Lab Guide Hematology Section Lab Guide

Transcription

1 Lab Guide 2018 Hematology Section Lab Guide

2 Anti - Xa level Anti - Xa level One tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%). Blood should be collected between 3 to 4 hrs after the injection of heparin. Request form should include type of anticoagulation, time of last dose and time of sample collection. *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Daily Turnaround time STAT: 4hr Routine: one working day. Automated. Factor Xa is inactivated by Anti-thrombin during the incubation phase of the test. This reaction is catalyzed by heparin. Dextran Sulfate (DS) releases heparin which has bound to interfering factors and thus makes it accessible to the assay.the quantity of F Xa remaining after the incubation phase is determined via the increase in absorbance at 405 nm, using a chromogenic substrate in a kinetic test. Adults 18 years -UFH (Therapeutic Range): IU/mL - LMWH (Therapeutic Range): IU/mL for twice daily dosing or 1.00 to 2.00 IU/mL for once daily dosing. <18 years: UFH (Therapeutic Range): 0.30 to 0.70 IU/mL LMWH (Therapeutic Range): IU/mL LMWH (prophylactic): 0.10 to 0.30 IU/mL Monitoring of LMWH therapy Hematology Main Lab, HGH (ext. # )

3 Activated Partial Thromboplastin Time (APTT) Activated Partial Thromboplastin Time (APTT) One tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) For pediatric patients below 1 year: One Tube 1.0 ml pediatric tube filled up to the mark on the tube label (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Daily Turnaround time STAT: 1hr Routine: 4hrs Automated sec for adult Normal Range: Reported with each patient s result. This may vary with the reagent lot and also from one hospital to the other depending on the machine/reagent combination in service. s for below 6 months of age are reported with patients results. Heparin therapeutic range This range in seconds corresponding to an unfractionated heparin (UFH) concentration of 0.3 to 0.7 U/mL as assessed by anti Xa assay. The test is useful to monitor patients under UFH therapy. Heparin therapeutic range is (50 70sec) The APTT is an assessment of the intrinsic and common pathways of blood coagulation. It is prolonged in deficiency of prekallikrein, HMWK (High Molecular Weight Kininogen), factors: VIII, IX, XI, XII, X, V, II and fibrinogen, or by inhibitors directed against any of these factors. Hematology Main lab, HGH (ext. # )

4 Activated Protein C Resistance (APCR) Test Activated Protein C Resistance (APCR) Test One tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once a week Turnaround time STAT: 8hrs. Routine: 10 working days Automated. When activated PC (APC) is added to plasma and an APTT is performed, there is normally a prolongation of the clotting time as a result of factor V and factor VIII degradation. The sample is pre diluted with factor V deficient plasma to overcome possible deficiency of other coagulation factors. Presence of factor V Leiden will lead to shortening of the clotting time. Normalized ratio: As a screening test for FV Leiden mutation Hematology Main lab, HGH (ext. # )

5 Antithrombin Activity Antithrombin Activity One tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once a week Turnaround time STAT: 8hrs. Routine: 10 working days. Automated. The Antithrombin in the sample is converted by heparin into an immediate inhibitor and inactivates the thrombin present. The residual thrombin content is determined in a kinetic test measuring the increase in absorbance at 405 nm % for adult. s for below 6 months of age are reported with patients results. Screening for thrombophilia Hematology Main lab, HGH (ext. # )

6 Bone Marrow Bone Marrow Bone marrow aspirate, trephine biopsy (in addition to peripheral smear) all prepared at the bed side Sunday to Thursday until 12:00 p.m. Turnaround time For Emergency cases, after 12:00 PM and in Holidays/Weekend : Physician/Nursing personnel are required to ask an approval from the Laboratory Pathologist and Cytogenetics personnel before the procedure Routine 7 working days. A preliminary verbal or written report will be issued on aspirate for urgent cases ONLY. Aspirate: Smears are immediately prepared and air dried. The rest of the aspirate is allowed to clot for preparation of clot sections. If immunophenotyping or cytogenetic studies are needed, parts of the marrow aspirate will be added to the appropriate containers. --- Biopsy: Using a Jamshidi needle, the physician obtains a biopsy which is sent in formalin to the Histopathology lab for processing. Examination of Bone marrow is done to rule out marrow pathology. Final interpretation is done through integration of peripheral blood, bone marrow aspirate and trephine biopsy findings, together with the results of supplementary tests such as immunophenotyping, cytogenetic analysis and molecular genetic studies as appropriate, in the context of clinical and other diagnostic finding Hematology Main lab, HGH (ext. # )

