Preanalytical-Haematology

Sample collection:

• 1. Use venous blood samples. 

  2. Please avoid sampling from indwelling lines. 

  3. Hematology test samples must be collected in EDTA tube or 3.2 % Trisodium Citrate tube unless stated otherwise.

  4. Blood sampling should be done with minimal venostasis (immediately release the tourniquet once the blood started to flow into the syringe). 

  5. Do not apply the tourniquet for more than 1 minute.

  6. To ensure a consistent and accurate result, follow strictly the blood volume required for the type of test specified, or fill the blood sample to the mark on the tube to ensure adequate anticoagulant-to-blood ratio. 

  7. The minimum sample volume for FBC is 1mL.

  8. Anticoagulant for hemostasis tests must be adjusted for cases with a high hematocrit value of more than 55% to maintain correct anticoagulant-to-blood ratio. Please call hematology lab Ext 7329 / 8241 for further clarification.

  9. To prevent hemolysis: 

     • Use recommended needle size i.e. 21-23G needles.

     • Avoid vigorous mixing.

     • Send the specimen to the laboratory as soon as possible after collection.

  10. Avoid clot formation by:

      • Ensuring a smooth venipuncture and steady flow of blood into the syringe.

      • Introduce the blood into the anticoagulant bottle as soon as the blood has been drawn (according to “Order of Draw’).

      • Mix immediately by gently inverting the tube at least 3-4 (sodium citrate tube) or 8-10 times (EDTA tube).

  11. Please make sure the labels on the specimen and form are correct with legible writing.

  12. Sample must be sent immediately after blood collection for all routine hematology tests.

  13. Retesting interval

      • BCR-ABL quantitative assay: Six monthly.

      • CD4/CD8 enumeration: Six monthly.

      • FBC: Once daily.

      • Hb analysis: Do not repeat unless suggested by the Pathologist.

      • PBF: 7 days.

1. Bone Marrow Aspirate (BMA)

1. 1. Please call ext: 7329 to arrange for an appointment (working days only).

   2. PBF sample is required to be sent together.

   3. Fill in the request forms with relevant CLINICAL INFORMATION for immunophenotyping, cytogenetic and molecular study for all cases.

   4. For BMA cases from OUTSTATION,  please include

      1. Minimum 5 unstained slides.

      2. Recent FBC and PBF slide.

      3. Sample for immunophenotyping analysis (if required)

2. Specialised hemostasis tests

2. 1. It is MANDATORY to fill in the form with relevant clinical history and drug history (i.e. anticoagulant usage/ factor replacement therapy including date and time of last dose given). Bleeding assessment can be done using  ISTH-SSC Bleeding Assessment Tool.

2. 2. Patient on anticoagulant or certain drugs should be discontinued as below:

      1. Warfarin 2 weeks

      2. Un-fractionated heparin (UFH) 24 hours

      3. Low molecular weight heparin (e.g; clexane) 12-24 hours

      4. Direct oral anticoagulant (e.g; dabigatran, apixaban) 72 hours

      5. Emicizumab 6 months (Further reading)

2. 3. For thrombophilia testing, please use this guideline

      1. Testing should be delayed until at least 6 weeks after the acute event.

      2. Arterial thrombotic event is not an indication for heritable thrombophilia study.

      3. Testing for heritable thrombophilia study is not recommended in patient with CVC-related venous thrombosis.

      4. Lupus anticoagulant testing during pregnancy does not exclude or confirm a diagnosis of APS. Further read.

      5. Anti-coagulant should be discontinued as above.

2. 4. Sample must be sent immediately or within 30 minutes after blood collection to the Haematology Laboratory during office hours.

2. 5. For sample from OUTSTATION LABORATORIES, 

      1. Sample MUST be filled to the mark on the tube to ensure adequate anticoagulant-to-blood ratio. 

      2. The sample MUST be double spun immediately to produce platelet poor plasma.

         • Procedure to produce platelet poor plasma

           1. Centrifuge sample at 3500RPM for 10 minutes.

           2. Transfer the plasma to transfer tube.

           3. Recentrifuge sample at 3500RPM for 10 minutes.

           4. Transfer the plasma to transfer tube.

              1. For adult sample: Transfer to 4 separate tubes (minimum 1ml each tube).

              2. For pediatric sample: To discuss with hematopathologist for determination of the number of tubes to be sent (minimum 1ml each tube). 

