HAEMATOLOGY INTRODUCTION

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HAEMATOLOGY INTRODUCTION Haematology Laboratory offers a comprehensive range of routine and specialised haematological investigations.

SPECIAL INSTRUCTIONS ON SAMPLE COLLECTION AND HANDLING 1. Follow standard precaution guidelines. Treat all samples as potentially hazardous. 2.

1 HAEMATOLOGY INTRODUCTION Haematology Laboratory offers a comprehensive range of routine and specialised haematological investigations. The laboratory also offers Routine Urine Sediment Microscopy and molecular testing for diagnosis of haematological disorders such as Leukaemia and Haemophilia. SPECIAL INSTRUCTIONS ON SAMPLE COLLECTION AND HANDLING 1. Follow standard precaution guidelines. Treat all samples as potentially hazardous. 2. Venous blood should be used as it has a lower margin of error. 3. Samples should be labelled with at least two separate identifying items (e.g. patient s name and NRIC) and must be accompanied by a requisition form. 4. Sample collection time should be written on the requisition form. 5. The anticoagulant used should be as denoted by the test procedure written under. Samples collected in anticoagulant should be well mixed by inversion at least four times. The tubes should not be shaken as mechanical trauma can induce platelet activation and/or red cell lysis. The following tubes containing certain anti-coagulants have to be noted: (a) Blood for routine examinations such as Full Blood Counts and ESR (b) Blood for coagulation studies (c) Hb Electrophoresis 3 ml tubes containing potassium EDTA as anti-coagulant Tubes containing 0.3 ml 3.2% sodium citrate for 2.7 ml blood, respectively 3 ml tubes containing potassium EDTA 6. Samples for haemostasis/coagulation tests must be collected in a way that preserves the integrity of easily activated or denatured proteins, enzymes and cofactors. A clean venipuncture with adequate blood flow into tubes or syringe provides the best specimen. The tubes should be filled to at least 90% full volume. In practice, ± 10% of the stated draw is acceptable. The correct order of draw should be followed to eliminate the risk of cross-contamination of additives during venipuncture and to reduce the risk of clotting in EDTA tubes. The recommended order of draw is: _PathoH_SL.indd 141 4/3/08 1:02:44 PM

2 SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS 142 (a) blood culture tubes (b) sodium citrate tubes (c) serum and serum gel tubes (d) heparin and heparin gel tubes (e) EDTA tubes (f) fluoride tubes 7. Collection of blood for coagulation testing through intravenous lines that have been previously flushed with heparin should be avoided, if possible. If the blood must be drawn through an indwelling catheter, possible heparin contamination and specimen dilution should be considered. When obtaining specimens from indwelling lines that may contain heparin, the line should be flushed with 5 ml of saline, and the first 5 ml of blood or 6-times the line volume (dead space volume of the catheter) be drawn off and discarded before the coagulation tube is filled. Prior to PT/INR testing or Factor VII assay, specimens should be maintained at room temperature to avoid cold activation of Factor VII and platelet disruption. 8. Urine Samples for Urine Sediment Microscopy, Urine Haemosiderin and Urine for fat globules should be collected as described in Clinical Microbiology Section and despatched to Haematology Lab as soon as possible as the urine sample must be examined within 1 2 hours of collection. REFERENCE VALUES IN HAEMATOLOGY There is no reliable method of defining normal values and the term reference range is used. 95% of healthy individuals have results within the reference range. The following factors may affect the haematological values: The sex, age, occupation, body build, genetic and ethnic background and geographical location, especially latitude. The environmental and physiological conditions under which the samples are obtained, including the subject s diet, and his posture when the sample was taken. The technique and timing of sample collection, transport and storage. Variation in the analytical method used. Some tests e.g. blood film and marrow morphology are observer-dependent. CALL-BACK VALUES OF CRITICAL ANALYTES Critical haematological analytes and their call-back values are listed below. Results are relayed by telephone to the requestor and recorded in our laboratory information system. Haemoglobin < 5.0 or > 20.0 g/dl (Adult) < 5.0 or > 25.0 g/dl (Newborn) WBC < 1.0 or > /L Platelets < 20 or > /L APTT > 100 seconds INR > 5.0 Fibrinogen < 0.9 or > 7.8 g/l Malaria Parasites or other Blood Parasites: Positive New cases of leukaemia Combination of strong positives (++) for glucose and ketones when dipstick is ordered _PathoH_SL.indd 142 4/3/08 1:02:45 PM