7 Body Fluids Analysis (CSF, Pleural, Pericardial And Synovial) Days test is performed Turnaround time Body Fluids Analysis (CSF, Pleural, Pericardial And Synovial) 2-5 ml body fluids collected in glass or plastic tubes, using appropriate anticoagulant, if necessary, for analysis (as those containing EDTA or Heparin). CSF samples preferably collected in sterile glass or plastic tubes numbered according to order of draw (preferably tube# 3 for cell count), EDTA tube can be used for bloody samples. /Ref Daily 4-8 hrs Macroscopic and microscopic evaluation of body fluid with total and differential cell counting. CSF RBC 0-2/ μl WBC Adults 0-5/ μl Neonates 0-30/ μl Pleural RBC 0-10,000/ μl WBC < 1000/ μl Peritoneal/ Pericardial RBC 0-10,000/ μl WBC < 500/ μl Synovial

8 RBC / μl WBC < 200/ μl Normal range for the differential cells of the body fluid CSF Synovial All other fluids Adults Neonates Lymphocytes 40-80% 5-35% Monocytes/Macrophage 15-45% 50-90% Neutrophills 0-6% 0-8% PMN (polymorphs) - Less than 25% Mononuclear cells, including lymphocytes, monocytes, macrophages and synovial lining tissue cells are the primary cells seen in normal synovial fluid. PMN -Less than 25% Macrophages and mesothelial cells may be present. Fluid analysis is done to assist in the diagnosis or exclusion of diseases and subarachnoid hemorrhage (CSF) Hematology Main lab, HGH (ext. # ).

9 Bone Marrow Iron Stain Turnaround time Bone Marrow Iron Stain Air dried bone marrow aspirate smears. As approved by haematopathologist Reported within the BM report Prussian Blue reaction. Present in the store and erythroblast Reduced or depleted iron store is seen in iron deficiency anaemia while it is increased in anaemia of chronic disorders and haemosiderosis. Abnormal sideroblasts and ringed sideroblasts are seen in myelodysplastic syndrome and sideroblastic anaemia. Increased sideroblasts are seen in megaloblastic anemia, alcoholism and following splenectomy Hematology Main lab, HGH (ext. # )

10 Complete Blood Count (CBC) Complete Blood Count (CBC) 3 ml in EDTA Tube. /Refrigerated (specimens are stable for 24 hours if stored at room temperature and 48 hours if stored at 2-8 C) Daily Turnaround time STAT- 1 hr Routine - 4 hrs Automated. This comprises estimation of Hemoglobin (Hb), Hematocrit (Hct), Red Blood Cells (RBC) count, White Blood Cells (WBC) count, RBC indices; platelet count ± automated differential count ± reticulocyte count. See table 1 An essential test for the diagnosis and follow up of various hematological and non-hematological diseases. Hematology Main lab, HGH (ext. # ). Day 0-2 Day 3-6 Day 7-13 Day Day Day M 7M - 2 Yrs 2-6Y 6-12Y Adults RBCs M F Hb M F PCV MCV M F MCH MCHC Retic WBCs Neutro Lympho Mono Eoso Baso Platelets