      3. Plasma is to be kept in a frozen state (for storage) and send to Hematology Unit, Pathology Department, QEH in a cool box packed with ice. 

      4. Warm/thawed samples upon arrival will be rejected. 

3. Hb analysis

3. 1. Hb Electrophoresis is NOT indicated as first-line test for investigation of anaemia or hypochromic microcytic anaemia in adults, except in cases with hepatosplenomegaly to suggest thalassemia intermedia. 

   2. Please exclude iron deficiency first either biochemically or by trial of iron therapy in younger patients.

   3. In accordance with the National screening policy, Hb electrophoresis will only be performed in cases with MCV <80 fl or MCH <27. Hence, rare milder cases of silent or MCH or MCV normal Thalassemia will not be detected.

   4. Post transfusion sample will not give accurate result. Kindly send Hb analysis 3 months post-transfusion.

   5. Neonatal less than 1 year of age has physiologically high HbF. Kindly discuss with pathologist if Hb analysis is required.

   6. For samples from OUTSTATION, laboratory personnel must attach 

      1. Peripheral blood film smear.

      2. Recent FBC result labelled with 2 identifiers (Name and IC/Passport number).

   7. All Hb analysis requests must be registered in iThal system.

4. DNA analysis for alpha and beta-thalassemia

4. 1. Please use the latest form. 

   2. Recent FBC (within 3 months) and a copy of Hb analysis report must be attached. The attached FBC result must be labelled with 2 identifiers (name and IC/passport number).

   3. All paediatric (<12 y.o) samples to be referred to IMR, MUST be accompanied by both parents' FBC results and Hb analysis reports. Parents' samples are not required.

   4. The consent form must be filled and signed by the patient or parents/guardian if the patient is less than 18 years old.

   5. The request and consent form must be signed by a medical officer.

   6. For cascade or family screening, please state the index case's name, IC number/lab number, diagnosis, and relation with the index case.

5. DNA analysis for haemophilia A and B

5. 1. Please use the latest form. 

   2. All carrier screening must be accompanied by an index sample with a separate request form.

   3. A copy of the index and relative genetic test result (if available).

   4. Family tree.

   5. The consent form must be filled and signed by the patient or parents/guardian if the patient is less than 18 years old.

   6. The request and consent form must be signed by a medical officer.

6. BCR-ABL quantitative assay

   1. BCR-ABL 1, Major (p210), Quantitative.

   2. For chronic myeloid leukaemia treatment monitoring.

   3. Sample must be send immediately by 3.30pm, Tuesday.

   4. The number of cases is LIMITED to ONLY 6 cases per week. 

   5. The scheduling of appointments and the selection of appropriate cases are the responsibility of the on-duty Clinical Haematologist.

   6. The test application form must be completed in full (please specify the diagnosis date, the start date of TKI treatment, and the date of the last BCR-ABL1 molecular result), and must include the applicant’s signature and stamp.

   7. Ensure that the DATE and TIME of specimen collection are clearly written on the test application form.

   8. Ensure that the requested test is not less than 6 months from the previous test submitted.

7. G6PD screening and quantitative assay

   1. The measurement of G6PD (Glucose-6-Phosphate Dehydrogenase) enzyme activity can be significantly influenced by conditions like severe anemia, recent transfusion, acute hemolysis, and reticulocytosis. Here's how each condition affects G6PD activity:

      1. Severe Anemia: G6PD is predominantly found in red blood cells (RBCs). In severe anemia, the total number of RBCs is reduced, leading to a decrease in the overall G6PD activity. 

      2. Recent Transfusion: Transfusions introduce donor RBCs, which may have normal G6PD levels. The test may falsely indicate normal or borderline enzyme activity.

      3. Acute Hemolysis: During acute hemolysis, older, enzyme-deficient RBCs are destroyed preferentially, leaving behind younger RBCs and reticulocytes. These younger cells have relatively higher G6PD activity, which can give a falsely normal or borderline result during enzyme testing.

      4. Reticulocytosis: Reticulocytes have inherently higher G6PD activity than mature RBCs. In cases of reticulocytosis (e.g., following hemolysis), the proportion of reticulocytes increases, potentially leading to overestimation of G6PD enzyme activity in the test.