ANTIBODY SCREEN
Detection of allo- or autoantibodies directed against red blood cell antigens in the settings of pre-transfusion testing, haemolytic anaemia, pregnancy and transplantation (donor/ recipient).
Transfusion and pregnancy are the primary means of sensitization to red cell antigens. In a given population, 2% to 4% of the general population possess irregular red cell alloantibodies. Such antibodies may cause haemolytic disease of the newborn or haemolysis of transfused donor red blood cells.
If the antibody screen is positive or the negative control is invalid when tested by Awanui, then an antibody identification will be referred to either New Zealand Blood service (NZBS) or North shore hospital (NSH) for further testing. This will be clearly indicated on the Awanui report.
Clinical evaluation of antibodies identified is necessary to determine their potential for harm to the patient at this time and to assess appropriate action to be taken in the future.
APTT TEST
The APTT is used to screen for abnormalities of the intrinsic and common pathways of the coagulation cascade. Furthermore, an APTT may also be used for monitoring of heparin therapy.
A prolonged APTT may be due to: Haemophilia A (factor VIII deficiency), haemophilia B (factor IX deficiency), von Willebrand Disease, factor XII deficiency (not usually associated with abnormal bleeding), factor XI deficiency, liver disease, disseminated intravascular coagulation, heparin therapy, factor inhibitors (most commonly against F VIII), lupus anticoagulant.
If the APTT is prolonged, then a 1:1 mix with normal plasma will be performed.
Short APTT usually reflects factor activation from a difficult collection or recent trauma/surgery.
The APTT may not be prolonged if the setting of mild clotting factor deficiency and is more sensitive in cases of moderate to severe clotting factor deficiency. If mild disorder is suspected based on the clinical, bleeding or family history, even in the presence of a normal APTT, specific factor assays or von Willebrand testing should be performed.
Collection artefacts may result in a spuriously prolonged APTT, such as: heparin contamination, clots, incompletely filled tubes (excess citrate), polycythaemia (citrate/plasma ratio inappropriate), high lipid levels and TPN.
APTT 1+1
An APTT 1+ 1 is performed when the APTT is prolonged as the first step in the investigation. It is a mix of patient and normal pool (patient) plasma in a 1:1 ratio.
Complete correction of the APTT with 1+1 suggests a clotting factor deficiency. No or partial correction suggests the presence of an inhibitor e.g. heparin, direct thrombin inhibitors, lupus anticoagulant or a specific factor inhibitor (antibody).
BLOOD FILM
There are certain situations where a blood film is automatically generated in our laboratory. All CBC samples are run on our analyser and the results of each patient pass through an algorithm which considers current CBC findings, cumulative results on the specific patient and analyser ‘flags’. This algorithm will determine whether a blood film is automatically generated or not.
Blood films will also be assessed if specifically requested by the clinician and a reason is provided with any useful clinical information.
The laboratory scientist reviews the blood film manually to confirm cell counts, enumerate cell populations and to describe red cell, white cell and platelet morphology. Interpretative comments are then added when appropriate. Each laboratory has predetermined criteria for referral of the blood film to a haematopathologist for further assessment. In such cases, an additional comment may be made by the pathologist providing more detailed information, a specific diagnosis and management/investigation advice.
Some of the commonly described morphological features are detailed in the links below.
https://bpac.org.nz/Supplement/2008/May/docs/bpac_cbc_in_primary_care.pdf
Red cell abnormalities and terminology:
Dimorphic refers to the presence of two distinct red cell populations on the blood film. This most commonly occurs after red cell transfusion or iron therapy. It can also be seen in myelodysplasia; refractory anaemia with ringed sideroblasts; haemolytic processes involving a reticulocyte response; and erythropoietin therapy.
Echinocytes are red blood cells with numerous, fine, uniform spicules along the periphery. They are often an artefact of due to exposure to EDTA prior to smear preparation but may be physiologic, commonly occurring in association with renal disease / uraemia.
Elliptocytes/ Oval cells are abnormally shaped red blood cells that appear oval or elongated, from slightly egg-shaped to rod or pencil forms. Elliptocytes are commonly associated with hereditary elliptocytosis. However, they may also be seen in iron deficiency anaemia, megaloblastic anaemia and other dyserythropoetic states.
Spherocytes are round RBCs that are smaller in diameter than normal RBCs, lack central pallor, and have a denser (hyperchromic) staining quality. This is due to a loss of membrane and change in surface area to volume ratio. This is prominent in the setting of immune-mediated haemolysis or congenital red cell membrane defects such as hereditary spherocytosis. They also can be seen in snake bites and thermal injury.