11 D-Dimer D-Dimer One tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) For pediatric patients below 1 year: One Tube 1.0 ml pediatric tube filled up to the mark on the tube label (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Daily Turnaround time STAT: 1hr Routine: 4hrs Automated (0.0 up to 0.44 mg/l FEU Normal range is reported with each patient s result. This may vary with the reagent lot and also from one hospital to the other depending on the machine/reagent combination in service. D-dimer is produced by the digestion of cross-linked fibrin by plasmin. A positive test is seen in thrombosis, PE and many other situations including DIC, post-surgery, trauma, infection, malignancy, pregnancy, atherosclerosis and in the elderly D-Dimer test has been cleared by the Food and Drug Administration (FDA) to exclude Venous Thromboembolism (VTE) and pulmonary embolism (PE) at a cut-off of 0.50 mg/lfeu in patients where a physician's Pretest Probability assessment (PTP) indicates a non-high probability of pulmonary embolism. (<0.50 mg/lfeu is considered negative). If D-Dimer test is used for clinical conditions other than exclusion of Venous Thromboembolism (VTE) or pulmonary embolism (PE), then the test should be used as an aid in the diagnosis. *Cut off value may vary between labs operating different instrument/reagent. This is noted in patients' reports. Hematology Main lab, HGH (ext. # ).

12 Eosinophils in Urine /Sputum Eosinophils in Urine Minimum 5.0ml of midstream urine (first morning collection) collected in sterile plastic urine container without any preservative. Sputum :collected in sterile plastic container Turnaround time Daily 1 day Wright stained smear Negative Non-invasive test to aide in the diagnosis of Acute Interstitial Nephritis

13 Erythrocyte Sedimentation Rate Turnaround time Erythrocyte Sedimentation Rate 2ml in EDTA tube (may be part of the CBC). Microtainer tubes should not be used for Automated ESR test Daily 1 day Automated Age Sex Reference Range 0 14 years W / M 2 34 mm/hr years W 2 37 mm/hr years M 2 28 mm/hr years W 2 39 mm/hr years M 2 37 mm/hr > 70 years W / M 3 46 mm/hr W: Women; M: Men ESR is useful in disorders associated with an increased production of acute-phase proteins. It is non-specific and will be raised in any inflammatory condition. Low normal results are obtained in cases of polycythemia

14 Factor II, V, VII and X (Bio assays based on the one-stage Prothrombin Time) Factor II, V, VII and X (Bio assays based on the one-stage Prothrombin Time) 3 tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%). *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once week Turnaround time STAT: 4hrs. Routine: 10 working days Automated. Plasma deficient in any of the factors comprising the extrinsic and common pathways will result in a prolonged prothrombin time (PT). Coagulation factor deficient plasma can be used to confirm a factor deficiency, in general, and to identify and quantify coagulation factor deficiency in patient plasma. A mixture of the factor deficient plasma and the patient plasma is tested in the PT assay and the result is interpreted using reference curve obtained with dilutions of Standard Human Plasma or normal pooled plasma mixed with deficient plasma. Patient plasma deficient in a specific factor will not be able to overcome the absence of the factor in the corresponding coagulation factor deficient plasma and therefore result in a prolonged PT. Factor assays are preceded by PT/APTT and mixing study to detect a possible inhibitor % s for below 6 months of age are reported with patients results. Diagnosis of factor deficiency Hematology Main lab, HGH (ext # ).

15 Factors VIII, IX, XI, XII Factors VIII, IX, XI, XII 3 tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%). *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once a week. Turnaround time STAT: 4hrs. Routine: 10 working days Automated. A mixture of commercial factor deficient plasma and the patient plasma is tested using APTT. The result is interpreted using reference curve obtained with different dilutions of calibration plasma mixed with deficient plasma. Factor assays are preceded by PT/APTT and mixing study to detect a possible inhibitor. FVIII=70-150% FIX=70-120% FXI=70-120% FXII=70-150% s for below 6 months of age are reported with patients results. Diagnosis of factor deficiency

16 Factor XIII Activity in Plasma Factor XIII Activity in Plasma 1 tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%). *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once a week. Turnaround time STAT: 4hrs. Routine: 10 working days Automated. FXIII in the sample is converted by the action of thrombin into FXIIIa. The latter cross-links a specific peptide substrate to glycine ethyl ester, thereby releasing ammonia which is determined in a parallel enzymatic reaction. The decrease in NADH is measured by monitoring the absorbance at 340 nm % s for below 6 months of age are reported with patients results. Congenital or acquired deficiency of Factor XIII Very low fibrinogen (<0.8 g/l) or very high (>6g/l) may lead to false results.