3 HAEMATOLOGY ALPHABETICAL TEST LISTING HAEMATOLOGY ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Photo-optical Clot Detection : seconds (CA 1500) : 3 4 hours : Monday Sunday ANTI-HEPARIN PF4 ANTIBODIES : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : ELISA : 3 days ANTI-THROMBIN III, FUNCTIONAL PLASMA : 3 ml blood containing 0.3 ml of 3.2% sodium citrate Note: Please indicate if patient has recently been treated with heparin. Heparin may lower plasma anti-thrombin III. : Chromogenic assay : % : 1 week : Once a week ANTI Xa ASSAY (FOR MONITORING LOW MOLECULAR WEIGHT HEPARIN) Please indicate the type of heparin given. : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Chromogenic assay : Prophylactic range: IU/mL : Therapeutic range: IU/ ml : 3 4 days : Monday Friday (office hours) AUTOHAEMOLYSIS Includes incubation with/without glucose for 48 hours. : By appointment only (Tel No.: ) Specimen to be collected by arrangement with the laboratory staff : Spectrophotometry : % % (with glucose) : 3 days : Monday Wednesday (office hours) _PathoH_SL.indd 143 4/3/08 1:02:45 PM

4 SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS B-CELL GENE REARRANGEMENT Please specify : (a) t(11:14) bcl-1/jh (b) t(14;18) bcl-2/jh (c) IgH Gene Rearrangement : 10 ml EDTA marrow or 15 ml EDTA blood : One week BCR/ABL (QUALITATIVE) BCR/ABL (QUANTITATIVE) : 10 ml EDTA marrow or 15 ml EDTA blood : 2 weeks : 10 ml EDTA marrow or 15 ml EDTA blood : Real Time Polymerase Chain Reaction : 2 4 weeks BLEEDING TIME : Test to be done by technician by appointment. Note: Please indicate if patient has recently ingested aspirin. Aspirin will prolong the bleeding time. Test should be done only after stopping the medication for at least one week. : Template : 1 day 5 months : minutes 5 months 15 years : minutes > 15 years : 2 8 minutes : 1 hour BLOOD FOR BASOPHILIC STIPPLING : 3 ml EDTA blood : Microscopy : 3 4 hours BLOOD FILM REPORT Includes RBC morphology, WBC and Platelets estimations : 3 well made peripheral blood smears or 3.0 ml EDTA blood : Microscopy : 3 4 hours _PathoH_SL.indd 144 4/11/08 12:52:54 PM

5 HAEMATOLOGY BLOOD FOR MALARIA PARASITE : 3 ml EDTA blood or thick film : Microscopy : 3 4 hours : Monday Sunday BLOOD FOR MICROFILARIA : 3 ml EDTA blood or thick films To be taken preferably around midnight. : Microscopy : 3 4 hours BONE MARROW EXAMINATION : 2 well made peripheral blood films and 8 bone marrow smears. All slides should be air-dried, unfixed and unstained. Another 8 10 slides are required for cytochemical stains for the diagnosis of new leukaemia and lymphoma cases. Note: All bone marrow reports include a blood film report. Iron stains are done routinely on all marrow aspirates. : Microscopy : 3 4 days CD 34 HSC ENUMERATION CD 4/8 ASSAY : 3 ml EDTA blood : Flow Cytometry : 1 2 days : 3 ml EDTA blood : Flow Cytometry Cell markers Unit 50 years > 50 years CD 4 % CD 4 absolute # X10 9 /L CD 8 % CD 8 absolute # X10 9 /L CD 4/CD 8 ratio : 1 2 days _PathoH_SL.indd 145 4/11/08 12:53:28 PM