Stomatocytes: Stomatocytes are erythrocytes with an elongated (mouth-like) area of central pallor. An occasional cell of this type might be seen as a non-specific finding in a variety of situations, such as regenerative anaemias, neoplasms, cardiovascular and hepatobiliary disease, alcoholism, and therapy with drugs. It can be seen in congenital disorders such as membrane disorders
Polychromasia refers to the grey blue appearance of immature red cells released prematurely into the blood stream. This can be in response to a drop in Hb as in bleeding or haemolysis or can be due to a response to treatment such as iron therapy in iron deficiency anaemia.
Acanthocytes are dense, shrunken, and irregularly shaped red blood cells with spikes on the outside. These cells form from changes in the fats and proteins on red blood cells’ outer layers. They are associated with both inherited and acquired diseases. Many adults have a small number of acanthocytes in their blood. Common causes include:
• Severe liver dysfunction.
• Abetalipoproteinemia.
• Post-splenectomy.
• Hypothyroidism.
• Myelodysplastic syndromes.
• Anorexia nervosa.
• Medications.
Howell Jolly bodies
Howell-Jolly bodies are nuclear remnants found in red blood cells (erythrocytes) under various pathological states. They are most commonly present in patients with absent or impaired function of the spleen; this is because one of the spleen’s functions is to filter deranged blood cells and remove the intracellular inclusions left by the erythrocyte precursors
White cell abnormalities and terminology:
Leucoerythroblastic blood film Presence of nucleated red cells and early myeloid cells with or without anaemia. This can be seen in a marked bone marrow response, especially due to severe acute blood loss, acute haemolysis or rebound recovery of the bone marrow. Other causes include severe infection, severe megaloblastic anaemia, bone marrow infiltration, myelofibrosis, acute myeloid leukaemia and myelodysplasia.
Reactive lymphocytes Reactive lymphocytes are large, immune-stimulated lymphocytes they can be seen in infection, drug reactions, stress and asplenia.
Monomorphic population this described a population of cells left shift
Left shift is a term used to describe an increase in more immature forms of a cell line seen in a blood sample it is commonly used in the context of the granulocyte lineage. This may be physiologic in response to a stress on the body such as pregnancy, infection or inflammation; it may occur with administration of granulocyte colony stimulating factor (G-CSF); or it may be a sign of bone marrow infiltration.
Immature granulocytes Causes of a raised Immature Granulocyte Count include sepsis, inflammation, haematological or other malignancies, and conditions where there is a leucoerythroblastic picture.
Smear cells are lymphocytes with membranes that have ruptured primarily when a blood smear is prepared. They are found in conditions where the lymphocytes are more fragile and are commonly seen in Chronic lymphocytic leukaemia. They may also be seen in infectious conditions.
Platelet changes
Large and Giant Platelets: Large and giant platelets are seen when platelet turnover is increased (e.g. auto-immune disease, infections (incl. H pylori), splenomegaly and drug induced thrombocytopenia. Other causes of giant platelets may be due to myelodysplasia or myeloproliferative conditions or more rarely congenital thrombocytopenia syndromes.
Platelet clumping: The presence of platelet clumps on blood smear, whether platelet count is normal or low, it has no clinical significance and does not represent a disease state. The most common cause of platelet clumping in blood is EDTA (the anticoagulant used in test tubes for complete blood counts). Follow up is only required if an accurate platelet count is required in which case a Sodium citrate (blue) tube should be sent for a platelet count (Please state Citrate tube for platelets due to previous platelet clumping).
Platelet satellitism: a rare in vitro phenomenon presenting with platelets rosetting around neutrophils (PMN). It occurs when an IgG antibody forms in the presence of EDTA, but not in blood samples treated with heparin or sodium citrate. Follow up is only required if an accurate platelet count is required in which case a Sodium citrate (blue) tube should be sent for a platelet count (Please state Citrate tube for platelets due to previous platelet satellitism in the clinical details).
BLOOD GROUP ONLY
Blood grouping is performed to:
- determine the patient’s red cell type prior to blood transfusion/transplantation
- in pregnancy to assess the risk of haemolytic disease in the newborn.
BLOOD GROUP AND ANTIBODIES
Blood group
Antibodies
BONE MARROW
A bone marrow biopsy may be used to:
- diagnose and stage hematologic disorders
- detect infiltration of a marrow by a nonhematologic malignancy
- to determine the cause of cytopenias
- to confirm or exclude metabolic diseases or infectious
The bone marrow biopsy is performed by a pathologist at our Awanui Carbine road site and can only be requested by a clinical haematologist. We take a bone marrow aspirate and trephine biopsy.