17 Factors VIII & IX Inhibitors Factors VIII & IX Inhibitors Pre-factor infusion samples (2 tubes 2.7 ml citrated blood sample; blue top tube 3.2%) and one tube collected 30 min post infusion are needed. *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once a week Turnaround time STAT: 8hrs. Routine: 10 working days Automated. The Bethesda assay is the method used to quantitate the level of factor VIII inhibitor in patient plasma. It is calculated by incubating the patient's plasma with normal pooled plasma at 37 ⁰C for 2 hours. This will allow any antibody in the test plasma to consume factor VIII in normal plasma. Then by performing a factor VIII assay, residual factor VIII activity is measured. Inhibitors are measured in Bethesda Unit. APTT and mixing study will be performed before assaying factor inhibitors. Factor IX inhibitors are measured according to the same principle. Negative The inhibitor is an antibody directed against factor VIII and behaves as an anticoagulant producing a bleeding disorder of varying severity.

18 Fibrinogen Fibrinogen One tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%). For pediatric patients below 1 year: One Tube 1.0 ml pediatric tube filled up to the mark on the tube label (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Days test is performed Daily Turnaround time STAT: 1hr Routine: 4hrs Automated. Fibrinogen determination by Clauss method. An excess of thrombin converts fibrinogen to fibrin. The time taken for the clot to form is directly proportional to the concentration of fibrinogen in the sample g/l Normal Range Reported with each patient s result. This may vary with the reagent lot and also from one hospital to the other depending on the machine/reagent combination in service. s for below 6 months of age are reported with patients results. Diagnosis of hypofibrinogenaemia and DIC screen

19 Hemoglobin Electrophoresis Hemoglobin Electrophoresis 2 ml in EDTA tube (may be part of the CBC). Daily Turnaround time 7 working days, Automated HPLC and manual electrophoresis Adult: HbA: % HbA2: % HbF: %. Pediatric reference ranges will be released with the patient s report according to the age.. Hb electrophoresis allows the detection of abnormal hemoglobin patterns in cases of hemoglobinopathies. Hematology Main lab, HGH (ext. # ).

20 Hemoglobin H Inclusion Hemoglobin H Inclusion 3ml in EDTA tube (may be part of the CBC) Turnaround time Daily 2 working days Supravital stain Negative As adjuvant in the diagnosis of alpha thalassaemia.

21 Hemosiderin in Urine /BAL Hemosiderin in Urine Minimum 5.0ml of freshly voided urine (preferably,first morning collection) collected in sterile plastic urine container without any preservative BAL: Collected in sterile plastic urine container without any Preservative. The cells remain viable in BAL fluid for up to 4 hours when.stored at 25 C Days test is performed Turnaround time Daily 1 working day. Prussian blue reaction Negative Diagnosis of intravascular hemolysis Hematology Main lab, HGH (ext # ).

22 Heinz Body Inclusions Heinz Body Inclusions 2 ml in EDTA tube (may be part of the CBC) /Refrigerated Daily Turnaround time 1 day Supravital stain. Negative Heinz bodies represent precipitated denatured Hb and appear as single or multiple, round/oval bodies in RBC. Heinz bodies are found in the presence of unstable Hbs (such as Hb Zurich), in splenectomized patients, in some hemolytic disorders. e.g. G6PD deficiency and glutathione deficiency.

23 Heparin Induced Thrombocytopenia (HIT) Heparin Induced Thrombocytopenia (HIT) 3 ml whole blood freshly collected in PLAIN tube (without gel) red top. After appointment Turnaround time STAT: 4hrs. Routine: 1 working day. Particle Gel Immuno Assay (PaGIA) Negative( No antibody detected) Positive result: Indicates presence of antibodies specific to HPF4 complex. Negative result: Indicates absence of detectable antibodies to HPF4 complex. Disclaimer: To evaluate the diagnosis of HIT, the results of laboratory testing have to be assessed in direct combination with the pre-test score for the clinical probability of HIT. Commercial HIT immunoassays have high sensitivity (> 99%); however, these assays have low specificity (40-70%) due to the frequency of asymptomatic seroconversions (seropositivity without HIT).