6 SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS CYTOCHEMICAL STAINS : Unstained and unfixed bone marrow smears or peripheral blood smears : Microscopy : 1 2 days D-DIMER TEST QUANTITATIVE : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Immunoturbidimetry : ug/l : 1 day EOSINOPHIL COUNT : 3 ml EDTA blood : Microscopy : ( ) 10 9 /L : 4 hours ERYTHROCYTE SEDIMENTATION RATE : 3 ml EDTA blood : Westergren : Male : 1 10 mm/hour Female : 50 years 3 15 mm/hour > 50 years 3 20 mm/hour : 3 4 hours FACTOR ASSAYS Specific Coagulation factor assays are available individually. Each assay utilises the same sample requirement. Order the specific factor required. : 3 ml blood containing 0.3 ml of sodium citrate Patient must not be on heparin. : Clotting Assay _PathoH_SL.indd 146 4/11/08 3:05:34 PM

7 HAEMATOLOGY Factor assay I(Fibrinogen) g/l II(Prothrombin) % V % VII % VIII % IX % X % XI % XII % XIII % FACTOR INHIBITOR, SPECIFIC : 2 days : Monday Friday (office hours) : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Clotting Assays : 1 day : Monday Friday (office hours) FIBRINOGEN ASSAY (See Factor Assay) FACTOR V LEIDEN ANALYSIS : 3 5 ml EDTA blood/citrated blood : PCR and Restriction Endonuclease Digestion : 1 2 weeks : Monday Friday (office hours) FIP1L1-PDGFRA FUSION GENE : 10 ml EDTA bone marrow or 15 ml EDTA blood : 2 weeks _PathoH_SL.indd 147 4/3/08 1:02:45 PM

8 SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS FLT 3 ITD MUTATIONS Norma Test result : 10 ml EDTA bone marrow or 15 ml EDTA blood : 2 3 weeks FULL BLOOD COUNT Includes WBC, RBC, Hb, HCT, MCV, MCHC, PLT and Differential Count. : 3.0 ml EDTA blood : Impedance Counting/Flow Cytometry Test Unit Newborn Adult (M) Adult (F) WBC X 10 9 /L RBC X /L HB g/dl HCT % MCV fl MCH pg MCHC % PLT X 10 9 /L NEUTROPHIL # X 10 9 /L LYMPHOCYTE # X 10 9 /L MONOCYTE # X 10 9 /L EOSINOPHIL # X 10 9 /L BASOPHIL # X 10 9 /L : 3 4 hours : Monday Sunday HAEMATOCRIT (PCV) : 3 ml EDTA blood : Computation from Indices : 0 7 days : 44 64% 7 days 3 years : 32 44% 3 6 years : 36 44% > 6 years : M : 38 52% F : 36 46% : 2 hours : Monday Sunday _PathoH_SL.indd 148 4/11/08 12:56:18 PM

9 HAEMATOLOGY HAEMOGLOBIN (HB) : 3 ml EDTA blood : Photometric : 0 7 days : g/dl 7 days 1 month : g/dl 1 month 1 year : g/dl 1 year 12 years : g/dl > 12 years (Male) : g/dl > 12 years (Female) : g/dl : 2 hours : Monday Sunday HAEMOGLOBIN ELECTROPHORESIS : 3 ml EDTA blood : Cellulose Acetate Electrophoresis Hb A, Hb A 2 and Hb F by HPLC. May include agar gel electrophoresis if results so warrant : Hb A 0 30 days : 10 40% > 6 months : 95 98% Hb A days : < 0.1% > 1 year : % Hb F 0 30 days : 70 90% > 2 years : % : 2 3 days (identification of abnormal variants will take longer) HAEMOGLOBIN F (KLEIHAUER TEST) : 3 ml EDTA blood : Acid Elution Test : < 1% fetal cells (excluding cord blood and neonates) : 1 day HAEMOGLOBIN H INCLUSION BODIES : 3 ml EDTA blood : Brilliant Cresyl Blue Incubation : 3 4 hours HAEMOGLOBIN, UNSTABLE : By appointment only. 3 ml EDTA blood Note: False positive results will be obtained in blood specimens that contain > 5% fetal haemoglobin or in specimens that are received more than a week after the blood has been drawn _PathoH_SL.indd 149 4/11/08 12:58:30 PM

