Steps Categories Of Health Hazards Categories Of Physical Hazards Laboratory Haematology Investigations Summary – Liesl Butler Page | 8 General Guidelines For Safe Work Practices, Personal Protective Equipment & Engineering Controls Laboratory Haematology Investigations Summary – Liesl Butler Page | 9 Biosafety Level 2 Precautions Chemical Categories & How To Work Safely With Them Laboratory Haematology Investigations Summary – Liesl Butler Page | 10 Incident Spill Response Laboratory Haematology Investigations Summary – Liesl Butler Page | 11 Managing Hazardous Chemical Spills Process Management Systematic Errors In Analyses Controls Out-Of-Range Review all components of the test system to ensure correct functioning o Assay conditions o Reagents o Calibration o Integrity of sample Quality control (QC) chart is reviewed and confirmed Check reagent levels, control batch numbers and age of controls Re-run controls (if possible also using second analyser) Re-run fresh controls (if possible also using second analyser) Change buffer Renew reagents Laboratory Haematology Investigations Summary – Liesl Butler Page | 12 Perform a rinse probe Attempt to centrifuge controls (if appropriate) Must be recorded in the problem log (including action taken) If problem unable to be rectified, the senior scientist should be notified o Unable to use instrument until resolved o Deauthorise results up until last QC was in Documents & Records Format For A Standard Operating Procedure (SOP) Laboratory Haematology Investigations Summary – Liesl Butler Page | 13 Management Of Nonconforming Events Components Of An Internal Event Report Example Of An Event Classification Laboratory Haematology Investigations Summary – Liesl Butler Page | 14 Monitoring & Assessment Comparison Of Remedial, Corrective & Preventive Actions Example Of A Cause-And-Effect Diagram National Pathology Accreditation Advisory Council (NPAAC) The National Pathology Accreditation Advisory Council (NPAAC) advises the Commonwealth, state and territory health ministers on matters relating to the accreditation of pathology laboratories. NPAAC plays a key role in ensuring the quality of Australian pathology services and is responsible for the development and maintenance of standards and guidelines for pathology practices. NPAAC is comprised of representatives from all states and territories, nominees from peak professional bodies and the Department of Health. NPAAC is a ministerially-appointed Council that was originally established in 1979 by an Order made by the GovernorGeneral (known as an Order in Council), which sets out NPAAC’s role, structure and function. The authority to create such orders is outlined in subsection 9(1) of the National Health Act 1953. The Order in Council was updated in 2003. In 1986, the Commonwealth introduced a compulsory accreditation system in relation to Medicare benefits for pathology. In order to be accredited, a pathology laboratory must meet specified quality standards. The Health Insurance (Accredited Pathology Laboratories – Approval) Principles 2002 set out the specifics of pathology accreditation and its requirements. The Principles include a Schedule that lists accreditation materials (or the standards) that relate to the actual process. The accreditation material is developed and maintained by NPAAC. Medicare benefits are only payable for pathology services if: o Approved services are performed in a laboratory within an appropriate Accredited Pathology Laboratory (APL) category o The service is rendered by or on behalf of an Approved Pathology Practitioner (APP) o The proprietor of the laboratory is an Approved Pathology Authority (APA) The Health Insurance Act 1973 includes a provision for the Federal Minister to determine the principles for accreditation of a pathology laboratory (s23DNA). Department of Human Services (formerly Medicare Australia) manages the administration of the pathology laboratory accreditation. This includes tracking the accreditation status of laboratories and managing the arrangements with organisations that conduct accreditation checks. If patients of a laboratory wish to claim Medicare benefits for services rendered, the laboratory must have APL status approved by Department of Human Services (formerly Medicare Australia). The views of an endorsed assessing body are considered by Medicare when granting APL status. Currently, NATA is the only endorsed assessing body for pathology accreditation. Laboratory Haematology Investigations Summary – Liesl Butler Page | 15 Process Improvement Examples Of Laboratory Audit Other Laboratory Set-Up Determine the aims of the laboratory: o Diagnostics o Blood banking, e.g. ability to manage MTP/critical bleeding scenarios o Obstetrics – capacity for FMH quantification and anti-D administration Engage key stakeholders: o Laboratory staff: ▪ Executive/administration (e.g. if private laboratory) ▪ Haematologists ▪ Scientists o Hospital staff: ▪ Executive/administration ▪ Medical staff ▪ Nursing staff o Patients o Department of Health (if applicable) Establish business plan and organisational committee: o Assign specific roles o Costs of tests, equipment, consumables, staff Determine specifics of laboratory function: o Tests to perform o Laboratory information system (LIS) o Staff employment/training o Compliance with standards – ISO, NPAAC, Australian Standards (AS) +/- TGA Setup and implementation Quality control and assurance: o Manuals, SOPs o Participation in QAPs – internal/external o Audits, records Laboratory Haematology Investigations Summary – Liesl Butler Page | 16 o NATA accreditation o User/staff feedback Ongoing management: o Occupational health and safety o Continuing staff education Development Of A New Diagnostic Test In The Laboratory Suitability of the instrument or clinical usefulness Therapeutic Goods Administration (TGA) – a proposal for new regulartory framework for in vitro diagnostic devices Strategy Cost Funding Implementation practicalities Trial Proficiency testing or analytic reliability of the new diagnostic method o Evaluation of performance for ▪ Specimen ▪ Request ▪ Reference ranges ▪ Accuracy ▪ Sensitivity and specificity ▪ Receiver operating characteristic curves ▪ Precision (calculating ranges on the sensitivity and specificity of a test ▪ Post-test probability Predictive values o Positive predictive value o Negative predictive value o 1-Negative predictive value Likelihood ratios o