24 Kleihauer Test Turnaround time Kleihauer Test 3ml in EDTA tube (may be part of the CBC) Daily 72 hrs. This is a quantitative cytochemical test for the detection of HbF- containing red cells. (acid elution) The percentage of fetal hemoglobin in adults is normally < 0.05 % The test is used to detect fetal RBCs in maternal circulation, thus estimating the amount of trans placental hemorrhage (TPH) in cases of feto-maternal Rh incompatibility or unexplained fetal anemia.

25 Lupus Anticoagulant Lupus Anticoagulant 2 tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%). *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once a week Turnaround time STAT: 8hrs. Routine: 10 working days Automated. Dilute Russell viper venom test (DRVVT-LAS and LAC) LAS= sec : LA is not detected. Positive result in case of Thrombophilia. The test should be repeated after 12 weeks to exclude transient occurrence which is seen in the course of many illnesses

26 Malaria and Other Blood parasite blood film. Blood film for Malaria and Other Blood parasite 3ml in EDTA tube (may be part of the CBC) Daily Turnaround time STAT: 3hrs Routine: 1 day Thin and thick smears. The thick smear is used as a screening test to check for the presence of parasite and the thin smear is used to identify species. Negative Diagnosis of malaria infection and other blood parasite Hematology Main lab, HGH (ext # ).

27 Microfilaria Turnaround time Microfilaria 3ml in EDTA tube (may be part of the CBC). Sample to be collected between 10pm and 3am Daily STAT: 3hrs Routine: 1 day Direct visualization of the parasite using Wright stained smear. Negative Filariasis involving the lymphatics is the cause of elephantiasis. It is caused by the filarial worms Brugia malayi, Wuchereria bancrofti & Brugia timor, whereas filarial infection of the subcutaneous tissues is caused by Loa Loa. The larvae are transmitted by mosquito to humans where they can be found in the blood and where they show periodicity with fluctuating levels at different times of the day.

28 Manual Differential count Turnaround time Manual Differential count Smears spread from EDTA (may be part of the CBC) Daily 2 working days Examination of Wright stained smear See table (1) in CBC test for Differential analytes As a re-check for the automated count. Interpreted by lab technical staff/ Hematopathologist

29 Mixing study (using PT or APTT) Mixing study (using PT or APTT 3 tube 2.7 ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%). *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Daily Turnaround time STAT: 4hrs. Routine: 2 working days The test is performed by mixing the patient s plasma with Pooled Normal Plasma (PNP) and repeating the clotting test (PT and/or APTT) in question immediately and after an established incubation period. Mixing is done when prolongation of PT is >3.0 seconds or APTT >5.0 seconds above the upper limit of the reference range. Mixing for shorter prolongation will follow the pathologist s decision. --- Investigation of an unexplained prolongation of PT or APTT. Correction indicates a possible factor deficiency, whereas failure to correct suggests the presence of inhibitor. If both the PT and APTT are prolonged, the problem is likely to be in the final common pathway. If only APTT is prolonged, the problem lies in the intrinsic pathway. If the APTT is normal and PT is prolonged then the problem lies in the extrinsic pathway.

30 Neutrophil Alkaline Phosphatase Score (NAP score/lap) Turnaround time Neutrophil Alkaline Phosphatase Score (NAP score/lap) Direct smears spread immediately after collection. After appointment 2 working days Cytochemical methods applied to hematopoietic cells allow the demonstration of specific enzymes or other substances in individual cells. Alkaline phosphatase activity is found predominantly in mature neutrophils and to some extent in metamyelocytes. An overall score is obtained by assessing the stain intensity in 100 consecutive neutrophils which are scored from 0-4 Score Low level in CML (almost zero) and PNH. High scores in leukaemoid reactions

31 Osmotic Fragility Turnaround time Osmotic Fragility 2-3 ml in lithium heparin tube (green top) accompanied with 2 ml blood in EDTA tube. After appointment. 3 working days The blood sample is pre-incubated at 37 C for 24 hrs. before testing. The test determines the resistance of the red cells to hemolysis in varying concentrations of hypotonic saline. MCF: g/l NaCl This test is done for the diagnosis of hereditary spherocytosis.