10 SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS : Isopropanol Solubility : 1 day : Monday Friday (office hours) HAEMOPHILIA LINKAGE ANALYSIS (FS) : 5 10 ml EDTA blood of patient and family members : 2 weeks HAEMOPHILIA LINKAGE ANALYSIS (CVS) : Chorionic Villus Sampling (CVS) & 5 ml EDTA blood Note: By appointment only. Please call Molecular Lab (Tel: ) before the CVS procedure to ensure the availability of the informative marker. : 1 week HAEMOPHILIA A INTRON 1 INVERSION : 10 ml EDTA blood : 1 2 weeks HAEMOPHILIA A INTRON 22 INVERSION : 10 ml EDTA blood : Long distance Polymerase Chain Reaction : 1 2 weeks HEINZ BODIES : 3 ml EDTA blood : Methyl Violet Staining : 3 4 hours _PathoH_SL.indd 150 4/11/08 2:22:28 PM

11 HAEMATOLOGY IMMUNOPHENOTYPING Please specify : (a) Acute Leukaemia Panel (b) CLL Panel (c) Myeloma Immunophenotyping (d) PNH Screening (e) PNH Full Panel (f) General Lymphoid Panel (g) LPD (T & NK Cells) (h) Other Markers (Please specify) : 3.0 ml EDTA blood/bone marrow : Flow Cytometry : 1 week INR (See Prothrombin Time) LUPUS ANTICOAGULANT JAK2 MUTATION : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Clotting : 1 week : Monday Friday (office hours) : 10 ml EDTA blood or bone marrow : 2 3 weeks NEUTROPHIL ALKALINE PHOSPHATASE (NAP SCORE) : 3 well made peripheral blood films which should be airdried, unfixed and unstained : Azo dye Coupling : : 1 day : Monday Friday (office hours) NITROBLUE TETRAZOLIUM TEST (NBT TEST) : 2.5 ml lithium heparin blood Note: Specimens to be sent to the laboratory immediately for viable cells : PMA Stimulation : 63 90% : 1 day : Monday Friday (office hours) _PathoH_SL.indd 151 4/11/08 12:59:31 PM

12 SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS NPM1 MUTATIONS : 10 ml EDTA bone marrow or 15 ml EDTA blood : 2 3 weeks OSMOTIC FRAGILITY By appointment only. The blood should be sent within 2 hours of collection. : 3.0 ml EDTA blood NaCl (g/l) % Lysis (before incubation) % Lysis (after 24 hours incubation) : 1 2 days : Monday Friday (office hours) PLASMINOGEN ACTIVATOR INHIBITOR : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Chromogenic Assay : 0 10 Au/mL : 2 weeks : Fortnightly PLASMINOGEN ASSAY : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Chromogenic Assay : % : 1 week : Weekly _PathoH_SL.indd 152 4/3/08 1:02:58 PM

13 HAEMATOLOGY PLATELET COUNT : 3 ml EDTA blood : Impedance Counting : /L : 2 3 hours : Monday Sunday PLATELET FUNCTION TEST Include PML/RAR-ALPHA : Aggregation with 5 reagents Note: To make appointment with laboratory (Tel: / ). Patient to fast overnight prior to test and should not be on aspirin for at least 2 weeks : 3 ml blood containing 0.3 ml of 3.2% sodium citrate 6 tubes. Specimen should be kept at room temperature and should not be kept in ice. : Platelet Aggregation with: (ADP) % (Collagen) % (Epinephrine) % (Ristocetin) % (Arachidonic Acid) % : 1 day : Monday Friday (office hours) : 10 ml EDTA marrow or 15 ml EDTA blood : 1 2 weeks PROTHROMBIN ASSAY (See Factor Assays) PROTHROMBIN G20210A ASSAY : 3 5 ml EDTA blood/citrated blood : 1 2 weeks PROTHROMBIN TIME (INR WILL BE GIVEN UPON REQUEST) : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Photo-optical Clot Detection : seconds : 3 4 hours : Monday Sunday _PathoH_SL.indd 153 4/11/08 1:00:03 PM