The aspirate sample is prepared and stained for the pathologist to assess on the same day where possible. From the initial screening, additional special tests may be requested (e.g. flow cytometry, standard cytogenetics) and sample is then sent to our referral laboratory (currently LabPlus) for processing.
The trephine biopsy is sent to Anatomical pathology services (HNZ) for processing which is a long process that can take up to 5 working days.
The aspirate and trephine are reported together in a single report. If the findings are delayed, it is usually a result of the pathologist requiring supplementary stains/tests to provide a diagnosis.
COAGULATION PROFILE/ STUDIES
The Coagulation screen is useful in a number of scenarios including:
- A pre-operative screen
- To monitor bleeding disorders
- To monitor medication (e.g. warfarin)
- To assess patients with a history of bleeding/ bruising
- To assess patients with a history of thrombosis history
- To assess a patient with a known family history of bleeding/bruising
It must be noted that these parameters are helpful but do not detect all haemostatic abnormities, thus clinical history is still of the utmost importance to aid in the diagnosis of a condition.
Clinical relevance
A raised PT or APTT result, without a known cause or with clinical symptoms (e.g. bleeding, bruising), should always be further investigated. In cases where both the PT and APTT are prolonged, common factor pathway deficiency, warfarin/superwarfarin, vitamin K deficiency, DIC and liver disease should be considered.
COMPLETE BLOOD COUNT
The complete (full) blood count is performed for a wide range of indications. It is used to:
- Confirm or exclude the reduction or increase of a particular cell line eg. Anaemia or a high neutrophil count
- Diagnosis of a medical condition (e.g. leukaemia, malaria)
- Monitoring of a patient on treatment (post-op, chemotherapy)
Please note the ranges for adults do not change with age it should never be assumed that a decrease in the blood count is due to ageing. BPAC resource in the link has information on individual parameters.
https://bpac.org.nz/Supplement/2008/May/docs/bpac_cbc_in_primary_care.pdf
D-DIMER
A D-dimer is requested:
- To rule out a deep vein thrombosis (DVT).
- For the work-up of other medical conditions e.g. DIC.
Due to the incidence of false negatives, a D-dimer should ONLY be used in the setting of LOW suspicion for pulmonary embolism (PE) or LOW suspicion for DVT.
Increased D. Dimer levels occur in any situation where excess clotting is followed by fibrinolysis e.g. recent thrombosis or thromboembolism but also in bleeding, surgical operations (up to 8 weeks post op), haematoma formation, DIC and thrombolytic therapy. D-dimers can also be increased in a variety of clinical situations e.g. malignancy, pregnancy, renal failure, liver disease, snake bite, or infection/inflammation.
A D-dimer has a LOW positive predictive value for VTE.
Wells score for DVT : https://www.mdcalc.com/calc/362/wells-criteria-dvt
Wells score for PE: https://www.mdcalc.com/calc/115/wells-criteria-pulmonary-embolism
DILUTE THROMBIN CLOT TIME
The Thrombin Clotting Time (TCT) reflects inhibitors to thrombin e.g. heparin, FDP and fibrinogen abnormalities (low levels, dysfibrinogenaemia) and direct thrombin inhibitors e.g. Dabigatran.
TCT is most commonly used as part of the evaluation of a prolonged APTT to check for the presence of heparin which markedly prolongs the TCT.
Interpretation:
- Prolonged TCT is common in liver disease and newborn infants (dysfibrinogenaemia). Clinically, there are rarely associated bleeding problems.
- The TCT is markedly prolonged in the presence of direct thrombin inhibitors, novel anticoagulants
DIRECT ANTIGLOBULIN TEST/ DIRECT COOMBS TEST
The direct antiglobulin test (DAT) is used to detect immunoglobulin and/or complement on the surface of red blood cells. The utility of the DAT is to distinguish between immune and non-immune aetiology. As with all tests, DAT results must be viewed in light of clinical and other laboratory data. A low-level positive DAT is a common finding in elderly and hospitalised patients and is a non-specific finding.
DIC SCREEN
No single test is sufficiently specific and robust to confirm the presence of a DIC. A diagnosis of DIC is therefore made on clinical suspicion supported by appropriate laboratory tests grouped together in a DIC screen. Identification of the underlying precipitating clinical disorder is key to appropriate investigation and management.
ESR
The ESR is an indirect measure of inflammation; it does not measure an analyte but rather a physical phenomenon that depends on a large number of physiological, pathological and analytical (test) variables.