32 Prothrombin Time (PT) Prothrombin Time (PT) One tube 2.7ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) For pediatric patients below 1 year: One Tube 1.0 ml pediatric tube filled up to the mark on the tube label (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Daily Turnaround time STAT: 1hr Routine: 4hrs Automated sec for adult. Normal range is reported with each patient s result. This may vary with the reagent lot and also from one hospital to the other depending on the machine/reagent combination in service. s for below 6 months of age are reported with patients results. Assessment of the extrinsic and common pathways of blood coagulation. It is prolonged in deficiency of factor II, V, VII, X and fibrinogen or in presence of an inhibitor to any of these factors. Monitoring of Warfarin therapy. Rarely reagents used in different labs may show high sensitivity to lupus anticoagulant in the patient s plasma. It is recommended that such patients are monitored in the same lab.

33 Peripheral Smear Peripheral Smear Smears spread from EDTA blood. Turnaround time Daily 2 working days This is a thin film examined for morphological assessment of RBC, WBC and Platelets. Peripheral smears are prepared from CBC samples according to criteria set by the lab. NA Peripheral smear examination is of unquestionable value in sorting out hematological abnormalities.

34 Platelet Aggregation Turnaround time Platelet Aggregation 5 tube 2.7ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. On appointment 2 working days Platelet rich plasma (PRP) is prepared by centrifugation of the patient s citrated sample at low speed. Measurement of platelet aggregation is done by monitoring the transmission of light through stirred platelet-rich-plasma (PRP) to which an aggregating agent has been added. As the platelets aggregate, the transmission of light increases and is proportional to the amount of aggregation occurring. Generally aggregation of 70 % or greater will occur in response to ADP, Arachidonic Acid, Ristocetin and Collagen utilizing normal PRP. The test is usually preceded by an abnormal PFA test. Response of platelets to different agonists in vitro yields different aggregating patterns that are interpreted by the pathologists, taking into consideration platelet morphology, clinical history and other coagulation test results. NB: Ristocetin is tested in 2 concentrations (high and low) for the detection of type IIB vwd.

35 Platelet Estimation in Peripheral Smear Turnaround time Platelet Estimation in Peripheral Smear Smears spread from EDTA blood. Daily 2 working day Platelets are estimated in Wright- stained peripheral smear See table (1) in CBC test for platelet Re-checking platelet count in questionable automated result Hematology Main lab. HGH (ext. # ).

36 Platelet function Test -PFA-200 (in Vitro Bleeding time) Platelet function Test -PFA-200 (in Vitro Bleeding time) Two tube 2.7ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) + one EDTA tube *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Daily Turnaround time STAT: 4hrs. Routine: 2 working days Automated. Whole blood is incubated at 37 C and subjected to high sheer within a capillary tube. The blood flows through an aperture within a membrane coated with the platelet agonists collagen and ADP/Epinephrine. Platelets adhere to the membrane and aggregate to form a plug within the aperture. The change in flow rate of the blood with time is recorded as a closure time in seconds EPI: sec ADP: sec Prolonged PFA closure times in different patterns are seen in patients under anti-platelet medications, hereditary and acquired platelet disorders.

37 Protein C Protein C One tube 2.7ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once a week Turnaround time STAT: 8hrs. Routine: 10 working days Protein C in the patient sample is activated by a specific snake venom activator. The resulting Activated Protein C is assayed in a kinetic test by measuring the increase in absorbance at 405 nm % for adult. s for below 6 months of age are reported with patients results. Determination of the activity of protein C, especially in cases suspected of thrombophilia

38 Protein S Protein S One tube 2.7ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%). *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once a week Turnaround time STAT: 8hrs. Routine: 10 working days Automated. Protein S enhances the anticoagulant action of activated protein C. This enhancement is reflected by the prolongation of the clotting time of a system enriched with factor Va which is the physiological substrate for activated Protein C *M: 72 >126 % * F: 56.1 >126% * for adult. s for below 6 months of age are reported with patients results. Screening for thrombophilia