14 SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS PROTEIN C ASSAY (FUNCTIONAL) : 3 ml blood containing 0.3 ml of 3.2% sodium citrate Note: Coumadin will lower the Protein C level. Assay cannot be performed if patient is on heparin. : Chromogenic Assay : % : 1 week : Weekly PROTEIN C RESISTANCE (ACTIVATED) : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Clotting : : 1 week : Weekly PROTEIN S (FUNCTIONAL) : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Clotting : % : 1 week : Weekly PROTEIN S (FREE) : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : ELISA : % : 2 weeks : Fortnightly PROTEIN S ANTIGEN, TOTAL : 3 ml blood containing 0.3 ml of 3.2% sodium citrate Note: Coumadin will lower Protein S level. : ELISA : % : 2 weeks : Fortnightly REPTILASE TIME : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Clotting : seconds : 3 4 hours : Monday Saturday _PathoH_SL.indd 154 4/3/08 1:02:59 PM

15 HAEMATOLOGY RETICULOCYTE COUNT : 3 ml EDTA blood : Supravital Staining with Brilliant Cresyl Blue : 1 8 days : % > 8 days : % : 2 3 hours : Monday Sunday RISTOCETIN CO-FACTOR : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Platelet Aggregation : % : 1 week : Weekly SICKLING TEST : 3 ml EDTA blood : Sodium Metabisulphite Reduction Test : 2 days : Monday Friday (office hours) SOLUBLE FIBRIN/FIBRINOGEN MONOMER : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Haemagglutination : 3 4 hours TCR GENE REARRANGEMENTS : 10 ml EDTA bone marrow or 15 ml EDTA blood : 1 week THROMBIN TIME : 3 ml blood containing 0.3 ml of 3.2% sodium citrate : Clotting : seconds : 3 4 hours : Monday Sunday _PathoH_SL.indd 155 4/11/08 1:00:38 PM

16 SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS URINE SEDIMENT MICROSCOPY : 12 ml fresh urine : Kova Glasstic Slide : RBC : 0 3 /ul WBC : 0 6 /ul Epithelial cell : 0 4 /ul : 4 hours : Monday Sunday URINE HAEMOSIDERIN URINE FOR FAT GLOBULES VNTR ANALYSIS : 12 ml fresh urine : Prussian Blue Reaction : Negative : 4 hours : 12 ml fresh urine : Sudan III Staining : Negative : 4 hours : 10 ml EDTA bone marrow or 15 ml EDTA blood (donor & recipient) (PCR) : 1 week VON-WILLEBRAND FACTOR ANTIGEN : 3 ml blood containing 0.3 ml of 3.2% sodium citrate Note: Test for ristocetin co-factor and Factor VIII activity is recommended in conjunction with the above test. : Immunological ELISA : % : 1 week : Once a week WHITE BLOOD CELL COUNT (WBC) : 3 ml EDTA blood : 1 7 days : /L 7 days 1 month : /L 1 month 2 years : /L > 2 years : /L : 2 3 hours : Monday Sunday _PathoH_SL.indd 156 4/11/08 1:00:55 PM

Schedule of Accreditation issued by United Kingdom Accreditation Service 2 Pine Trees, Chertsey Lane, Staines-upon-Thames, TW18 3HR, UK

Schedule of Accreditation issued by United Kingdom Accreditation Service 2 Pine Trees, Chertsey Lane, Staines-upon-Thames, TW18 3HR, UK Laboratory locations: 2 Pine Trees, Chertsey Lane, Staines-upon-Thames, TW18 3HR, UK Blood Sciences Pathology Building Hull Royal Infirmary Anlaby Road Hull HU3 2JZ Contact: Dr Andrew Botham Tel: +44 (0)1482