Positive likelihood ratio o Negative likelihood ratio Conversion of pre-test probability into post-test probabilities o Converting pre-test probability to odds o Nomogram ▪ Linearity and dynamic range ▪ Precision ▪ Carry over ▪ Sample stability ▪ Freedom from interference Overall work value o Turnaround time o Through put time Reagents, controls and calibration Key operator survey – staff acceptability Maintenance issues Method manual Data controlled systems Commissioning Staff training Assessment of safety Quality Laboratory Haematology Investigations Summary – Liesl Butler Page | 17 Techniques/Equipment Light Microscopy Kohler illumination o If the microscope requires an external light source, using the mirror at the base, direct the light into the condenser; if the illumination is built in, make sure that the lamp voltage is turned down before switching on the microscope, then turn up the lamp until it is at c 70% of maximum power o Place a slide of a blood film with a coverslip on the stage o Lower the condenser, open the iris diaphragm fully and bring the preparation on the slide into focus with the 10x objective o Check that the eyepieces are adjusted to the operator’s interpupillary width and that the specimen is in focus for each eye by rotating the focusing mechanism on the adjustable eyepiece o Close the diaphragm and raise the condenser slowly until the edge of the circle of light comes into sharp focus and there is a faint blue tinge at the edge of the diaphragm o Using the condenser centering screws, adjust its position so that the circle of light is in the centre of the field o Open the diaphragm completely so that light fills the whole field of view o Remove the eyepieces, so that the upper lens of the objective is seen to be filled with the circle of light; close the diaphragm slowly until the circle of light occupies about 2/3 of the surface o Replace the eyepieces, refocus the specimen and if necessary readjust the condenser aperture and lamp brightness to obtain the sharpest possible image Phase Contrast Microscopy Electron Microscopy Photo Electric Colorimeter Automated Cell Counter Automated Staining Machine Automated Or Semi-Automated Coagulation Instruments Automated Blood Bank Instruments Techniques/Equipment For Blood Transfusion Testing Electrophoresis (Serum Proteins, Haemoglobin & For Molecular Studies) Laboratory Haematology Investigations Summary – Liesl Butler Page | 18 Ph Meter Weighing Machines Centrifuge (Including Cytocentrifuge) Spectrophotometer Calibration & Use Of Diluters And Pipettes Flow Cytometry Technologies Immunoassays High Pressure Liquid Chromatography Instruments For Molecular Techniques Refrigeration/Cold Chain Equipment Laboratory Haematology Investigations Summary – Liesl Butler Page | 19 Morphology Preparation & Differential Count On Blood & Bone Marrow Aspirate Films Quality control (QC) o External ▪ RCPA Morphology o 3 samples, 4 times a year Manual differential o 1 sample, 12 times a year Paediatric morphology (virtual) o 2 cases, 2 times a year Bone marrow morphology o 2 cases, 2 times a year Selection Of Blood Films For Review And/Or Retention According To Laboratory Guidelines Scientist film review criteria o Auto-authorised ▪ Low or high reticulocyte count ▪ Addition of the following test sets within 24 hr of collection Malaria Direct Coomb test (if positive) Screening test for infectious mononucleosis (if positive) Erythrocyte sedimentation rate >100 mm o Not auto-authorised ▪ Requesting doctor is on the film exceptions list in Apollo and has requested film review ▪ Suspect or ‘?’ instrument flags not mentioned in the previous table ▪ Certain test sets requested (i.e. malaria, bone marrow and positive infectious mononucleosis screen) ▪ Cases presenting for the first time with moderate to markedly abnormal numeric results ▪ When there is an unexplained significant change from previous results ▪ Where the patient is 10 ▪ Monocytes >1.5 ▪ Eosinophils >3 ▪ MCV >106 o Previously undiagnosed ▪ Leukaemia ▪ Myelodysplasia ▪ Leucoerythroblastic blood film ▪ Lymphoma ▪ Myeloproliferative neoplasm ▪ Neutropenia (106) ▪ Moderate cytopenias for 2 consecutive FBEs ▪ Thrombocytopenia in a pregnant patient o First time patient with a stated haematological diagnosis o Morphological abnormalities ▪ Blasts – new presence or significant increase in numbers ▪ Lymphoma cells – new presence or significant increase in numbers ▪ Severely toxic/degenerative/artefacts ▪ Positive malaria ▪ Acute leukaemia in remission ▪ Fragmentation/haemolysis (hereditary or acquired)/disseminated intravascular coagulation (DIC) ▪ Marked anisopoikilocytosis or unusual morphologic change not previously noted Laboratory Haematology Investigations Summary – Liesl Butler Page | 20 ▪ Abnormal cells, reactive atypical lymphocytes and unidentified cells Other ▪ Scientist unsure of a particular cell type present ▪ Scientist is unsure or unable to determine the significance of results ▪ Referring doctor requests that the film is to be reviewed by a haematologist ▪ Sudden marked/unexpected change in ≥1 parameters ▪ Any film the scientist wishes for review Retention of films o Clinically significant (pathologist review) ▪ Collected daily ▪ Indexed by date ▪ Not confidential ▪ Boxed for 1 yr minimum and long term storage for 1 yr maximum o Not clinically significant ▪ Collected daily ▪ Indexed by date ▪ Not confidential ▪ Boxed for 1 mo minimum and long-term storage not required o Manual Leucocyte Count Sample o Well-spread film Method o Count cells using a x40 objective in a strip running the whole length of the film ▪ Avoid the lateral edges of the film ▪ Inspect the film from the head to the tail and if fewer than 100 cells are encountered in a single narrow strip examine ≥1 additional strips until at least 100 cells have been countered o Results can be recorded using a multiple manual register or can be directly entered into a computer Calculations o Differential count should be expressed as the percentage of each type of cell, should be related to the total leucocyte count and results reported in absolute numbers o Correcting the count for nucleated red blood cells ▪ Corrected WBC = total WBC – [(total WBC - %NRBC) / 100] Limitations o Variance dependent on artefactual differences in distribution owing to spreading and ‘random’ distribution Laboratory Haematology Investigations Summary – Liesl Butler Page | 21 Manual Platelet Count, Using Phase