39 Reptilase Time (RT) Reptilase Time (RT) One tube 2.7ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Daily Turnaround time STAT: 1hr Routine: 4 hrs Automated. Batroxbin (Reptilase) Reagent is added to plasma and the clotting time recorded sec Prolonged Reptilase Time is seen in hypo- and dysfibrinogenaemia, DIC and post thrombolytic therapy (high FDPs) Reptilase time is rarely performed separately. Since it is not affected by heparin, it is useful in ruling out heparin contamination of the sample when thrombin time is prolonged.

40 Reticulocyte Count Turnaround time Reticulocyte Count Whole blood (1 ml) in EDTA tube or as part of the CBC specimen. /Refrigerated Daily 4 hrs A supravital stain is used to stain remnants of RNA in the cytoplasm of young RBCs (reticulocytes). The number of reticulocytes in 1000 RBCs is determined and reported as %. Currently automated CBC analyzer is used for reticulocyte counting. Adults % Pediatric reference ranges will be released with the patient s report according to the age. Reticulocyte count is an index of red cell production by the bone marrow. Increased reticulocyte count occurs in compensated anemias e.g. haemolysis, bleeding etc; while decreased reticulocyte count occurs in marrow failure as in aplastic anemia. Hematology Main lab HGH (ext. # )

41 Sickling Test Sickling Test 3ml in EDTA tube (may be part of the CBC) /Ref Turnaround time Daily 2 working days Sickling test may be requested alone or is done as part of Hb electrophoresis. SICKLEDEX method: A strong reducing agent reduces the hemoglobin. Reduced Hb-S is insoluble in the concentrated phosphate buffer and forms a cloudy, turbid suspension. Negative Positive Sickling test occurs in Hb S disease, sickle cell trait and Hb S/C double heterozygosity. The test is done to confirm/rule out sickling, or any time when S peak/band is detected by HPLC or Hb electrophoresis.

42 Thrombin Time (TT) Thrombin Time (TT) One tube 2.7ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Daily Turnaround time STAT: 1hr Routine: 4 hrs Automated, based on Clauss method. Thrombin is added to plasma and the clotting time recorded sec Prolonged TT is seen in hypo- and dysfibrinogenaemia, heparin contamination and high FDPs as in DIC and liver diseases.

43 Von Willebrand Factor (VWFAg assay) Von Willebrand Factor (VWFAg assay) One tube 2.7ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%) *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. One a week Turnaround time STAT: 4hrs. Routine: 10 working days Automated. Small polystyrene particles to which specific antibodies to VWF have been attached by covalent bonding are aggregated when mixing with samples containing von Willebrand antigen. This aggregation is then detected turbidimetrically via the increase in turbidity, which is proportional to the antigen level present in the test sample. Blood Group O: % Non-O Blood Group: % s for below 6 months of age are reported with patients results. Diagnosis of vwd. It also aids in the differential diagnosis of hemophilia A along with other tests such as mixing study, bleeding time (by PFA-200), Ricof and factor VIII activity.

44 Von Willebrand Factor (VWF) Activity Von Willebrand Factor (VWF) Activity One tube 2.7ml citrated blood sample filled up to the mark on the tube label. (Light blue top tube, citrated 3.2%). *In case of high hematocrit (>55%) contact the lab before extracting blood for all coagulation testing because special tube(s) will be provided. Once a week Turnaround time STAT: 4hrs. Routine: 10 working days Automated. The assay principle makes use of the binding of VWF to its receptor Glycoprotien Ib(GPIb). (GP Ib) is the main VWF receptor on platelets. Polystyrene particles are coated with an antibody against (GPIb).Recombinant GPIb (two gain-of-function mutations incuded) is added and binds to the antibody as well as to the VWF of the sample. Due to the gain-of function mutations, VWF binding to GPIb does not require ristocetin. This VWF binding induce a particle agglutination which can be measured as increase in extinction by turbidimetric measurements Blood Group O: % Non-O Blood Group: % Diagnosis of von Willebrand disease (vwd)