Technical factors which may give erroneous results and affect the ESR’s validity include specimen age (>4hrs), improper filling of the ESR tube, inadequate anticoagulants, and ambient room temperature.
From a quality perspective there is considerable variation between results obtained by different methods, very poor precision, and lack of suitable material for quality assurance. Thus, results performed at different laboratories are not comparable.
The ESR may be used in the initial assessment of the conditions listed below. Thereafter the CRP is recommended for ongoing monitoring, except in the rare instance when only the ESR is raised.
- Paediatric inflammatory bowel disease: ulcerative colitis/Crohn’s disease (initial presentation only)
- Systemic lupus erythematosus (SLE)
- Connective tissue disorder
- Vasculitis/arteritis
- Temporal (Giant cell) arteritis
- Juvenile idiopathic arthritis
- Rheumatoid arthritis
- Polymyalgia Rheumatica
- Kawasaki Disease
- Rheumatic fever
- Hodgkin lymphoma
- Suspected prosthetic joint inflammation
Both ESR and CRP should be requested simultaneously in the initial workup of patients with possible Giant Cell Arteritis (GCA), because rarely one or other marker may be raised in isolation.
The ESR should not be used to screen for plasma cell dyscrasias. If these conditions are suspected, protein electrophoresis and/or serum free light chains should be used.
Reference intervals:
| Male upper limit of normal | Female upper limit of normal |
| ≤20 years <10 mm/h | ≤20 years <10 mm/h |
| 21-50 years <20 mm/h | 21-50 years <25 mm/h |
| 50 years <25 mm/h | 51-65 years <30 mm/h |
| 65 years <40 mm/h |
FIBRINOGEN
Is a glycoprotein complex produced by the liver and is converted to fibrin in the final part of the clotting cascade. (This final step is measured by the Dilute Thrombin Clotting Time (TCT).
Increased fibrinogen
As part of acute phase response. e.g. acute and chronic inflammation, liver disease, pregnancy, compensated intravascular coagulation, nephrotic syndrome.
Decreased
In congenital hypofibrinogenemia (rare), DIC, advanced liver disease, lytic therapy, L-asparaginase therapy. Acquired dysfibrinogenaemia occurs mainly in association with liver and renal disease. Bleeding is rarely a problem. Typically, thrombin clotting time is long and /or fibrinogen is low.
Result interpretation can be difficult in the presence of dysfibrinogenaemia, high FDP and high dose heparin, when fibrinogen can appear (falsely) low.
NOTE: In consumptive processes serial fibrinogen levels are of more use than single point assays.
FILARIA PARASITES/ FILARIAL PARASITES
This Is a wet prep slide test to identify microfilariae in the peripheral blood.
Microfilariae are occasionally found in patients from tropical areas, especially the Pacific Islands, where the parasite is endemic. It causes blockage of the lymphatic system. W bancrofti (var. Pacifica) is most common.
Diagnostic sensitivity is increased with the number of smears evaluated. Brugian filariasis tend to show nocturnal periodicity (greatest number of parasites found in the blood between 10pm and 2 am) whereas W Bancrofti appear in the blood in the daytime (diurnal periodicity). The timing of blood specimen collection is important for diagnosis via blood smear examinations.
G6PD ASSAY / SCREEN
A G6PD assay is used in the investigation of haemolytic anaemia, particularly with features of oxidative haemolysis or a clinical history suggestive of G6PD deficiency (ingestion of certain foods, medications, stress or infection causing haemolysis or family history). G6PD deficiency should be excluded prior to commencing treatment with certain medications including primaquine, tafenoquine and rasburicase.
- Reticulocytosis can mask a G6PD deficiency. If reticulocytosis is present and G6PD deficiency is suspected, suggest a repeat screen when the reticulocyte count returns to normal.
- A normal screen test does not exclude the presence of a female carrier state. If clinically indicated, family studies and enzyme assays can be requested.
HAEMOSIDERIN
Urine haemosiderin is done to look for the presence of haemoglobin in the glomerular filtrate. This is broken down into haemosiderin. The presence of haemosiderin in the uroepithelial cells indicates chronic intravascular haemolysis. Haemosiderin is not found in the urine at the onset of a haemolytic attack even if this is accompanied by haemoglobinaemia and haemoglobinuria, as the haemoglobin is initially absorbed by the tubular cells of the kidney. Haemosiderin may persist in the urine for weeks after a haemolytic episode.
HAEMOGLOBINOPATHY SCREEN
Includes:
- Capillary electrophoresis (CE)
- Alpha ICT (not in babies under 6 months and if HbF > 5%) – Positive in >90% of alpha thalassaemia phenotypes especially two alpha chain deletions in same chromosome and Three alpha deletions(HbH disease).