Contrast Microscopy Principle o Performed by visual examination of diluted, lysed whole blood using a Neubauer or Improved Neubauer counting chamber Sample o Venous blood in ethylenediaminetetra-acetic acid (EDTA) Method o Examine sample for blood clots (if present, a fresh specimen should be requested) o Make a 1 in 20 dilution of well-mixed blood in the diluent by adding 0.1 ml of blood to 1.9 ml of ammonium oxalate dilulent (10 g/l) o Mix the suspension on a mechanical mixer for 10-15 min o Fill a Neubauer counting chamber with the suspension, using a stout glass capillary or Pasteur pipette o Place the counting chamber in a moist Petri dish and leave untouched for at least 20 min (allowing platelets to settle) Calculations o Platelet count per litre = (# cells counted x dilution x 106) / volume counted (μL) o If N is the number of platelets counted in an area of 1 mm2 (0.1 μL in volume), the number of platelets per litre of blood is ▪ N x 10 x 20 (dilution) x 106 = N x 200 x 106 Interpretation o Examine the preparation with the x40 objective and x6 or x10 eyepieces ▪ Platelets appear under ordinary illumination as small, highly refracticle particles (if viewed with the condenser racked down) o Number of platelets in ≥1 areas of 1 mm2 should be counted (total number of platelets counted should exceed 200 to ensure a coefficient of variation of 8-10%) Limitations o All equipment must be scrupulously clean to avoid introducing dirt particles into the chamber that might be mistaken as platelets Calculation Of Red Cell “Absolute Values” Mean corpuscular volume (MCV) o MCV (fL) = = [HCT% / RBC (x 10-12/L)] x 10 Mean cell haemoglobin (MCH) o MCH (pg) = [Hb (g/L) / RBC (x 10-12/L)] x 10 Mean cell haemoglobin concentration (MCHC) o MCHC (g/dl) = [Hb (g/L) / HCT%] x 10 Full Blood Examination (FBE) Principle o Employs 3 primary analysis principles ▪ Fluorescent flow cytometry – using a semiconductor laser at 633 nM ▪ Hydro dynamic focusing (DC detection) – red blood cell and platelet analysis ▪ Sodium lauryl sulphate (SLS) haemoglobin – cyanide-free haemoglobin analysis Sample o Venous or capillary blood in ethylenediaminetetra-acetic acid (EDTA) ▪ Method o Automated cell counter (Roche Sysmex XN-9000) ▪ SLS detection Reagent haemolyses red blood cells and white blood cells in the sample Chemical reaction begins by altering the globin and then oxidising the haeme group, enabling the SLS’ hydrophilic groups can bind to the haem group and form a stable, coloured complex (SLSHGB), which is analysed using a photometric method An LED sends out monochromatic light and by moving through the mixture light is absorbed by the SLS-HGB complexes; the extinction is measured by a photo sensor and is inversely proportional to the haemoglobin concentration of the sample ▪ DC sheath flow detection A portion of blood is separated from the aspirated whole blood and mixed with the diluent in a pre-set ratio Of this dilution a defined amount is sent to the detection chamber and passed through a small opening, known as the aperture Laboratory Haematology Investigations Summary – Liesl Butler Page | 22 o ▪ There are electrodes on each side of the aperture, and direct current passes through these electrodes o The direct current resistance between the electrodes changes as blood cells suspended in the diluent pass through the aperture; this resistance causes an electrical pulse change proportional to the size of the blood cell o These electrical data are converted into graphical displays of volume distribution curves, or histograms Once the cells leave the sample nozzle exit they are surrounded by a sheath flow of diluent; they are aligned and moved to the centre of the orifice o This reduces interference errors and the possibility of abnormal cell pulse detection, which could be caused by cells passing through the transducer off-centre As soon as the cells have passed the orifice, they are seized by another, inverse flow and immediately led to the drain o This prevents renewed circulation and a change in the platelet count Fluorescence flow cytometry A blood sample is aspirated and proportioned, then diluted to a pre-set ratio and labelled with a proprietary fluorescence marker that binds specifically to nucleic acids The sample is transported into the flow cell The sample is illuminated by a semiconductor laser beam, which can separate the cells using 3 different signals o Forward scattered light (forward scatter or FSC) – indicates cell volume o Side scattered light (side scatter or SSC) – indicates intercellular information such as granularity and lobularity o Side fluorescence light (side fluorescence or SFL) – indicates type and amount of nucleic acids and cell organelles including FNA/DNA content of the cell Cells with similar physical and chemical properties form a cluster in a graph known as a scattergram Applied to several channels o WBC analysis (WNR) ▪ Used to count the WBCs and NRBCs ▪ 2-dimensional scattergram is plotted with the X-axis representing the intensity of SFL and Y-axis representing the intensity of the FSC ▪ Reagents – LYSERCELL WNR, FLUROCELL WNR ▪ Parameters – WBC, NRBC#, NRBC%, BASO#, BASO% ▪ Flags – PLT clumps, NRBC present, WBC abnormal scattergram o Differential analysis (WDF) ▪ Cell scatter properties are classified by the Sysmex adaptive cluster analysis (ACAS) as well as Sysmex adaptive flagging algorithm based on shaperecognition (SAFLAS) ▪ 2-dimensional scattergram is plotted with the X-axis representing the intensity of SSC and Y-axis representing the intensity of the SFL ▪ Reagents – LYSERCELL WDF, FLUROCELL WDF ▪ Parameters – NEUT#, NEUT%, LYMPH#, LYMPH%, MONO#, MONO%, EO#, EO%, IG#, IG%, WBC-BF ▪ Flags – atypical lymph, blast/abn lymph, IG present, PLT clumps, WBC abnormal scattergram o White precursor cells (WPC) ▪ Used for detection of immature cells ▪ 2-dimensional scattergram is plotted with the X-axis representing the intensity of SSC and Y-axis representing the intensity of the SFL ▪ Reagents – LYSERCELL WPC, FLUOROCELL WPC ▪ Flags – abnormal lymph, blasts o Reticulocyte (RET) ▪ Classifies reticulocytes and their sub-populations; fluorescent optical platelet count (PLT-O) is also derived ▪ 2-dimensional scattergram is plotted with the X-axis representing the intensity of SFL and Y-axis representing the intensity of the FSC ▪ Reagents – CELLPACK DFL, FLUOROCELL RET ▪ Flags – RET abnormal scattergram, fragments o PLT-F ▪ Classifies PLT and immature platelet fraction (IPF) Laboratory Haematology Investigations Summary – Liesl Butler Page | 23 ▪ ▪ ▪ 2-dimensional scattergram is plotted with the X-axis representing the intensity of SFL and Y-axis representing the intensity of the FSC Reagents – CELLPACK DFL, FLUOROCELL PLT Flags – PLT abnormal scattergram, PLT clumps Calculations o RET% = [particle count in reticulocyte zone / (particle count in mature RBC zone + particle count in reticulocyte zone)] / x 1000 o RET# = (RET% x RBC) / 100 o IPF = particle count in IPF zone / particle count in PLT zone) / x 1000 Quality control (QC) o Internal (reviewed weekly and 5 weekly) ▪ Third party Full set (low, medium, high) twice daily Drift (normal, medium) 4 hourly ▪ X-bar M 20 samples plotted on a Levy Jennings chart If 2 consecutive runs ‘out’ then perform QC o If QC normal then compare results with other analysers ▪ Comparison sample with regional laboratories monthly o External ▪ RCPA Full blood count (FBE) (WBC, RBC, Hb, HCT, MCV, MCH, MCHC, RDW-CV, PLT, MPV) o 2 samples, 12 times a year Reticulocyte o 2 samples, 4 times a year Automated differential o 2 samples, 4 times a year Limitations o SLS detection ▪ Turbidity (lipaemia or leucocytosis) ▪ No international reference standard available Spun Micro-Haematocrit Principle o Centrifuge used for the capillary tubes provides a centrifugal force of c 12000 g and 5 min centrifugation results in a constant PCV (further 5 min centrifugation may be necessary if PCV >0.5) Sample o Capillary blood directly collected (heparin-coated tubes) or venous blood in K2-EDTA (plain tubes) ▪ K3-EDTA causes shrinking of the red cells, reducing the PCV by about 2% o 6 hr at RT or 20% of the container volume Laboratory Haematology Investigations Summary – Liesl Butler Page | 24 o o Variation of the bore of the tubes may cause serious errors if they are not within the narrow limits of defined specifications that should be met by manufacturers: length 75 ± 0.5 mm; internal diameter 1.07-1.25 mm; wall thickness 0.18-0.23 mm; and bore taper not exceeding 2% of the internal diameter over the entire length of the tube Amount of plasma trapped between red cells, especially in the lower end of the red cell column, and red cell dehydration during centrifugation generally counterbalance each other and the error caused by trapped plasma is usually not more than 0.01 PCV units Erythrocyte Sedimentation Rate (ESR) Principle o Defined as the rate (mm/hr) at which erythrocytes suspended in plasma settle when placed in a vertical tube o Indirect measure of acute phase response o Elevated by increased fibrinogen, increased immunoglobulins and increased cholesterol Sample o Anticoagulated whole blood ▪ Sodium citrate ▪ EDTA or VES-TEC Dilute accurately in the proportion of 1 volume of citrate to 4 volumes of blood o Perform within 4 hr or collection or 6 hr if stored at 500 nm Dichroic mirror ▪ Short pass transmits 8 at 4°C o Thermal Amplitude Laboratory Haematology Investigations Summary – Liesl Butler Page | 60 ▪ ▪ Anti-I, Anti-i and Auto titres are performed at temperatures of 37°C, 30°C and 4°C to determine the thermal amplitude of the cold autoagglutinin Thermal amplitude of Anti-I, Anti-i or the cold autoagglutinin is the highest temperature at which the agglutinin is reactive Cold autoagglutinin reactivity at ≥ 30°C or above is considered clinically significant ▪ Sample o Cold agglutinin screen and titre – 10 ml plain clotted specimen (white cap) maintained strictly at 37⁰ until separated o Direct antiglobulin test (DAT) – 6 ml EDTA Method – group O adult cells (I positive) 3% screen cells (pooled), group O cord cells (i positive) cord cells 3% o Screen ▪ Label 2 x 12mm glass tubes with patient’s surname, episode number and ¼ and 1/32 ▪ Add 600uL of 5% BSA to the tube labelled ¼ and add 700uL of 5% BSA to the tube labelled 1/32 ▪ Add 200uL of patient’s serum to the tube labelled ¼ to make a ¼ dilution then mix ▪ Add 100uL of the ¼ dilution to the tube labelled 1/32 to make a 1/32 dilution then mix ▪ Label 4 x 10mm glass test tubes with patient’s surname, episode number and: OI ¼ OI 1/32 Oi ¼ Oi 1/32 ▪ Add 100uL of the 1/32 dilution to each of the 2 test tubes labelled 1/32 and add 100uL of the ¼ dilution to each of the 2 test tubes labelled ¼ ▪ Add 1 drop of O adult cells (I positive) to each of the 2 test tubes labelled OI and add 1 drop of O cord cells (i positive) to each of the 2 test tubes labelled Oi ▪ Incubate the 2 test tubes labelled ¼ at room temperature for 30 minutes and incubate the 2 test tubes labelled 1/32 at 4⁰ C for 30 minutes ▪ Centrifuge at high speed (900-1000rcf) for 15 sec and read results promptly for agglutination or haemolysis o Titre ▪ Incubate 1mL of O adult cells (I positive) and 1mL of O cord cells (i positive) at 37⁰ C for 5 min ▪ Wash autologous red cells 6 times in warm buffered saline then prepare a 3% suspension of washed autologous red cells in buffered saline by adding 40uL of washed red cells to approximately 1mL of warm buffered saline and maintain at 37⁰ C ▪ Label 12 x 12mm glass tubes with the patient’s surname, episode number and Neat (1), 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048 in which to prepare doubling dilutions of serum in 5% BSA ▪ To each tube except the neat tube, add 800uL of 5% ▪ Add 800uL of patient serum to the tubes labelled Neat and 2 ▪ Mix tube labelled 2 (½ dilution) then sample 800uL of this ½ dilution and add to the tube labelled 4 to make a ¼ dilution ▪ Mix tube labelled 4 (¼ dilution) then sample 800uL of this ¼ dilution and add to the tube labelled 8 to make a 1/8 dilution ▪ Continue in this manner making doubling dilutions to a dilution of 1/2048, using a clean pipette tip for each dilution ▪ Label 3 sets (OI, Oi, auto) of 12 x 10mm glass test tubes with the patient’s surname, episode number and Neat (1), 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048 ▪ Starting from the highest dilution (1/2048), transfer 100uL of the master titration to the corresponding tube in each of the 3 sets of test tubes ▪ Label one additional 12 x 10mm glass test tube BSA control and add 100uL of 5% BSA to this tube ▪ Incubate the 3 sets of test tubes and the BSA control tube at 37°C for 5 min ▪ Add 1 drop of warm O adult cells (I positive) to each of the OI set of test tubes, 1 drop of warm O cord cells (i positive) to each of the Oi set of test tubes, 1 drop of warm 3% autologous red cells to each of the auto set