- Isopropanol (only performed where an abnormal band is detected)- also useful to detect unstable Hb variants, which may not show in Capillary EPG
- Sickle solubility test (if a band is detected in the S zone of CE is detected on Capillary EPF or requested for immigration/ refugee screening)
- HbH bodies (only in babies where Alpha ICT is not appropriate)
Haemoglobinopathy screening is performed on the Capillarys system with the Capillarys Haemoglobin (E) kit and demonstrates separation of the normal haemoglobins (A, A2 and F) in human blood samples and detection of the major haemoglobin variants (S, C, E and D) and HbH and alpha variants by capillary electrophoresis in alkaline buffer (pH 9.4).
The haemoglobinopathy screen is used to determines the presence and relative amounts of abnormal forms of haemoglobin in order to screen for and/or diagnose a haemoglobin disorder
These screens are commonly performed for: prenatal screening, symptomatic individuals and in family studies. Where screening will not affect management in children this should be done at sexual maturity (after 16 years of age). Thalassaemia screening is not usually useful in people after their childbearing years unless it is going to change management or if it is required for family studies.
INTERNATIONAL NORMALISED RATIO (INR)
An INR is used to monitoring warfarin therapy and also forms part of broader testing of the coagulation cascade.
The therapeutic interval for oral anticoagulant therapy varies according to the clinical indication. Using a computer-based algorithm may be useful for dosing warfarin and the frequency of testing should be adjusted to minimise phlebotomy when the patient is in the therapeutic range.
https://www.hqsc.govt.nz/assets/Our-work/Improved-service-delivery/Primary-care/Publications-resources/Example-of-Primary-Care-Guidelines-for-Warfarin-Maintenance.pdf
LUPUS ANTICOAGULANT
A lupus anticoagulant study is used to detect the presence of a lupus anticoagulant (LA) in the blood. LA is an autoantibody that can be transient with no clinical significance or can lead to antiphospholipid syndrome with an increased risk of arterial or venous thromboembolism.
LA testing may be used to help determine the cause of:
- Arterial or venous thromboembolism
- Recurrent miscarriages
- An unexplained prolonged APTT test
A Lupus Screen includes an APTT, PTT-LA, dilute Russell Viper Venom Time and correction studies. Patients should not be tested whilst on anticoagulant therapy as this may give rise to false positive results.
MALARIAL PARASITES
The malaria screen consists of a thick and thin films and where malaria is detected a malaria ICT.
Microscopy is more sensitive than the ICT and therefore it is only done where malaria is identified. In smaller centres with less expertise the ICT is done in conjunction with films.
The malaria screen is used to detect malarial parasites and if present help with speciation.
NEONATAL GROUP AND COOMBS
Refer to Blood Group and Direct Coombs
NEONATAL SCREEN
Refer to Antibody Screen
PROTHROMBIN TIME/ PROTHROMBIN RATIO
The PT is also a screening tool to detect deficiencies of the extrinsic clotting pathway (Factors: I, II, V, VII and X ) due to: hereditary coagulation disorders, liver disease or Vitamin K deficiency.
The PT is used to calculate the INR for warfarin monitoring.
RETICULOCYTE COUNT
The reticulocyte count reflects the erythropoietic activity of bone marrow and is used to monitor bone marrow activity. A high reticulocyte count in the setting of anaemia indicates the bone marrow is functioning appropriately whereas a normal or low reticulocyte count in the setting of anaemia may indicate the bone marrow is unable to respond either to a nutritional deficiency or bone marrow cause.
The Reticulocyte haemoglobin (Ret Hb) is always reported together with the Reticulocyte count
RET HB
Reticulocyte haemoglobin equivalent (Ret-He) is the measure of haemoglobin content in reticulocytes produced by Sysmex FBC analysers. Ret-He is a more sensitive reflection of available iron for haemoglobinisation and may be more clinically relevant than traditional red cell parameters, such as MCV and mean cell haemoglobin, as mature red blood cells can persist in circulation for up to 120 days compared to 3–5 days for reticulocytes. Studies suggest a RET-He is useful in distinguish iron deficiency anaemia from anaemia of chronic disease. In iron deficiency anaemia the RET-He is usually <25 pg. In anaemia of chronic disease and reduced tissue iron store without anaemia the RET-He will be low-normal or slightly reduced but usually >25 pg. RET-He does not distinguish between iron deficiency anaemia and thalassemia trait, another cause of hypochromic microcytic anaemia. In thalassemia trait the RET-He is low also (<25 pg).