of test tubes, and 1 drop of warm 3% autologous red cells to the BSA control tube ▪ Mix and incubate the 3 sets of test tubes and the BSA control tube at 37°C for 30 min ▪ Remove tubes from the waterbath one set at a time then centrifuge at high speed (9001000rcf) for 15 sec and commencing with the highest dilution (1/2048), gently resuspend the cell button and read results promptly for agglutination or haemolysis (important to work quickly to avoid lowering the temperature of the tubes); score results using the CSL Haemagglutination Grading Chart and record results ▪ Remove the BSA control tube from the waterbath then centrifuge at high speed (9001000rcf) for 15 seconds, gently resuspend the cell button and read result promptly for agglutination or haemolysis; score results using the CSL Haemagglutination Grading Chart and record results ▪ Mix and incubate the 3 sets of test tubes and the BSA control tube at 30°C for 30 min then repeat above steps ▪ Mix and incubate the 3 sets of test tubes and the BSA control tube at 4°C for at least 2 hrs (can be left overnight); remove tubes from the refrigerator one set at a time and do not centrifuge; commencing with the highest dilution, gently resuspend the cell button and read results promptly for agglutination or haemolysis (important to work quickly to avoid lowering the temperature of the tubes); score results using the CSL Haemagglutination Grading Chart and record results Laboratory Haematology Investigations Summary – Liesl Butler Page | 61 ▪ Remove the BSA control tube from the refrigerator and do not centrifuge; gently resuspend the cell button and read result promptly for agglutination or haemolysis o Direct antiglobulin test (DAT) ▪ Must be performed if the cold agglutinin screen is positive to determine the immune specificity of the agglutinin attached to the patient’s red cells Calculations o Titre ▪ The cold agglutinin titre results are measured at 4°C ▪ The cold agglutinin titre result is the reciprocal of the endpoint dilution ▪ The endpoint of the titration the highest dilution showing a score 3 reaction ▪ If there is agglutination in the last dilution tube (1/2048), the endpoint has not been reached; report titre as > 2048 or ≥ 2048 Interpretation o Positive: agglutination or haemolysis (+) indicates presence of the corresponding agglutinin in the patient’s serum o Negative: no agglutination (0) indicates the absence of demonstrable agglutinins in the patient’s serum o 5% BSA control ▪ Should be negative ▪ Where agglutination is observed in the 5% BSA Control tube, a valid interpretation of the Auto titre result cannot be made ▪ If the 5% BSA control tube is positive, autoagglutinins were not completely removed from the patient’s red cells during washing (further washing of the patient’s red cells with warm buffered saline is required prior to repeating the Auto titre) o Reference range (titres at 4⁰ C) ▪ Anti-I 1-32 ▪ Anti-I 0-8 ▪ Auto pyruvate + ATP Pyruvate + NADH + excess LDH = lactate + NAD ▪ LDH is added in excess to the reaction mixture to ensure that the secondary reaction is not rate limiting ▪ Activity is measured by the rate of fall of absorbance at 340 nm over 5 min Calculations o Activity of the enzyme in micromoles of NADH reduced/min/ml haemolysate is calculated as follows ▪ (∆A/min / 6.22) x 10 6.22 is the millimolar extinction coefficient of NADH at 340 nm Quality control (QC) o A blank should be carried out to be certain that the lactate dehydrogenase (LDH) is free of PK activity Limitations o Important to remove as many contaminating leucocytes and platelets as possible because these cells may be unaffected by a deficiency affecting the red cells and may have high activities of PK o False-negatives with reticulocytosis o Low-substrate concentrations may be required to identify unusual variants Red Cell Membrane Tests Osmotic Fragility Principle o Small volumes of blood are combined with a large quantity of buffered saline solution at a range of concentrations o Fraction of red cells lysed at each saline concentration is assessed colourimetrically Acidified Glycerol Lysis-Time Test (AGLT) Principle o Glycerol is added to the hypotonic buffered saline solution in an effort to improve ‘time taken for lysis’ Cryohaemolysis Test Principle o Specific for hereditary spherocytosis (HS) o Dependent on molecular defects of the red cell membrane rather than just surface area-to-volume ratio Autohaemolysis Principle o Incubation of aliquots of blood with and without glucose solution at 37⁰C for 48 hours o Quantity of spontaneous haemolysis is measured using a colourimeter Membrane Protein Analysis Sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis o Qualitative and quantitative abnormalities in particular proteins Densitometry of protein o General profile Haemoglobin Studies Laboratory Haematology Investigations Summary – Liesl Butler Page | 65 Foetal Haemoglobin – Kleihauer Test Principle o Identification of cells containing haemoblobin F depends on the fact that they resist acid elution to a greater extent than do normal cells Sample o Fresh air-dried blood films (preferably within 24 hr of collection Method o Acid-elution cytochemical method ▪ Immediately after drying, fix the films for 5 min in 80% ethanol ▪ Rinse the slides rapidly under running tap water ▪ Immerse the slides in McIlvaine’s buffer (pH 3.2) for 11 min at RT ▪ Rinse the slides rapidly under running tap water ▪ Immerse the slides in Erythrosin B for 5 min ▪ Wash slides in running tap water until water runs clear ▪ Immerse slides in Harris Haematoxylin for 5 min ▪ Wash slides in running tap water until water runs clear ▪ Allow to dry Calculations o At x10 magnification, scan a minimum of 50 fields of approximately 200 red cells (10,000 red cells) for ≥4 foetal red cells ▪ 4.0 ▪ Repeat +/- repeat with venepuncture INR (action if >20% difference) ▪ Report immediately to referring doctor Quality control (QC) o Internal ▪ Code chip The code chip contains information about the test method and assay calibration; this provides the monitor with important information needed to perform the coagulation test A code chip is supplied with each box of test strips When starting each new lot number of test strips, remember to remove the old code chip from the monitor and replace it with the new code chip supplied with the strips Each time a strip is inserted in the monitor, the display shows the number of the code chip that is presently inserted; always compare the code number on the display to the number printed on the test container ▪ Inbuilt A check of the electronic components and functions every time the monitor is turned on A check of the test strip temperature while test is in progress A check of the expiry date and lot confirmation of the test strip A quality control function is incorporated into the test strip ▪ Roche Coaguchek XS and XS Plus Control With each new batch of consumbles, an operator lacks confidence in a patient result, ≥1 mo, substantial maintenance procedures have been carried out on the device, device has suffered a physical insult ▪ Patient comparison specimens With each new batch of consumbles, an operator lacks confidence in a patient result, ≥1 mo, substantial maintenance procedures have been carried out on the device, device has suffered a physical insult Acceptable limits o Individual specimens +/- 20% o R2 for all data >0.8500 o External ▪ RCPA 2 samples, 6 time a year Prothrombin Time (PT) & International Normalised Ratio (INR) Principle o Measures the clotting time of recalcified plasma when combined with an optimal concentration of thromboplastin o Assesses the extrinsic coagulation pathway, in particular the action of factor II, factor V, factor VII, factor X and fibrinogen Sample o Venous blood collected in 3.2% of sodium citrate, centrifuge immediately post collection (3000rpm for 10 min) and separate plasma o RT 40 secs Review medications then check for clots if not on anticoagulant Repeat test Laboratory Haematology Investigations Summary – Liesl Butler Page | 74 ▪ If receiving heparin o Release result If not receiving heparin and >3 sec above reference range o Perform mixing studies >200 secs Review medications and check source of specimen Release result if quality of specimen confirmed Maintenance o Daily ▪ Perform shutdown ▪ Start-up the system and computer ▪ Check analyser status ▪ Check printer paper ▪ Check +/- replace DI water ▪ Check +/- trap chamber fluid ▪ Check +/- reaction tubes ▪ Empty +/- clean reaction tube trash (reset software counter) ▪ Check +/- remove condensation from reagent table and cover ▪ Check +/- prepare reagents ▪ Check stores and write on whiteboard o Weekly ▪ Instrument clean ▪ Rinse tank clean o Monthly ▪ Left and right filter clean Quality control (QC) o Internal ▪ Reagents Controls (Biorad Lyphocheck Level 1 and Level 2) o Preparation and testing performed at start-up (morning) and at various intervals throughout the day ▪ 2 – 8 hrly ▪ Every new vial o Pre-acceptance testing ▪ Every day for 5 days a vial of the new lot of Biorad Lyphocheck 1 and 2 are reconstituted and added to QC file ▪ PT, APTT, TCT and fibrinogen are tested on both analysers for both levels of controls 4 times during the day ▪ Data is collated and appropriate target ranges entered following discussion with the senior scientist (generally assigned at mean +/- 2SD) ▪ Lot changed from new to current when satisfactory o Data saved electronically monthly and checked by senior scientist weekly ▪ Instrument Data saved electronically at the beginning of the month ▪ Staff Competency o External ▪ RCPA Haemostasis (PT, INR, APTT, fibrinogen, TCT) o 2 samples, 8 times a year Limitations o Haemolysed or clotted sample o Poor collection technique o Incorrect temperatures o Age of sample or reagents o Incorrect anticoagulant, volume or contamination o Timing in relation to heparin therapy o Lipaemic, polychthaemic and hyperbilirubinaeic samples o Poor removal of platelets Laboratory Haematology Investigations Summary – Liesl Butler Page | 75 Fibrinogen Plasma Fibrinogen Measurement Principle o Measures the amount of fibrinogen in normal plasma o Enzyme thrombin converts the soluble protein, fibrinogen, into an insoluble polymer, fibrin o Clauss technique ▪ Employs diluted plasma, which has a low concentration of inhibitors (such as FDPs and heparin) ▪ When a strong thrombin solution is added to diluted plasma, the clotting time is inversely proportional to the fibrinogen concentration Strong solution enables a clotting time to be established over a broad range that is independent of the thrombin level ▪ Patient’s clotting time is compared with that on a calibration curve (plotted using a standardised fibrinogen preparation) to determine the amount of fibrinogen Sample o Venous blood collected in 3.2% of sodium citrate, centrifuge immediately post collection (3000rpm for 10 min) and separate plasma o Place on ice until testing (including during transport) o 4°C ≤24 hrs (separated plasma) Method o Optical detection (660 nm) – Siemens Sysmex CS-5100, Siemens Thrombin, Owrens Veronal Buffer ▪ Patient samples are recorded in the COA workstation ▪ Patient sample centrifuged for 10 min at 3000rpm (if required) ▪ Load patient sample, control samples and reagents ▪ Automatic process by which the instrument downloads the test request and processes the sample Detects the change in turbidity of the sample-reaction mixture during the coagulation process; this is reflected in the change in scattered light intensity when fibrinogen is converted into a fibrin mesh Light strikes the reaction mixture, after the scattered light intensity is received by a photodiode; this converts the light intensity into electrical signals and is used to find the coagulation time ▪ Automatic reanalysis of high or low fibrinogens ▪ Review results then unload the samples Calculations Interpretation o Abnormal or unexpected result ▪ Check for clots, haemolysis & filling then repeat test ▪ Review by haematologist if reproducible Maintenance o Daily ▪ Perform shutdown ▪ Start-up the system and computer ▪ Check analyser status ▪ Check printer paper ▪ Check +/- replace DI water ▪ Check +/- trap chamber fluid ▪ Check +/- reaction tubes ▪ Empty +/- clean reaction tube trash (reset software counter) ▪ Check +/- remove condensation from reagent table and cover ▪ Check +/- prepare reagents ▪ Check stores and write on whiteboard o Weekly ▪ Instrument clean ▪ Rinse tank clean o Monthly ▪ Left and right filter clean Quality control (QC) o Internal ▪ Reagents Controls (Biorad Lyphocheck Level 1 and Level 2) o Preparation and testing performed at start-up (morning) and at various intervals throughout the day Laboratory Haematology Investigations Summary – Liesl Butler Page | 76 o ▪ ▪ o ▪ 2 – 8 hrly ▪ Every new vial Pre-acceptance testing ▪ Every day for 5 days a vial of the new lot of Biorad Lyphocheck 1 and 2 are reconstituted and added to QC file ▪ PT, APTT, TCT and fibrinogen are tested on both analysers for both levels of controls 4 times during the day ▪ Data is collated and appropriate target ranges entered following discussion with the senior scientist (generally assigned at mean +/- 2SD) ▪ Lot changed from new to current when satisfactory Data saved electronically monthly and checked by senior scientist weekly o Instrument Data saved electronically at the beginning of the month Staff Competency External ▪ RCPA Haemostasis (PT, INR, APTT, fibrinogen, TCT) o 2 samples, 8 times a year Limitations o Haemolysed or clotted sample o Poor collection technique o Incorrect temperatures o Age of sample or reagents o Incorrect anticoagulant, volume or contamination o Lipaemic, polychthaemic and hyperbilirubinaeic samples Fibrinogen/Fibrin Degradation Products (FDP) & Cross-Linked Fibrin Assays Principle o A suspension of latex particles is sensitised with specific antibodies to the purified FDP fragments D and E; the suspension is mixed on a glass slide with a dilution of the serum to be tested o Agglutination indicates the presence of FDP in the sample o By testing different dilutions of the unknown sample, a semiquantitative assay can be performed Sample o Venous blood collected in a special tube containing antifibrinolytic agent and thrombin Method o Latex agglutination ▪ Allow the tube with blood to stand at 37⁰ C until clot retraction commences; then centrifuge the tube and withdraw the serum for testing (it is important that the fibrinogen in the sample is completely clotted or this will be detected by the test) ▪ Make 1 in 5 and 1 in 20 dilutions of serum in glycine buffer ▪ Mix 1 drop of each serum dilution with 1 drop of latex suspension on a glass slide; rock the slide gently for 2 min while looking for macroscopic agglutination ▪ If a positive reaction is observed in the higher dilution, make doubling dilutions form the 1 in 20 dilution until macroscopic agglutination can no longer be seen Interpretation o Agglutination with a 1 in 5 dilution of serum indicates a concentration of FDP in excess of 10 μg/ml o Agglutination in a 1 in 20 dilution indicates FDP in excess of 40 μg/ml o Reference ranges ▪ 40 μg/ml – systemic fibrinolysis associated with disseminated intravascular coagulation (DIC), thrombolytic therapy with streptokinase Limitations o Presence of heparin or a dysfibrinogenaemia or high levels of FDPs can prevent complete clotting of fibrinogen in the sample (addition of thrombin may help) Cross-Linked Fibrin Assays Principle Laboratory Haematology Investigations Summary – Liesl Butler Page | 77 o A suspension of latex particles is sensitised with specific antibodies to the purified fibrin D-dimer; the suspension is mixed on a glass slide with a dilution of the serum to be tested Agglutination indicates the presence of fibrin D-dimer in the sample By testing different dilutions of the unknown sample, a semiquantitative assay can be performed o o Method o Latex agglutination ▪ The manufacturer’s protocol should be followed ▪ Undiluted plasma is mixed with 1 drop of latex suspension on a glass slide and the slide is gently rocked for the the length of time recommended in the kit Interpretation o Agglutination with the undiluted plasma indicates a concentration of D-dimers in excess of 200 mg/l o The D-dimer level can be quantified by multiplying the reciprocal of the highest dilution showing a positive result by 200 to give a value in mg/l o Reference ranges ▪ 20 μg/ml ▪ Reported as >20 μg/ml Quality control (QC) o Internal ▪ Innovance D-dimer Controls 1 and 2 Every run o External ▪ RCPA QAP ▪ ECAT Limitations Laboratory Haematology Investigations Summary – Liesl Butler Page | 78 o o o o o o o o Haemolysed or clotted sample Poor collection technique Incorrect temperatures Age of sample or reagents Incorrect anticoagulant, volume or contamination Lipaemic, polychthaemic and hyperbilirubinaeic samples Turbidity and particles may interfere with determination Falsely elevated or depressed result with heterophilic antibodies Euglobulin Clot Lysis Time (ECLT) Principle o Reflects the overall fibrinolytic activity of plasma o When plasma is diluted and acidified, the precipitate (euglobulin) that forms contains plasminogen activator [mostly tissue plasminogen activator (t-PA)], plasminogen and fibrinogen; most of the plasmin inhibitors are left in the solution o Precipitate is redissolved, the fibrinogen is clotted with thrombin and time for clot lysis is measured Sample o Venous blood in trisodium citrate o Transported on ice Method o Centrifuged at 4⁰ C then plasma collected and diluted with acetic acid and incubated on ice for 15 min o A precipitate forms (euglobulin fraction of plasma), which contains plasminogen, plasminogen activators (primarily t-PA) and fibrinogen o Supernatant is collected by centrifugation in a refrigerated centrifuge at 4⁰ C then discarded and the precipitate dissolved in buffer o Clotted with thrombin and the time to clot lysis determined by inspection every 15 min Interpretation o Normal range is 90-240 min o Hyperfibrinolysis 240 min Quality control (QC) o Internal ▪ Control plasma sample Run in parallel Limitations o Failure to maintain a low temperature throughout all stages of the test may lead to an artefactually long lysis time o In disseminated intravascular coagulation (DIC), a low fibrinogen concentration in the patient’s plasma gives a wispy clot, which dissolves rapidly and results in a falsely short lysis time Coagulation Factor Assays & Inhibitor Studies Factor VIII, IX, XI & XII Assays (One-Stage) Principle o Based on the ability of test plasma to correct the prolonged clotting time of plasmas with known factor deficiencies o Activity of test plasma is expressed as % of activity in normal control plasma o Activated partial thromboplastin time (APTT) is employed to assess clotting time Sample o 4.5 mL blood added to 0.5 mL citrate Method o One-stage clotting assay – Siemens Coagulation Factor Deficient Plasmas ▪ Clotting times obtained for 1 in 5, 1 in 10, 1 in 20 and 1 in 40 dilutions (Owren and Kohler buffer) of test and standard plasma Calculations ▪ Using a 1 in 10 dilution of the standard plasma as a potency of 100 IU/dL, plot the clotting times of the test and standard against the concentration of factor VII on semi-log paper ▪ Draw a horizontal line from test plasma’s 1 in 10 dilution point to the equivalent point on the standard plasma reference line, then from this point draw a vertical line to the x-axis, which will correspond to the potency of the test plasma Interpretation o Factor VIII 70-150% of the norm o Factor IX 70-120% of the norm Laboratory Haematology Investigations Summary – Liesl Butler Page | 79 o Factor XI 70-120% of the norm o Factor XII 70-150% of the norm Quality control (QC) o Internal o External ▪ Factors VIII and IX 2 samples, 6 times a year ▪ Factors II, VI, VII, X, XI and XII 2 samples, 3 times a year Limitations o Therapeutic doses of hirudin or other direct thrombin inhibitors lead to an erroneously lower factor activity o Specific inhibitors against plasmatic coagulation factors can modify the real factor activity o Partial activation of coagulation factors due to incorrect sample handling can lead to falsely elevated single factor results o Lupus anticoagulant can affect the apparent factor activity in single factor determination; non-parallelism upon dilution may also occur when a lupus anticoagulant is present Factor VIII Assay (Two-Stage & Chromogenic) Principle o Attempt to eliminate the possible interference of sample preactivation o Preactivate all the available factor VIII and assay it in a separate system by its ability to generate Xa ▪ Chromogenic assay – measured using a chromogenic Xa substrate ▪ Two-stage assay – measured by a clotting endpoint Factor II, V, VII & X Assay Principle o Based on the ability of test plasma to correct the prolonged clotting time of plasmas with known factor deficiencies o Activity of test plasma is expressed as % of activity in normal control plasma o Prothrombin time is employed to assess clotting time Sample o Venous blood collected in 3.2% of sodium citrate, centrifuge immediately post collection (3000rpm for 10 min) and separate plasma Method o One-stage clotting assay – Siemens Coagulation Factor Deficient Plasmas ▪ Clotting times obtained for 1 in 5, 1 in 10, 1 in 20 and 1 in 40 dilutions (Owren and Kohler buffer) of test and standard plasma Calculations ▪ Using a 1 in 10 dilution of the standard plasma as a potency of 100 IU/dL, plot the clotting times of the test and standard against the concentration of factor VII on semi-log paper ▪ Draw a horizontal line from test plasma’s 1 in 10 dilution point to the equivalent point on the standard plasma reference line, then from this point draw a vertical line to the x-axis, which will correspond to the potency of the test plasma Quality control (QC) o Internal o External ▪ Factors II, VI, VII, X, XI and XII 2 samples, 3 times a year Limitations o Specific inhibitors against plasmatic coagulation factors can modify the real factor activity o Partial activation of coagulation factors due to incorrect sample handling can lead to falsely elevated single factor results o Values will be reduced in patients with vitamin K deficiency and in those on oral vitamin K antagonists such as warfarin o Some F7 gene mutations (e.g. FVII Padua) can give rise to varying factor levels depending upon the source of tissue factor used; wherever possible, human recombinant tissue factor should be employed as this gives a result that more closely relates to the situation found in vivo Bethesda Assay Principle o To quantitate factor VIII (FVIII) inhibitory antibodies (inhibitors) in patients with classical, inherited haemophilia or acquired haemophilia Laboratory Haematology Investigations Summary – Liesl Butler Page | 80 o Sample o Method o o o May be used to estimate the level of inhibitors to human FVIII or adapted to measure inhibitors for Factors IX, XI, X, V etc 4.5 mL blood added to 0.5 mL citrate Heat-inactivated test plasma is mixed with an equal volume of buffered normal pooled plasma (NPP) Control is prepared by mixing equal volumes of commercial factor VIII deficient plasma and buffered NPP Serial dilutions (using commercial factor VIII deficient plasma) of heat inactivated test plasma are mixed in an equal volume of buffered NPP o Incubated at 37⁰C for 2 h (5 min if factor IX) and the FVIII activity of each mixture is measured o FVIII of the test plasma is compared to that of the control and the percentage of residual FVIII is calculated; dilutions of patient plasma are also tested Calculations o Bethesda unit is defined as the amount of inhibitor that will neutralise 50% of 1 unit of factor VIII in normal plasma after 2 h of incubation at 37⁰C o Residual FVIII % = (FVIII level of test plasma dilution / FVIII level of control plasma) x 100 o Inhibitor units/mL = [corresponding point to residual FVIII % plotted on graph (25-70%)] x (denominator of the dilution) o Dilution of the test plasma that gives the residual FVIII % nearest to 50% (30-60%) is chosen for calculating the strength of the inhibitor o Antibody is displaying a prozone effect if increasing dilutions do not give acceptable duplicates Interpretation o Residual FVIII 80-100% - no inhibitor o Residual FVIII (FXIIIa) > conjugate + NH3 Ammonia level is quantified by a parallel reaction in which a decrease in NADH is measured by absorbance at 340 nm NH3 + NADH + α-ketoglutarate > (GIDH) > NAD + glutamate Limitations o Underestimation of FXIII if elevated ammonium levels in the sample (>0.5 mmol/L) o Very low (6.0 g/L) fibrinogen concentrations may lead to falsely low FXIII activity Platelets Laboratory Haematology Investigations Summary – Liesl Butler Page | 82 Platelet Function Analyser-100 (PFA-100) Principle o Measures platelet-related primary haemostasis in citrated whole blood o Employed as an initial platelet function test in patients with a normal prothrombin time (PT), activated partial thromboplastin time (APTT) and platelet count who are suspected of having platelet-mediated bleeding Sample o Venous blood collected in 3.2% of sodium citrate, NOT centrifuged o Store at RT, process within 4 hr Method o Dade Behring PFA-100, Dade PFA Collagen/Epinephrine test cartridge, Dade PFA Collagen/ADP test cartridge, Dade PFA Trigger solution ▪ Uses two disposable cartridges that contain a membrane with a central aperture coated with aggregation agonists [collagen-epinephrine and collagen-adenosine diphosphate (ADP)], through which platelets are passed at high shear rates ▪ Measures the ‘closure time’ required for platelets to adhere to the membrane, aggregated and occlude the aperture Interpretation o Reference ranges ▪ Adult Collagen-epinephrine CT 82-150 sec Collagen-ADP CT 62-100 sec ▪